Sven Laming

Duperron Sébastien

Marina R Cunha

Gaudron Sylvie
email: sylvie.gaudron@snv.jussieu.fr

ADAPTIVE DEVELOPMENTAL ANATOMY IN THE DEEP-SEA, CHEMOSYMBIOTIC MUSSEL IDAS MODIOLAEFORMIS

accompaniment to wine is my marvellous wife Clare. Thank you so much for following me on this journey. I ① so ① fo ① all① the① st ess,① o plai i g① a d① despai .① I ① g ateful① fo ① all① the① jo ,① e hila atio ① a d① ou ① everlasting patience and love. I know it has been an un-royal mess at times but I never have anything other tha ①fu ① he ①I ① ith① ou.①②t①so e①poi t①i ①the①futu e①I①hope①to①look① a k①a d①s ile①at①these①da s.①He e s① to our future.

It s been a hard time for family, and when members of mine had much bigger things going on they still took the time to ask how it was all going. Thanks for the support! To my supervisors: I could not, in all honesty, have asked for a better compliment of people to have as a network of support, as colleagues and lifelong friends, I should imagine. It has been through this holy trinity that① I e① a ed① ① u o e tio al① path① th ough① this research. You gave me the freedom to work in my own peculiar way, for which I am very grateful. With each of you I have had the most enlightening conversations, both in science and out of it and I cannot express enough how that has enriched this whole experience. To Sébastien, I wish to express my gratitude for putting a roof over my head when no-one else could, and for the highly engaging coffee breaks, guitar music (I hear you play the Bossa Nova...?), and above all else your infectious positivity (excluding the special type of positivity you reserve for the trains...). To Sylvie, thanks for the phone calls to ask if I had remembered X or Y, and for the level of personal trust you placed in me. I ①pa ti ula l ①g ateful①fo ①so eo e① ho① ill①laugh①at① ①jokes,①so① u h①app e iated.① Marina, above all else, I wish to thank you for wise council and quiet support of my methods.... and duck rice... I had the most wonderful time in Portugal and UA, and can truly say that Clare and I both felt welcome whilst there.

To my colleagues and friends: To my friends back in Norn Irooon, we are both so grateful for the love and support we have received through undoubtedly difficult times. I look forward to a few drinks once all this is behind me! To the folks in Team Amex, UPMC, but especially Kamil and Clara, a whopping big thank you for the support you have all shown me through the course of the PhD. To Kamil thanks for your Polish perspective on the French language. Always refreshing, and the school-boy humour will never get old! Clara, a huge thank you for taking the time out of your busy life to help me when I asked: including the abstract! To others, thanks to Paul for lodgings when I was homeless! To Nadine Le Bris, Katell Guizen, and the whole team at Banyuls, my sincerest thanks. Cheers to Ghisaine Frebourg, Catherine Pierre, Anne-Sophie Martinez, Chiara Romano, Justine Thubaut, Nelly, Juliette, Magali and Bruce Bossman: thanks for just being in the right place at the right time! To the MARES family, thanks for a great bunch of friends and various and unexpected training sessions! A big thank you in particular to people in Aveiro, notably Ana Hilarío, Clara (again) and São Ravara but also to everyone in the lab too.

It been a hard road, but well enjoyed. If①I e① issed①a o e①out,①thanks to you too (I at the end of a long 2-day sleepless night)

Patterns in adaptive developmental biology and symbioses of small-sized deep-sea chemosymbiotic mussels (Bathymodiolinae)

An array of deep-sea habitats are characterised by thermo-and/or biogenic production of chemically edu ed① o pou ds.①Be thi ① o u ities①th i e①at①these① edu i g①ha itats ,①due①to①t ophi ①li ks①to①f eeliving and symbiotic chemosynthetic bacteria which derive energy from electron donors (sulphides, hydrocarbons) and acceptors (O 2 ), at reduction-oxidation boundaries. The bathymodiolin mytilids (sensu lato) are a keystone taxon in these habitats; all-but-one species host gill-associated symbiotic Gammaproteobacteria as adults, but data are scarce on the remaining lifecycle, particularly at organic falls.

To understand how mussels are adapted to these habitats, this research characterises the reproductive, developmental and nutritional lifecycle biology of two species, Idas modiolaeformis and I. simpsoni. Anatomical, histological and molecular analyses on post-larval-to-adult size spectra are complemented with live observations. Contrasting and converging aspects of their biology and symbioses are presented. In both species, aposymbiotic post-larvae confirm strict, larval heterotrophy. While still very small, environmental symbiont infection is extracellular and initially non-specific, becoming progressively isolated to lateroabfrontal gill surfaces in adults. Maturation is rapid, in parallel with a transition from heterotrophy to chemosymbiotic mixotrophy: symbioses, filter-feeding, and the retention of a complete gut are observed si ulta eousl .①I te spe ifi ① a d①ha itat① diffe e es①a e①dis ussed①i ①the① o te t①of① oth①spe ies ①e ide t① evolutionary success in adapting to ephemeral, chemically reduced habitats in the deep sea.

Aspects adaptatifs de la biologie du développement et des symbioses chimiosynthétiques chez les petites oules Bath odioli ae de l'O éa profo d

Plusieurs habitats de①l O a ①p ofo d sont caractérisés par la présence de composés chimiques réduits. Les communautés benthiques prospèrent dans ces «habitats réducteurs», en raison de liens trophiques avec des bactéries chimiosynthétiques, qui tirent l'énergie de donneurs (sulfures, hydrocarbures) et accepteurs (O 2 ) d'électrons. Les moules bathymodiolines sont un taxon clé dans ces habitats. À l'âge adulte, presque toutes possèdent des bactéries symbiotiques, mais les données sont rares concernant leur cycle de vie. Cette recherche explore la biologie de la reproduction, le développement et la nutrition dans le cycle de vie des espèces Idas modiolaeformis et «I.» simpsoni. Les analyses anatomiques, histologiques et moléculaires à divers stades du développement sont associées à des observations in vivo. Une comparaison est proposée entre ces espèces. Chez les deux, les post-larves o t①pas①de①symbiotes, suggérant des larves strictement hétérotrophes. L'infection par les symbiotes est environnementale, extracellulaire et initialement nonspécifique, se restreignant aux surfaces latéro-abfrontales des filaments branchiaux chez les adultes. La maturation est rapide, de même que la transition de l'hétérotrophie à la mixotrophie chimiosymbiotique: les symbioses, l'alimentation par filtration, et la① te tio ① d u ① s st e① digestif① o plet① oï ide t.① Les différences entre espèces et habitats sont discutées dans le contexte de leur évidente réussite évolutive à s'adapter aux habitats réduits et éphémères de①l O a ①p ofo d.

Padrões na biologia do desenvolvimento adaptativo e simbiose em pequenos mexilhões (Bathymodiolinae) quimiossimbioticos de profundidade

Vários habitats de profundidade são caracterizados pela produção termogénica e / ou biogênica de compostos quimicamente reduzidos. Nestes habitats-reduzidos, as comunidades bentônicas prosperam, devido a ligações tróficas a bactérias quimiossintéticos de vida livre ou simbióticas. Estas bacterias derivam a energia a partir de dadores de elétrons (sulfetos, hidrocarbonetos) e receptores (O 2 ), nos limites de oxidação-redução. Os mitilídeos Bathymodiolinae (sensu lato) são um táxon importante nestes habitats; todos, excepto uma espécie, tem simbiontes Gammaproteobacteria associados aos adultos, mas os dados para o restante ciclo de vida são escassos, particularmente em acumulações de matéria orgânica. Para entender como os mexilhões estão adaptados a estes habitats, esta tese caracteriza a biologia reprodutiva, do desenvolvimento e nutricional do ciclo de vida de duas espécies: Idas modiolaeformis e I. simpsoni.

Análises anatómicas, histológicas e moleculares de diversos tamanhos de pos-larva a adulto foram complementadas com observações ao vivo. Aspetos contrastantes e convergentes da biologia e simbiose são apresentados. Em ambas as espécies, a ausência de simbiontes em pós-larvas confirmam a heterotrofia larval estrita. Quando juvenil, a infecção pelo simbionte ambiental é extracelular, e inicialmente nãoespecífica, tornando-se① p og essi a e te① ais① isolada① a① supe fi ie① a f o tal ① dos① fila e tos① das① brânquias dos adultos. A maturação é rápida, em paralelo com uma transição de heterotrofia para mixotrofia quimiossintética: simbiose, com alimentação por filtração demonstrada, e com um intestino completo e em pleno funcionamento. Diferenças interespecíficas e entre habitats, são discutidas para ambas as espécies no contexto do seu evidente sucesso evolutivo na adaptação aos habitats efêmeros, quimicamente reduzidos no fundo do mar.

. Deep-sea research still remains in its infancy, despite notable technological advances in the last hundred years and a considerable expansion of our scientific understanding (German et al. 2011). Accordingly, empirical observations and discoveries of deep-sea habitats have generally been as a result of technological advances in deep-water surveying [START_REF] Koslow | The silent deep: the discovery, ecology, and conservation of the deep sea[END_REF]Ramirez-Llodra et al. 2010). During an intense period of exploration between the mid-19 th century and early-20 th century, advances in sea navigation, sounding equipment for measuring depth and dredging apparatus saw several independent discoveries of marine life at depths famously argued to be devoid of life by Edward Forbes previously [START_REF] Koslow | The silent deep: the discovery, ecology, and conservation of the deep sea[END_REF]). The first global oceanographic expedition aboard the HMS Challenger, was from 1872-1876 during which physical and chemical characteristics of the oceans were documented, alongside hundreds of newly described species (Murray et al. 1891). The sounding equipment, though primitive, also permitted the discovery of continental shelf breaks and continental slopes, and highlighted variability in the topography of the northern Atlantic (1853[START_REF] Koslow | The silent deep: the discovery, ecology, and conservation of the deep sea[END_REF]Ramirez-Llodra et al. 2010). The first evidence for submarine ridges, trenches and seamounts were collected soon after using both the Thomson and Sigsbee sounding machines [START_REF] Koslow | The silent deep: the discovery, ecology, and conservation of the deep sea[END_REF], just prior to the beginning of the 20 th century. Both the Juan de Fuca ridge and Gorringe ridge were identified during this period. With the advent of the acoustic sounder in 1914 (patented by Reginald Fessenden, [START_REF] Koslow | The silent deep: the discovery, ecology, and conservation of the deep sea[END_REF], mapping of ocean depths became possible. From World War I (1914-1919) to the present day, oceanic acoustic research has seen intensive international military investment with notable developments such as radio acoustic ranging, sonar and radar that paved the way for presentday global positioning systems (GPS) and sounding equipment with greater coverage, reliability and precision. The use of sensitive magnetometers in the mid-20 th century provided critical evidence for platetectonic theory in the form of crustal magnetism striping, and therefore faulting, in the crust (Vine and Matthews 1963). Remote sensing has emerged as a critical tool in advancing our understanding of largescale hydrodynamics [START_REF] Kerr | From space to species: ecological applications for remote sensing[END_REF]. Advances in deep-sea exploratory equipment have been staggering, as global enterprises invested heavily into research and development in telecommunications, offshore drilling (and piping) for oil and gas and an impending rise in deep-sea mineral extraction (Van Dover 2011a). The consequences for deep-sea habitat conservation are thus bitter-sweet, as advances in underwater high-pressure technology provide the means to further understand, and exploit, deep-sea environments simultaneously. 1-1.2. Key advances and the current state of the art Critical advances in deep-sea exploration can be broadly separated into several categories. These have been in navigation, depth-sounding approaches, means of profiling environmental and chemical water characteristics, underwater exploratory innovations and habitat and faunal sampling techniques. Note that fo ① the① pu poses① of① this① a us ipt,① u less① stated① othe ise,① deep ① e i o e ts① a e① those① >① ① ,① beyond the continental break, and in which sunlight is at such low intensities as to be of little direct ecological significance (material and nutrients of shallow-water origin, which may be photosynthetically derived, are considered indirect).

LIST OF TABLES

1-1.2.1 Navigation and positioning

Advances in navigation techniques within the last century have been from the use of hand-drawn maps, compasses, sextants and callipers (and of course, a cloudless sky) with >100-km margins of error, to the application of global positioning systems (GPS), informed by a network of 32 mid-orbiting satellites in space, ground-based control stations and antennae, with an accuracy of 10 metres or much less with landreferenced Differential GPS [START_REF] Parkinson | Differential GPS[END_REF]. GPS-positioning has been incorporated into nearly all deep-sea measuring devices since, permitting the geo-referencing of any data to coincidental, potentially explanatory, environmental data. This is of particular importance when data is to be integrated into geographic information systems (GIS), a conceptual quantitative visual-mapping tool developed initially for use in multidisciplinary terrestrial geographic studies [START_REF] Hatcher | The role of alterations in membrane lipid composition in enabling physiological adaptation of organisms to their physical environment[END_REF].

1-1.2.2 Sounding to assess depth

In one of the first attempts to estimate depths in the 18 th century, the French mathematician and astronomer Pierre-Simon Laplace argued (rather accurately) that the Atlantic ocean ought to be around 4000m deep on average, based on observed tidal patterns in the southern Atlantic (Gillispie 2000).

However, attempts to actually assess water depths definitively would be made almost a hundred years late ,①e plo i g① asi ① sou di g ①appa atus① Koslo ① .①I ①sta k① o t ast,①s ie tists① o ①e plo ①a① ide① array of remote-sensing approaches including high-resolution bathymetric track data, Shuttle Radar Topography Mission data [START_REF] Becker | Global bathymetry and elevation data at 30 arc seconds resolution: SRTM30_PLUS[END_REF]) and gravitational anomalies in the sea-surface geoid, identifiable when examining high resolution gravity field data (satellite radar altimetry data, [START_REF] Smith | Global sea floor topography from satellite altimetry and ship depth soundings[END_REF]. These measurements are then interpolated in suitable GIS software in order to create continuous high-resolution bathymetric data (e.g. Figure 1.1C) fo ①the① o ld s①o ea s:①a①po e ful①tool①fo ① predicting the locations of currently undocumented topographic features and thus potentially associated fauna (e.g. [START_REF] Marks | An Evaluation of Publicly Available Global Bathymetry Grids[END_REF][START_REF] Kim | New global seamount census from altimetry-derived gravity data[END_REF].

1-1.2.3 Environmental profiling

In terms of environmental data, the first attempts to map global temperatures and salinities both at the surface and at depth were during the Challenger Expedition [START_REF] Dittmar | Report on researches into the composition of ocean-water collected by HMS Challenger during the years 1873-76[END_REF][START_REF] Thomson | Report on the scientific results of the voyage of HMS Challenger during the years 1873-76 under the command of Captain George S. Nares[END_REF].

Approximately 300 profiles were recorded using pressure-protected thermometers and stopper bottles (for salinity) attached to sounding lines, subsequently demonstrating for the first time the relatively constant salinity of offshore marine waters, the presence of oceanic thermoclines, and inadvertently providing a baseline record for future global-warming studies (e.g. [START_REF] Roemmich | 135 years of global ocean warming between the Challenger expedition and the Argo Programme[END_REF]). These samples required lengthy post-expedition analyses however [START_REF] Koslow | The silent deep: the discovery, ecology, and conservation of the deep sea[END_REF]. In contrast, today such measurements can be made in real time using conductivity-temperature-depth (CTD) profilers, while current-velocity profiles are constructed from acoustic Doppler current profiler data (ADCP), both typically being mounted on a submerged platform (Ramirez-Llodra et al. 2010). Additional data on turbidity and light intensity within the euphotic zone (0-200 m, [START_REF] Gage | Deep-sea biology: a natural history of organisms at the deep-sea floor[END_REF]) can be measured using dedicated instruments. Remote sensing in the deep-sea can provide broad scale environmental data at the surface (e.g. temperature and chlorophyll anomalies, [START_REF] Kerr | From space to species: ecological applications for remote sensing[END_REF], identifying zones of upwelling and the presence of largescale eddies potentially important for long-distance larval dispersal [START_REF] Pineda | Larval transport and dispersal in the coastal ocean and consequences for population connectivity[END_REF]). Some of this equipment is also used in the deep sea, and where appropriate, can be installed on remotely operated vehicles (ROVs, Figure 1.1A), on landers, or at cabled observatories. The latter often has a dedicated array of technology at its disposal (e.g the MARS benthic rover Figure 1.1D). The exploration of deep-water environments where the water chemistry is of particular interest (e.g. chemically reduced end-member fluid emission in vent fields) has fuelled further innovations in measuring devices for assessing water chemistries in situ and at great depths. Many compounds which would formerly have needed to be analysed at the surface in a laboratory, can now be recorded in situ using highly sensitive potentiometric sensors (Le [START_REF] Bris | Autonomous potentiometric sensor for in situ sulfide monitoring in marine sulfidic media[END_REF] or pressure-housed spectrometers (e.g. the Raman spectrometer, [START_REF] Brewer | Development of a laser Raman spectrometer for deep-ocean science[END_REF][START_REF] Zhang | Development and deployment of a deep-sea Raman probe for measurement of pore water geochemistry[END_REF]; Figure 1.1G), removing the effects of alterations in water chemistry during water sample recovery.

1-1.2.4 Underwater exploration

Accessing the deep-sea directly was simply impossible prior to the advent of submersible exploratory equipment in the 20 th century [START_REF] Koslow | The silent deep: the discovery, ecology, and conservation of the deep sea[END_REF]Ramirez-Llodra et al. 2010). This was demonstrated to be possible through the pioneering work of Edward Beebe (1934,923 to document a deep-sea habitat in its entirety, using the systems already described. With geo-referenced data, this can then be coupled with remote sensing data to provide a complete picture of the environment in question.

1-1.2.5 Biological sampling

While all these developments in technology have placed deep-sea research at the cutting edge of marine sciences, methods for sampling the benthos and pelagos remains limiting in deep-sea research. Deep-sea benthic sampling is still relatively cumbersome, patchy and extremely expensive as it is necessarily vesselbased and evermore ROV-orchestrated. Although deep-sea trawling/dredging is best avoided being highly dest u ti e,①it s①use①is①still①p e ale t,①pa ti ula l ①fo ①deep-sea science in developing countries or when no access to an ROV is available (e.g. Samadi et al. 2010). Cores and grabs have been optimised with the use of remotely-guided operation. Additional means of sampling are stationary deployments such as nets, traps [START_REF] Beaulieu | Comparison of a sediment trap and plankton pump for time-series sampling of larvae near deep-sea hydrothermal vents[END_REF]) and nascent substrata for colonisation (e.g. the European CHEMECO project, Gaudron et al. 2010;①INDEEP s①"e pe t①P oje t①e.g.①Higgs et al. 2014), alongside direct sampling using corers, suction dredges and articulated robotic arm. Deep-sea research has historically been hindered by poor sampling resolution with limited comparability across studies, as sampling approaches are often not uniform. The maintenance of international collaborations, with sustained active communications between experts, remains of prime importance. The whole field begins to benefit from the up-scaling of time-series multiplesite sampling where habitat surveillance is routinely performed, such as in cabled observatories. Innovative techniques that can be performed by ROV are now being developed for the deep sea (e.g. burrow casting, Seike et al 2012, or calcein staining for growth measurements, Nedoncelle et al. 2013; see also Figure 1.1).

1-1.2.6 Live maintenance of deep-sea organisms

Pressurised conditions present several problems, not least the requirement for pressure-resistant equipment with which to carry out scientific work, but also the singular most difficult aspect of the study o ga is s① e i o e t① to① sustai ① i ① the① la o ato ① Ma sh① et① al. 2001). Even with the use of hypobaric chambers, reproductive and larval biology studies remain highly challenging, though possible (e.g. [START_REF] Young | Embryos of the deep-sea echinoid Echinus affinis require high pressure for development[END_REF][START_REF] Young | Embryology of vestimentiferan tube worms from deepsea methane/sulphide seeps[END_REF][START_REF] Eckelbarger | Gametogenesis, spawning behavior, and early de elop e t① i ① the① i e o ① Hesio ae a① etha i ola① Pol haeta:① Hesio idae ① f o ① etha e① hydrates in the Gulf of Mexico[END_REF][START_REF] Marsh | Larval dispersal potential of the tubeworm Riftia pachyptila at deep-sea hydrothermal vents[END_REF][START_REF] Pradillon | Influence of environmental conditions on early development of the hydrothermal vent polychaete Alvinella pompejana[END_REF]). An alternative, for species with a less constrained depth range, is to bring them to the surface and maintain them alive for reproductive experiments at atmospheric pressure [START_REF] Mortensen | The functional morphology of the organs of feeding and digestion of the hydrothermal vent bivalve Calyptogena magnifica (Vesicomyidae)[END_REF]Young and Cameron 1989;Young et al. 1989;Colaço et al. 2006;Dixon et al. 2006;Arellano and Young 2009;Arellano and Young 2011). Such examples include slope-based communities which can also be the sites of reducing conditions (e.g. species from cold seeps) or reducing habitats which can occur over large depth-ranges (e.g. sunken wood). Keeping these animals alive long enough for larval studies to be performed in the laboratory either requires fully equipped aquaria on the research vessel, or more typically, facilities at least equipped to transport organisms to land-based aquaria.

1-1.2.7 Perspectives in deep-sea

Deep-sea sciences have gained momentum over the last half-century with technical advances and relative reductions in cost. Exploration efforts increased with the discovery of highly productive chemosynthetic communities thriving in the immediate vicinity of hydrothermal vents in 1977 at the Galapagos Rift, a spur of the East Pacific Rise (Corliss and Ballard 1977;Lonsdale 1977;[START_REF] Corliss | Submarine thermal springs on the Galapagos rift[END_REF]. If a research vessel is fully-equipped, it is now possible to collect sufficient data in order to document those environmental parameters and biogeochemical characteristics which define different deep-sea habitats, as well as interactions between habitats (e.g. fluxes, migration and the formation of environment boundaries which may promote allopatric speciation). The bathymetry, hydrodynamics and water or substrate chemistry may be documented using remotely-operated vehicles, gliders or autonomous underwater vehicles (AUV), coupled with multibeam arrays, ADCPs, CTD profilers and chemical picosensors with perhaps in the future, in-situ-mounted equipment for inductively-coupled plasma mass-spectrometry (ICP-MS) or gas chromatography (GC). Biological data can be obtained through geo-referenced video-surveying, the use of temporally-sequential multicorers (e.g. Figure 1.1E), continuous plankton recorders, permanent deep-sea monitoring stations, manipulative experimental installations. The use of naturally occurring (or experimentally-derived) chemical tracers to directly assess growth (e.g. [START_REF] Tada | In situ fluorochrome calcein marking of deep-sea molluscs using a new growth chamber[END_REF]Nedoncelle et al. 2013), transport, connectivity and nutrition in-situ in deep-sea benthic species, has already proved informative in shallow-water environments [START_REF] Becker | Spatial and temporal variation in trace elemental fingerprints of mytilid mussel shells: A precursor to invertebrate larval tracking[END_REF][START_REF] Becker | Complex larval connectivity patterns among marine invertebrate populations[END_REF]Cowen and Sponaugle 2009). However, these techniques require considerable investment in terms of resources as well as overcoming the logistical difficulties of the environment in question ( Van Dover 2011b). The sheer expanse of deep-sea environments also presents a daunting prospect for marine scientists. If an ephemeral habitat is under investigation, much time and expenses must be spent in order to simply locate the habitat (Rogers et al. 2012). By definition, these habitats are also the most challenging habitats to sample temporally (and for studies examining temporal variations in recruitment for example), though they may also be the most informative (e.g. Mullineaux et al. 2010). In habitats that occur over large spatial scales (such as abyssal plains, oceanic trenches, and continental slopes), researchers may be tempted (inappropriately) to interpret findings in isolated studies and make wide-sweeping conclusions about larger regions (e.g. [START_REF] Thorson | Reproductive and larval ecology of marine bottom invertebrates[END_REF]).

U de sta di g① su h① ha itats① is① esse tial① ho e e ,① if① the① o e① at pi al ① d a i s① of① so e① deep-sea habitats are to be placed in an appropriate biological context.

Cost-effective innovations for the future to address the historical imbalance might include remoterelease sampling methods, combined with permanent high-resolution remote monitoring stations (e.g. NSF-

Ocean Observatories Initiative, U.S.A; NEPTUNE, Canada; DELOS project, Scotland; the European Seas Observatory NETwork or ESONET; DONET, Japan) and periodic geo-referenced video surveys using autonomous underwater vehicles (AUV).

1-2 Deep-sea benthic habitats 1-2.1. Habitat diversity and productivity in the deep-sea Despite historical opinion to the contrary, it appears that the deep-sea①is① ot①a① dese t①a ss .①This①is①all①the① more impressive considering approximately 75% of extant species are thought to have evolved following the Cretaceous-Paleogene (K-Pg) mass extinction event [START_REF] Jacobs | Oxygen and evolutionary patterns in the sea: Onshore/offshore trends and recent recruitment of deep-sea faunas[END_REF][START_REF] Schulte | The Chicxulub asteroid impact and mass extinction at the Cretaceous-Paleogene boundary[END_REF], possibly subsequent to a currently hypothesised reinvasion by species from shallow-water environments.

Even in the abyssal plains, traditionally thought to be highly conservative, stable environments (and thus not sufficiently dynamic to peak scientific interest), growing evidence suggests that environmental and organismal processes act to maintain highly levels of biodiversity (with low species densities) through macro-and mesoscale habitat disturbance (e.g. formation of massive burrow complexes by deep-sea bivalves, [START_REF] Seike | Novel use of burrow casting as a research tool in deep-sea ecology[END_REF]; mound formation through bioturbation, [START_REF] Kukert | Disturbance, colonization and succession in a deep-sea sediment community: artificial-mound experiments[END_REF]. In habitats where a persistent REDOX boundary occurs (reducing habitats), symbiotic adaptations have evolved in deep-sea metazoans to harness the energy available from reduced electron-acceptor compounds (e.g. H 2 S and S 2 O 3 -, and CH 4 ) by hosting bacteria and archaea capable of either chemoautotrophy or -heterotrophy, using inorganic minerals (chemolithotrophy) or organically derived reduced compounds (chemoorganotrophy), reviewed in Vrijenhoek (2010). This results in highly productive, specialised communities in the absence of sunlight, though with relatively poor species diversity [START_REF] Grassle | Species diversity in deep-sea communities[END_REF].

Between the discovery of habitats that can support highly productive communities in the absence of①light① e.g.①h d othe al① e t①fields,①h d o a o ① old ①seeps①a d①o ga i ①falls ①a d①the①asso iated① edia① i te est①i ①su h① alie ①ha itats,①a① e e ed①i te est①i ①the①deep-sea has emerged in recent decades. With technical advances in the field continually emerging, discovery rates of new habitat types in the deep sea have consequently accelerated exponentially over the last hundred years (Figure 1.3). Such interest is justified; the deep-sea①is①a gua l ①the①la gest① io e①o ①Ea th.①Of①the①pla et s① ① illio ①k 2 surface, 71% is coastal and ocean seafloor where approximately 90% is classified as deepi.e. beyond continental shelf breaks (Ramirez-Llodra et al. 2010; Van Dover 2011b). Global interactions between plate-tectonics and hydrological, meteorological, biogeochemical, and geological processes combine to sculpt ocean-floors and, together with organic fluxes to the seabed, determine habitat characteristics. The level of habitat diversity on a global scale in the deep sea is astounding, but the horizontal coverage of various formations and habitats is extremely asymmetric, according to the most current data available from bathymetric topography (Ramirez-Llodra et al. 2010, the values cited in this section henceforth are from this review, unless stated otherwise).

Descending down the continental slopes, depths range from around 150 m (with a few exceptions) at the shelf break to depths of around 3500m as the slope gradient flattens out at the continental rise. The continental slopes and rises together account for about 10% of the deep-sea ocean floor. In terms of surface-area however, the vast soft-sediment seascapes that extend beyond these margins, the abyssal plains, dominate the deep-sea (76%). Studies during the last half-century (reviewed in [START_REF] Gage | Food inputs, utilization, carbon flow and energetics[END_REF] have revealed previously hidden levels of dynamism in the ecology of abyssal plains. Localised macro-and mesoscale processes, both organic and environmental (Ramirez-Llodra et al. 2010), act to maintain high levels of diversity in these soft-sediment environments, but with typically low resource-limited productivity (Ramirez-Llodra et al. 2010). These community characteristics are assumed to apply for the majority of the abyssal soft-sediment deep-sea, forming what scientists studying chemically reduced systems refer to as the① a kg ou d ① fau a① o ① o u ities.① Though① p edo i a tl ① flat① a d① featu eless,① these① e pa ses① a e① punctuated by other deep-sea structures. The most extensive are the mountain ridges along tectonic boundaries, being almost equivalent to continental margins in their coverage (9%). The formation of nascent oceanic crust occurs at mid-ocean spreading centres, flanked either side by semi-contiguous volcanic mountain ranges that extend along their entire lengths (depending on the rate of spread).

Relatively isolated submarine mountains (seamounts) make up around another 2.6% (though estimates range up to 19.6%, or 67. Although Ramirez-Llodra et al. (2000) include whale-fall data in their estimates of global habitat coverage in the deep-sea, they do not account for sunken vegetative organic matter. This may because estimates of coverage by sunken wood and other debris in the deep sea are not currently feasible; it is not even clear whether sunken wood in the deep sea is more or less abundant than in coastal systems, as data on the volume and fate of wood exported to the open oceans have never been quantitatively assessed (though qualitative records suggest that they could be considerable, [START_REF] Maser | From the forest to the sea: a story of fallen trees[END_REF]. The influx of sunken wood to deep environments may be in the form of numerous but small fragments of submerged woody debris deposited in and exported down continental slopes (e.g. via submarine canyons, [START_REF] Josselyn | Composition, export and faunal utilization of drift vegetation in the salt river submarine canyon[END_REF], right up to whole trees weighing several tonnes that can take many months to become waterlogged and sink to the bottom [START_REF] Maser | From the forest to the sea: a story of fallen trees[END_REF][START_REF] Maser | From the forest to the sea: the ecology of wood in streams, rivers, estuaries, and oceans[END_REF]. The distribution of vegetative debris already submerged on the continental shelf is likely to be restricted to continental margins, while the largest pieces of floating wood could sink practically anywhere on the ocean floor. In the presence of suitable offshore currents, the rate at which wood becomes waterlogged and sinks likely dictates its final settle e t① site,① ith① i te ediate ①sizes①of① ood①ope ating somewhere along this spectrum of transport potential. The distribution of wood in the deep-sea is probably asymmetric, favouring stretches of the continental margin that are strewn with canyons, and subject to fluvial discharge from rivers with flood plains in heavily forested areas, particularly if the region is subject to storms. The maximum known depth for any vertebrate, is currently 7.7 km for a hadal liparid fish [START_REF] Jamieson | Hadal trenches: the ecology of the deepest places on Earth[END_REF]. Invertebrates however appear less constrained by pressures at depth (except decapods <7 km, [START_REF] Jamieson | Hadal trenches: the ecology of the deepest places on Earth[END_REF] in the absence of large gaseous cavities (e.g. lungs or swim bladders) and being released from hypoosmotic constraints associated with the protein-stabilising molecule trimethylamine, present in hadal fish [START_REF] Yancey | Marine fish may be biochemically constrained from inhabiting the deepest ocean depths[END_REF]. One of the adaptations proposed for deep-sea species is of a cellular nature. Phospholipid-membrane proteins, Na + /K + -ATPases, appear to be more resilient to increases in pressure and decreases in temperature in deep-sea species than in their shallow-water counterparts, owing to greater cell membrane fluidity at a given depth [START_REF] Cossins | The adaptation of biological-membranes to temperature and pressurefish from the deep and cold[END_REF][START_REF] Hatcher | The role of alterations in membrane lipid composition in enabling physiological adaptation of organisms to their physical environment[END_REF]. Other adaptations to elevated pressures include chemically stabilised structuring in enzymes, adaptive functioning in nervous and musculoskeletal systems, differing osmolytes, and the loss of unsustainable carbonate skeletal structures, where applicable [START_REF] Somero | Adaptations to high hydrostatic pressure[END_REF]). Some of these adaptations are even orchestrated by pressure-resistant operons within DNA (Li et al. 1998). Put simply, for species that ha e①e ol ed①i ①deep①e i o e ts,①su h①p essu es①a e①a①ha itual① o po e t①of①thei ①li es.①It s①also① o th① noting that changes in pressure with depth are actually most abrupt at the surface; a transition from surface waters to 100-m depth results in a ten-fold increase in ambient pressure, whereas a transition of 5 km from 5000-10000 m, only equates to a doubling in pressure.

1-3.1.2 The absence of light and photosynthesis

In the absence of light, prey and predators alike have developed capacities for bioluminescence, employed complex and ingenious visual signalling (reproduction), distraction (predator evasion) and attraction (visualising and luring prey) in the deep sea, where a predator-prey arms race is ongoing [START_REF] Herring | How to survive in the dark: bioluminescence in the deep sea[END_REF].

The absence of light from the deep sea was also once thought to be the single-most limiting attribute of this e i o e t s①p odu ti it ①fo ①all①o ga is s①that①li ed① elo ① ①depth.①E e ①at①the①sta t①of①the① s, eminent scientists were still of the opinion that the deep sea was decoupled spatially and temporally from primary production in surface waters [START_REF] Sanders | Ecology of the Deep-Sea Benthos[END_REF]. However, evidence of some deep-sea organisms timing their spawning events so as to take advantage of spring blooms in surface waters (e.g. Dubilier et al. 2008).

1-3.1.3 Vertical flux of food supply

In non-reducing deep habitats, benthic assemblages are thought to be dependent wholly on the vertical flux of organic material from proximal surface waters. This comprises of organic aggregatessome derived from plankton bloomsslowly sinking through the water column (collectively alled① a i e①s o Turner 2002, i.e. macrozooplankton, fish faecal pellets and sinking phytoplankton). The vertical flux thus varies in quality, on account of inherent variability in contributing sources and due to nutrient recycling and repacking during descent (Turner 2002). Since the composition of vertical organic flux is derived from primary productivity in the euphotic zone, it is worth considering how this can vary from coastal systems to the open oceans and with increasing depth, since the underlying soft-sediments will mirror the productivity of proximal surface waters, but an increase in the depth of descent reduces the volume of material that can reach the seafloor [START_REF] Levin | Environmental Influences on Regional Deep-Sea Species Diversity[END_REF].

The productivity of surface waters will depend upon seasonal levels and latitudinal gradients of irradiance and the availability of nutrients; rising irradiance drives photosynthesis while the nutrient supply sustains algal growth, resulting in a surge in planktonic densities (a bloom Turner 2002 and references therein), with general proportionality between surface primary productivity and net downward organic carbon flux for the first 2000 m at least (Suess 1980;[START_REF] Lampitt | Particle flux in deep seas: regional characteristics and temporal variability[END_REF].

The arrival of particulates to the seabed is known to be seasonal even in deep environments [START_REF] Gooday | Responses by Benthic Organisms to Inputs of Organic Material to the Ocean Floor: A Review [and Discussion}[END_REF][START_REF] Lampitt | Particle flux in deep seas: regional characteristics and temporal variability[END_REF]Turner 2002), evidenced by seasonality in soft-sediment community oxygen consumption rates (e.g. [START_REF] Smith | Seasonal fluctuations in deep-sea sediment community oxygen consumption: central and eastern North Pacific[END_REF] and in cyclical reproductive patterns of some responding species (summarised in [START_REF] Gooday | Responses by Benthic Organisms to Inputs of Organic Material to the Ocean Floor: A Review [and Discussion}[END_REF]). Bacteria and protozoan flagellates and foraminifera rapidly settle upon and proliferate in phytodetritus when it settles, while deposit feeding organisms appear to consume it directly (reviewed in [START_REF] Gooday | Responses by Benthic Organisms to Inputs of Organic Material to the Ocean Floor: A Review [and Discussion}[END_REF]). Larger organic falls also transport food to the seafloor, but with habitat-specific responses by benthic organisms (discussed in section 1-5.3). The nature of arriving taxa appears to depend on the form of the organic material [START_REF] Gooday | Responses by Benthic Organisms to Inputs of Organic Material to the Ocean Floor: A Review [and Discussion}[END_REF]). The exceptions are large-s ale①sa pli g① a paig s①u de take ①o ①the① o ld s① o ti e tal①slopes①to①asses① biodiversity in the deep sea [START_REF] Sanders | Ecology of the Deep-Sea Benthos[END_REF][START_REF] Hessler | Abyssal community analysis from replicate box cores in the central North Pacific[END_REF][START_REF] Grassle | Deep-sea species richness: regional and local diversity estimates from quantitative bottom samples[END_REF][START_REF] Snelgrove | A riot of species in an environmental calm: The paradox of the species-rich deep-sea floor[END_REF]. Due to the operational costs of performing detailed ecological studies in deep-sea environments, ecologically significant biological interactions are poorly defined (other than commensal and symbiotic associations), even in chemosynthetic environments [START_REF] Glover | Temporal change in deep-sea benthic ecosystems: a review of the evidence from recent time-series studies[END_REF]Ramirez-Llodra et al. 2010). Genetic connectivity (e.g. [START_REF] Bors | Patterns of Deep-Sea Genetic Connectivity in the New Zealand Region: Implications for Management of Benthic Ecosystems[END_REF], biogeography (Bachraty et al. 2009;Olu et al. 2010;Rogers et al. 2012) and levels of endemism (Howell et al. 2010) have formed the focus of recent studies. Within single communities, data is fragmented concerning interactions within and between cohabitant species and their environment. This fundamentally limits our ability to assess the sensitivity of communities to environmental disturbances both in terms of physiological tolerance and resilience through inter-population connectivity. Basic data concerning the physiological flexibility and the life-histories of deep-sea organisms are scarce [START_REF] Tyler | Reproduction and dispersal at vents and cold seeps[END_REF]. Whether observed ecological patterns in shallowwater habitats are even applicable to the deep-sea remains to be seen. Behavioural data on deep-sea organisms is extremely limited. Evolutionary alterations to the mytilid bauplan are generally inferred from shallow-water species and preserved-tissue analyses only. On an organismal level, a basic understanding of the developmental, reproductive and nutritional biology of select model species throughout its lifecycle could provide insights into the origin and maintenance of high biodiversity in deep-sea assemblages. What troubles researchers presently, is the rampant pace at which commercial interests in the deep sea moves forward with plans to make full use of deep-sea ecosystem services. As the forerunners, with the means to explore the deep sea extensively, they outpace scientists, management authorities and legislators whose aim it is to control and regulate the exploitation of deep-sea services. Not included in the assessment performed by [START_REF] Ramirez-Llodra | Man and the Last Great Wilderness: Human Impact on the Deep Sea[END_REF] is the range of large-sized organic falls that reach the deep-ocean floor. Organic falls are highly ephemeral with somewhat less predictable distributions than other reducing habitats. Their conservation status is therefore poorly defined. However, larger organic falls may provide intermediary settlement sites for long range multigenerational dispersal in species that bridge these, and other reducing habitats. In addition, wood accumulations in submarine canyons appear to be the preferred habitat context for deep-sea wood-boring bivalves Xylophaga spp. [START_REF] Romano | Submarine canyons as the preferred habitat for wood-boring species of Xylophaga (Mollusca, Bivalvia)[END_REF]Fagervold et al. 2014), which frequently occur at staggering densities, in conjunction with an array of other opportunistic organisms, chemoautotrophic free-living material mats and chemosymbiotic bivalves from the genus Idas (e.g. Gaudron et al. 2010;Bienhold et al. 2013;Cunha et al. 2013;Thubaut et al. 2013a).

They represent production hotspots at the juncture between coastal-and deep-sea-dominated systems, both in terms of hydro-geography and their ecology.

Human activities are placing mounting ecological pressure on deep sea habitats, where the severity of these impacts will likely worsen with time. If humankind is to exploit the deep seabed now and increasingly in the future, as seems inevitable [START_REF] Ramirez-Llodra | Man and the Last Great Wilderness: Human Impact on the Deep Sea[END_REF]Van Dover 2011a;b), then the One critical line of research lies in describing deep-sea invertebrate lifecycles in relation to the physical, chemical and biological processes that impact upon their development, as this will help us understand and predict which factors will control spatial and temporal variability in populations (i.e. distribution patterns, composition, stability, migration, resource partitioning and larval recruitment). This goal can be achieved in part through the design, validation and application of population dynamics models informed by crucial lifecycle metrics from the community [START_REF] Eckman | Closing the larval loop: linking larval ecology to the population dynamics of marine benthic invertebrates[END_REF]. This requires a full account of the Coastal systems are far more complex, where habitat heterogeneity is high and dependent on both hydrodynamics and depth. These are discussed in detail.

1-5.2. Hydrocarbon seeps and hydrothermal vents

Since the processes that form hydrocarbon seeps are linked intrinsically to the offshore export of coastal detritus, they are distributed predominantly along continental margins, in areas with a history of organicsrich sedimentary deposition. At passive margins, deep layers of sediment have formed due to the transport of terrigenous and marine material to the slope and deep-sea ocean basins, delivered by turbidity flows, sedimentary slumps and offshore detrital deposition [START_REF] Boetius | Seafloor oxygen consumption fuelled by methane from cold seeps[END_REF]. Thus continental slopes and oceanic basins that are coupled with passive continental margins are frequently subject to high degrees of organic-enriched sediment loading. When sedimentary deposition continues in this way over geologically significant periods of time, the buried organic matter undergoes chemical maturation during lithification (Figure 1.8:① POC① u ial ,① aused① ① a ① ele atio ① i ① geothe al① te pe atu es① a d① the① considerable compressive forces exerted by millions of tonnes of sediment and water [START_REF] Didyk | Organic geochemical indicators of palaeoenvironmental conditions of sedimentation[END_REF].

These processes alter interstitial pore-waters chemically, forming hydrocarbon-laden fluids, which readily percolate towards seabed surfaces when destabilised (Figure 1.8 .① These① e issio s① a ifest① as① old ① hydrocarbon seeps, frequently with methane as a principal component [START_REF] Sibuet | Biogeography, biodiversity and fluid dependence of deep-sea cold-seep communities at active and passive margins[END_REF], while processes such as the anaerobic oxidation of methane along with sulphate-reduction can generate local enrichments of sulphide (Duperron 2010). When gas-hydrates occurnatural gases such as methane dissolved in seawater, p e iousl ①su je ted①to①p essu es①o e ①≈ ①kPa,① Mai i①a d①Bish oi① ①their destabilisation can provide high-flux seepage [START_REF] Mienert | Gas hydrates along the northeastern Atlantic margin: possible hydrate-bound margin instabilities and possible release of methane[END_REF]. In other instances the flows can be viscous or even semi-solid, as in oil seeps (e.g. [START_REF] Sassen | Association of oil seeps and chemosynthetic communities with oil discoveries, upper continental slope, Gulf of Mexico[END_REF]), asphalt volcanism (e.g. MacDonald et al. 2004), mud volcanoes (e.g. Vogt et al. 1997) and brine pools [START_REF] Mapelli | Microbial diversity in deep hypersaline anoxic basins[END_REF]. Hydrocarbon seepage can occur at active margins such as sedimented vent/seep sites on the Juan de Fuca Ridge, or at the unusual CO 2 -vent systems of the Mediterranean, however this is not so common.

These sites of hydrocarbon seepage are an example of reducing environments which are all characterised by the release of chemically reduced fluids which interact with oxygenated seawater at the reduction-oxidation (REDOX) interface. These reducing compounds can support free-living and symbiotic chemosynthetic bacteria, which derive their energy from the metabolic oxidation of reduced compounds.

Typically, they employ hydrogen, or sulphidic compounds as electron donors and gain their carbon from CO 2 (i.e. chemoautotrophic primary production) though several other electron donors have also been implicated [START_REF] Anantharaman | Evidence for hydrogen oxidation and metabolic plasticity in widespread deep-sea sulfur-oxidizing bacteria[END_REF]). Sulphides at hydrocarbon seeps can be generated in large amounts at sites where the anaerobic oxidation of methane occurs [START_REF] Boetius | A marine microbial consortium apparently mediating anaerobic oxidation of methane[END_REF][START_REF] Treude | Anaerobic oxidation of methane above gas hydrates at Hydrate Ridge, NE Pacific Ocean[END_REF]. Some bacteria can also derive their energy from the metabolic oxidation of methane (methanotrophs, methane supplies a portion of their carbon also). The result is a myriad of bacterial biofilms and mats comprised of chemoautotrophic and methanotrophic bacteria, as well as bacterivorous primary consumers, specialist secondary consumers and a number of chemosymbiotic metazoan species that are seep-or reducinghabitat specialists. At times, biofilms are so ubiquitous and dense that they act as a biofilter, significantly lowering the concentrations of reducing compounds exiting the mats as free-fluid production (e.g. up to 40% reduction, [START_REF] Niemann | Novel microbial communities of the Haakon Mosby mud volcano and their role as a methane sink[END_REF]. Those chemosymbiotic organisms which can survive and prosper at seeps include Bathymodiolinae mussels and Pliocardiinae clams (Figure 1.9) forming dense beds, and narrow, elongate Lamellibrachia siboglinid tubeworms, aggregating in bushes (Le [START_REF] Pennec | Nutritional strategies of the hydrothermal ecosystem bivalves[END_REF]). In additio ① to① this① o e① o u it ,① oppo tu isti ① a kg ou d ① fau a① o u .① ②① high① di e sit ① of① gast opod① species also appears to reside at mud volcanoes in the eastern Atlantic, in the Gulf of Cadiz [START_REF] Génio | The snails' tale in deep-sea habitats in the Gulf of Cadiz (NE Atlantic)[END_REF].

The seepage of chemically reduced fluids capable of supporting specialised communities is not restricted to hydrocarbon cold seeps. In fact, the acknowledgement that cold seeps were a habitat type in their own right (Paull et al. 1984) came almost a decade after another chemically reduced habitatthe hydrothermal ventwas described for the first time (Corliss and Ballard 1977;Lonsdale 1977). By Vents are most numerous within the valleys and upon the flanks of spreading mid-ocean ridges, but also occur on subducting back-arc basins, on volcanic arcs and on active (volcanic) seamounts (Figure 1.6-1.5). The chemosynthetic basis for primary production is analogous to that of hydrocarbon seepage, driven generally by free-living and chemoautotrophic symbionts (though not always exclusively, e.g. Duperron et al. 2006a). Vent communities vary depending on the end-member fluid composition (e.g.

across different vent fields), the ratio of mixing of these fluids with ambient oxygenated seawater within a single field, and on the geographic setting. Based on the current data available, a complex global biogeography for vent communities is known to exist (discussed in section 1-5.4, Van Dover et al. 2002;Bachraty et al. 2009;Rogers et al. 2012). Generally however, mid-ocean spreading ridges in the Atlantic and Pacific are both characterised by the presence of chemosymbiotic mytilids and large vesicomyid clams, forming extensive aggregations around diffuse vent flows. In addition on the Mid-Atlantic ridge species of hydrothermal vent shrimp, move around in search of suitable crevices with diffuse flow, and together these

Figure 1.9 Chemosymbioses in different marine habitats

Chemosynthetic symbioses occur in a multitude of habitats. Aside from shallow-water sediments, these habitats include continental slope sediments, submarine canyons and seamounts, mainly as reduced sediments but also due to the accumulation of organic matter, which enriches localised patches of sediment [1]; hydrocarbon seeps [2], nektonic megafaunal carcasses and wood falls [3], and hydrothermal vents [4]. Some host groups are restricted to one habitat (such as Osedax on megafaunal bones), whereas others are pervasive in several different environments (such as thyasirid clams, which are found in shallowwater sea-grass sediments, deep sea cold seeps, whale falls and hydrothermal vents), while the bathymodiolins, occur in the three deepest habitats, at vents, seeps and organic falls. Animals are not to scale. Adapted from Dubilier et al. 2008. Photo acknowledgements are cited on photos when known (see electronic version, with high resolution). three taxa dominate macrofaunal community compositions (Figure 1.9). In the Pacific, such shrimp do not occur, however in their place are perhaps the most extraordinary chemosymbiotic organisms to occur in the marine environment, the vestimentiferan (Sibogliniae) tubeworm Riftia pachyptila. These worms display the fastest growth rates in any marine metazoan [START_REF] Lutz | Rapid growth at deepsea vents[END_REF]. Smaller but equally impressive polychaetes from the Alvinellidae, tolerate some of the hottest temperatures of any multicellular marine organism, spiking at 80 °C maximums, but typically around 40 -45 °C.

Figure 1.10 The biogeochemistry of hydrothermal-vent fluid formation

Above is a schematic of the hydrothermal circulation in a basalt-hosted mid-oceanic ridge vent system. Seawater infiltrates the semiporous igneous rock, is heated and compressed. The chemistry of the evolved fluid is determined by the minerals available but also the temperature reached at the HT reaction zone. For details see main text. Diagram is adapted from an open domain figure. 1-5.3. Organic falls: megafauna and plant-derived material Since the discovery of communities at hydrothermal vent and hydrocarbon seeps, additional habitats have ee ①esta lished①as①p odu ti it ① hotspots ①i ①the①deep-sea, including communities that settle and thrive on, in or around large megafaunal carcasses at various stages of their decomposition and accumulations of sunken vegetation on the deep-sea floor ( Van Dover et al. 2002). Unlike vents and seeps, organic falls are not linked inextricably to biogeochemical or geothermal processes, but rather to the relative productivity of overlying eutrophic waters as feeding grounds (e.g. in the case of baleen whales) or the localised import of coastal and terrestrial vegetative material to the deep sea (e.g. wood and kelp). By far the best understood of these are whale-falls (Baco and Smith 2003;Smith 2006) aggregations may provide conduits for multi-generational dispersal in the endemic fauna (Levin 2007).

Like vents, these habitats are ephemeral and ultimately become sulphidic (e.g. Treude et al. 2009).

In the case of megafaunal nektonic carcasses, four stages of community succession have been recognised

during decomposition, of which the first three are expedited by biological processes (Bennett et al. 1994).

Depending on the carcass size, some of the stages may be truncated (Baco and Smith 2003;Smith 2006).

Initially however, the carcass must first settle on the seafloor: given that sinking rates for naturally deceased megafauna are thought to be rapid, it is argued that relatively little tissue removal occurs during the① a ass ① des e t① e plai ed① i ① detail① i ① " ith① .① O e① the① a ass① a i es① a d① settles① o ① the① sea① floor, the first stage is effectively a feeding frenzy where bacteria and mobile necrophagous scavengers feed at① high① de sities① a d① eakdo ① o ① e o e① the① ulk① of① soft① tissues① o ile① s a e ge , or necrophage stage ,①Be ett①et①al.① .①

Particles of soft-tissue and faecal matter that sink to the seabed during scavenger feeding, organically enrich the underlying sediments up to 3 metres from the carcass, hosting microbial mats and within 4 months, densities of heterotrophic and chemosymbiotic macrofauna an order magnitude higher than in background communities (enrichment-opportunist stage, Bennett et al. 1994;Baco and Smith 2003;[START_REF] Bernardino | Comparative composition, diversity and trophic ecology of sediment macrofauna at vents, seeps and organic falls[END_REF]. Many of the taxa present during this stage are documented at other organicenriched habitats and may be considered as opportunistic, but a select number appear to be specialist to carcass-falls (Smith 2006), though not necessarily whales only (e.g. bone-eating siboglinid polychaete, Osedax spp., Jones et al. 2008;Rouse et al. 2011). The end of this stage is marked by the depletion of all organic tissue, other than those internal to the skeleton, and the disappearance of the opportunist community indicative of these sediment enrichment conditions.

Internal lipid-rich bone marrow within the skeleton probably begins to undergo sulphate-reductive anaerobic degradation soon after external tissue putrefaction begins. Once bones are exposed, the resultant efflux of sulphides (Treude et al. 2009) can fuel the metabolism of chemoautotrophic bacteria, both free-living (e.g. microbial mats) and as symbionts in specialist chemosymbiotic metazoans (e.g. the siboglinid polychaete Osedax spp.; the mussels, Idas washingtonius, Baco and Smith 2003;and Adipicola pelagica, Fujiwara et al. 2007;Figure 1.9). This stage has been described as the sulphidic stage (Bennett et al. 1994), and has been documented to last for at least two years in 4 whale skeletons on the California slope (Bennett et al. 1994;Smith et al. 2002). The proportion of natural whale falls found at this stage of degradation, in comparison to the initial two stages described by Bennett et al. (1994), suggests that the sulphidic stage may in fact be the most extended of the biodegradation phases (e.g. Smith et al. 2002;Baco and Smith 2003;[START_REF] Glover | World-wide whale worms? A new species of Osedax from the shallow north Atlantic[END_REF]Smith 2006;Treude et al. 2009;Tyler et al. 2009;[START_REF] Bernardino | Comparative composition, diversity and trophic ecology of sediment macrofauna at vents, seeps and organic falls[END_REF][START_REF] Amon | The discovery of a natural whale fall in the Antarctic deep sea[END_REF]. Radiochemical methods based on 210 Pb/ 226 Ra disequilibria have identified sulphidiccommunity colonised skeletons up to 85 years old (Schuller et al. 2004). Exceptions may be in juvenile whale falls (Smith and Baco 2003), and whale falls at shallower depths (e.g. [START_REF] Fujiwara | Three-year investigations into sperm whale-fall ecosystems in Japan[END_REF], which can deviate from the described timings and patterns of succession. In practice, evidence suggests that no clear temporal delineation exists between any of the biodegradation stages; in particular, characteristics of the enrichment-opportunist stage and the sulphidic stage are often observed simultaneously (Lundsten et al. 2010).

With the depletion of internal lipids, all organic tissues within and upon the skeleton are consumed, after which only hollow bones remain. Frequently, a proportion of these bones are elevated from the seafloor. Consequently they could provide a hard substrate for filter-feeding organisms adapted to low particulate flux to the deep-sea floor for as long as the bones persist (coined the final reef stage, Bennett et al. 1994).

Like whale falls, wood and macroalgae settling on the sea floor deliver organic material in bulk to sediments, ultimately enriching them via similar avenues (vegetative fragments and faecal matter, [START_REF] Wolff | Magrofaunal utilization of plant remains in the deep sea[END_REF]Bernardino et al. 2010;Bienhold et al. 2013) and initiating reducing conditions both within the wood (Yücel et al. 2013) and to a limited extent in the sediments (Bernardino et al. 2010). However, estimating the supply of vegetative material that enters deep-sea marine environments is complicated byand likely dependent uponconditions during their long journeys from source locations. Environmental transitions through which these materials must pass are more diverse for terrestrial wood than coastal macroalgae:

the two principal types of plant debris to reach deep-sea environments (Bernardino et al. 2010).

Macroalgae are highly labile and are consumed and degraded quickly in coastal and deep sea systems (Bernardino et al. 2010). Being most prevalent in the intertidal rocky shore, they are predominantly exported to the deep-sea floor by tidal currents and wave-driven hydrodynamics (e.g. down-slope flushing)

and are most abundant during severe storm events, particularly when higher densities of kelp grazers and encrusting epibionts compromise kelp-blade integrity [START_REF] Josselyn | Composition, export and faunal utilization of drift vegetation in the salt river submarine canyon[END_REF]Vetter 1994;Vetter and Dayton 1998;[START_REF] Krumhansl | Production and fate of kelp detritus[END_REF]. It is only during storms and down-slope flushing that transport rates will be sufficiently rapid to deliver largely un-degraded material to the deep sea floor providing high quality nutrient input to the deep-sea benthos (Bernardino et al. 2010;[START_REF] Krumhansl | Production and fate of kelp detritus[END_REF]. Based on the① li ited① data① a aila le① Be a di o① et① al.① ,① odest ① a o ① a d① it oge ① e i h e t① i ① sedi e ts① impacted by kelp parcels actually reflect rapid rates of utilisation by specialised infauna, which outstrip rates of carbon and nitrogen loading in the sediment (Bernardino et al. 2010). By comparison, the arrival of wood to the deep-sea floor does not result in immediate carbon-enrichment of sediments; the initiation of deep-sea sediment enrichment by refractory woody substrata is markedly delayed (>1.8 years) compared with labile macroalgal enrichment (Bernardino et al. 2010). However, levels of enrichment at 3 years far exceeded rates at which they could be utilised, resulting in massive carbon enrichment 3-6 times higher than background sediments. The differences in C/N ratios between the two types of plant material reflect the relatively nitrogen-rich composition of kelp versus the extremely nitrogen-poor composition of wood.

The physical breakdown of wood is a consequence of intensive burrowing activity by deep-sea wood-boring bivalves (e.g. Xylophaga spp., Pholadidae) and to a lesser extent through grazing by other groups including the limpet genus Pectinodonta. Both these taxa host cellulolytic symbiotic bacteria [START_REF] Distel | Bacterial Endosymbionts in the Gills of the Deep-Sea Wood-Boring Bivalves Xylophaga atlantica and Xylophaga washingtona[END_REF][START_REF] Zbinden | Bacterial o u ities① asso iated① ith① the① ood-feedi g① gast opod① Pe ti odo ta① sp. Patellogast opoda,① Mollusca)[END_REF]) that aid in the assimilation of cellulose as a carbon source and in some cases contribute to nitrogen fixation. This also facilitates burrowing activity, which greatly accelerates wood degradation in the deep sea (Voight 2009). Fragments of wood and vast volumes of faecal matter then settle on the surrounding sediment, enriching the underlying seabed locally (<0.5m from wood, Bernardino et al. 2010). Simultaneously, anaerobic sulphate-reducing heterotrophic bacteria deriving their a o ①f o ①the① ood s① o e①p odu e①sulphides①as①a① -product (Treude et al. 2009;Khelaifia et al. 2011;Bienhold et al. 2013;Yücel et al. 2013). These sulphides sustain chemoautotrophs occurring both free-living in microbial films and mats and as symbiotic bacteria within hosts that belong to similar higher taxonomical ranks to those at seeps or whale falls (depending on the species: e.g. small bathymodiolin mussel Idas modiolaeformis, Duperron et al. 2008b;siboglinid polychaetes, Dando et al. 1992).

I ① the① past,① o ga i ① falls① ha e① ee ① p oposed① as① pote tial① e olutio a ① steppi g① sto es ① fo ① the① ancestral invasion and eventual adaptation of bivalve species to other reducing habitats, such as vents and seeps (Smith et al. 1989;Distel et al. 2000). The most up-to-date extant phylogenies of wood-fall species (Jones et al. 2006;Miyazaki et al. 2010;Lorion et al. 2013;Thubaut et al. 2013b) appear to support this hypothesis for Bathymodiolinae (s.l.) living on wood. More controversial however, is the notion that organic falls may be used as conduits for dispersal in contemporary vent and seep species across consecutive However in this study, the authors acknowledge some potential bias in relation to sampling effort. In addition, flaws in the choice of statistical approach have been inferred in a subsequent study casting doubt upon the number of provinces proposed, which are postulated to be far more numerous than originally believed; 11 with additional data from vent-field communities discovered in the Southern Ocean (Rogers et al. 2012;Figure 1.11).

In contrast, any trends that may exist in the faunal biogeography of seeps along the Atlantic Equatorial Belt (AEB) were overwhelmingly dominated by depth-dependent clustering (Bray-Curtis and Hellinger similarities), particularly in specialist seep fauna (Olu et al. 2010). Similar depth-dependent trends have been identified in cold seeps of the Gulf of Mexico [START_REF] Cordes | Cold seeps of the deep Gulf of Mexico: Community structure and biogeographic comparisons to Atlantic equatorial belt seep communities[END_REF]. Relatively giant species of the Bathymodiolinae are conspicuously absent from the Mediterranean Sea, where small-sized members of this subfamily and siboglinid Lamellibrachia sp. are the dominant chemosymbiotic species found on carbonate concretions at seeps, with additional sediment-based Vesicomyidae, Lucinidae and Thyasiridae bivalves.

Identifying biogeographic patterns for organic falls is confounded by the infrequency with which they have been discovered. Whale-fall assemblages may have more restricted biogeographic ranges, but the degree to which this trend is biased towards inherently low sampling effort, as opposed to constrained gene flow in spatially and temporally fragmented habitats, remains unclear (McClain and Hardy 2010).

Similarly, for wood, the majority of studies to date have been on experimentally deployed wood substrata, and about three-quarters of the limited number of natural wood studies appear to originate from a single sampling region around the Pacific Islands (e.g. [START_REF] Palacios | Microbial ecology of deepsea sunken wood: quantitative measurements of bacterial biomass and cellulolytic activities[END_REF]Pailleret et al. 2007a;Lorion et al. 2010; Samadi et al. 2010;[START_REF] Zbinden | Bacterial o u ities① asso iated① ith① the① ood-feedi g① gast opod① Pe ti odo ta① sp. Patellogast opoda,① Mollusca)[END_REF], making biogeographic assertions difficult. That said, several reducing-habitat genera appear to be widely distributed among deep-sea habitats, including siboglinid polychaetes and chemosymbiotic bivalves ( [START_REF] Dover | The ecology of deep-sea hydrothermal vents[END_REF][START_REF] Cordes | Cold seeps of the deep Gulf of Mexico: Community structure and biogeographic comparisons to Atlantic equatorial belt seep communities[END_REF]). These patterns of large deep-sea species ranges appear to hold true for vents, seep, seamounts, and soft-sediments also [START_REF] Bisol | Ecological and genetical survey on two deep-sea holothurians: Benthogone rosea and Benthodytes typica[END_REF]. Global biogeographic patterns of genetic heterogeneity across bathymetric gradients of a few kilometres only, are recurrent on slopes, at seeps and vents. In view of the fact that deep-sea chemosynthetic habitats continue to be discovered [START_REF] Domack | A chemotrophic ecosystem found beneath Antarctic ice shelf[END_REF]Rogers et al. 2012), the resolution of biogeographic complexity can only increase in the coming years.

1-6 Adaptations to reducing environments: A benthic lifecycle perspective 1-6.1. Environmental constraints

In addition to the constraints of the deep sea, specific characteristics of reducing habitats make them challenging places to live. First, chemosynthetic environments are typically ephemeral over short timescales, normally decades (Tunnicliffe et al. 1997;Smith et al. 2002;[START_REF] Snelgrove | A riot of species in an environmental calm: The paradox of the species-rich deep-sea floor[END_REF]Smith and Baco 2003;Smith 2006;Gaudron et al. 2010;Mullineaux et al. 2010;Gaudron et al. 2012 except perhaps seeps, von [START_REF] Von Cosel | Gigantism in Mytilidae. A new Bathymodiolus from cold seep areas on the Barbados accretionary Prism[END_REF]. This may be caused by geological instability (e.g. fast-spreading vent-ridges or changes in fluid supply at back-arc basins due to continental subduction) or it may relate to the finite nature of the organic supply of reduced compounds (lipids in whale-bone, sulphidic core of sunken, decomposing wood, Metaxas and Kelly 2010). Second, these habitats are not continuous (occasionally separated by up to 1000 km, Mullineaux et al. 2010) interspersed among a seemingly unsuitable background soft-sediment habitat (e.g. Figure 1.6; Figure 1.7). Third, endemicity (or at the very least strict habitat specificity) is a common feature, which can place species adapted to these habitats at high risk of local extinction. Finally, the dominant species that successfully colonise chemosynthetic environments rely on larval dispersal to populate new habitat, being generally sessile or of limited mobility (Paull et al. 1984;[START_REF] Olu | Structure and distribution of cold seep communities along the Peruvian active margin: relationship to geological and fluid patterns[END_REF][START_REF] Metaxas | Behaviour in flow: perspectives on the distribution and dispersion of meroplanktonic larvae in the water column[END_REF]Ramirez-Llodra et al. 2010), Therefore, in order to exist and persist in the more ephemeral, patchy, chemically reduced habitat isla ds ,① e thi ① o ga is s① i ① these① ha itats① ust have initially had (or subsequently evolved) advantageous physiological, behavioural, and life-history traits. Physiological and behavioural adaptations would permit the identification and colonisation of sparse suitable, habitat, with mechanisms in place to tolerate and even harness the reducing conditions of said habitat indirectly. Life-history traits which would be well adapted to overcome geographic isolation and ephemeral timescales, are probably those that favour dispersal phases capable of reaching the nearest suitable habitats, and rapid rates growth, maturation and reproduction respectively. Given such life history traits are energetically demanding, a reliable supply of nutrition would be required. Thus biological communities that thrive in reducing habitats are faced with specific obstacles above and beyond those of soft-sediment environments that dominate the deep-sea in surface area (Figure 1.12). 1-6.2. Biology, behaviour and critical junctures in the benthic lifecycle Benthic invertebrates found in deep-sea reducing habitats usually exhibit complex life cycles that include a planktonic larval phase, a demersal or benthic juvenile phase, and a benthic adult phase (Figure 1.12, see also Figure 1.13). Critical junctures in development occur throughout the benthic in e te ate s① life le① including metamorphoses, a period of small-sixed developmental, reproductive activities, and transitions between the pelagic and benthic environment (Figure 1.13). Such junctures carry high levels of mortality (e.g. export of larvae off-habitat, developmental abnormalities during complex ontogenetic changes, and post-settlement mortality) and represent some of the most vulnerable phases in the lifecycle [START_REF] Morgan | The larval ecology of marine communities[END_REF]. On top of this are multiple stressors typical of benthic and pelagic environments alike (Figure 1.13, core of schematic). At reducing habitats, juveniles are prone to predation on account of their small size, while competition for space and access to reducing fluids is probably fierce in juveniles and adults.

1-6.3. Adult reproduction

Reproductive traits of adult deep-sea benthic invertebrates are well-documented in comparison with larval characteristics, but represent a small fraction of what is known for shallow-water species. Most reproductive processes identified in deep-sea metazoans appear to be phylogenetically conservative, even

i ① e t e e ① e t①a d①seep①ha itats① T le ①a d①You g① . Species in the deep-sea have been identified as being gonochoristic; including many annelids, most gastropods, nearly all bivalves and crustacea and the majority of echinoderms and octacorals (Young 2003, Figure 1.12). Exceptions exist of course, such as protandry in gastropods (e.g. Haliella stenostoma, [START_REF] Warén | A generic revision of the family Eulimidae (Gastropoda, Prosobranchia)[END_REF], bivalves (e.g. Idas spp., Tyler et al. 2009;Gaudron et al. 2012) and pandalid shrimp (Young 2003) and simultaneous hermaphroditism in holothurians [START_REF] Young | Seasonal breeding aggregations in low-density populations of the bathyal echinoid Stylocidaris lineata[END_REF]. The advantages of being either gonochoristic or hermaphroditic in the deep-sea are unclear, other than constraints upon phylogeny, but probably depend upon the evolutionary history of a species distribution and densities.

The move from benthic to pelagic occurs subsequently to spawning or hatching (Figure 1.13). By this stage, gonads have ripened and gametes are mature in the chemosymbiotic adult, prior to the release of gametes (in broadcast spawners). The timing and frequency of spawning in reproductive adults in the deep-sea are known to strongly influence the direction of transport and therefore dispersal with consequences for marine connectivity (reviewed in [START_REF] Carson | Reproductive timing alters population connectivity in marine metapopulations[END_REF]), be it seasonal (e.g. bathyal echinoids, [START_REF] Young | Seasonal breeding aggregations in low-density populations of the bathyal echinoid Stylocidaris lineata[END_REF], or continuous (e.g. polychaetes in reducing habitats, [START_REF] Rouse | Spawning and development in Osedax boneworms (Siboglinidae, Annelida)[END_REF]scleractinian coral, Waller et al. 2002). The timing will dictate the water mass into which the gametes or larvae begin their journey, whilst a higher frequency (number of cohorts per unit time) will mitigate the impact of incidents of mass-mortality during a single spawning event. For example, high rates of fecundity and continuous spawning in the genus

Osedax and several bathymodiolins are thought to support their capacity to populate nascent habitats on the seafloor [START_REF] Rouse | Spawning and development in Osedax boneworms (Siboglinidae, Annelida)[END_REF]Mullineaux et al. 2010). In fact, semi-continuous reproduction is a common strategy in deep-sea fauna (reviewed in Young 2003), particularly in polychaetes but with examples within the molluscs [START_REF] Olabarria | Reproductive strategies of two deep-sea gastropod species from the Porcupine Seabight (Northeast Atlantic)[END_REF]Villanueva 1992). Seasonal spawning has been documented in bathyal environments (e.g. [START_REF] Young | Seasonal breeding aggregations in low-density populations of the bathyal echinoid Stylocidaris lineata[END_REF]) and particularly in the Bathymodiolinae ( Van Dover et al. 2002;Van Dover et al. 2003;Colaço et al. 2006;Dixon et al. 2006;Tyler et al. 2007;but not B. thermophilus Tyler and Young 1999), and even alternating spawning strategies, correlated with habitat depth [START_REF] Mercier | Depth-related shift in life history strategies of a brooding and broadcasting deep-sea asteroid[END_REF]. Behaviour in deep-sea broadcast-spawners has generally been attributed to constraints relating to sperm concentration and thus lowering encounter and fertilisation rates in gametes with increasing diffusion [START_REF] Levitan | Sperm limitation in the sea[END_REF][START_REF] Powell | Effect of sperm concentration and sperm ageing on fertilisation success in the Antarctic soft-shelled clam Laternula elliptica and the Antarctic limpet Nacella concinna[END_REF]. Other hypotheses cite a combination of extended gamete longevity, coupled with the increased viscosity and capacity for retention at the benthic boundary layer (e.g. echinoids, [START_REF] Yund | Sea urchin spawning in benthic boundary layers: Are eggs fertilized before advecting away from females?[END_REF]. Documented examples of adaptive behaviour compensating for these problems include pairing in echinoderms (e.g. [START_REF] Young | Seasonal breeding aggregations in low-density populations of the bathyal echinoid Stylocidaris lineata[END_REF][START_REF] Young | Seasonal breeding aggregations in low-density populations of the bathyal echinoid Stylocidaris lineata[END_REF][START_REF] Mercier | Depth-related shift in life history strategies of a brooding and broadcasting deep-sea asteroid[END_REF] or relocation to a bathyal habitat for spawning in mesopelagic cephalopods (e.g. [START_REF] Seibel | Life history of Gonatus onyx (Cephalopoda: Teuthoidea): deep-sea spawning and post-spawning egg care[END_REF]. Timing adaptations may include taking advantage of environmental cues (e.g. snow crabs, [START_REF] Starr | Larval release in a sub-euphotic zone invertebrate triggered by sinking phytoplankton particles[END_REF] or the presence of mating partners in close proximity (e.g. [START_REF] Young | Seasonal breeding aggregations in low-density populations of the bathyal echinoid Stylocidaris lineata[END_REF] .① What s① o e,① spa i g① a d① ep odu ti e① iolog can be depth-dependent (and presumably resource-specific) in starfish Henrica lisa [START_REF] Mercier | Depth-related shift in life history strategies of a brooding and broadcasting deep-sea asteroid[END_REF].

Figure 1.13 Critical junctures in the lifecycle of chemosymbiotic organism

Centre: Selective pressures which impact on physiological health and mortality, for both pelagic and benthic lifecycle phases. Surrounding white region: key junctures in the benthic and pelagic periods of the lifecycle (pelagic phases may be demersal in select species). Emigration is away from the source habitat and immigration is towards the sink, however during self-recruitment, sink and source habitats are the same geographic location. The peripheral coloured graphic relates to symbiont densities (width of band = density) in the tissue of the host, where zero density indicates the aposymbiotic state (absence of symbionts in a typically symbiotic species). In vertical transmission (green), symbionts are passed down the germ-line in gametes①f o ①pa e t①to①offsp i g① ). Thus the organism is never strictly aposymbiotic. In horizontal transmission (two shades of blue), symbionts are either acquired laterally, through parental non-germ-line, conspecific or interspecific host transmission, or environmentally, as free-living bacteria. This is likely to occur at source or at sink habitats ( and respectively), where environmental conditions favour the presence of symbiotic bacterial candidates. Where a uisitio ① is① at① the① sou e①

), only a very brief aposymbiotic period exists prior to acquisition. In contrast, acquisition at the sink habitat ① enforces a longer aposymbiotic period during larval development and dispersal. Intermediate acquisition during larval dispersal is thought to be unlikely due to a lack of symbiont supply in the pelagos. In demersal larvae, this assumption may not apply. Adapted from the lifecycle of benthic organisms in [START_REF] Morgan | The larval ecology of marine communities[END_REF] Invertebrates achieve fertilisation in various ways from releasing gametes at high densities (broadcast spawning) with pelagic embryos (Porifera, echinoderms, bivalves and corals (Levinton 2001), to internal fertilisation via copulation, with viviparity (e.g. some crustaceans, polychaetes, and ascidians (Levinton 2001), or external egg masses/capsules (some anthozoans, nemerteans, polychaetes, and gastropods (Levinton 2001 .① Most① spe ies ① ode① of① fe tilisatio ① ill① eside① ithi ① this① spe t u .① The① method of fertilisation and the dominant larval mode employed typically co-evolve, and the costs and benefits of both stages in development are intertwined [START_REF] Morgan | The larval ecology of marine communities[END_REF]. Fertilisation success in some situations will be limited by access to gametes of the opposing sex [START_REF] Levitan | The ecology of fertilization in free-spawning invertebrates[END_REF][START_REF] Levitan | Sperm limitation in the sea[END_REF], but in others, the reverse is true with fertilisation success curtailed by incidents of polyspermy (Franke et al. 2002). After hatching, a period of larval transport takes places, the duration of which depends on the mode of development and physical oceanographic conditions. 1-6.4. The larval phase Following successful fertilisation, the development of larvae is typically in the water column (Figure 1.13, except in larval-brooding species) where several modes of development are known to exist (Figure 1.12).

The most common are planktotrophic larvae (represented by the widest arrow in Figure 1.12), which can actively feed and are thus assumed to be capable of remaining in the plankton for longer than non-feeding larvae that rely on a maternally-derived yolk reserve (lecithotrophy); both have far greater larval durations than brooded larvae (few seconds to hours). Note that, though planktotrophy permits higher fecundity in adults due to minimal energy investment per gamete, it is also believed to result in higher rates of mortality when compared with yolk-nourished larvae [START_REF] Strathmann | Feeding and nonfeeding larval development and life-history evolution in marine invertebrates[END_REF]. Logically, the larval mode will reflect cost to benefit trade-offs between the number of larvae that can be produced and the actual success of dispersal in species-specific situations. These developmental modes are often ranked in terms of a hierarchy of increasing larval duration as follows (assuming identical conditions during transport): brooding (directdevelopment) << lecithotrophy < planktotrophy (which includes extreme teloplanic1 examples, Scheltema 1971) and is either pelagic or demersal. However, when environmental conditions differ between similar larval modes, the afore-stated hierarchy in duration regularly breaks down (e.g. [START_REF] Mercier | Pelagic propagule duration and developmental mode: reassessment of a fading link[END_REF].

Temperature increases are known to have a universal impact upon all modes of development increasing rates of development presumably through increased metabolism [START_REF] Reitzel | Relationships between spawning date and larval development time for benthic marine invertebrates: a modeling approach[END_REF]). Consequently, even though a lack of feeding in lecithotrophy places an upper limit upon energy resources, many extreme coldate ① spe ies ① le ithot ophi ① la ae① displa ① p olo ged,① eta ded① la al① de elop e t① due,① it s① elie ed,① to① metabolic suppression [START_REF] Young | Biogeographic and bathymetric ranges of Atlantic deepsea echinoderms and ascidians: the role of larval dispersal[END_REF]). In addition, at least in the echinoderms, a synergistic influence of phylogeny and climate has greater influence on larval dispersal than larval mode [START_REF] Mercier | Pelagic propagule duration and developmental mode: reassessment of a fading link[END_REF].

Given a large enough yolk reserve, lecithotrophy may even be competitively comparable to planktotrophy [START_REF] Marshall | Variation in the dispersal potential of non-feeding invertebrate larvae: the desperate larva hypothesis and larval size[END_REF] and potentially the more reliable strategy, if local planktonic nutritional resources are unpredictably patchy [START_REF] Pearse | Reproduction of Antarctic benthic marine invertebrates: tempos, modes, and timing[END_REF]. Extreme examples of unexpectedly far-reaching dispersal have been documented in larvae undergoing direct development (brooding), where otherwisei e pli a l ① oad① adult① dist i utio s① a e① ai tai ed① ① afti g ① o ① flotsa ① o e ① g eat① dista es① [START_REF] Johannesson | The paradox of Rockall: why is a brooding gastropod (Littorina saxatilis) more widespread than one having a planktonic larval dispersal stage (L. littorea)?[END_REF][START_REF] Helmuth | Long-distance dispersal of a subantarctic brooding bivalve (Gaimardia trapesina) by kelp-rafting[END_REF][START_REF] Foighil | Trans-Pacific range extension by rafting is inferred for the flat oyster Ostrea chilensis[END_REF]. Two additional considerations must be the hydrodynamics that determine the transport environment for larvae, and the active mediation of transport by larvae themselves.

1-6.4.1 Physical oceanography: Hydrodynamics and its impact on dispersal Hydrodynamic processes that are known to affect benthic invertebrates (Figure 1.12) operate on a vast temporal and spatial scale. Large-scale but slow-moving hydrodynamic processes, in particular thermohaline (Figure 1.12 ,① o ① o e e ① elt ① i ulatio ① esults① f o ① te pe atu eand salinity-driven density differences in oceanic water masses, due to wind-driven Eckman transport, solar heating and evaporation. These large-scale movements transport energy (kinetic and heat) and substances in suspension and dissolution around the globe, with residency times in the order of 1000s of years.

Mesoscale processes such as the formation and movement of meanders, upwelling and downwelling currents, mesoscale eddies, frontal boundaries and complex coastal operate on regional km-wide scales.

Smaller-scale interactions then act from the kilometre-wide scale to the molecular level, such as the conditions that favour either laminar or turbulent flow (the basis for frontal formation) and dictate how fluids in the deep oceans interact with abiotic and biotic processes (e.g. the initiation of fluid-driven lift in grains of sediment). Together these hydrodynamic processes can result in very low and high-energy environments, with far-reaching ecological consequences from zones of permanent hypoxia on some continental margins to the complex energetic environment of submarine canyons and upwelling zones respectively. In the context of reducing habitats, hydrodynamics govern the delivery of sunken wood the deep sea and provide the oxygenated ambient water present at all anoxic-oxic interfaces, critical to chemosynthetic life. Propagule dispersal, typically associated with the earliest life history stage (as gametes, fertilised eggs, larvae), is assumed to be the principal means by which the distribution of benthic species is maintained in chemosynthetic deep-sea habitats (Mullineaux et al. 2010). For these organisms including those from deep-sea reducing habitats, hydrodynamics provide a vector for planktonic propagule transport at scales of dispersal that would otherwise be unachievable.

Examples of current-aided larval dispersal from coastal (mainly) and deep-sea environments include the transport of larvae entrained in and conveyed by mesoscale eddies [START_REF] Lobel | Transport and entrapment of fish larvae by ocean mesoscale eddies and currents in Hawaiian waters[END_REF][START_REF] Limouzy-Paris | Translocation of Larval Coral Reef Fishes Via Sub-Mesoscale Spin-Off Eddies from the Florida Current[END_REF][START_REF] Siegel | Lagrangian descriptions of marine larval dispersion[END_REF][START_REF] Aiken | Along-shore larval dispersal kernels in a numerical ocean model of the central Chilean coast[END_REF][START_REF] Paris | Surfing, spinning, or diving from reef to reef: effects on population connectivity[END_REF]Christie et al. 2010); complex but predictable circulations generated by tides, such as salt intrusions in estuarine systems [START_REF] Shen | A modeling study of a tidal intrusion front and its impact on larval dispersion in the James River estuary, Virginia[END_REF][START_REF] Shanks | Observations on the distribution of meroplankton during a downwelling event and associated intrusion of the Chesapeake Bay estuarine plume[END_REF][START_REF] Hsieh | Effects of semidiurnal tidal circulation on the distribution of holo-and meroplankton in a subtropical estuary[END_REF][START_REF] Murray | Report on deep-sea deposits based on the specimens collected during the voyage of HMS Challenger in the years 1872 to 1876. Printed for HM Stationery off[END_REF]; net transport during long-shore drift [START_REF] Richards | Larval dispersion along a straight coast with tidal currents: complex distribution patterns from a simple model[END_REF][START_REF] Siegel | Lagrangian descriptions of marine larval dispersion[END_REF]; the action of internal waves [START_REF] Wing | Breaking internal waves on a Florida (USA) coral reef: a plankton pump at work?[END_REF][START_REF] Epifanio | Larval transport on the Atlantic continental shelf of North America: a review[END_REF]; peripheral larval transport during the formation of oceanic gyres (Lee et al. 1992); upwelling zones [START_REF] Shanks | Demonstration of the onshore transport of larval invertebrates by the shoreward movement of an upwelling front[END_REF] and the particular case of geothermal-based plume circulation (e.g. [START_REF] Kim | Larval dispersal via entrainment into hydrothermal vent plumes[END_REF][START_REF] Marsh | Larval dispersal potential of the tubeworm Riftia pachyptila at deep-sea hydrothermal vents[END_REF][START_REF] Bailly-Bechet | A modeling approach of the influence of local hydrodynamic conditions on larval dispersal at hydrothermal vents[END_REF]. Hyper-evaporative processes such as deep-water cascading may represent an additional means of dispersal [START_REF] Canals | Flushing submarine canyons[END_REF][START_REF] Canals | Cascades in Mediterranean submarine grand canyons[END_REF]. With exception to geothermal systems (and even then occasionally; [START_REF] Marsh | Larval dispersal potential of the tubeworm Riftia pachyptila at deep-sea hydrothermal vents[END_REF], most of these phenomena can be partially or wholly attributed to wind-or tide-driven currents, interacting with topography where relevant.

Conversely, interactions between larvae and counter-dispersive features can assert sub-maximal dispersal range limits (i.e. oceanographic or topographic barriers) such as in coastal boundary layers (Lee et al. 1992;[START_REF] Gaylord | Temperature or transport? Range limits in marine species mediated solely by flow[END_REF] Meroplankters have historically been thought of as almost entirely passive: insufficiently motile to overcome currents which drive local and regional water circulation; the physical environment that will largely dictate the direction and speed of larval transport from source to sink. However, growing evidence suggests that while this is true for horizontal water mass speeds, it may not apply to vertical migration between two horizontal water masses. If these act in opposing directions, it is possible to preferentially navigate between favourable currents. This is well-documented for larval marine fish (reviewed in [START_REF] Neilson | Diel vertical migrations of marine fishes: an obligate or facultative process?[END_REF]; with examples more recently in [START_REF] Cowen | Connectivity of marine populations: open or closed?[END_REF][START_REF] Kingsford | Sensory environments, larval abilities and local self-recruitment[END_REF][START_REF] Gerlach | Smelling home can prevent dispersal of reef fish larvae[END_REF]) and often enables high levels of retention at natal sites for endemic species [START_REF] Jones | Coral reef fish larvae settle close to home[END_REF][START_REF] Almany | Local replenishment of coral reef fish populations in a marine reserve[END_REF][START_REF] Gerlach | Smelling home can prevent dispersal of reef fish larvae[END_REF]. Tendencies towards natal larval retention have also been postulated for hydrothermal vent species (e.g. vent tubeworm Riftia pachyptila, [START_REF] Shank | Toward a mechanistic understanding of larval dispersal: insights from genomic fingerprinting of the deep-sea hydrothermal vent tubeworm Riftia pachyptila[END_REF].

Incidents of vertical migration have also been documented to a lesser extent in marine invertebrates (e.g. in response to light and food availability, [START_REF] Starr | Larval release in a sub-euphotic zone invertebrate triggered by sinking phytoplankton particles[END_REF][START_REF] Gallager | Ontogenetic changes in the vertical distribution of giant scallop larvae, Placopecten magellanicus, in 9-m deep mesocosms as a function of light, food, and temperature stratification[END_REF][START_REF] Manuel | Veligers from different populations of sea scallop Placopecten magellanicus have different vertical migration patterns[END_REF]; or dielmigration in reverse to counteract offshore-export in an upwelling zone, [START_REF] Poulin | Avoiding offshore transport of competent larvae during upwelling events: the case of the gastropod Concholepas concholepas in central Chile[END_REF]. In fact, examples from upwelling zones are documented so frequently as to suggest that this is the norm, employing various documented or assumed migrations [START_REF] Shanks | Demonstration of the onshore transport of larval invertebrates by the shoreward movement of an upwelling front[END_REF][START_REF] Kingsford | Sensory environments, larval abilities and local self-recruitment[END_REF][START_REF] Shanks | Upwelling, downwelling, and cross-shelf transport of bivalve larvae: test of a hypothesis[END_REF][START_REF] Dubert | Influence of vertical migration pattern on retention of crab larvae in a seasonal upwelling system[END_REF]Morgan et al. 2009a;[START_REF] Morgan | Nearshore larval retention in a region of strong upwelling and recruitment limitation[END_REF]). Larvae will even perform migrations that place them at higher risk of predation, if it can result in finding suitable habitat and prevent exportation to unsuitable environments (e.g. inverse vertical migrations during daylight hours in estuaries [START_REF] Morgan | Behavioral tradeoff in estuarine larvae favors seaward migration over minimizing visibility to predators[END_REF].

Thus, although far-reaching dispersal necessitates a long larval duration in order to accommodate extensive larval transport, extensive duration will not necessarily equate to a far-reaching dispersal (see Figure 1.14).

This is because larval transport is the accumulative distance covered along the path of motion during the entire planktonic existence prior to settlement in the form of a translocation between two points (Figure 1.14; blue path in A and B), and is a direct function of planktonic duration [START_REF] Pineda | Larval transport and dispersal in the coastal ocean and consequences for population connectivity[END_REF]. It is subject both to larval behavioural mediation and physical mechanistic processes, where the spatiotemporal scales of variability are vast (Shanks 2009). In contrast, the distance dispersed is effectively a measure of linear spread from the source [START_REF] Pineda | Larval transport and dispersal in the coastal ocean and consequences for population connectivity[END_REF]). The concept of the pre-competent duration not correlating with the dispersal distance on account of these phenomena is an increasingly pervasive one in marine science; larval durations of less than a day predict dispersal capacity well, whereas the true dispersal distance realised by larvae with extended larval durations, frequently falls well-short of predictions (Shanks 2009). Given the importance that larval motility appears to play in mitigating unfavourable physical transport processes in shallow-water systems, a better understanding of larval motility and behaviour in the deep sea is of fundamental importance. Categorising motility remains challenging however. Larval-tracking in the deep-sea is not tractable, so larval studies must be performed ex situ with the associated difficulties of recreating pressurised conditions. In addition the requirements for larval pilotage and settlement remain untested, in oceanographic current regimes which are poorly described. [START_REF] Metaxas | Behaviour in flow: perspectives on the distribution and dispersion of meroplanktonic larvae in the water column[END_REF]. These could include micro-turbulence at physical and biological discontinuities in the water column, behaviour at the benthic boundary layer during demersal transport and all settlement behaviour; maintaining location (retention) or changing direction (mediated transport) in laminar flow and transport in internal waves, eddies and meanders. The difficulties are compounded by the likelihood that larvae might also respond to other directional stimuli such as electro-magnetic energy (intensity and frequency e.g. phototaxis) or display capacities for chemotaxis (particulates and solutes), thermotaxis, galvanotaxis, magnetotaxis or engage in spatial orientation (gravitaxis and linear acceleration) and respond to the kinematic viscosity of seawater. One of the most widely acknowledged applications of motility in shallow-water species is in vertical migration through the water column. The degree to which deep-sea invertebrate larvae mediate their transport with swimming behaviour is unclear, with limited information available concerning actual larval trajectories (e.g. Arellano et al. 2014). The high densities of larvae found in coastal current regimes which have permitted reliable interpretations of mediated transport, are not reflected in the open ocean where larvae of deep-sea species are rarely collected in large numbers. This is particularly true for reducing habitats. However it is known that, with the exception of siboglinids and vesicomyids, many of the organisms that dominant vent and seep habitats including bathymodiolin mussels, alvinocaridid shrimps and vent crabs, have either indirectly or directly, have been shown to employ long-lived planktotrophic larvae. The demonstrated ability to colonise nascent sites following natural disturbances by organisms adapted to these habitats (e.g. [START_REF] Shank | Temporal and spatial patterns of biological community development at nascent deep-sea① h d othe al① e ts① ① ′① N,① East① Pa ifi ① Rise)[END_REF]Mullineaux et al. 2010), where supply is known to be from eigh ou i g ① distant sites, provides evidence that the larvae of deep-sea chemosymbiotic invertebrates are capable of long-range dispersal, requiring long planktonic duration times. Other than apparently long larval durations [START_REF] Scheltema | Larval dispersal as a means of genetic exchange between geographically separated populations of shallow-water benthic marine gastropods[END_REF]Arellano and Young 2009), the means by which long-ranging dispersal is achieved, specifically with regard to mediated behaviour, remains unknown. In the deep-sea only limited examples exist that successfully identify a larval trait as having acted to influence dispersal: the possible effects of positive buoyancy in Riftia pachyptila in vent-plumes [START_REF] Marsh | Larval dispersal potential of the tubeworm Riftia pachyptila at deep-sea hydrothermal vents[END_REF]) and negative buoyancy in another siboglinid genus, Osedax sp., found on sunken carcasses [START_REF] Rouse | Spawning and development in Osedax boneworms (Siboglinidae, Annelida)[END_REF]).

The apparent level of connectivity over long distances maintained by some species from reduced habitats (e.g. Olu et al. 2010, Rogers et al. 2012), is even more extraordinary when one considers the effect that extended larval transport could have on densities of surviving larvae in the deep sea, by the time they are competent to settle (Figure 1.15). It is evident that from the moment of spawning, larval transport is subject to a plethora of forcing variables (Figure 1.12-15) which dictate dispersal capacity, successful settlement on a suitable substrate and survival through to recruitment (reproductive maturity). [START_REF] Pawlik | Settlement of a marine tube worm as a function of current velocity: interacting effects of hydrodynamics and behavior[END_REF][START_REF] Krug | Developmental Dimorphism: Consequences for Larval Behavior and Dispersal Potential in a Marine Gastropod[END_REF], while other responses to cues include active sinking in turbulent flow to facilitate retention [START_REF] Fuchs | Sinking behavior of gastropod larvae (Ilyanassa obsoleta) in turbulence[END_REF][START_REF] Fuchs | Effects of turbulence-mediated larval behavior on larval supply and settlement in tidal currents[END_REF]) and settlement and metamorphosis in response to conspecific cues (e.g. [START_REF] Tamburri | Chemical induction of larval settlement behavior in flow[END_REF].

The delaying of the onset of metamorphosis subsequent to reaching competence has also been reported (e.g. [START_REF] Bishop | Interspecific variation in metamorphic competence in marine invertebrates: the significance for comparative investigations into the timing of metamorphosis[END_REF]. Presumably however, in deep sea reducing habitats, larvae must precisely identify chemical traces which indicate suita le①su st ate,① hile① eighi g①up ① o petiti e①a d①fa ilitati e①i t aand interspecific interactions and environmental habitability [START_REF] Levin | Recent progress in understanding larval dispersal: new directions and digressions[END_REF], since settling in high-output fluid emissions may be sub-lethal due to fluid toxicity. Examples of chemotaxis towards reduced fluids as cues for larval settlement are limited to chemical traces of conspecific or interspecific exudates (e.g. Bathynerita naticoidea veligers cueing to Bathymodiolus childressi, [START_REF] Dattagupta | Deep-sea hydrocarbon seep gastropod Bathynerita naticoidea responds to cues from the habitat-providing mussel Bathymodiolus childressi[END_REF]. Chemotaxis towards sulphides though inferred in adult shrimp [START_REF] Renninger | Sulfide as a chemical stimulus for deep-sea hydrothermal vent shrimp[END_REF] has not been identified in the larvae of deep-sea reducing habitats. Evidence for cueing to chemical plumes does exist in opportunistic polychaete Capitella sp. larvae (sulphides, Cuomo 1985). Cueing to physical parameters in deep-sea invertebrates is also documented (e.g. thermotaxis in Bythograea thermydron megalopae, [START_REF] Epifanio | Development and behavior of megalopa larvae and juveniles of the hydrothermal vent crab Bythograea thermydron[END_REF]. Analogous parasite-host scenarios provide examples of the level of cueing precision that is possible, where

Heterosaccus dollfusi naupliar settle e t ① ues① a e① highl ① lo alised① the① od ① of① the① brachyuran crab Charybdis longicollis) and involve complex mediated swimming responses [START_REF] Pasternak | Host location by larvae of a parasitic barnacle: larval chemotaxis and plume tracking in flow[END_REF]. For chemosymbiotic species which engage in environmental acquisition of symbionts, the presence of freeliving chemoautotrophic bacteria rather than reduced compounds may be of fundamental importance and a powerful cue for settlement.

Once cues have been identified, larvae are assumed to sink rapidly towards the identified signature and begin trying to locate the habitat while minimising predator encounters, by actively mediating their position in the environment. If contact is made, then proximal, directional environmental cues are thought to further dictate microhabitat selection. Assuming that a suitable microhabitat has been identified, remaining energy reserves within each larva must be sufficient in order to complete metamorphosis and sustain the newly formed post-larva until it can independently acquire energy for growth. Once larvae have successfully metamorphosed into juveniles, metamorphosed individuals face multiple sources of postsettlement mortality (Figure 1.15) through predation, competition for resources (i.e. starvation, smothering and exclusion), immuno-incompetence with death from bacterial, viral and parasitic disease, environmental pressures such as toxic fluid exposure or sediment loading and anthropogenic disturbance (Levinton 2001).

Thus, recruitment in to the adult population will reflect the net contributions of larval supply, successful settlement and metamorphosis, and rates of post-settlement mortality. Recruitment rates for a given species population (when not due to larval retention) are a major determinant of between-population dynamics and when combined with birth and death rates, will dictate population connectivity within a metapopulation (Cowen and Sponaugle 2009). This universal process ultimately governs global biogeography.

1-6.6. Resource availability and environmental cues

The availability of resources and the concept of environmental cues are not necessarily independent (Figure 1.12 .①I ① oastal①s ste s①it①is①u de stood①that①su essfull ① uei g ①to① esou e①a aila ilit ,① e①it① ut itio al① or habitat-based, is fundamental to effective dispersal and in mitigating mass-mortality [START_REF] Levin | Recent progress in understanding larval dispersal: new directions and digressions[END_REF].

Habitat availability certainly impacts on dispersal success, since patchiness of habitat will reduce substrate encounter rates for sessile species. The availability of suitable nutrition is evidently pivotal in the survival of planktotrophic larvae, particularly in environments where available nutrition are limiting [START_REF] Starr | Larval release in a sub-euphotic zone invertebrate triggered by sinking phytoplankton particles[END_REF].

In chemosynthetic-based deep-sea environments where scarcity in habitat and food are linked intrinsically, the relative importance of photosynthetically derived organic material arriving seasonally (e.g. Tyler et al. 2007) and nutrition derived directly from symbioses with chemoautotrophic bacteria (e.g. [START_REF] Childress | Sulfide and carbon dioxide uptake by the hydrothermal vent clam, Calyptogena magnifica, and its chemoautotrophic symbionts[END_REF]Robinson and Cavanaugh 1995;[START_REF] Marsh | Larval dispersal potential of the tubeworm Riftia pachyptila at deep-sea hydrothermal vents[END_REF]Duperron et al. 2008a;Duperron 2010;Vrijenhoek 2010;[START_REF] Duperron | Relative abundances of methane-and sulphur-oxidising symbionts in the gills of a cold seep mussel and link to their potential energy sources[END_REF]) remains unclear for many reducing habitats. It is likely that at least in some adult metazoans, changes in the availability or quality of either of these nutritional resources (or both) may instigate the onset of gametogenesis in preparation for spawning (Colaço et al. 2006;Gaudron et al. 2012), the initiation of spawning itself [START_REF] Fujiwara | In situ spawning of a deep-sea vesicomyid clam: Evidence for an environmental cue[END_REF] or a change in the dominant means of energy acquisition in adults (Tyler et al. 2007). Given the ephemeral nature of vent, seep (to a lesser degree) and organic-fall habitats, it is believed that some species must have evolved a means to detect certain anomalies during larval dispersal, cueing for substrate selection and settlement (discussed previously). The alternative is that larvae are ubiquitous and omnipresent which, while feasible given the high productivity of these ecosystems [START_REF] Olu | Structure and distribution of cold seep communities along the Peruvian active margin: relationship to geological and fluid patterns[END_REF][START_REF] Mccollom | Geochemical constraints on primary productivity in submarine hydrothermal vent plumes[END_REF], would incur heavy energy investments reproductively. Given these considerations, the availability of suitable habitat or nutritional resources (or potentially both) in the vicinity of source populations could affect larval transport pathways, assuming that larvae can actively respond to these directional environmental cues and, alter their behaviour accordingly in order to take advantage of those currents orientated favourably. Finally, mediated selection of bacteria during infection and the ensuing symbiont acquisition (in the case environmental transmission, as in Figure 1.12) is suspected, given the prevalence for exclusively symbiotic, and frequently monophyletic, associations in many chemosymbiotic species (Dubilier et al. 2008;[START_REF] Bright | A complex journey: transmission of microbial symbionts[END_REF] 1-7 Chemosynthetic bacteria as symbionts in marine systems 1-7.1. Symbioses: definitions and application

Symbiosis and its definition have evolved conceptually over time, as new data comes to light describing symbiotic function both extrinsically within the context of the surrounding environment, and intrinsically between the host (usually ascribed to the larger or more complex organism in terms of anatomy) and the symbiont. A common interpretation of symbiosis① is① ei g① of① utual① e efit ① to① host① a d① s io t.① However symbioses are often asymmetric, where benefit is difficult to demonstrate, as partner(s) can be constrained physiologically-speaking (as in some vertically transmitted bacterial symbionts). Examples are available throughout nature, frequently when a symbiont organism must derive its nutrient supply by means of host intervention (e.g. symbiotic bacteria in some insects have a limited ability to synthesise nucleotides and amino acids, [START_REF] Zientz | Metabolic interdependence of obligate intracellular bacteria and their insect hosts[END_REF]).

Symbioses are not completely mutualistic and between-partner conflict is a widespread feature, though it is generally managed and contained [START_REF] Douglas | Conflict, cheats and the persistence of symbioses[END_REF]Figure 1.16). Reciprocated mutualisms may in fact be host-enforced in cases where one partner stops investing in the symbiotic exchange of resources but continues to receive the benefit of it. In other words the symbiosis is no longer co-operative. This viewpoint presumes some asymmetry in the association, with one partner exacting control over the other(s), e fo i g① good① eha iou ① i ① its① pa t e s .① The① as et ① p ese ts① a① ea s① ① hi h① the① e fo i g partner might provide incentives for cooperation, while punishing cheats. This process is described well in [START_REF] Douglas | Conflict, cheats and the persistence of symbioses[END_REF], from which Figure 1.16 is derived, and takes examples founded on known plant physiology or known animal behaviour. The concept is quite intuitive: take a two-partner symbiosis where each gains so e①ad a tage①f o ①the①othe ,①the① e efit ①out eighi g①the①e e geti ① ost①i ① oth① ases (Figure 1.16b).

The① isk① to① ea h① pa t e ① is① that① the① opposi g① pa t e ① a ① heat ① the① s ste ① ① ot① o itti g① a ① energetic investment, while benefiti g①f o ①its①pa t e s①suppl :①this① a ① esult①i ①fu the ① et①e e g -gain for the cheat, while the partner that continues to co-operate incurs a net loss through unreciprocated investment (Figure 1.16c). Recent modelling studies investigating symbiotic conflict (see [START_REF] Douglas | Conflict, cheats and the persistence of symbioses[END_REF] have found that the initially co-ope ati e①pa t e ① a ① pu ish ①the① heat① ①i posi g①sa tio s①o ①suppl ① Figure 1.16d), which forces the supplicant partner to reinitiate investment, so resetting conditions to those depicted in Figure 1.16b. Repeated or persistent interactions of this sort are thought to temper conflict in reciprocal interactions [START_REF] Douglas | Conflict, cheats and the persistence of symbioses[END_REF], and create a steady state. Examples of such situations may be drawn from the 450-million year-old symbioses between most land plants and arbuscular mycorrhizal fungi, where positi e①feed a k①is①th ough①a ①e ha ge①of①su plus① lu u ① esou es① et ee ①pa t e s,① egulated①th ough① sanctions by one or both partners [START_REF] Kiers | Host sanctions and the legume-rhizobium mutualism[END_REF][START_REF] Kiers | Mutualistic stability in the arbuscular mycorrhizal symbiosis: exploring hypotheses of evolutionary cooperation[END_REF].

Figure 1.16 The cost of reciprocal benefit between two organisms (A and B).

a) The cost of reciprocal benefit: each organism provides a service at a cost of 10 arbitrary units, which has a value of 30 units to the recipient, yielding a net benefit of 20 u its①fo ①ea h①o ga is .① ①The① te ptatio ①to① heat:①o ga is ①②①does① ot①p ovide the service and obtains increased net benefit (30 units) to the detriment of organism B, which derives a net cost of 10 units. Note that for the heati g ① o ga is ① ②,① the① e efit① of① ei g① i effe ti e① as① a① pa t e ① i ①a① s iosis①is①a① gai ① of① ①additional arbitrary units. Adapted from [START_REF] Douglas | Conflict, cheats and the persistence of symbioses[END_REF].

The① o ept①of① utual① e efit ①is①e t e el ①diffi ult①to①test①e pi i all2 since experiments would first have to identify an unequivocal advantage to both partners; and then importantly, that their integ atio ① i to① a① si gle,① olle ti e① o ga is ① is① a① supe io ① a a ge e t① tha ① if① the① t o① pa t e s① li ed① independently. In theory, this might be achieved by applying with-, versus without-association experimental t eat e ts①a d①e plo i g①a ①i de ①of① e efit ①(e.g. changes in rates of growth, reduced mortality, etc), but in practice, symbioses are often so physiologically entangled that this is impossible. Therefore, many scientists have explicitly abandoned the traditional view that symbioses are mutually beneficial relationships, in favour of symbioses representing vehicles by which many organisms have garnered complex metabolic capabilities [START_REF] Douglas | Symbiotic interactions[END_REF], providing augmented fitness for the host as a minimum. For the purposes of this research, concepts from [START_REF] Douglas | Symbiotic interactions[END_REF] and [START_REF] Zook | A new symbiosis language[END_REF]) may be combined to form a loose definition. Thus, symbiosis is considered herein to be the association of one (or more) organism(s) to anotherhost -o ga is ① fo ① all① o ① pa t① of① the① host s① life le,① g a ti g① the① host① o el metabolic capabilities and providing such fitness advantage that the symbiosis persists in this way throughout the course of natural selection.

1-7.2. Evolutionary transition hypotheses

Intracellular endosymbiosis, wherein the internalised organism resides non-dest u ti el ① ithi ① its① host s① tissue beyond the barrier of epithelia, represents the most integrated of interspecific organismal interactions [START_REF] Petersen | Origins and evolutionary flexibility of chemosynthetic symbionts from deep-sea animals[END_REF]. In this sense, bacterial symbioses have been pivotal in eukaryote evolution and diversity, since mitochondria and chloroplasts are thought to have arisen from endosymbiotic i lusio ① of① p oka oti ① ells① so ethi g① si ila ① to① a ① α-p oteo a te ia,① G a ① et① al.① ① a d① α-proteocyanobacteria transitional organism respectively, [START_REF] Moreira | The origin of red algae and the evolution of chloroplasts[END_REF][START_REF] Yoon | Tertiary endosymbiosis driven genome evolution in dinoflagellate algae[END_REF]; Figure 1.17). The exact mechanisms involved in Endosymbiotic Theory are still a matter of considerable ongoing debate (e.g. [START_REF] Moran | Lifestyle evolution in symbiotic bacteria: insights from genomics[END_REF][START_REF] Rivera | The ring of life provides evidence for a genome fusion origin of eukaryotes[END_REF][START_REF] Wächtershäuser | From volcanic origins of chemoautotrophic life to Bacteria, Archaea and Eukarya[END_REF][START_REF] Poole | Evaluating hypotheses for the origin of eukaryotes[END_REF].

Genomic studies which have sought to resolve the Tree of Life have employed novel genomic analyses, revealing that one possible origin of the eukaryotic genome may be through the ancient ancestral fusion of two diverse prokaryotic genomes, linking prokaryotes and eukaryotes and the deepest levels of the tree of life,① hi h①i ①this① o te t①is① o e①aki ①to①a① i g①of①life ① Rivera and Lake 2004). Thioautotrophic bacteria in marine invertebrates (e.g. [START_REF] Kuwahara | Reduced Genome of the Thioautotrophic Intracellular Symbiont in a Deep-Sea Clam, Calyptogena okutanii[END_REF]) display certain traits that have been proposed as the beginnings of a new eukarya lineage, the thiotrophic eukaryotes (Vetter 1991;[START_REF] Lallier | Thioautotrophic symbiosis: towards a new step in eukaryote evolution[END_REF]). However, organelles as we understand them currently display bidirectional gene exchange with their hypothetical hosts, and are present in many types of tissue. Thiotrophic bacteria have not yet been shown to display either of these characteristics. Thus, currently it is difficult to imagine a situation in which this will arise in multicellular organism, though it does not rule out the possibility of such a novel eukarya in single-celled organisms. The obligatory, vertically (maternally) transmitted symbionts of Vesicomyid clams display some of the most reduced genomes (1,022,154 bp with 31.6% guanine + cytosine content, the smallest reported genome in autotrophic bacteria at the time of publication, [START_REF] Kuwahara | Reduced Genome of the Thioautotrophic Intracellular Symbiont in a Deep-Sea Clam, Calyptogena okutanii[END_REF]. This is coupled with a loss of both essential genes (e.g., ftsZ for cytokinesis) for functioning and genes that are superfluous for an intercellular existence. The genomes of other chemosymbiotic deep-sea marine invertebrates symbionts (horizontally and vertically transmitted) are currently being sequenced in order to understand the broader context of endosymbiosis from both a physiological and evolutionary perspective [START_REF] Lallier | Thioautotrophic symbiosis: towards a new step in eukaryote evolution[END_REF].

Figure 1.17 Endosymbiotic theory

The diagram depicts a hypothesised evolution of multicellular Eukaryotes, where the origins of plastids and mitochondria in contemporary eukaryotes were through incidences of endosymbiosis of prokaryotes/archea. Source: Benjamin Cummings, Pearson education 1-7.3. Known interactions: free-living bacteria, to obligate symbionts One of the indicative features of reducing habitats is the prevalence of metazoans such as siboglinid polychaetes and several bivalve families, which have evolved intrinsic and complex symbiotic relationships with chemosynthetic bacteria, most notably sulphur-or methane-oxidisers [START_REF] Childress | Sulfide and carbon dioxide uptake by the hydrothermal vent clam, Calyptogena magnifica, and its chemoautotrophic symbionts[END_REF]Dufour 2005;[START_REF] Dechaine | Symbioses of methanotrophs and deep-sea mussels (Mytilidae: Bathymodiolinae)[END_REF][START_REF] Nussbaumer | Horizontal endosymbiont transmission in hydrothermal vent tubeworms[END_REF]Gros et al. 2007;Duperron et al. 2008a;Duperron et al. 2008b;[START_REF] Thornhill | Siboglinid-bacteria endosymbiosis: A model system for studying symbiotic mechanisms[END_REF]Lorion et al. 2009;[START_REF] Bright | The biology of vestimentiferan tubeworms[END_REF][START_REF] Bates | Phylogenetic characterization of episymbiotic bacteria hosted by a hydrothermal vent limpet (Lepetodrilidae, Vetigastropoda)[END_REF][START_REF] Duperron | Relative abundances of methane-and sulphur-oxidising symbionts in the gills of a cold seep mussel and link to their potential energy sources[END_REF][START_REF] Oliver | Chemosymbiotic bivalves from the mud volcanoes of the Gulf of Cadiz[END_REF][START_REF] Rodrigues | Distinct symbiont lineages in three thyasirid species (Bivalvia: Thyasiridae) from the eastern Atlantic and Mediterranean Sea[END_REF]Rodrigues et al. 2011;[START_REF] Stewart | Metatranscriptomic analysis of sulfur oxidation genes in the endosymbiont of Solemya velum[END_REF][START_REF] Bernardino | Comparative composition, diversity and trophic ecology of sediment macrofauna at vents, seeps and organic falls[END_REF]) though examples of methylotrophic-type bacteria (Duperron et al. 2008a) and hydrogen-oxidisers [START_REF] Petersen | Hydrogen is an energy source for hydrothermal vent symbioses[END_REF]) are also documented. The energy from these reduced compounds permits the synthesis of ATP through oxidative phosphorylation, used in cellular metabolism [START_REF] Dechaine | Symbioses of methanotrophs and deep-sea mussels (Mytilidae: Bathymodiolinae)[END_REF]. Courtesy of the interaction, bacteria are believed to provide a sizeable fraction of their hosts ① ut itio al① e ui e e ts① th ough① the① sustai a le① p odu tio ① of① o ga i ① a o ① othe ise① inaccessible to metazoans (e.g. [START_REF] Conway | The role of endosymbiotic bacteria in the nutrition of Solemya velum: evidence from a stable isotope analysis of endosymbionts and host[END_REF][START_REF] Duperron | Relative abundances of methane-and sulphur-oxidising symbionts in the gills of a cold seep mussel and link to their potential energy sources[END_REF]. Bacteria are thought to benefit from physical shelter and increased access to oxygen and reduced compounds, delivered collectively by the host (Duperron 2010). This removes the oxidative constraints normally placed upon aerobic free-living chemosynthetic bacteria when metabolising reduced compounds typically produced in anoxic environments. However, for those symbionts who live part of their life-cycle freely outside the host, metazoan symbiosis is evidently just one of several suitable niches available to them in the deep-sea environment. Consequently, for these host-symbiont associations, the interaction is assumed to be obligatory for the host, but requiring some level of mediation in order to secure their symbionts. Symbioses may also mitigate reduced-fluid toxicity, imparting a greater tolerance in the host, and so extending the ecological niche for the host (and possibly the bacteria). The formation of symbioses between deep-sea metazoans and chemosynthetic bacteria thus allows the metazoan hosts to tolerate (initially) and then ultimately thrive in reducing habitats which would have been uninhabitable otherwise, where contributions from photosynthetically derived organic material can be low ( Van Dover et al. 2002).

Within most deep-sea chemosymbiotic metazoa, details regarding the contribution of chemosynthetic bacterial eta olis ①to①the①host s①e e g ①de a ds①du i g①de elop e t①a e①u k o .①I ① fact, even the means by which this association is initiated (i.e. maternal or environmental symbiont t a s issio ① a d① i teg ated① at① all① stages① of① the① host s① de elop e t① i.e.① ti ing of bacterial symbiont acquisition and patterns of spread) remain elusive for many if not most, deep-sea chemosymbiotic species (Dubilier et al. 2008).

1-7.3.2 Vertical transmission of symbionts (via the germ-line)

The acquisition of symbionts by offspring through vertical transmission is via one or both of the parental organisms [START_REF] Bright | A complex journey: transmission of microbial symbionts[END_REF][START_REF] Bright | The biology of vestimentiferan tubeworms[END_REF]Figure 1.13). Consequently, bacteria are present throughout the dispersal of larvae (Figure 1.13; Figure 1.18B, red bacterium). In the deep sea, the largest group known to employ vertical transmission are the contemporary vesicomyid clams. Symbiont and host alike depend upon their symbioses; the consequences for either party if the symbiosis was somehow terminated, is assuredly death. Vertical transmission in the vesicomyid subfamily Pliocardiinae, occurs during gametogenesis (i.e. maternally, e.g. [START_REF] Endow | Occurrence of bacteria in the primary oocytes of vesicomyid clam Calyptogena soyoae[END_REF][START_REF] Cary | Transovarial inheritance of endosymbiotic bacteria in clams inhabiting deepsea hydrothermal vents and cold seeps[END_REF], with evidence of resulting host-symbiont co-speciation (Peek et al. 1998a;Peek et al. 1998b;[START_REF] Hurtado | Coupling of bacterial endosymbiont and host mitochondrial genomes in the hydrothermal vent clam Calyptogena magnifica[END_REF]. This form of transmission creates evolutionary bottlenecks in the bacterial genome, with farreaching consequences for their survival. These include the increased frequency of deleterious (nearly neutral) substitutions (Peek et al. 1998b) right up to a genome-wide reduction through deletions in the thioautotrophic symbiont of the vesicomyid bivalve Calyptogena okutanii, causing the loss of genes that code for proteins essential for their survival [START_REF] Kuwahara | Reduced Genome of the Thioautotrophic Intracellular Symbiont in a Deep-Sea Clam, Calyptogena okutanii[END_REF]). These proteins, which remain necessary even as a symbiont, are believed to be provided by the host directly. Likewise, C. okutanii is constrained nutritionally, having neither the means to filter feed (extreme hypertrophy of gills that accommodate bacteria intracellularly, results in ciliated surfaces being dwarfed), nor a true digestive system. This extreme interaction is in fact quite rare, even vesicomyids have been shown to supplement maternal symbionts with chemosynthetic bacteria acquired from the environment [START_REF] Kuwahara | Reduced Genome of the Thioautotrophic Intracellular Symbiont in a Deep-Sea Clam, Calyptogena okutanii[END_REF], and transmission may often be a combination of the two (Figure 1.18).

1-7.3.3 Horizontal transmission of symbionts

For the majority of chemosymbiotic associations currently documented to occur in reducing habitats in the deep sea, the mode of transmission is partially or entirely horizontal (e.g. as in the lecithotrophic larva of Riftia pachyptila, [START_REF] Nussbaumer | Horizontal endosymbiont transmission in hydrothermal vent tubeworms[END_REF]. Horizontal transmission may be at some initial period during larval development (at the source habitat), while settling at the sink location, or following metamorphosis as a post-settlement larva [START_REF] Bright | A complex journey: transmission of microbial symbionts[END_REF]Figure 1.13). Bacteria are likely acquired environmentally from free-living bacterial pool or laterally from spatially proximal hosts (Figure 1.13; Figure 1.18). For the former, the host-based symbiotic existence is not obligatory for the bacteria, but may be for the host (e.g. Riftia pachyptila, [START_REF] Nussbaumer | Horizontal endosymbiont transmission in hydrothermal vent tubeworms[END_REF] or perhaps facultative (evidence for filter feeding in B. thermophilus and B. azoricus, Page et al. 1990;Page et al. 1991;Dixon et al. 2006). Once infection has occurred, some level of mediation on the part of the host may occur, as bacterial densities are often actively constrained. Examples of free-living populations which are also symbiotic in the marine environment are known to occur in both coastal waters [START_REF] Lee | Detection of the light organ symbiont, Vibrio fischeri, in hawaiian seawater by using lux gene probes[END_REF][START_REF] Gros | Detection of the free-living forms of sulfide-oxidizing gill endosymbionts in the lucinid habitat (Thalassia testudinum environment)[END_REF][START_REF] Aida | Distribution and population of free-living cells related to endosymbiont a harbored in Oligobrachia mashikoi (a Siboglinid Polychaete) inhabiting Tsukumo Bay[END_REF]) and the deep-sea [START_REF] Harmer | Free-living tube worm endosymbionts found at deep-sea vents[END_REF]. While known occurrences are limited, they may not be isolated, in a discipline which for now remains in its infancy. This is despite the obvious advances that might be gained from better understanding horizontal, and potentially environmental, acquisition [START_REF] Bright | A complex journey: transmission of microbial symbionts[END_REF][START_REF] Bright | The biology of vestimentiferan tubeworms[END_REF]. Intracellular endosymbioses are known to occur in many bivalves from reducing environments including the Vesicomyidae (e.g. [START_REF] Fujiwara | Phylogenetic characterization of the endosymbionts of the deepest-living vesicomyid clam, Calyptogena fossajaponica, from the Japan Trench[END_REF][START_REF] Hurtado | Coupling of bacterial endosymbiont and host mitochondrial genomes in the hydrothermal vent clam Calyptogena magnifica[END_REF][START_REF] Szafranski | Direct evidence for maternal inheritance of bacterial symbionts in small deep-sea clams (Bivalvia: Vesicomyidae)[END_REF], Lucinidae (e.g. [START_REF] Distel | Endosymbiosis in the lucinid clams Lucinoma aequizonata, Lucinoma annulata and Lucina floridana: a reexamination of the functional morphology of the gills as bacteria-bearing organs[END_REF], Thyasiridae (e.g. [START_REF] Brissac | Characterization of bacterial symbioses in Myrtea sp.(Bivalvia: Lucinidae) and Thyasira sp.(Bivalvia: Thyasiridae) from a cold seep in the Eastern Mediterranean[END_REF] with most documented in Mytilidae, e.g.

Bathymodiolus sp. (though in the genera Idas and Adipicola, symbionts are generally to be extracellular 3 , when present, [START_REF] Halary | Direct image-based correlative microscopy technique for coupling identification and structural investigation of bacterial symbionts associated with metazoans[END_REF]. In polychaetes, known symbioses are not only endosymbiotic but sometimes highly integrated physiologically (e.g. in siboglinids which lack a digestive tract, bacteria are housed in a dedicated organ, the trophosome: Vestimentifera, [START_REF] Nussbaumer | Horizontal endosymbiont transmission in hydrothermal vent tubeworms[END_REF]Frenulata and Monilifera, Lösekann et al. 2008). Siboglinid symbionts are acquired horizontally following settlement and metamorphosis [START_REF] Nussbaumer | Horizontal endosymbiont transmission in hydrothermal vent tubeworms[END_REF]. They enter the host by infecting the epidermis, and subsequently the adjacent muscle-layer and underlying mesodermal tissue, ultimately becoming enclosed in the vacuoles of mesodermal cells which eventually develop into a dedicated chemosynthetic organ, the trophosome 3 Except Idas (s.s.) washingtonius [START_REF] Nussbaumer | Horizontal endosymbiont transmission in hydrothermal vent tubeworms[END_REF]. Bacterial ectosymbioses (reviewed in Goffredi 2010) in chemosynthetic deep-sea habitats are known to occur in association with nematodes [START_REF] Polz | Phylogenetic analysis of a highly specific association between ectosymbiotic, sulfur-oxidizing bacteria and a marine nematode[END_REF], the thermo-tolerant polychaetes Alvinella pompejana and Paralvinella grasslei [START_REF] Chevaldonné | Thermotolerance and the'Pompeii worms[END_REF], the highly productive Mid-Atlantic vent-shrimp Rimicaris exoculata [START_REF] Zbinden | Distribution of bacteria and associated minerals in the gill chamber of the vent shrimp Rimicaris exoculata and related biogeochemical processes[END_REF]), the recently described yeti crab (Kiwa spp.; e.g. [START_REF] Goffredi | Epibiotic bacteria associated with the recently discovered Yeti crab, Kiwa hirsuta[END_REF] and various examples of limpet (e.g. [START_REF] Bates | Phylogenetic characterization of episymbiotic bacteria hosted by a hydrothermal vent limpet (Lepetodrilidae, Vetigastropoda)[END_REF]).

1-8 Symbiotic bivalves 1-8.1. The ubiquity of bivalves in reducing environments

1-8.1.1
The bivalve bauplan

In terms of abundance, functional ecology, distribution and adaptation to varying habitats, bivalve molluscs represent a remarkably adaptable class in the animal kingdom, often representing dominant or important groups in most benthic ecosystems [START_REF] Mikkelsen | Speciation in Modern Marine Bivalves (Mollusca: Bivalvia): Insights from the Published Record*[END_REF]. The evident success of Bivalvia in reducing habitats is believed to be attributable to several governing class-specific characteristics. The class is known to inhabit a wide variety of physical substrata with an extensive depth range worldwide [START_REF] Barnes | K,① Hashi oto① J① .① Metha e-ased① symbiosis in a mussel, Bathymodiolus platifrons, from cold seeps in Sagami Bay, Japan[END_REF]. Flexibility in habitat use across symbiotic bivalves owes its roots to the considerable plasticity of the bivalvian (and more generally molluscan) bauplan. This has given rise to wide variability in species body size, diverse shell morphologies and variation in their anatomy. Depending on the family and the selective pressures imposed by the surrounding environment, diverse life habits have been adopted both within and across families. The net result is that bivalves are prolific in habitats within reducing environments.

1-8.1.2 Body size

The range of sizes displayed in bivalves both generally, but particularly within the chemosymbiotic families is worthy of note; variability in maximum body sizes has allowed bivalves to make maximal use of resources in the context of bodily demand, under differing environmental conditions. At hydrothermal vents and methane seeps the abundant availability of electron donors for chemosynthesis-driven productivity has led to frequent cases of bivalve gigantism. Gigantism is particularly apparent in species in the Mytilidae (e.g. [START_REF] Lutz | A comparison of bivalve (Calyptogena magnifica) growth at two deepsea hydrothermal vents in the eastern Pacific[END_REF], while it has been suggested that the relative temporal stability of less productive hydrocarbon seeps which can persist for many decades to millennia, permits extended life spans at slower growth rates (e.g. B. boomerang, as suggested by von [START_REF] Von Cosel | Gigantism in Mytilidae. A new Bathymodiolus from cold seep areas on the Barbados accretionary Prism[END_REF]. Gigantism seems to have been a relatively ubiquitous trait in extinct species from several of the above families [START_REF] Kelly | Early Cretaceous giant bivalves from seep-related limestone mounds, Wollaston Forland, Northeast Greenland[END_REF][START_REF] Campbell | Hydrocarbon seep and hydrothermal vent paleoenvironments and paleontology: Past developments and future research directions[END_REF]) and may in fact be more prevalent in extant soft-sediment taxa than current records suggest, as these habitats are severely underrepresented in the literature (e.g. the genus Meganodontia, Bouchet and von Cosel 2004). At the other end of the size spectrum, a large number of the described Heterodonta (includes most of the burrowing species from the Thyasiridae, Lucinidae and Vesicomyidae) are in the range of a few to tens of millimetres in length. For example thyasirids display size ranges of 1-110 mm (upper limit is for Conchocele), but the majority are relatively tiny, at <10 mm (Taylor and Glover 2010); similar sizes are reported for most members of the vesicomyid subfamily Vesicomyinae, in contrast to the larger Pliocardiinae. The diversity of smaller-sized representatives from each of the families is likely to be highly underestimated particularly in poorly explored biogeographic regions, since these habitats are patchy and many of the families are infaunal.

A wide range of shell lengths also characterises the Bathymodiolinae (Mytilidae), which display shell lengths from less than 20 mm to around 50 mm in the smaller bathymodiolin species predominantly found on wood-and whale falls (Idas, Adipicola, Benthomodiolus, Tamu), 80-316 mm at vents (B. and references therein) have an extensive bathymetric range to 6,000 m depth [START_REF] Fujikura | Estimated population densities of megafauna in two chemosynthesis-based communities: a cold seep in Sagami Bay and a hydrothermal vent in the Okinawa Trough[END_REF] and include two extant genera Solemya (from shallow shelf to bathyal depths) and Acharax (bathyal), distributed world-wide often at low densities, except for the most extreme Polar Regions where they are absent [START_REF] Taviani | Chemosynthetic bivalves of the family Solemyidae (Bivalvia, Protobranchia) in the Neogene of the Mediterranean Basin[END_REF]. The two genera are clearly distinct genetically and to a lesser extent morphologically (mainly due hinge internalisation in Solemya, and its smaller size) with evidence of an ancient divergence in their evolutionary history [START_REF] Neulinger | Presence of two phylogenetically distinct groups in the deep sea mussel Acharax (Mollusca, Bivalvia, Solemyidae)[END_REF]).

1-8.2.3

The Heterodonta: Lucinidae, Thyasiridae and Vesicomyidae

The family Lucinidae①also①o u s①u i uitousl ①th oughout①the① o ld s①o ea s,① ei g①pa ti ula l ①p e ale t①i ① tropical and temperate seagrass beds (Taylor and Glover 2000, and references therein). Having been formerly classified along with Thyasiridae, up-to-date molecular evidence has revealed that common taxonomic features to both families are convergent; the two families are only distantly related [START_REF] Taylor | Functional anatomy, chemosymbiosis and evolution of the Lucinidae[END_REF]. Lucinoma currently represents the prevalent genus of this family in the deep sea [START_REF] Cary | Habitat characterization and nutritional strategies of the endosymbiontbearing bivalve Lucinoma[END_REF][START_REF] Okutani | A new species of lucinid bivalve (Heterodonta: Lucinidae) from Kanesu-no-Se Bank near the Mouth of Suruga Bay, with a review of the recent species of the chemosynthetic genus Lucinoma from Japan[END_REF][START_REF] Salas | Lucinoma kazani n. sp. (Mollusca: Bivalvia): evidence of a living benthic community associated with a cold seep in the Eastern Mediterranean Sea[END_REF]Olu-Le Roy et al. 2004;[START_REF] Holmes | A new species of Lucinoma (Bivalvia : Lucinoidea) from a methane gas seep off the southwest coast of Chile[END_REF][START_REF] Holmes | A new species of Lucinoma (Bivalvia: Lucinoidea) from the oxygen minimum zone of the Oman margin, Arabian Sea[END_REF][START_REF] Von Cosel | Taxonomy of tropical West African bivalves. VI. Remarks on Lucinidae (Mollusca, Bivalvia), with description of six new genera and eight new species[END_REF][START_REF] Von Cosel | Tropical deep-water lucinids (Mollusca: Bivalvia) from the Indo-Pacific: essentially unknown, but diverse and occasionally gigantic[END_REF][START_REF] Cordes | Macro-ecology of Gulf of Mexico cold seeps[END_REF]).

Thyasirids are found from coastal intertidal habitats to hadal depths and from both the poles to the equatorial latitudes in sediments often with hydrocarbons-enriched, of varying grain size including mud, silt and sand (Dufour 2005;Taylor and Glover 2010). Thyasirids can also be found at oxygen minimum zones [START_REF] Oliver | A new species of the family Thyasiridae (Mollusca: Bivalvia) from the oxygen minimum zone of the Pakistan Margin[END_REF], and in the vicinity of high-productivity reducing habitats including hydrocarbon seeps [START_REF] Clarke | New mollusks from undersea oil seep sites off Louisiana[END_REF][START_REF] Fujikura | The deepest chemosynthesis-based community yet discovered from the hadal zone, 7326 m deep, in the Japan Trench[END_REF][START_REF] Kamenev | Conchocele bisecta (Conrad, 1849)(Bivalvia: Thyasiridae) from cold-water methane-rich areas of the Sea of Okhotsk[END_REF][START_REF] Gebruk | Methane seep community of the Håkon Mosby mud volcano (the Norwegian Sea): composition and trophic aspects[END_REF]Olu-Le Roy et al. 2004;[START_REF] Holmes | A new species of Lucinoma (Bivalvia : Lucinoidea) from a methane gas seep off the southwest coast of Chile[END_REF][START_REF] Rodrigues | Thyasiroidea (Mollusca: Bivalvia) from the mud volcanoes of the Gulf of Cadiz (NE Atlantic)[END_REF][START_REF] Oliver | Spinaxinus (Bivalvia: Thyasiroidea) from sulfide biogenerators in the Gulf of Mexico and hydrothermal vents in the Fiji Back Arc: chemosymbiosis and taxonomy[END_REF], and hydrothermal vents [START_REF] Holmes | A new species of Lucinoma (Bivalvia: Lucinoidea) from the oxygen minimum zone of the Oman margin, Arabian Sea[END_REF][START_REF] Oliver | A new species of Axinus (Bivalvia: Thyasiroidea) from the Baby Bare Seamount, Cascadia Basin, NE Pacific with a description of the anatomy[END_REF]. Extant thyasirids are divided into approximately 12 genera, based principally upon rather poorly defined morphometrics which have led to taxonomic difficulties, with frequent arbitrary placement of species in the genus Thyasira in particular (see [START_REF] Oliver | The Thyasiridae (Mollusca: Bivalvia) of the British continental shelf and North Sea oil fields: an identification manual. National Museums & Galleries of Wales, Cardiff, Oliver PG, Holmes AM[END_REF]. The family is thus in need of revision.

The Vesicomyidae consists of over 100 described species, which occur worldwide within an impressive depth-range of (100-9500m, latter limit is for Vesicomya (s.l.) sergeevi, currently the deepest recorded bivalve living in abundance at hadal depths, Krylova and Sahling 2010), although within-genus depth ranges vary, some being relatively narrow (see table 1 of Krylova and Sahling 2010). The depth ranges of large-bodied vesicomyid genera of the subfamily Pliocardiinae appear to be restricted to deeper habitats, in contrast to the small-bodied pliocardin genus Isorropodon and the subfamily Vesicomyinae (which includes the genus Vesicomya only) which are eurybathic and transoceanic (Krylova and Sahling 2010).

Vesicomyinae and Pliocardiinae can be found worldwide and a little less than a third of known species have panthalassic-type distributions (from the genera Vesicomya, Abyssogena, Laubiericoncha, Pliocardia and Phreagena, Krylova and Sahling 2010). That said, the deep-sea subfamily Vesicomyinae are mainly known from trenches and abyssal plains, whilst the Pliocardiinae appear to be highly specialised for sulphide-rich environments and are so far documented to occur principally on continental margins and along mid-ocean ridges (Krylova and Sahling 2010). The phylogenetics of this family has seen several revisions (Krylova and Sahling 2010;[START_REF] Krylova | A new genus of large Vesicomyidae (Mollusca, Bivalvia, Vesicomyidae, Pliocardiinae) from the Congo margin, with the first record of the subfamily Pliocardiinae in the Bay of Biscay (northeastern Atlantic)[END_REF][START_REF] Decker | Phylogeny and diversification patterns among vesicomyid bivalves[END_REF] where even today polyphyletic genera persist, These small-sized mussels have historically been placed in the subfamily Modiolinae (having been described

prior to the last century in some cases) and are usually found on organic falls, though some species also occur at seeps (e.g. Gustafson et al. 1998;Olu-Le Roy et al. 2004;[START_REF] Rodrigues | Chemosynthetic bacteria found in bivalve species from mud volcanoes of the Gulf of Cadiz[END_REF]Ritt et al. 2012;Rodrigues et al. 2013) and in rare instances, vents (e.g. McKiness et al. 2005). Current understanding of the systematics of these two subfamilies (Jones et al. 2006;Kyuno et al. 2009;Miyazaki et al. 2010;Lorion et al. 2013;Thubaut et al. 2013b), undoubtedly warrants the inclusion of all known chemosymbiotic Mytilidae into a single subfamily (be it the Bathymodiolinae [s.l.] or Modiolinae [s.l.]), including the additional genera Idas, Adipicola (formerly Teru/Myrina), Benthomodiolus, Tamu and Vulcanidas). The subfamily name Bathymodiolinae (sensu lato), though incorrect, is used to collectively discuss these genera from here on in.

Within the subfamily Bathymodiolinae (s.l.), 55 species are currently described in the World

Register for Marine Species (Bouchet 2014c;a;b;[START_REF] Bouchet | Idas Jeffreys[END_REF]Sartori 2014; Table 1.1).

However, only a fraction of these species have undergone molecular analysis, particularly lacking in the smaller mussel genera Idas and Adipicola for which species descriptions are sometimes restricted to shell characteristics. That said, the multigene Bayesian trees of Lorion et al. (2013) and Thubaut et al. (2013b) generally appear to corroborate one another, thus bringing a great deal of new information to the phylogeny.

Of the 55 species in Bathymodiolinae (s.l.), three are Benthomodiolus spp., presently known from the Pacific only (but, with possible addition of two more species from Mid Valley, Juan de Fuca Ridge and an unspecified locality in the South Atlantic, Thubaut et al. 2013b, Figure 1.19 Cosel and Marshall 2010). Additional undescribed elongate congenerics may exist (Thubaut et al. 2013b, Figure 1.19). The second genus, Tamu fisheri, occurs on relatively shallow sulphidic hydrocarbon seeps on the Louisiana slope, Gulf of Mexico (Gustafson et al. 1998; Table 1.1).

The① e ai i g① odioli ① s all-mussel genera, Adipicola and Idas, predominantly live on sunken wood or whale skeletons (Table 1.1). They are represented by 9 and 16 described species respectively (Bouchet 2014c;[START_REF] Bouchet | Idas Jeffreys[END_REF] for Idas distribution, Figure 1.20). Both genera remain contentious, as each has a long history of existence and both are evidently polyphyletic (Lorion et al. 2013;Thubaut et al. 2013b), where nucleotide sequences are not available for either holotype (A. pelagica and I.

argenteus respectively). The genus Idas (s.l.) remains poorly resolved phylogenetically; many species were iwaotakii in a study describing this species general biology (Thubaut et al. 2013a) with phylogenetic analysis that suggests it does indeed belong in the clade within which most Idas (s.l.) spp. appear to occur (Thubaut et al. 2013a;Thubaut et al. 2013b). However, this①spe ies ① official classification remains within the genus Adipicola (perhaps incorrectly). Remaining morphospecies were described recently from the Mediterranean include: I. ghisottii, I. cylindricus, I. cristiani, I. emmae, I. filippoi, and I. jaclinae, whose morphoanatomies though quite distinct, bear some resemblance to type specimens of both I. modiolaeformis and I. simpsoni that the azoricus-puteoserpentis complex is more divergent than the thermophilus-antarcticus complex, but the opposite is the case when considering nuclear genes [START_REF] Johnson | A hybrid zone between Bathymodiolus mussel lineages from eastern Pacific hydrothermal vents[END_REF]). An additional species, B. (s.l.)

(
marisindicus occurs at vents in the Indian Ocean (e.g. Kairei field, 2460m): both this species and B. (s.l.)

aduloides, warrant placement in their own genus (Lorion et al. 2013;Thubaut et al. 2013b) platifrons (Kyuno et al. 2009;Miyazaki et al. 2010;Lorion et al. 2013;Thubaut et al. 2013b).

1-8.3. Habitats and life habits: driven by symbiosis

The habitats in which chemosymbiotic bivalves live tend to reflect adaptations that have permitted maximal reducing-habitat exploitation. These ha e① defi ed① he os ioti ① i al ia ① odes① of① e iste e① o ① life① ha its ① "ta le ① ,① i.e.① eha io al① a d① ph siologi al① adaptatio s① e plo ed① i ① elatio ① to① feedi g,① interactions with their habitat substratum and gaining access to reduced compounds.

1-8.3.1 Characteristics of the physical substratum

Chemically-reduced pore-waters in the deep-sea are most common in sedimentary substrata, occurring in interstitial sub-surface sediments below the REDOX boundary layer, in organically enriched fine silt and mud including those near whale-and wood-falls, and in zones of soft-bottom hydrocarbon seepage (e.g. mud volcanoes). For this reason the greatest diversity of bivalve genera are found at these habitats. In relatively rare instances, substrata can also be solid, associated with seepage of percolating reduced fluids, such as in venting chimneys and diffuse flows from fissured igneous rocks at hydrothermal vents, on the solid surfaces of decomposing nektonic megafaunal skeletons and wood, and on authigenic carbonate crusts or asphalt concretions formed at some hydrocarbon seeps. Bivalve assemblages that occur on solid substrata, subjected to reducing conditions, are dominated by the chemosymbiotic Mytilidae (with a few exceptions from the Pliocardiinae), which are well adapted to these habitats on account of being byssate (a common neotenous post-larval feature of substratum attachment by byssus threads retained in adults of Mytilidae, [START_REF] Morton | The evolution and success of the heteromyarian form in the Mytiloida[END_REF]).

1-8.3.2 Feeding modes and reducing fluid access

Feeding modes appear to provide the means to exclusively target reduced compounds for nourishing chemosymbiosis on which the host depends, while in the chemosymbiotic Mytilidae, diet may be supplemented significantly by heterotrophy (e.g. particle assimilation in B. azoricus and B. thermophilus, Page et al. 1990;Page et al. 1991). Thus feeding modes depend upon the species location in the substratum and in relation to the REDOX boundary in particular, with contributory roles from hydrodynamics and sea surface processes in some instances (e.g. vertical flux of organic material).

The Solemyidae are infaunal and make U-or Y shaped burrows that permit simultaneous access to aerated seawater at the sediment boundary layer, and interstitial pore-waters in reducing sediments [START_REF] Stanley | Relation of shell form to life habits of the Bivalvia (Mollusca)[END_REF][START_REF] Reid | Aspects of the biology of a gutless species of Solemya (Bivalvia: Protobranchia)[END_REF]Fisher 1990;[START_REF] Stewart | Metatranscriptomic analysis of sulfur oxidation genes in the endosymbiont of Solemya velum[END_REF]. Despite weak filter feeding capabilities [START_REF] Krueger | Suspension feeding on phytoplankton by Solemya velum, a symbiont-containing clam[END_REF]), symbiotic nutrition is presumed to be obligatory and the principal source of nutrition (Duperron et al. 2013), evidenced by the radical reduction (e.g. Solemya velum), or complete loss of a digestive system, with only basic labial palps remaining, as in S. pervernicosa (formerly S. reidi).

The most diverse of the chemosymbiotic bivalve families 12 , Lucinidae, are also infaunal, found in a multitude of soft sediment habitats with high plant-derived organic enrichment, with some additional species from hydrocarbon seeps and vents (Taylor and Glover 2010). Lucinidae can often burrow deeply into sulphidic, anoxic sediments using their foot (e.g. [START_REF] Cary | Habitat characterization and nutritional strategies of the endosymbiontbearing bivalve Lucinoma[END_REF]) and typically live in proximity to the REDOX boundary layer. While orientated vertically in the sediment with the hinge-line uppermost, aeration and sulphide acquisition are both performed by the foot, the former by maintaining a mucous-reinforced inhalant tunnel linked to the surface, the latter by probing down into anoxic reducing layers [START_REF] Taylor | Functional anatomy, chemosymbiosis and evolution of the Lucinidae[END_REF].

Second only to the Lucinidae, Thyasiridae are the most diverse, most varied in their morphology and most extensive in their habitat range (Taylor and Glover 2010). The Thyasiridae are infaunal, burrowing into suboxic-to-anoxic sediments (including those in oxygen minimum zones), in which the interstitial porewater is enriched with organic matter, hydrocarbons, or reducing fluids. Means of sulphide acquisition is presumed to be in line with that of other Heterodonta, although evidence suggests that many thyasirids do not rely on chemosymbiotic bacteria at all (summarised below). This family is poorly studied in the deep sea by comparison. Species which engage in symbiosis generally burrow deeper than non-symbiotic species and use their super-extensile foot, which can extend 30 times the length of the shell, to search for and extract sulphides from deeper reducing sediment patches, consequently creating a network of tunnels (the number being dependent on sulphide levels, [START_REF] Dufour | Sulphide mining by the superextensile foot of symbiotic thyasirid bivalves[END_REF]. The extraordinary length of the extended foot is made possible through having evolved dorso-ventrally elongate shells, which can accommodate a longer foot than those in the antero-posteriorly extended asymbiotic thyasirids [START_REF] Dufour | Sulphide mining by the superextensile foot of symbiotic thyasirid bivalves[END_REF].

Unlike other Heterodonta, the Vesicomyidae display two separate life habits. Most are partially or almost entirely infaunal (all Vesicomyinae, and Pliocardiinae associated with non-vent sedimentary habitats) inhabiting the abyssal plains in association with subsurface REDOX boundaries or pockets of hydrocarbon seepage that underlie surface sediments, from which their posterior half protrudes (Krylova and Sahling 2010. Pliocardiinae inhabiting vents however, orientate themselves along seeping fissures in igneous rocks. The Vesicomyidae frequently contribute to community composition in hydrocarbon seeps at continental margins [START_REF] Sibuet | Biogeography, biodiversity and fluid dependence of deep-sea cold-seep communities at active and passive margins[END_REF], at mid-ocean hydrothermal vents (Taylor and Glover 2010), and occur both on and in proximity to organic falls (e.g. whale bones and adjacent sediment, Bennett et al. 1994;Smith and Baco 2003). The sheer abundance and frequency of the Pliocardiinae discovered at multiple sites along continental margins are thought to be indicative of more common occurrences of reducing environments on both active and passive margins than previously realised (Krylova and Sahling 2010). Access to sulphides and oxygen are via two separate avenues. Sulphides are absorbed by the foot [START_REF] Childress | Oxygen and sulfide consumtion rates of the vent clam Calyptogena magnifica[END_REF], extending into reduced sediments or seeping fissures (as in the case of vents), transported to the gills by the blood [START_REF] Arp | Metabolic and blood gas transport characteristics of the hydrothermal vent bivalve Calyptogena magnifica[END_REF][START_REF] Goffredi | Species-specific variation in sulfide physiology between closely related Vesicomyid clams[END_REF] while oxygen uptake is via the gills directly. In pliocardiins, sulphide may be transported by zinc-based sulphide-binding molecules, sulphide oxidation maintaining sub-saturation levels [START_REF] Childress | The role of a zinc-based, serum-borne sulphide-binding component in the uptake and transport of dissolved sulphide by the chemoautotrophic symbiontcontaining clam Calyptogena elongata[END_REF], and so protecting host tissues from poisonous respiratory inhibition. Additional oxidation of unbound sulphides to thiosulphate in blood serum may occur also [START_REF] Childress | The role of a zinc-based, serum-borne sulphide-binding component in the uptake and transport of dissolved sulphide by the chemoautotrophic symbiontcontaining clam Calyptogena elongata[END_REF]. Sulphide absorption from water drawn in through the gills may present a secondary means of supply, as the gills of certain vesicomyid species are arranged in tubular bacteriocyte-lined channels (Fiala-Médioni and Le Pennec 1988; Krylova and Sahling 2010).

Collectively, the genera of the mytilid subfamily Bathymodiolinae have evolved to take advantage of a multitude of reducing habitats. In contrast to the other chemosymbiotic bivalve families discussed so far,

Figure 1.22 Habitat use in chemosymbiotic bivalves

Pictured are the five main bivalves discussed in this introduction. In the Solemyidae, Lucinidae and Thyasiridae anoxic sulphide rich sediments (darker regions are mined with a vermiform foot). In most Vesicomyidae, this remains the case, however the unusual Pliocardiinae reside on fissures of on the surface of sediments subject to diffuse flows loaded with reducing compounds. Mytilidae that are chemosymbiotic are almost always epibenthic (see text for exceptions), taking advantage of free fluids being released at vents, seeps, and organic falls. Blue①i te al①o ga s①a e①o e①of①the①o ga is s①t o①gills.①B o ①i te al①o ga s①a e①foot① e te ded ①a d① is e a① (internal). Images from Dubilier et al. 2008 the members of this byssate mytilid subfamily are generally not infaunal, living instead as epibenthos attached to hard substrata which can include authigenic carbonates and asphalt concretions at hydrocarbon seeps, igneous formations in the vicinity of hydrothermal vents, the outer walls of large polycheate tubes (Annelida: Siboglinidae, with accounts both at vents and seeps) or on the shells of other organisms including conspecifics (Duperron 2010). Since the upflux of subsurface reduced interstitial fluids to the sediment boundary layer in the vast majority of deep-sea habitats is effectively nil (except during large scale disturbance), the chemosymbiotic Mytilidae, which exclusively harbour symbionts in their gills and are not known to mine for sulphides with their foot, must rely on being bathed in reducing fluids, in the absence of direct access to anoxic sediments. To date, two exceptions to this life habit are found in Bathymodiolus boomerang and Gigantidas horikoshi (Hashimoto and Horikoshi 1989;[START_REF] Hashimoto | Microdistribution pattern and biogeography of the hydrothermal vent communities of the Minami-Ensei Knoll in the Mid-Okinawa Trough, Western Pacific[END_REF][START_REF] Desbruyères | Handbook of deep-sea hydrothermal vent fauna[END_REF][START_REF] Von Cosel | Gigantism in Mytilidae. A new Bathymodiolus from cold seep areas on the Barbados accretionary Prism[END_REF], who bury themselves by up to two-thirds of their shell length

i ①sedi e ts①su je t①to①sulphide①a d① etha e①seepage① though①it s①thought①that①the①fo e ①still①atta hes① ① byssus threads to subsurface solid-mud accretions, von [START_REF] Von Cosel | Gigantism in Mytilidae. A new Bathymodiolus from cold seep areas on the Barbados accretionary Prism[END_REF]. Even these two partially infaunal species, appear to be restricted to seeping sediments however. Consequently, combined with the almost universal requirement for byssate attachment, it is only in association with active reduced-fluid seepage at vents and seeps that the relatively giant chemosymbiotic mussels occur. Remaining smallersized chemosymbiotic Mytilidae, whose nutritional demands per individual are presumably much reduced, are capable of colonising decomposing organic substrates (particularly nektonic megafaunal carcasses and accumulations of sunken wood) where reduced fluid production is less dramatic but where hard substrate for attachment is available, although select species colonise seeps and vents (Gustafson et al. 1998;Olu-Le Roy et al. 2004;[START_REF] Rodrigues | Chemosynthetic bacteria found in bivalve species from mud volcanoes of the Gulf of Cadiz[END_REF]Ritt et al. 2012).

1-8.3.3 Chemosymbioses in bivalves

Chemosymbiosis has been identified in all species of Lucinidae, Solemyidae and Vesicomyidae studied to date, suggesting it has an obligatory role in the biology of these families (Taylor and Glover 2010), though weak filter-feeding capacity is retained in Solemya velum [START_REF] Krueger | Suspension feeding on phytoplankton by Solemya velum, a symbiont-containing clam[END_REF]). In the Thyasiridae, while many species engage in symbioses, including the deepest known chemosymbiotic species, Maorithyas hadalis [START_REF] Fujiwara | Dual symbiosis in the cold-seep thyasirid clam Maorithyas hadalis from the hadal zone in the Japan Trench, western Pacific[END_REF], others do not (Dufour 2005). In Mytilidae, chemosymbiosis is confined to members of the subfamily Bathymodiolinae while all other mytilids are believed to be asymbiotic, though the two remaining deep-sea mytilid genera have not been examined for symbioses in detail (i.e.

Amygdalum and Dacrydium, [START_REF] Salas | Brooding and non-brooding Dacrydium (Bivalvia: Mytilidae): a review of the Atlantic species[END_REF][START_REF] Oliver | Functional morphology and description of a new species of Amygdalum (Mytiloidea) from the oxygen minimum zone of the Arabian Sea[END_REF]. Symbioses almost invariably involve chemoautotrophic (typically sulphur-oxidising) bacteria and often, in the case of the chemosymbiotic Mytilidae, additional methane-oxidising methanotrophs (Dubilier et al. 2008). Rare instances of other bacterial phylotypes are detailed subsequently, pertaining to mytilids specifically. Most symbioses in chemosymbiotic bivalves involve Gammaproteobacteria, though rare instances of possible Epsilonproteobacteria, Spirocheates, Bacteroidetes subdivisions have also been suggested, though their functional significance remains speculative (e.g. Duperron et al. 2006b;[START_REF] Rodrigues | Chemosynthetic bacteria found in bivalve species from mud volcanoes of the Gulf of Cadiz[END_REF][START_REF] Brissac | Characterization of bacterial symbioses in Myrtea sp.(Bivalvia: Lucinidae) and Thyasira sp.(Bivalvia: Thyasiridae) from a cold seep in the Eastern Mediterranean[END_REF]).

In the Solemyidae, endosymbiotic coccoid-to-rod-shaped Gammaproteobacterial phylotypes predominate and cluster cladistically with those of lucinid and thyasirid bivalves (Taylor and Glover 2010), excluding Acharax, whose symbionts are phylogenetically distinct [START_REF] Imhoff | 16S rDNA-based phylogeny of sulphur-oxidising bacterial endosymbionts in marine bivalves from cold-seep habitats[END_REF]. Strong host mediation over bacterial division is suspected, as bacteria undergoing cell division are rarely witnessed (Duperron et al. 2013), crammed into bacteriocytes occupying the abfrontal regions of the individual filaments of the large posterior gills (Taylor and Glover 2010). Symbiont transmission is thought to be vertical, at least in the genus Solemya (symbiont DNA found in ovaries, oocytes and larvae of two species [START_REF] Cary | Vertical transmission of a chemoautotrophic symbiont in the protobranch bivalve, Solemya reidi[END_REF][START_REF] Krueger | Vertical transmission of chemoautotrophic symbionts in the bivalve Solemya velum (Bivalvia: Protobranchia)[END_REF].

Lucinidae gills are usually thickened and optically opaque, representing about one-third of the a i al s① io ass,①a d① egio s①of① oi ide t①gill①fila e ts①a e①f e ue tl ①fused①to① eate① ha els,①de sel ① populated by bacteriocytes [START_REF] Distel | Endosymbiosis in the lucinid clams Lucinoma aequizonata, Lucinoma annulata and Lucina floridana: a reexamination of the functional morphology of the gills as bacteria-bearing organs[END_REF]. Much like the Solemyidae, host species are thought to exert considerable physiological restraint upon their symbiont densities; curiously, bacteria appear unable to escape host tissues once acquired [START_REF] Brissac | Lack of endosymbiont release by two Lucinidae (Bivalvia) of the genus Codakia: consequences for symbiotic relationships[END_REF]) and while genomic replication can take place, bacteria do not actively divide within hosts cells [START_REF] Caro | Characterization of the population of the sulfuroxidizing symbiont of Codakia orbicularis (Bivalvia, Lucinidae) by single-cell analyses[END_REF]). Lucinid symbionts cluster in the Gammaproteobacteria, in a clade that also includes Solemya and vestimentiferan tubeworm symbionts (Cavanaugh et al. 2006;Dubilier et al. 2008). Based on tropical shallow-water research, symbiont transmission is believed to be horizontal following settlement and metamorphosis [START_REF] Gros | Embryonic, larval, and post-larval development in the symbiotic clam Codakia orbicularis (Bivalvia: Lucinidae)[END_REF][START_REF] Gros | Embryonic development and endosymbiont transmission mode in the symbiotic clam Lucinoma aequizonata (Bivalvia: Lucinidae)[END_REF], acquired in some instances from a pool of free-living bacteria present in the sink environment (e.g. [START_REF] Gros | Detection of the free-living forms of sulfide-oxidizing gill endosymbionts in the lucinid habitat (Thalassia testudinum environment)[END_REF], where ongoing acquisition may occur as adults (Le [START_REF] Pennec | Endocytosis and lysis of bacteria in gill epithelium of Bathymodiolus thermophilus, Thyasira flexuosa and Lucinella divaricata (Bivalve, Molluscs)[END_REF].

In stark contrast to the other families discussed, a notable number of the Thyasiridae examined for bacterial symbionts, have been shown to be asymbiotic (Dufour 2005), with varying densities of and nutritional dependencies upon symbiosis in those that are symbiotic [START_REF] Dando | Varying nutritional dependence of the thyasirid bivalves Thyasira sarsi and T. equalis on chemoautotrophic symbiotic bacteria, demonstrated by isotopic ratios of tissue carbon and shell carbonate[END_REF]). The family is thus considered to be at an earlier stage of evolution in the context of symbiosis (Dufour 2005). Across the family, for species in which symbiosis has been examined to any degree, a spectrum of gill characteristics appears to exist that roughly place into three types. They pass from a typical homorhabdic filibranch gill with inter-filamental fusion dorsoventrally (along 70% of filament length) and only a few (if any) bacteria nestled between the microvilli that are located abfrontally, through a state where bacteria are numerous but filaments are thin and unmodified abfrontally, up to a state where gill filaments are abfrontally thickened and the greater non-ciliated epithelial surface area houses distinct bacteriocyte zones adorned with microvilli, among which extracellular bacteria are dispersed. In one species (Maorithyas hadalis) where bacteria are intracellular however, internalised bacteria-filled vacuoles are often located apically such that they remain partially open to external water [START_REF] Fujiwara | Dual symbiosis in the cold-seep thyasirid clam Maorithyas hadalis from the hadal zone in the Japan Trench, western Pacific[END_REF]. This fourth state is considered intermediate between extracellular and intracellular symbiosis where only bacteria in vacuoles close to the apical end of bacteriocytes are in contact with seawater [START_REF] Endow | Occurrence of bacteria in the primary oocytes of vesicomyid clam Calyptogena soyoae[END_REF]. Dual, spatially partitioned symbioses occur in this species between a thioautotrophic bacteria and a second unknown phylotype, distantly related to the Thiomicrospira and Hydrogenovibrio [START_REF] Fujiwara | Dual symbiosis in the cold-seep thyasirid clam Maorithyas hadalis from the hadal zone in the Japan Trench, western Pacific[END_REF]. In thyasirids, symbiont acquisition is preceded by the abfrontal development of filaments, which expands colonisable non-ciliated surfaces (Dufour 2005). Transmission though not rigorously examined, is thus suspected to be horizontal.

Studies into symbioses in the Vesicomyidae have focussed on those of the subfamily Pliocardiinae Current evidence available indicates that when examined, symbiont transmission is vertical with maternal origins, where primary oocytes are known to be already infected with symbiotic phylotypes in Phreagena (Calyptogena) soyoae [START_REF] Endow | Occurrence of bacteria in the primary oocytes of vesicomyid clam Calyptogena soyoae[END_REF]. Further putative evidence for vertical transmission is in apparent host-symbiont co-speciation based on mirrored phylogenies (Peek et al. 1998a;Peek et al. 1998b), and in reported genome reduction in the symbiotic bacteria, in comparison to free-living relatives [START_REF] Kuwahara | Reduced Genome of the Thioautotrophic Intracellular Symbiont in a Deep-Sea Clam, Calyptogena okutanii[END_REF]), indicative of vertical transmission and the absence of a free-living mode (e.g. as in some insects, [START_REF] Gil | Bacterial endosymbionts of insects: insights from comparative genomics[END_REF]). In addition to the vertical transmission of symbionts, rare incidents of both intraspecific variability in symbiont lineages (e.g. Calyptogena valdiviae, Duperron et al. 2013) and non-parental lateral transmission are documented in species occurring in proximity to one another [START_REF] Stewart | Lateral symbiont acquisition in a maternally transmitted chemosynthetic clam endosymbiosis[END_REF][START_REF] Decker | Physical proximity may promote lateral acquisition of bacterial symbionts in vesicomyid clams[END_REF]); these phenomena may be linked.

As the most documented taxa to date, more is known about symbioses in the Bathymodiolinae then perhaps any other bivalve group employing symbionts for their nutrition (Duperron 2010). Part of their evident success is through the flexibility of their symbiotic associations. One notable exception may be Idas argenteus a deep-water, wood-associated mussel which has been described as a larviphage, ingesting larvae of the small wood-boring bivalve Xyloteredo (Ockelmann and Dinesen 2011), with no obvious bacterial symbionts in its gills. Otherwise, bacterial symbioses are at their most diversified within the bivalve family Bathymodiolinae based on symbiosis studies to date. These assemblages, like the monophylotypic symbioses of other bivalves discussed, almost invariably contain thiotrophic Gammaproteobacteria. However, the Bathymodiolinae (s.l.) are noted in particular for their incorporation of methanotrophic Gammaproteobacteria that employ methane monooxygenase in order to harness methane that both drives metabolic processes (by oxidation) and provides a source of carbon (Cavanaugh et al. 2006) [START_REF] Raggi | Bacterial symbionts of Bathymodiolus mussels and Escarpia tubeworms from Chapopote, an asphalt seep in the southern Gulf of Mexico[END_REF]). Bathymodiolus (s.s.) heckerae as a species demonstrates the adaptability that multiple symbioses can afford a host, having different symbionts assemblages depending on its location. The species is known to harbour two distinct sulphur-oxidisers, a methanotroph and then either methylotroph-related symbiont in the northern Gulf of Mexico, or hydrocarbon-degrading bacteria in the southern Gulf of Mexico [START_REF] Childress | The role of a zinc-based, serum-borne sulphide-binding component in the uptake and transport of dissolved sulphide by the chemoautotrophic symbiontcontaining clam Calyptogena elongata[END_REF][START_REF] Distel | Intracellular coexistence of methano-and thioautotrophic bacteria in a hydrothermal vent mussel[END_REF]Duperron et al. 2005;[START_REF] Dechaine | Symbioses of methanotrophs and deep-sea mussels (Mytilidae: Bathymodiolinae)[END_REF][START_REF] Duperron | Diversity, relative abundance and metabolic potential of bacterial endosymbionts in three Bathymodiolus mussel species from cold seeps in the Gulf of Mexico[END_REF][START_REF] Raggi | Bacterial symbionts of Bathymodiolus mussels and Escarpia tubeworms from Chapopote, an asphalt seep in the southern Gulf of Mexico[END_REF]).

The resulting advantages of geneticallyand perhaps more importantlynutritionally and metabolically diverse symbiotic assemblages are two-fold. The first, is that such assemblages bring an unrivalled level of nutritional flexibility with regards to the variety of reduced compounds present at reducing habitats, in environments where relative proportions fluctuate temporally (Dubilier et al. 2008).

This permits the Bathymodiolinae (s.l.) to colonise habitats which are dominated by methane production, such as periodite-hosted vents and many methane seeps [START_REF] Tivey | Generation of seafloor hydrothermal vent fluids and associated mineral deposits[END_REF]). In addition, the relative dominance of co-occurring sulphur or methane oxidisers are known to reflect (Fiala-Médioni et al. 2002;[START_REF] Bris | Chemosynthetic communities and biogeochemical energy pathways along the Mid-Atlantic Ridge: The case of Bathymodiolus azoricus[END_REF], and respond to [START_REF] Halary | 3D FISH for the quantification of methane-and sulphur-oxidizing endosymbionts in bacteriocytes of the hydrothermal vent mussel Bathymodiolus azoricus[END_REF]Riou et al. 2010b) changes in relative sulphide and methane concentrations within their environment. Astoundingly, bacterialor host-mediatedresponses to alterations in the proportional availability of reducing compounds are within the scale of hours (S.

Duperron, unpublished data). The second, perhaps less evident advantage is that inter-bacterial competition is reduced (except perhaps, for vacuolar space), since the segregation of reduced-fluids across nutritionally independent bacterial populations with differing metabolic pathways allow resources to be partitioned (Dubilier et al. 2008). In some instances it is thought that such dual (or multiple) symbioses may even be synergistic, such as methanotrophs generating inorganic carbon by-products, which can be used by neighbouring thiotrophs (Dubilier et al. 2008). The level of symbiont integration in host organisation within the Bathymodiolinae varies across, and within genera. Bacteria although always documented to be endosymbiotic, may be either arranged extracellularly among microvilli that differentiate from the apical membranes of bacteriocytes (with one account of bacteria being partially housed open superficial pseudopodium-like structures, Fujiwara et al. 2010), or arranged intracellularly in the vacuoles of bacteriocytes that from part of the gill epithelium in close association with a central blood lacuna. In both cases putative lysosomes are often present, with bacteria restricted to the non-ciliated lateral-to-abfrontal regions of gill filaments in adult specimens (Duperron 2010;Duperron et al. 2013). In the small-sized mussels which are often able to colonise several types of reducing habitat, extracellular bacteria are documented for Tamu fisheri (Nelson and Fisher 1995) while both intra-and extracellular endosymbioses have been described in the genera Idas (s.l.) and Adipicola (s.l.); the former genus (s.l) is discussed in more detail in Chapter 2. No conclusive data is available for the genus Benthomodiolus. In the larger bathymodiolins (e.g. Gigantidas, Bathymodiolus), bacteria have been exclusively intracellular for those examined in detail, where several species of Bathymodiolus (s.l.) may in fact belong in the genus Gigantidas (s.s.), according to recent assertions by Thubaut et al. 2013b, though at the time of writing these had yet to be published in a formal nomenclature revision. As the phylogeny currently stands, localisation of thiotrophic symbionts in the genus Gigantidas has yet to be assessed.

Patterns of co-speciation seen in some other chemosymbiotic bivalves and their symbionts (e.g. the vesicomyids) are not reflected in the Bathymodiolinae, where transmission is suspected to be predominantly horizontal i.e. acquired from the environment or from adult host organisms laterally at any point in the lifecycle after the initiation of dispersal (i.e. post-spawning). Evidence for horizontal transmission have been posed for Idas modiolaeformis from the NDSF, based on the absence of bacteria in gonadal tissues in adults where densities of methanotrophic symbionts in gills were high (Gaudron et al. 2012), visualised using specific oligonucleotide probes and Fluorescence in situ hybridisation (FISH). Studies that have examined the ultrastructure of gill filaments in Bathymodiolus (s.l.) spp. have identified that intracellular bacteria are proximal to the bacteriocytes apical microvilli, where plasma membrane invaginations are visible at various stages of bacteria isolation within endocytotic vacuoles (Le [START_REF] Pennec | Endocytosis and lysis of bacteria in gill epithelium of Bathymodiolus thermophilus, Thyasira flexuosa and Lucinella divaricata (Bivalve, Molluscs)[END_REF]Won et al. 2003b). This suggests that bacteria are internalised directly from the environment.

1-8.3.4 Acquisition of horizontally transmitted symbionts

Whilst there is growing molecular evidence that transmission in mussels is horizontal (Won et al. 2003b;Won et al. 2008;Fontanez and Cavanaugh 2014), limited data exists about the biological processes that govern infection, particularly in terms of host-symbiont recognition and dynamics of cellular communication, the incorporation of symbionts, and ultimately their assimilation. Apparent symbiont specificity in or on host tissues suggests mediation of bacterial distribution or resource partitioning. The timing of symbiont acquisition and the degree to which hosts depend upon symbionts at various stages of development are not well defined. Horizontal transmission invariably raises the question of timing of symbiont acquisition, given the nutritional consequences for being aposymbiotic at some point in the lifecycle of a chemosymbiotic species. Settling siboglinid tubeworms and bathymodiolin mussels that acquire locally adapted microorganisms might profit from the greater flexibility needed to take advantage of a wider range of reducing environments, when compared with vertically transmitted symbionts in Vesicomyidae hosts, occurring much less frequently in Indian and Atlantic vent sites (Won et al. 2003b). As bacteria are most likely to be present at either the source (spawning) or the sink (settlement) habitats being reducing environments, it is during these periods that acquisition is suspected to take place (Figure 1.13).

However, information regarding the acquisition of symbionts is scarce for the deep sea generally. Data available for the Bathymodiolinae (s.l.) suggest that for B. (s.s.) azoricus, pediveligers are already symbiotic (Won et al. 2003b) where bacteria were almost certainly acquired horizontally, though perhaps with an unua tified① deg ee① of① leak ,① pa e tal① t a s issio ① du ing post-embryonic development, based on the mixture of symbionts identified in host hybrid zones. In B. (s.l.) childressi methanotrophic symbionts appear to be already present in juveniles, but with less specific infection, occurring on other epithelial surfaces as well as the non-ciliated abfrontal and lateral regions of the gill filaments (Streams et al. 1997) the remainder of the subfamily from the described biology of these highly-derivative evolutionary states.

Yet this remains the only course of①a tio ①i ①the①fa e①of①a① data①d ought :① u h①of①the①i fo atio ①gathe ed① which follows is, by default, derived from these sorts of end-state species. Much about the bathymodiolin lifecycle remains inferred, no more so than in small-sized species. Both highly variable and notably conservative traits have been identified in several members of Bathymodiolus and Idas. Details identified for the subfamily (or at least inferred from the literature) are summarised below. A schematic is also

Figure 1.23 Hypothetical lifecycle of a Bathymodiolin

Cycle is inferred based on analogous species from shallow water and on limited data for Bathymodiolinae. a) Spawning. b) Fertilisation is probably external. c) Embryonic development as a morula. d) Gastrulation leads to formation of trochophore larva with two depressions: one is the gastropore, coupled with ciliary bands in a simple alimentary system; the other forms the origin of the shell field, at the① gast ula s① animal pole. e) The organic matrix of the shell field extends ventrally around the late trochophore from the dorsal hinge line, and ultimately becomes calcified, as it encloses the larva forming the straight-hinged D ① veliger stage. The prototroch develops to form the ciliated velum, for locomotion and filter-feeding. f) The development of the prodissoconch II (in red) in a veliger larva represents the period of greatest planktotrophic growth (1: Planktotrophic or lecithotrophic). g) Competent to settle: the velum regresses, as filter-feeding and locomotion are adopted by the developing gills and foot respectively. The pediveliger probably responds rapidly to a positive cue for suitable habitat. h) Initial attachment to substratum. i) Dissoconch development begins (yellow shell margin). In some shallow-water species, byssus drifting is a means for pediveligers/plantigrades to relocate using bottom currents (not yet documented in deep-sea mussel species). j) Some level of mobility is likely retained in developing juvenile mussels prior to maturation. Schematic created by S.R. Laming presented in order to delineate the main junctures in the development of a generic bathymodiolin mussel (Figure 1.23). Some of these stages remain fundamentally hypothetical for deep-sea species. [START_REF] Kenk | A new mussel (Bivalvia, Mytilidae) from hydrothermal vents in the Galapagos Rift zone[END_REF], has since been reported as gonochoristic based on specimens from the Galapagos rift and 13°N [START_REF] Pennec | Ultrastructural characteristics of spermatogenesis in three species of deepsea hydrothermal vent mytilids[END_REF]. This study also identified B. puteoserpentis to be gonochoristic, while B. elongatus shows evidence of protandry (immature oocytes identified along the periphery of male acini). Thus for the smaller-sized species, at least from the genera Idas (s.s.) and Adipicola (s.l.), protandric hermaphroditism appears to be the norm, while in Bathymodiolus spp., both sequential hermaphroditism (apparently exclusively protandric) and gonochorism have been identified, where both processes of sex determination may exist within a single species (or an unidentified species complex), perhaps as a consequence of environmental drivers: the processes which determine the timing of sex-switching are not known, though they are thought to be epigenetic in origin (Tyler et al. 2009;Gaudron et al. 2012).

1-9. childressi [START_REF] Eckelbarger | Ultrastructure of gametogenesis in a chemosynthetic mytilid bivalve (Bathymodiolus childressi) from a bathyal, methane seep environment (northern Gulf of Mexico)[END_REF]Tyler et al. 2007;Arellano and Young 2009). The species is known to be gonochoristic, with gametogenic evidence to support a seasonal reproductive cycle (with spawning occurring sometime between December and March) and planktotrophic larvae that appear to be teloplanic (Arellano and Young 2009;Arellano et al. 2014). Gonads contained acini surrounded by inter-acinal tissue composed of adipogranular cells [START_REF] Eckelbarger | Ultrastructure of gametogenesis in a chemosynthetic mytilid bivalve (Bathymodiolus childressi) from a bathyal, methane seep environment (northern Gulf of Mexico)[END_REF], whose nutritive function suggests noncontinuous spawning (Le Pennec and Beninger 2000). A temporal sampling regime coupled with histological analysis established that, despite a continuous energy source in the form of methanotrophic chemosymbiosis, gametogenesis was seasonal at the Brine Pool (650 m depth, Louisiana slope). Gamete development between females and males was synchronous, with spawning beginning as early as November and continuing as far as February, with gametogenesis beginning in earnest again in March with evidence of oocyte growth and spermatozoan proliferation (Tyler et al. 2007). The shallower vent species B. (s.s.)

azoricus on the MAR also displays seasonality in gamete production (Comtet et al. 1999), based on skewed population structure [START_REF] Comtet | Population structure and recruitment in mytilid bivalves from the Lucky Strike and Menez Gwen[END_REF], and analyses of seasonally collected samples from Menez Gwen (Colaço et al. 2006), which revealed not only that spawning was an annual event (at a slightly latter time to B. (s.l.) childressi, in January), but that mussels maintained this spawning regime in aquaria (a trend mirrored in spermatogenesis in this species, Kádár et al. 2006). In the bone-colonising species Idas washingtonius, gametogenesis also appears to be non-continuous, though gametogenic patterns were not so obviously synchronised across all individuals, such that a putative spawning period from March to April could be identified (Tyler et al. 2009) Periodicity in gametogenesis is unexpected in species where food supply is assumed to be continuous, particularly in light of the fact that many deep-sea species are continuous spawners (Tyler et al. 2007) with the exception of some species on continental slopes with planktotrophic larvae (Young 2003). However, some bathymodiolin species are known to be capable of mixotrophy, supplementing a chemosymbiotic diet with filter-feeding; this may explain their tendency towards seasonal spawning (Le Pennec and Beninger 2000;Dixon et al. 2006), since chemosymbiotic nutrition is likely to be nitrogen limited (Tyler et al. 2007).

It s① also① possi le① that① ph loge eti ① o st ai ts act upon any capacity for alterations to gametogenic processes in the family Mytilidae [START_REF] Eckelbarger | Ultrastructure of gametogenesis in a chemosynthetic mytilid bivalve (Bathymodiolus childressi) from a bathyal, methane seep environment (northern Gulf of Mexico)[END_REF], such that deep-sea mytilid spawning behaviour mirrors that of littoral species, considered by some to be analogous to the hypothesised ancestral origins of deep-sea mytilids.

That said caution is needed when extrapolating spawning behaviour and developmental trends from related species. In the genus Mytilus, astounding variability in reproductive traits exists even within a single habitat. For example, along the west coast of America in regions where M. californianus and M. edulis cooccur, their reproductive and morphological characteristics are surprisingly different (Suchanek 1981;Seed and Suchanek 1992). M. californianus is a bigger, more robust, but slower growing mussel, better placed to deter predators effectively and is a better competitor for spatial resources. It actually spawns continually with a considerably reduced reproductive output (Suchanek 1981). Comparatively, M. edulis is a typical fugiti e① spe ies ① hi h① a el ① attai s① its① a i u ① k o ① size,① atu i g① ea l ① a d① e gagi g① i ① a① si gle① high-volume reproductive event per year (Suchanek 1981). Fertilization is external with mature oocytes around 60 -90 µm in diameter, with optimal conditions being from 5-22 °C and at salinities of 15 -40psu [START_REF] Lutz | Ecology and morphology of larval and early postlarval mussels[END_REF]. As in many molluscs, fecundity and reproductive effort increase with mounting age and size (though usually with an upper limit), as energy is invested mainly in growth as a young adult. Reproductive output tends to vary from year to year on account of temperature, resource availability and emersion.

In the deep-sea mytilids, limited data is available on fecundity and live-egg characteristics (as opposed to histological inferences), because of the invested effort required to keep deep-sea chemosymbiotic mytilids alive, both in terms of nutritional requirements and the physiological consequences for relocating these organisms to ambient conditions in the aquarium, which inevitably differ markedly from their local habitat.

Fe u dit ①appea s①to① e① high ①i ①Bathymodiolus (s.l.) spp. [START_REF] Tyler | Reproduction and dispersal at vents and cold seeps[END_REF] (s.l.) childressi [START_REF] Berg | Reproductive strategies of mollusks from abyssal hydrothermal vent communities[END_REF][START_REF] Hessler | Temporal change in megafauna at the Rose Garden hydrothermal vent (Galapagos Rift; eastern tropical Pacific)[END_REF][START_REF] Pennec | Ultrastructural characteristics of spermatogenesis in three species of deepsea hydrothermal vent mytilids[END_REF][START_REF] Comtet | Population structure and recruitment in mytilid bivalves from the Lucky Strike and Menez Gwen[END_REF]Colaço et al. 2006;Arellano and Young 2009), while egg diameters are smaller again in Idas spp., being exclusively <50 µm in both I. modiolaeformis and I. washingtonius (Tyler et al. 2009;Gaudron et al. 2012). In both cases, these oocyte diameters are considered too small to contain the energetic yolk reserve necessary for lecithotrophy (Lutz et al. 1980), leading to the conclusion that larval development is likely to be planktotrophic in the Bathymodiolus (s.l.) spp. and Idas spp. examined. for embryos to pass through the relatively brief trochophore stage (Figure 1.23d), develop a velum and secrete a larval shell and, at 8 days, become straight-hi ged,① D -shell veligers (Figure 1.23e). The trochophore was typical of mytilids: a multi-cellular embryo with a lateral perimeter of ciliary bands (the metatroch and prototroch). These cilia deliver food to the gastropore (a rudimentary mouth formed through gastrulation by invagination) and aid in locomotion (Bayne 1971). An opposing depression formed the site of shell field development: the origin of the larval shell. The shell field evaginated and extended ventrally from its dorsal origin around the embryo as it developed. This organic matrix folded to the right and left of the dorsal line, along which the two segments were connected by the thickened larval hinge-line (Arellano and Young 2009). The complete enclosure of the embryo by the shell field, once calcified, marked the formation of the two shell valves, and thus the D-shell veliger (Figure 1.23e). The prototroch was displaced by these valves ventrally, forming the velum. This ciliated circular membrane extended out from between the ventral shell margins, with an inner band of long cilia and a peripheral band of short cilia adorning the thi ke ed① a gi ① of① this① i g-like ① structure. The former cilia aid in propulsion and both bands act as a basic conveyer mechanism for filter feeding (Bayne 1976). It was at this stage that larval development halted in the study by Arellano and Young (2009). However, typical mytilid development probably ensues at similarly retarded rates to those of early development (Bayne 1971). The larva typically increases considerably in size as a veliger, after which time a muscular larval foot begins to develop (Bayne 1971; Figure 1.23f). In Bathymodiolinae, veliger larvae probably use energy derived from filter-feeding (i.e.

planktotrophy, Lutz et al. 1980). This last larval stage, the pediveliger, can then either continue to swim using its velum (which is eventually reabsorbed) or crawl using its developing foot, subsequent to initial settlement (larvae are thought to respond strongly to local settlement cues, descending rapidly, Figure 1.23g). While the veliger has relatively basic anatomical requirements as a pelagic dispersal phase, i.e.

swimming apparatus, feeding apparatus and a shell for protection, the needs of the pediveliger phase are, by comparison, much more complex. It must select and colonise a suitable habitat and then make the physiologically demanding transition towards a benthic mode of life. This is reflected in the array of sensory and nervous anatomy already evident in pediveligers, and the presence of the main components of the gillpalp feeding assemblage (Figure 1. 24). Thus by this stage, pediveligers already possess many juvenile characteristics including a muscular larval foot, photosensitive eye-spots, a fully developed mouth, oesophagus, stomach, intestine and anus.

Pediveligers select substrates on which to settle, and secrete byssal threads for attachment, undergoing post-larval metamorphosis to become settled plantigrades (Figure 1.23h). A process termed bysso-pelagic drifting or casting (Figure 1.23i) is known to occur in some post-larval mytilids, where a network of byssus monofila e ts① fo ① a① sail ① ith① hi h① pedi elige s① a ① o ti ue① to① dispe se① ithi ① the① su st atuseawater boundary layer [START_REF] Lane | Byssus drifting and the drifting threads of the young post-larval mussel Mytilus edulis[END_REF]. These simple monofilaments are distinct both in their form and function, from the attachment threads of settled individuals [START_REF] Lane | Byssus drifting and the drifting threads of the young post-larval mussel Mytilus edulis[END_REF]. Bysso-pelagic drifting permits two phases of settlement following dispersal: the pediveligers first settle on filamentous substrates adult① o spe ifi s① ei g① ota l ①a se t ①a d①the ① a igate①to① o e① pe a e t ①su st ata①afte ①a①pe iod①of① post-larval growth (up to 2 mm shell length in this species, [START_REF] Lane | Byssus drifting and the drifting threads of the young post-larval mussel Mytilus edulis[END_REF] In Mytilus edulis, planktotrophic larval development can pass through to the pediveliger stage (Figure 1.23g) in less than twenty days though mean time-to-competency is closer to 30 days (at 10 °C). A delay in metamorphosis can perhaps double this period, when substrate availability is poor (Bayne 1976).

The development in B. (s.l.) childressi is also believed to be planktotrophic, though feeding behaviour was not witnessed during the aforementioned culturing experiment (Arellano and Young 2009). The frankly astounding larval pediveliger sizes identified to be those of B. (s.l.) childressi in Arellano et al. 2014, suggests a lengthy planktotrophic development for this species, corroborated by the considerable disparity between sizes at hatching (approximated by the size of the prodissoconch I <100 µm) and the size of the pediveliger shell (prodissoconch II, can be >600 µm).

Such differences in hatching and settling sizes (and consequently, larval tissue volumes) is a recurring theme in other members of the subfamily, where pediveliger size has been inferred from plantigrade sizes and prodissoconch II shell margins that persist in adults. The pacific hydrothermal vent mussel, Bathymodiolus (s.s.) thermophilus, the bivalve species characteristic of the EPR, has a larval prodissoconch I (PI) of between 95 and 110 µm in length with indeterminate granular sculpture and a prodissoconch II (PII) of 400-470 µm where incremental concentric growth can be identified (Lutz et al. 1980;[START_REF] Berg | Reproductive strategies of mollusks from abyssal hydrothermal vent communities[END_REF]. These larval shell characteristics are indicative of planktotrophic development (Lutz et al. 1980). The minute hatching size (PI) prohibits the presence of a large yolk reserve, which necessitates the need to assimilate externally acquired food in order to meet energy demands required to sustain considerable period of growth, evidenced in the markedly larger PII.

In fact, planktotrophy is inferred in several species of Bathymodiolus (s.l.) based on these criteria Ritt et al. 2012) and the wood-colonising Idas14 (s.s.) iwaotakii, (Thubaut et al. 2013a). Similar prodissoconch II sizes are documented in several Mediterranean Idas (s.l.) morphospecies, however their status as species has yet to be confirmed using molecular analyses (also prodissoconch I sizes were not investigated, Giusti et al. 2012). Finally the sole member of the genus Tamu, Tamu fisheri also displays similar morphometric characteristics [START_REF] Gustafson | Molluscan Life History Traits at Deep-Sea Hydrothermal Vents and Cold Metha e/"ulfide①"eeps.①Reproduction, larval biology, and recruitment of the deep-sea benthos[END_REF]Gustafson et al. 1998).

No exceptions are known, suggesting that planktotrophy is a universal and probably ancestral trait in the Bathymodiolinae (s.l.), supported by current paleontological evidence (e. Planktotrophy furnishes larvae with the potential for an extended planktonic larval duration [START_REF] Gustafson | Molluscan Life History Traits at Deep-Sea Hydrothermal Vents and Cold Metha e/"ulfide①"eeps.①Reproduction, larval biology, and recruitment of the deep-sea benthos[END_REF]Arellano and Young 2009) and thus extended transport times (such as that for B. (s.l.) childressi, estimated to be from 13 -17 months, Arellano and Young 2009). As has already been discussed, while this does not guarantee long dispersal ranges (Shanks 2009), studies examining connectivity and the establishment of vent communities on nascent habitat provide indirect evidence that such far-reaching dispersal likely takes place (Craddock et al. 1995a;[START_REF] Vrijenhoek | Gene flow and genetic diversity in naturally fragmented metapopulations of deep-sea hydrothermal vent animals[END_REF]Won et al. 2003a;Plouviez et al. 2009;Mullineaux et al. 2010). While planktotrophy is apparently widespread in the subfamily, the dispersal environment may be more variable, not least because of the widely different environmental regimes that impact on vents, seeps, and organic falls. For example, only limited data exists identifying the relative importance of pelagic and demersal larval modes in the Bathymodiolinae, however both have been proposed. Larvae in this subfamily are thought to disperse in sub-surface waters following a relatively brief period of ascent (one of many principal assumptions for dispersal modelling for example), however little data exists to validate this assertion, though it is reasonable. It appears that for B. (s.l.) childressi dispersal is likely to be pelagic within the top 100m of the water column, based on rare occurrences of larvae in this area above seeps in the Gulf of Mexico, and experiments that demonstrated this to be perfectly feasible, physiologically (Arellano and Young 2011;Arellano et al. 2014). However the larvae of B. (s.s.) azoricus have been suggested to be demersal (Trask and Van Dover 1999), though this is based on potentially confounded stable isotopes data. Much has since been established concerning the mixotrophic nature of adults of the species, which calls some of the nutritional reasoning in this paper into question. Evidently this is an area which requires further investigation, though such studies are challenging to perform in these environments.

1-9.3.4 Settlement

Information regarding the processes that determine habitat selectivity in competent larvae has been examined earlier in this chapter, but suffices to say, a great deal remains speculative concerning which environmental cues most influence the timing of larval settlement in deep-sea chemosymbiotic bivalves generally, while rates of mortality during this critical juncture in the lifecycle are not evaluated. Given some of the developmental similarities between shallow-water mytilids and their deep-sea counterparts, it is likely that at least some of the scenarios known to induce settling in shallow-water mytilid pediveligers, probably reflect larval selectivity in reducing habitats. In coastal species from several phyla, it has become evident that rather than being stochastic, larval settlement and metamorphosis are driven both by chemosensory recognition of morphogenic and regulatory molecules in the environment [START_REF] Morse | Recent Progress in Larval Settlement and Metamorphosis: Closing the Gaps Between Molecular Biology and Ecology[END_REF], and larval behaviour which they use to mediate their position in the water column. The rare accounts of larval development chemosymbiotic bivalves focus on specific shallow-water species for which the entire development is known in detail. The settled plantigrade developmental stage of the lucinid clam Codakia orbicularis, whose entire developmental pathway differs considerably from that of mytilids, is heavily influenced by the presence of suitable sandy habitat to complete metamorphosis into a juvenile [START_REF] Gros | Embryonic, larval, and post-larval development in the symbiotic clam Codakia orbicularis (Bivalvia: Lucinidae)[END_REF]). Since the sand was sterilised, the presence of biological cues was however, non-essential (e.g.

biofilms, seagrass exudates or potential symbionts). However, since the absence of sandy habitat enforces a delay in metamorphosis in this species, it also directly affects the progression of gill development. Since the gills harbour thiotrophic symbionts in adults, this is thought to have negative fitness consequences for the developing plantigrades.

In shallow-water commercial mytilid species (the mussels most studied) interactions with the physical environment appear to be the most influential, both in terms of current flow and substratum characteristics (e.g. Mytilus spp. [START_REF] Pernet | Settlement success, spatial pattern and behavior of mussel larvae Mytilus spp. in experimental downwelling systems of varying velocity and turbulence[END_REF]. Compound effects with two or more factors are frequently observed. Variable boundary-layer hydrodynamics and either algal waterborne compounds [START_REF] Dobretsov | Larval recruitment of the blue mussel Mytilus edulis: The effect of flow and algae[END_REF] or elevated oxygen levels [START_REF] Alfaro | Effect of water flow and oxygen concentration on early settlement of the New Zealand green-lipped mussel, Perna canaliculus[END_REF] can induce unambiguously positive larval responses. In both cases, sustained reduced-flow regimes increased larval mortality but permitted la ae①to①a t①o ①settle e t① p efe e es ,① hile①high-flow regimes hampered re-settlement processes. The latter is of particular importance in M. edulis, M. trossulus, and Perna canaliculus [START_REF] Hunt | Physical and biological factors influencing mussel (Mytilus trossulus, M. edulis) settlement on a wave-exposed rocky shore[END_REF][START_REF] Gribben | Relative importance of natural cues and substrate o pholog ① fo ① settle e t① of① the① Ne ① )eala d① G ee shell™① ussel,① Pe a① a ali ulus[END_REF]). Gregarious settlement is well known in some coastal benthic species, however its role in mussel settlement is unclear. The biochemical environment can also strongly influence settlement, through the presence of particular biofilms [START_REF] Ganesan | The role of bacterial biofilms and exudates on the settlement of mussel (Perna canaliculus) larvae[END_REF][START_REF] Wang | Larval settlement and metamorphosis of the mussel Mytilus coruscus in response to natural biofilms[END_REF][START_REF] Yang | Larval settlement and metamorphosis of the mussel Mytilus coruscus in response to monospecific bacterial biofilms[END_REF], the presence of specific algae or their exudates (e.g. P. canaliculus [START_REF] Alfaro | Chemical cues promote settlement in larvae of the green-lipped mussel, Perna canaliculus[END_REF]; M.

galloprovincialis, [START_REF] Yang | Larval settlement and metamorphosis of the mussel Mytilus galloprovincialis on different macroalgae[END_REF], or an interactive effect between both algae and biofilms [START_REF] Dobretsov | Effects of macroalgae and biofilm on settlement of blue mussel (Mytilus edulis l.) larvae[END_REF]). Interactive effects have also been identified for the presence of a certain biogenic substratum (e.g.

filamentous substrate) and food (microorganisms, [START_REF] Dobretsov | Recruitment preferences of blue mussel spat (Mytilus edulis) for different substrata and microhabitats in the White Sea (Russia)[END_REF]. Rather more bizarrely some mussels, particularly biofouling species, have been shown to respond positively to sound (or vibrations) at a frequency typical of a large ocean-going vessel [START_REF] Wilkens | Induction of settlement in mussel (Perna canaliculus) larvae by vessel noise[END_REF]). Neurochemical signalling is probably involved in mediating behavioural responses during settlement since the use of targeted neuroactive compounds (e.g. neurotransmitters) have also been extremely effective in inducing [START_REF] Alfaro | Regulatory effects of mussel (Aulacomya maoriana Iredale 1915) larval settlement by neuroactive compounds, amino acids and bacterial biofilms[END_REF];

Sánchez-Lazo and Martínez-Pita 2012) and blocking [START_REF] Yang | Larval metamorphosis of the mussel Mytilus galloprovincialis Lamarck, 1819 in response to neurotransmitter blockers and tetraethylammonium[END_REF]) settlement behaviour. Thus, in shallow water species the intensity of flow has an overriding effect upon settlement where some minimum velocity is required for survival (presumably for the delivery of oxygen and food) but high flow regimes have a constricting effect on attempts to resettle after initial contact-i.e. if currents are excessive, larvae will not even attempt to relocate. If mediating their position is possible, larvae appear to respond to an array of cues to maximise their survival. At reducing environments in the deep sea however, the intensities of flow regimes are probably not in the same order of magnitude, and chemical cueing may be a more dominant feature, given the vast array of signature chemicals available to larvae approaching suitable habitat.

In addition to the effects of physical dispersal into unsuitable habitat, several overarching biological and physical factors act to reduce the number of larvae that settle and eventually recruit into the adult population, due to predator/prey interactions, availability of settlement habitat, and post-settlement survival (Cowen and Sponaugle 2009;refer to Figure 1.15). This is particularly relevant where densitydependent biological interactions operate, such as might occur in highly productive reducing environments, e.g. [START_REF] Lenihan | Biotic interactions at hydrothermal vents: Recruitment inhibition by the mussel Bathymodiolus thermophilus[END_REF]). These processes are all thought to be impacted upon by larval condition at time of settlement (i.e. carry-over effects, but also the larval age at the time of settlement, [START_REF] Gribben | Less inhibited with age? Larval age modifies responses to natural settlement inhibitors[END_REF]).

1-9.4. Post-larval development 1-9.4.1

Metamorphosis and post-settlement processes

There is a singular lack of information regarding the dynamics of metamorphosis in deep-sea mussels.

However, some classical work has been carried out in great detail in Mytilus spp. (particularly M. edulis).

Following successful identification of and settlement upon suitable habitat, several metamorphic biological processes are initiated in the larvae after which the organism is believed to be committed to their completion physiologically. At the time of settling as a pediveliger, much in the way of its future anatomy has already developed (Figure 1 .24). This includes most of the alimentary, excretory and central nervous systems: for example, a single digestive diverticulum communicates with the stomach along its anterior le gth① i ① pedi elige s① Ba e① ① a d① follo i g① eta o phosis,① the① ussel s① e t al① e ous① s ste ① already resembles the adult arrangement of ganglia [START_REF] Voronezhskaya | Neuronal development in larval mussel Mytilus trossulus (Mollusca: Bivalvia)[END_REF]. A metamorphic rearrangement of organs within the mantle cavity takes place to accommodate new levels of complexity in post-larval anatomy and a more organised feeding mechanism (Bayne 1971;1976, Figure 1.24a & b). Thus, several morphological changes occur following primary settlement and byssus secretion. The first is the rather rapid loss (e.g. within 48 hours after byssus secretion in M. edulis, Bayne 1971) of the velum and the network of muscles that support it, where some structures are adsorbed and others are re-orientated. The apical plate becomes part of the upper labial palps, and the mouth and oesophagus are retained, migrating dorso-anteriorly (Bayne 1971). The prior functional role of the velum is adopted instead by the ciliated, lower and upper labial-palps that form in pairs either side of the mouth, during this time. These will ultimately process inhalant particles transported to the buccal region by coordinated ciliary activity in the gills. However, at the very early stages of post-larval development, the movement of cilia along the ventral surface of the foot also plays an important role in particle transport (Bayne 1976). This is a period during which the larvae are particularly susceptible to mortality, as they tend not to feed, are exposed to predation, and are vulnerable to disease since most of their energy is invested in metamorphosis.

In M. edulis, pediveligers can delay metamorphosis for up to forty days at 10 °C or for up to six months in some cases [START_REF] Lutz | Ecology and morphology of larval and early postlarval mussels[END_REF]. In fact, delaying settlement and metamorphosis is a common trait in both asymbiotic and chemosymbiotic shallow-water species (e.g. [START_REF] Pechenik | Delayed metamorphosis by larvae of benthic marine invertebrates: Does it occur? Is there a price to pay?[END_REF][START_REF] Gros | Embryonic, larval, and post-larval development in the symbiotic clam Codakia orbicularis (Bivalvia: Lucinidae)[END_REF]). The extent of the delay is typically constrained by a lack of substrata [START_REF] Gros | Embryonic, larval, and post-larval development in the symbiotic clam Codakia orbicularis (Bivalvia: Lucinidae)[END_REF] or temperature, with longer delays possible at low temperature (effects of metabolism suppression), after hi h①la ae①appea ①to① e①less①sele ti e①of①su st ata① Ma shall①a d①Keough① ,①te ed①the① despe atela a① h pothesis . Interestingly, although it was not the goal of the study, [START_REF] Satuito | Survival, growth, settlement and metamorphosis of refrigerated larvae of the mussel Mytilus galloprovincialis Lamarck and their use in settlement and antifouling bioassays[END_REF] demonstrated that a period of refrigeration in M. galloprovincialis only acted to slow development rather than halt it (or kill more larvae), with survivability being comparable to controls even after a 2-month exposure to refrigeration. In the deep-sea, the concepts of physiological competency and an upper time limit for settlement are not defined. However, low temperatures and their effects on larval metabolism are probably not sufficient explanation for the markedly different rates of development seen with increasing habitat depth (particularly in larvae that ascend to the euphotic zone, e. Mytilid growth rates are subject to considerable variability (e.g. Mytilus spp. [START_REF] Holt | An overview of dynamic and sensitivity characteristics for conservation management of marine SACs[END_REF], due to variations in genotype, multilocus heterozygosity [START_REF] Gosling | The mussel Mytilus: ecology, physiology, genetics and culture[END_REF]) and environmental factors in particular (e.g. include temperature, salinity, resource availability, emersion stress, intraspecific competition for space and food, and parasitism). However growth rates can be 30 -40mm year -1 in length (Seed and Suchanek 1992). Mytilus spp. are gregarious, and in situations where larval supply is non-limiting they can form large beds or reefs (Bayne 1976). At high densities Mytilus beds become layered, with juvenile mussels taking advantage of the spatial heterogeneity of adult shells and thus further increasing it. The consequence is that the bed continually grows and develops with new recruits eventually replacing those being smothering by faecal waste beneath. These reefs provide complex niche-dense habitat for other species. Bathymodiolin mussels (particularly members of the genus Bathymodiolus) are known to form superficially similar reefs when conditions are optimal in diffuse-flow vent systems and in the vicinity of cold-seeps and methane hydrates [START_REF] Kiel | The Vent and Seep Biota[END_REF]).

Ontogenetic growth rates have rarely been examined in bathymodiolins [START_REF] Rhoads | Growth and predation activity at deep-sea hydrothermal vents along the Galapagos Rift[END_REF]Dean 1993;Nedoncelle et al. 2013). In vent and seep habitats, higher rates of growth (or greater longevity) are suspected, with some evidence in the literature that supports this (e. One other study has examined growth in a bathymodiolin species in the deep-sea, in the apparently asymbiotic, wood-inhabiting I. (s.l.) argenteus. Despite colonising sunken wood, this species appears to lack chemosynthetic symbionts in its gills (Ockelmann and Dinesen 2011). Its growth did not follow typical developmental growth-rate patterns (e.g. such as von Bertalanffy-type growth), which normally include greatly elevated growth rates in the first year, followed by a steep fall in growth by the second year (Dean 1993). Instead, growth was more diminutive in the first year, where rates saw only a slight decline over the following years (compared over 8 years). Consequently, rates of growth were lower in the first year but higher in later years in I. argenteus when compared with other species growth rates, available at the time of publication (fresh-water bivalves: Lampsilis radiata, Anodonta grandis; marine species Cerastoderma edule, Modiolus modiolus, Spisula solidissima, where one, M. modiolus is a mytilid).

Individuals of I. (s.l.) argenteus only matured a year after settlement, displaying lower maturation rates than shallow-water species, suggesting a degree of food limitation but greater stability in food supply over time (Jørgensen 1990;Cannuel et al. 2009). Simultaneously, the frontal cilia of each filament differentiate into three different forms which have differing roles to play during gill activity (Cannuel et al. 2009). Functionally, there is a transition from aeration only (in very young post-larvae) towards auto o ousl ① oo di ated① a es ①of① ilia,① hi h①o hest ate①pa ti le①t a spo t①to a ds①the① u al① egio ,① and facilitate aeration and respiration (Jørgensen 1981;[START_REF] Beninger | Labial palps of the blue mussel Mytilus edulis (Bivalvia: Mytilidae)[END_REF]Cannuel et al. 2009). At some point prior to sexual maturation, a definitive pair of gills is ultimately established. Each gill is composed of

Figure 1.25 Patterns of gill ontogeny in Mytilus galloprovincialis

The patterns of development of the gill in Mytilus edulis. Following settlement and metamorphosis a simple gill basket is already developed. With increasing size, new filaments are added at the posterior end of the inner demibranch. At < 1mm the second, as e di g① la ella① of① the① i e ① de i a h①has① egu ① to① fo .① ②t① ≈① . ① ,① the① des e di g① la ella① of① the①oute ① de i a h① the outer half of each gill) begins to develop. Finally at just < 2.16 mm, the ultimate ascending lamella of the outer demibranch has begun forming, wherein by 3.7 mm and larger, the gill is fully formed, and filaments continue to proliferate from the posterior budding zone. Dotted line, antero-posterior axis of symmetry. Note process of elongation-reflection in A-D. al, ascending lamella; dl, descending lamella; fdb, fused dorsal bend; ga, gill arch; gfb, gill filament bud; id, inner demibranch; od, outer demibranch; vg, ventral groove. From Cannuel et al. 2009 an innermedialdemibranch and an outerlateraldemibranch, in turn made up of a descending lamella that originates from the gill axis, and an ascending lamella, located along the leading edge of the descending lamella in reflexive juxtaposition (Figure 1.25). The ascending lamellar filaments proliferate in tandem with secondary (not post-larval) descending lamellar filaments, from the distal budding zone of the gill (Cannuel et al. 2009). Cilia eventually begin to form upon the abfrontal filament surfaces as well (i.e. the interior face of each demibranch lamella).

In chemosymbiotic mussels, the gills are thought to develop in much the same way as in shallow water species, though this has not been examined in the Bathymodiolinae. This is an unfortunate oversight, since such a study could also examine symbiotic dynamics in the context of gill development, being predominantly housed in the gills of adult chemosymbiotic mytilids (Wentrup et al. 2013). What is known is that unlike shallow-water species, cilia do not develop on the abfrontal surfaces (Duperron 2010), which are comparatively enlarged by hypertrophy. The increased surface area of non-ciliated epithelia permits the hosting of dense populations of bacteria either inside sub-epithelial bacteriocytes or among microvilli that differentiate from the epithelial surface of bacteriocytes (see previous section for details).

Sexual maturation and the development of gonads in shallow-water mytilids can be traced back to a provision of maternal primordial germ-line cells (PGCs), which are incorporated into oocytes during oogenesis [START_REF] Obata | The mechanisms of primordial germ cell determination during embryogenesis in molluscan species[END_REF]. In developing embryos, maternal germ-line-specific genes are transcribed within specific blastomeres, which ultimately differentiate into PGCs during development (termed PGC preformation, Obata and Komaru 2012). In M. galloprovincialis, these have been identified during the juvenile development of the species, tracking their apparent progress towards the ultimate formation of reproductively active tissues (Obata et al. 2010). In small juveniles (< 5 mm), two clumps of germ cells are distributed laterally within connective tissue found between the nephric tubules and posterior byssal retractor muscle (Obata et al. 2010), and along the pericardium. These aggregations appear to proliferate first as the mussels grow, and then migrate anteriorly along the mantle basement. In adults, Myvlg expression occurs in various gametogenic stages including spermatogonia, spermatocytes, oogonia, and oocytes, as well as germinal stem cells bordering the acini. In the last case, signals were stronger during the reproductive season (Obata et al. 2010). The preformation of PGCs seems to be a phylogenetically conservative trait in bivalves [START_REF] Obata | The mechanisms of primordial germ cell determination during embryogenesis in molluscan species[END_REF] suggesting similar patterns are likely to exist in deep-sea mytilids, though this remains to be examined experimentally. 1-9.4.5 Phenotypic plasticity during ontogenetic growth One of the pervasive traits of coastal mytilids is a certain degree of variability in growth rates and patterns of development, depending on the dynamics of their habitat, on the species, and on the age of the individual. The net result is that shell morphology displays considerable plasticity both intraspecifically across differing habitats and ages (e.g. [START_REF] Seed | Factors Influencing Shell Shape in the Mussel Mytilus Edulis[END_REF][START_REF] Akester | Shell shape, dysodont tooth morphology, and hinge-ligament thickness in the bay mussel Mytilus trossulus correlate with wave exposure[END_REF][START_REF] Peyer | Developmental plasticity of shell morphology of quagga mussels from shallow and deep-water habitats of the Great Lakes[END_REF], and interspecifically within the same habitat (Suchanek 1981).

Growing evidence in the Bathymodiolinae (s.l.) suggests that similar variability in shell morphology exists both within and across species. This is particularly evident in the smaller bathymodiolins (Lorion et al. 2010;Génio et al. 2012) where both cryptic species and monospecific morphospecies are prevalent [START_REF] Vrijenhoek | Cryptic species, phenotypic plasticity, and complex life histories: Assessing deep-sea faunal diversity with molecular markers[END_REF]. Such plasticity probably plays a role in habitat adaptation, such as compensating for constrained space in vacated wooden xylophaginid burrows. the influence of larval investment, transport and supply upon community dynamics, and the developmental challenges that post-larvae must subsequently face [START_REF] Levin | Recent progress in understanding larval dispersal: new directions and digressions[END_REF]). As a consequence some of the least studied aspects of deep-sea chemosymbiotic life are the water-borne phases in both the lifecycle of the host and its symbionts, and the earlier juvenile development of the host. In the host this spans the period of embryonic and larval growth to competency and by association, post-larval development prior to maturation. For the bacteria, this involves the free-living stage of their lifecycle which is assumed (and in instances, proven) to exist since they are not obliged to engage in symbiosis, unlike the host in most cases.

The temporal and spatial heterogeneity of physical and biogeochemical environmental parameters characteristic of reducing habitats enforce certain constraints upon the organisms adapted to living in them (discussed in section 1-6.1, p. 28). Reducing habitats are both ephemeral and spatially isolated, but they also provide a rich source of chemical energy, sustaining microbial metabolic processes and in turn, supporting dense aggregations of specialised metazoan life that derive their nutrition directly or secondarily from the abundant microbial assemblages present [START_REF] Kiel | The Vent and Seep Biota[END_REF]. The key to the success of the chemosymbiotic bivalves that inhabit reducing habitats in the deep-sea is likely to have been a combination of attaining the means to secure symbionts and benefit from their metabolic capabilities, and a predisposition for adaptable life-history traits. Within the chemosymbiotic bivalves it is the Bathymodiolinae (s.l.) that contain the greatest symbiont diversity, where examples of multiple chemosymbiotic lineages are frequent. They inhabit a wide range of reducing habitats courtesy of the (predominantly) thiotrophic and/or methanotrophic symbionts that are associated to their gill filaments.

Organisms that engage in bacterial symbioses have evolved to gain maximum benefit from such conditions. However, chemosymbiosis also appears to restrict their distribution to these reducing environments, which are interspersed among vast oligotrophic soft-sediment expanses. Consequently chemosymbiotic organisms must overcome several challenges within their lifecycle in order to persist (discussed in detail in section 1-3, p. 10). According to life-history theory, the spatial and temporal constraints of such reducing habitats (excluding seeps) are likely to favour organisms that mature rapidly, display early age at first maturity (and first reproduction), with high fecundity but minimal energetic investment per egg, short life-spans, and a limited number of reproductive events. Intrinsic to these attributes are elevated offspring mortality rates. Confirming or rejecting such assumptions about their biology requires that the lifecycle of these organisms be examined in full. Wentrup et al. 2013) and in the vesicomyid and solemyid clams, which appear to inherit their symbionts directly from parental organisms (e.g. [START_REF] Endow | Occurrence of bacteria in the primary oocytes of vesicomyid clam Calyptogena soyoae[END_REF][START_REF] Cary | Transovarial inheritance of endosymbiotic bacteria in clams inhabiting deepsea hydrothermal vents and cold seeps[END_REF][START_REF] Cary | Vertical transmission of a chemoautotrophic symbiont in the protobranch bivalve, Solemya reidi[END_REF][START_REF] Krueger | Vertical transmission of chemoautotrophic symbionts in the bivalve Solemya velum (Bivalvia: Protobranchia)[END_REF][START_REF] Szafranski | Direct evidence for maternal inheritance of bacterial symbionts in small deep-sea clams (Bivalvia: Vesicomyidae)[END_REF]. Thus, in order to fully appreciate the extent to which chemosymbiotic species depend upon these associations, the acquisition and role of symbionts throughout the lifecycle of these organisms must also be established.

More than any other aspect of their biology, details concerning the early developmental life-cycle stages of chemosymbiotic mussels are lacking for nearly all bathymodiolins. Most developmental biology has to be inferred directly from shallow-water species, in the absence of data for the deep sea. Given the degree of variability in life habits within the Mytilus genus alone, such extrapolations across more distant clades must be subject to systemic error. Gaps in our knowledge reveal a general lack of understanding regarding the interactive dynamics of development and symbiont acquisition together. Understanding how these prolific species persist in their environment requires that symbiont and host life cycles be considered together for those parts of their lifecycles which are intertwined.

Chapter 2 SCOPE OF RESEARCH

2-1 Research themes and aims

Since it has been suggested that organic falls in the deep sea may have played a part in the evolutionary transition of many of the species exclusive to deep-sea① h d othe al① e ts①a d①h d o a o ① old ①seeps① (e.g. Smith et al. 1989;Distel et al. 2000), the scope of this research was restricted to contemporary species found in association with decomposing vegetal and megafaunal material. The primary goal in developing The research presented herein sought to address three principal overlapping themes of organismal biology, which collectively comprise the complex lifecycles of chemosymbiotic mytilids colonising organic falls.

2-1.1. Theme 1: Reproductive biology

2-1.1.1 Research aims
This theme covers the adult period of the lifecycle, from the recruitment into the breeding population onwards. The primary aims of this theme were to describe aspects of reproductive biology recognised to be of particular importance in the context of organic falls. These included spawning patterns (output and frequency); gamete size, as a proxy both for propagule energy investment and larval mode (oocyte diameter) and; size-depe de t①se ① atios,① hi h① ight① la if ①the① ep odu ti e① st ateg ① ei g①e plo ed.①I ① addition, nutrition in adult specimens would be described within the framework of Theme 3.

2-1.1.2 A priori research objectives

To meet the above aim, the research needed to collate newly acquired data on gametogenic trends, sex ratios, fecundity and gamete sizes from live and preserved specimens, and consider these in the context of roughly analogous shallow-water species and in relation to end-state vent and seep fauna.

Larval development:

The primary aim of this subtheme was first to establish the feasibility of inducing gametes, implementing fertilisation and maintaining the embryos of an aquarium-based deep-sea chemosymbiotic organic-fall species, as they grow and develop into planktonic larvae. If this proved possible, the secondary aims were to examine aspects of early embryonic and larval developmental biology, both alive and in preserved subsamples. These would include embryonic cleavage patterns, rates of growth, larval developmental anatomy during its ontogeny, time until competency to settle, and any behavioural observations that could be performed 16 (under predefined environmental conditions). In addition, feeding behaviour and patterns in symbiotic associations were to be noted within the framework of Theme 3.

Post-larval and juvenile development:

The primary aims of this subtheme were to examine larval supply and settlement patterns, shell morphology, and both the detailed and gross anatomical changes taking place in post-larvae right through to pre-adult specimens. Emphasis was to be placed on tracking developing reproductive tissues, in order to characterise the rate at which the chosen taxon might reach sexual maturity, and developing tissues collectively involved in nutrition, within the framework of Theme 3.

Though it was not the principal focus of Theme 2, it was hoped that growth rates might be elucidated given the lack of data on growth in deep-sea, organic-fall-associated phyla.

2-1.2.2 A priori research objectives

Larval development: To meet the above aims, an ambitious live-animal experimental protocol and customised aquaria were to be designed and developed to accommodate the specific requirements of the chosen organic-fall species, based on what is known of these habitats. This would require deep-sea collection, wet-room facilities, and sample availability to all coincide. Depending on the level of success of this approach, incremental developmental stages were to be subsampled, recorded live and then preserved 16 No formal plans were made for tracking substrate selection or metamorphosis for an array of analyses that would best convey the developmental patterns observed. The data that resulted were to be compared with pre-existing data from roughly analogous shallow-water species and in relation to any larval data for end-state vent and seep fauna, be it experimentally acquired or inferred from adult biology. Any measurements that could be made on vestigial anatomy of larval origin were to be performed at this stage. Using the multiple sizes available, various aspects of the developing anatomy were to be examined following the embedding and sectioning of either whole animals or select organs if specimen size permitted this. For the formation of reproductive tissues, this would ideally have involved an tracing the migration and proliferation of the germ line into gonad tissues in recently matured adults (thus fusing aims in Theme 1 with this Theme). Size at first maturity was to be assessed based on the smallest size at which gametogenesis was identifiable. A rough minimal average estimate for growth rates was to be determined from substrate deployment length and the maximum specimen size recovered (making some pre-acknowledged assumptions). This entire approach was envisioned to be performed on relatively small organisms, thus requiring the development of techniques for micro-dissection dedicated to the model o ga is s① aupla ,① fo ① hi h① little information was available from the literature at the project design stage.

Post

2-1.3. Theme 3: Dynamics of symbiotic and filter-feeding based nutrition

This①The e① o e s①the① a ious① ea s① ① hi h① ut i e ts①a e①a ui ed① ithi ①the① odel①g oup s①life le.①

In deep-sea chemosymbiotic faunae that acquire their symbionts as free-living bacteria (or other microbial organisms) from the surrounding environment (i.e. horizontal transmission), ontogenetic variability in the dominant nutritional sources is often postulated, particularly during planktonic dispersal. Therefore, the principal aims sought to collect evidence for shifts in nutritional mode during development. These included identifying the approximate period within the lifecycle at which symbionts are first acquired, the sequence of infection (in terms of localisation and compartmentalisation in tissues, if applicable) within the host, changes in symbiont density (and thus their importance) with ongoing growth, and any observable succession in symbiont phylotypes (host/symbiont mediated nutritional adaptability). In parallel, data that a i als① a ① e① e o e ed① i ① good ① health ① fo ① the① ai te a e① of① ultiple① life le① stages,① gi e ① the① absence of detailed knowledge pertaining to the various environments experienced by organisms throughout its life. Collectively these confound all attempts to study lifecycles in their entirety in living specimens and make informed assertions much more difficult. , or by examining several cohorts within a single timeframe or better still; 3) incorporating these approaches synergistically.

2-2.1.2

The potential for lifecycle studies in chemosymbiotic organisms A suitable test organism, which meets the constraints listed, can permit certain questions to be answered that would otherwise be difficult to elucidate, such as those detailed in the research rationale previously (see 2-1, p. 107). From a chemosymbiotic lifecycle perspective, these ought to include aspects of the biology from each stage of development including the role symbionts play therein, so as to truly appreciate the degree to which lifecycles of chemosymbiotic species at reducing environments are adapted to their habitat(s).

If the chosen specimen can be kept alive in the aquarium, it is possible to induce gamete release and so collect data on adult spawning behaviour and timing, processes of gametogenesis, fecundity and fertilisation, and even on larval development. This can yield data on growths rates, developmental patterns, locomotor and feeding performance, the time until competency and thus planktonic larval duration (PLD) under predefined conditions. If competency can be reached, the complex array of behaviours and changes in physiology associated settlement and ultimately metamorphosis itself, may be investigated. In tandem, multiple behavioural aspects of breeding adults can be assessed. Such a study, if performed successfully, can furnish a wealth of data in a brief (but intensive) period of laboratory work.

As an alternative or better still, synergistic approach, studying a range of preserved specimens using standard histological approaches can be highly informative. The availability of multiple sizes (and presumably ages) presents a rare opportunity to examine various stages in a species ontogenetic development, wherein the specimens have been living in similar habitats. Comparisons across such size 17 Though this presents practical difficulties, particularly if the cohort size is low (subsampling will significantly change densitydependent population dynamics of the remaining individuals, confounding statistical independence) series from different habitats or species allow for the effects of habitat and evolutionary origin to be considered. For instance, one current area of interest for deep-sea research is in documenting basic iologi al①pa a ete s①i ① less-spe ialised ①spe ies①that① ight① ast①so e①light①o ①the①e olutio a ①o igi s①of① end-state contemporary species, whose adaptations now restrict them to living at hydrothermal vents and hydrocarbon seeps (e.g. Thubaut et al. 2013a).

2-2.1.3 A priori considerations

To be able to apply many of these approaches and maximise the data that can be collected from supplylimited samples, some criteria may prove pertinent when choosing an appropriate benthic, deep-sea chemosymbiotic study group. These restrict the suitable model group to: 1) organisms that as adults, are found upon reducing habitats exclusively, including incidences on organic falls; 2) organisms for which larval dispersal is the principal driver of connectivity between habitats; 3) organisms in which symbioses are suspe ted① o ① k o ① to① o u ;① ① o ga is s① that① a ① e① o side ed① less-spe ialised ① o ① o e① oppo tu isti ;① ① o ga is s① that① a ① e① kept① ali e① u de ① a ie t-pressure conditions in land-based aquaria (particularly organisms for which useful data exists in other related aquaculture species for defining these conditions) and; 6) organisms for which it is possible to acquire specimens at various stages of development, ideally from settlement size right through to maturation as adults.

2-2.1.4

Chemosymbiotic mytilids (Bathymodiolinae, s.l.)

The chemosymbiotic mytilids (Bathymodiolinae s.l.), which evidently include a considerable number of described and undescribed species, represent a relatively straight-forward model taxon for a lifecycle study for several reasons. First of all, the phylogenetic origins of the Bathymodiolinae are reasonably well understood (though not perfectly). This is a consequence of early molecular studies (e.g. Craddock et al. 1995b;Distel et al. 2000) and an intensive period of molecular systematics which have followed over the last decade performed by several researchers independently (e.g. Jones et al. 2006;Won et al. 2008;Kyuno et al. 2009;Lorion et al. 2010;Miyazaki et al. 2010;Schultz et al. 2011;Fontanez and Cavanaugh 2013;Lorion et al. 2013;Thubaut et al. 2013b). These studies have greatly improved our understanding of the su fa il s① possi le① e olutio a ① histo .① The① o pa iso ① of① these① ladisti s① ith① o phologi al① data① fo ① the subfamily, have revealed microstructural shell characteristics to be prohibitively conservative (Génio et al. 2012) and considerable intraspecific phenotypic plasticity (Lorion et al. 2010), perhaps due to neoteny and thus paedomorphism in smaller-sized genera of the subfamily (Génio et al. 2012). Thus alternative e ide e① o e i g①the①e olutio a ①status①of①the①su fa il s members (s.l.) would aid our understanding concerning the degree of divergence and true speciation in the Bathymodiolinae.

Secondly, as a consequence of their loose phylogenetic affinity with several shallow-water mytilids of commercial and/or ecological value (e.g. the genera Mytilus and Modiolus respectively), some limited information can already be gleaned concerning gross bathymodiolin biology from the literature and commercial aquaculture. This makes the interpretation of anatomy in juveniles feasible, for example. Thus, while the conservative nature of chemosymbiotic mytilid morphoanatomy in this subfamily presents difficulties for taxonomy (Lorion et al. 2010;Génio et al. 2012), shallow-water reference species already p o ide①putati e① aseli e ①data for informing hypothesis testing in members of their deep-water relatives.

Such hypotheses could assess anatomical divergence (and convergence) in the bathymodiolins in relation to the most robustly supported phylogenetics available. From a lifecycle point of view, this could include any biological aspect of their development. In addition, the presence of mussels from a multitude of substrates p o ides① o te t① fo ① e i o e tal① t a sitio s① p oposed① to① ha e① take ① pla e① du i g① the① su fa il s① evolutionary history (Distel et al. 2000). In this way, observed life-history traits in deep sea mussels can be formally assessed in relation to their ancestral background, likely from shallower seas (Jacobs and Lindberg 1998, though demonstrating this with molecular evidence has proven problematic, Jones et al. 2006;Lorion et al. 2013). Other important ways in which baseline data from commercial mussel species can be harnessed for deep sea mussel species is by providing a starting point when designing suitable experimental aquaria, and by providing some reference point for interpreting immature anatomy. Live observations evidently present several avenues for exploring the developmental biology of the Bathymodiolinae (as discussed previously).

Thirdly, let us return to the initial notion of identifying a sub-g oup① of① less-spe ialised ① ussels① f o ① reducing habitats to provide insight into the radiation of the subfamily. High degrees of habitat specialisation are common within the large-sized Bathymodiolinae (s.l.), but critically, the degree to which different genera are isolated to specific habitats appears to vary, where some genera are capable of invading several different reducing habitats, across wide depth ranges (e.g. Idas and Adipicola). The genus Bathymodiolus (s.l.) has seen the most detailed study both across habitat types and biological disciplines.

Paradoxically however, this genus, for which the phylogeny is still under revision (polyphyletic, currently split across 2 -3 clades depending on interpretation e.g. Lorion et al. 2013;Thubaut et al. 2013b), is comprised of some of the most recently emerged and specialised clades of the subfamily (Lorion et al. 2013). In reality however, with the exception of Gigantidas spp., the numerical majority of the Bathymodiolinae (s.l.) consist of a diverse range of smaller-sized genera occurring on multiple reducing habitats (Lorion et al. 2013). Some of these smaller-sized genera share recent evolutionary roots with the B. Several examples from this pool of small-sized mussels, frequently found thriving on organic falls, were cited by Distel et al. (2000) as extant evidence of an evolutionary habitat transition from wood and whale association to vent and seep specialisation in the relatively giant Bathymodiolus (s.l.) spp.. The deeper, ventand seep-endemic Bathymodiolus (s.s.) spp. and the shallower, more-opportunistic wood-, whale-, and occasionally seep-and vent-colonising Idas [s.s.] spp., probably began diverging as two lineages around 34-27 Ma ago from a common ancestor (Lorion et al. 2013). The radiation of known contemporary species of Idas (s.s.) is thought to have started around 24-17 Ma ago, while in Bathymodiolus (s.s.), contemporaryspecies radiation for the genus began more recently, around 15-11 Ma (Lorion et al. 2013). Members of the younger clade Bathymodiolus (s.s.) are thought to be approaching an evolutionary point of no return, as far as habitat specialisation is concerned (Jones et al. 2006;Thubaut et al. 2013b). In contrast, contemporary

Idas spp. can be found living on one or more types of decomposing organic material in the deep sea often occurring over shallower depth ranges (Lorion et al. 2010) Finally, having considered all the known biology of the possible model taxa, suitable sample availability had to meet the a priori aims of the research envisioned. Several candidates for less-derivative mussels exist in the basal genera of the Bathymodiolinae. Ultimately the genus Idas (s.l.) was chosen, since it met all of the criteria previously stipulated in 2-2.1.3. (page 112), including access to comprehensive series of postsettlement developmental stages in two species, and rarer still, live organisms for observation and experimental studies. Idas (s.l.) spp. have been cited previously in the literature as potential model organisms for investigating the evolutionary origin and dispersal capacity of chemosymbiotic mussels (Distel et al. 2000;Gaudron et al. 2012), as members of this genus (s.l.) have been documented to colonise vents, seeps, sunken-wood and whale-falls, sometimes within the same species [START_REF] Sturany | Zoologische Ergebnisse VII. Mollusken I (Prosobranchier und Opisthobranchier; Scaphopoden; Lamellibranchier) gesammelt von S.M. Schiff "Pola" 1890-1894[END_REF]Marshall 1900;Smith et al. 1989;Dean 1993;Tunnicliffe et al. 1997;Olu-Le Roy et al. 2004;Southward 2008;Lorion et al. 2009;Pelorce and Poutiers 2009;Gaudron et al. 2010;Lorion et al. 2010;Ockelmann and Dinesen 2011;Gaudron et al. 2012;Giusti et al. 2012;Ritt et al. 2012;Thubaut et al. 2013a). A summary of the current knowledge on this genus is thus given below and following that, the various means by which specimens were collected for this research.

2-3 The genus Idas

2-3.1. Shell plasticity and polyphyly

As evidenced by the numerous synonyms18 attributable to this genus over time and the arbitrary misallocation of certain related genera to the genus (for example AduIa), the history of Idas (s.l.) is punctuated with discourse and taxonomic errors (Giusti et al. 2012). It is thus a challenging genus to retrace, particularly since the distinction between Idas and Adipicola is not a clear one, both morphologically and phylogenetically (e.g. Lorion et al. 2013;Thubaut et al. 2013b). A detailed account of the convoluted phylogeny of this genus may already be found in 1-8.2.4 (page 49), but needless to say, an overhaul of the subfamily as a whole would need to pay particular attention to Idas (s.l.), which currently contains one of the highest number of putative species within the Bathymodiolinae (s.l.). The lack of concomitant genecology and malacology in the genus (e.g. Lorion et al. 2013;Thubaut et al. 2013b) may be due, in part, to phenotypic convergence within the entire family Mytilidae [START_REF] Distel | Phylogenetic relationships among Mytilidae (Bivalvia): 18S rRNA data suggest convergence in mytilid body plans[END_REF]Distel et al. 2000).

Issues more specific to Idas-like mussels have arisen because in some species, intraspecific shell morphology is fairly constant with growth, while in others species the shell outline and sculpture appears to (Génio et al. 2012). Several characteristics that were thought to be discriminative for the genus such as the microscopic structure of periostracal byssal hairs and the micro-taxodenticulate hinge in particular, now appear to be restricted to juveniles in some species, while seemingly persisting even as adults in others (Giusti et al. 2012), probably as a consequence of paedomorphism (Génio et al. 2012) and polyphyly (Thubaut et al. 2013b). Unfortunately, due to the polyphyly of the genus, it is unclear to what degree these apparent exceptions simply represent incorrectly placed species (Idas [s.l.] simpsoni being an excellent example). Thus any attempts to assign broad morphological characters to the genus are undermined. Species identification therefore ought to include as a minimum: intra-and interspecific anatomical and malacological comparisons, based on samples at comparable ages (or at comparable percentage maximum lengths, at least). This would involve the cataloguing of both inherent interspecific variability and intraspecific plasticity with increasing age, ideally assessing these characters in the context of the most thorough molecular phylogenies available. These are typically most reliable when variability across multiple gene regions is considered collectively in a concatenated tree (Lorion et al. 2013). [START_REF] Dell | Mollusca of the family Mytilidae (Bivalvia) associated with organic remains from deep water off New Zealand, with revisions of the genera Adipicola Dautzenberg, 1927 and Idasola Iredale[END_REF]Juniper et al. 1992;Deming et al. 1997;Smith and Baco 2003;Southward 2008;Tyler et al. 2009).

change

2-3.3. Nutrition and symbioses

The habitat coverage displayed by the genus is thought to be made possible courtesy of unrivalled levels of symbiont flexibility (Duperron 2010). Nutritionally, the limited data available on Idas spp. suggests that the genus nestles intermediately between heterotrophic shallow-water mytilids and the relatively giant, predominantly chemosymbiotic, deep-sea vent and seep specialists, Bathymodiolus (s.l.) spp. From a mytilid perspective, the complexity of the digestive system and labial palps in Idas spp. are somewhat basic (based on I. [s.s.] macdonaldi, Gustafson et al. 1998;and A.(s.l.) iwaotakii, Thubaut et al. 2013a), however they retain a higher level of organisation and anatomy-inferred functionality than in Bathymodiolus spp. (Gustafson et al. 1998). The exception as always being the larviphage, I. (d.f.) argenteus; the digestive system is typically mytilid-like and labial palps are enlarged and thought to be adapted to handling unusually large particle sizes for the family (Ockelmann and Dinesen 2011). Equally, although the abfrontal surfaces of gills are enlarged when compared with coastal mytilids [START_REF] Rodrigues | A complex picture of associations between two host mussels and symbiotic bacteria in the Northeast Atlantic[END_REF] Dinesen 2011), as such inferences would require the availability of some molecular data for this species (the type species for the genus). From the point of view of symbiosis, Idas (s.l.) thus has great potential in elucidating bathymodiolin evolution, especially since the genus (s.l.) may be undergoing an evolutionary transition from extracellular to intracellular endosymbiosis (Duperron et al. 2013). Extracellular endosymbioses are known to occur in I. modiolaeformis (Duperron et al. 2008a;Lorion et al. 2009), Idas (s.l.) simpsoni (Southward 2008), and several unidentified Idas spp. (Idas woodia and ESU C -D respectively, Gros et al. 2007;Lorion et al. 2009). Definitive intracellular endosymbiosis appears to be restricted to Idas (s.s) washingtonius, the only described species of the genus capable of colonising vents (Deming et al. 1997).

The timing and means of acquisition have not been described for any species within this genus. Yet, such processes present interesting areas of study for any chemosymbiotic relationship and especially for Idas,

given that acquisition is almost certainly horizontal and may differ in dynamic ways depending on species, ha itat①t pe,①lo alit ,①a d①the①s io t①ph lot pe s ① ei g① ta geted .

2-3.4. Growth and development

Bar a single paper examining growth in I. (d.f.) argenteus (Dean 1993), no research has ever examined growth or development in the genus Idas, despite the obvious benefits that rapid growth rates may have in their ephemeral habitats. Rates of growth for I. (d.f.) argenteus (see section 1-9.4.3, p. 77) were found to be roughly equivalent to those in B. (s.s.) thermophilus [START_REF] Kenk | A new mussel (Bivalvia, Mytilidae) from hydrothermal vents in the Galapagos Rift zone[END_REF], although rates of growth retarded rather less than growth curves would typically predict based on the post-larval rates of growth in I.

(d.f.) argenteus.

2-3.5. Reproductive and larval biology

Data concerning the reproductive biology of the genus is fragmented, though protandric sequential hermaphroditism has been identified in the three species in which size-specific sex ratios have been investigated (Tyler et al. 2009;Ockelmann and Dinesen 2011;Gaudron et al. 2012). Planktotrophy has been inferred for the larvae of I. modiolaeformis and I. argenteus (Dean 1993;Ockelmann and Dinesen 2011;Gaudron et al. 2012) based on deductions concerning prodissoconch malacology first postulated for bathymodiolins by Lutz et al. (1980). Egg sizes are small (< 60 µm diameter) and fecundity is thought to be high (Dean 1993;Tyler et al. 2009;Gaudron et al. 2012).

2-4 What remains to be discovered

2-4.1. A lack of basic biological characters

Basic biological understanding is lacking in the lifecycle of species of Idas (s.l.) including all aspects of anatomical development as post-larvae and juveniles, such as growth rates (except Dean 1993), patterns of tissue development and proliferation and, the origins of, and trends in, symbiont infection. It is also not known how these processes change (if at all) with increasing size prior to, and following maturation.

P o esses① that① togethe ① o p ise① the① ussel s① o e all① se ual① de elop e t① i ludi g① size① o ① age① at① fi st① maturity, sex determination and sex-ratio patterns, post-maturation gametogenesis, spawning behaviour, fecundity and species longevity are not rigorously assessed, or remain entirely unknown.

The genus Idas (s.l.) evidently displays diverse habitat use, sometimes within a single species. Yet, it is not known how the processes above behave across differing reducing habitats in a single species. Establishing vital baseline data for the various lifecycle components of these species will not only provide insight into chemosymbiotic mytilids that occur on organic falls, but also for the subfamily as a whole, particularly from a ① e olutio a ① poi t① of① ie .① B ① gathe i g① data① f o ① ultiple① ha itats① a d① f o ① diffe i g① i te ediate① e olutio a ①spe ies ① ithi ①the①Bath odioli ae① s.l.), our understanding of the evolution of for example, Bathymodiolus spp. and their symbioses, may be increased.

2-4.2. Study species for the current research

The three species that featured in this research were I. (s.s.) modiolaeformis, I. (s.l.) simpsoni, and the type species I. (d.f.) argenteus where the relative commitment of time and resources follow the same order.

Rather incidental, putative specimens of I. argenteus were found when examined wood substrates for the other two species. The role it played in this research, in collaboration with colleagues, was restricted to the provision of hitherto unavailable soft tissue for molecular confirmation of the type species of the genus, and extension of its known geographic distribution, juvenile morphoanatomy and further confirmation of its asymbiotic state. Outside of Chapter 6 in which this work is presented, it is not discussed further here. host is so far restricted to this region of the Nile Deep-sea Fan only. For other locations in which the species has been found and from which specimens have been examined, symbioses involve only a subset of this diversity (Duperron et al. 2013).

By way of contrast, only thiotrophic symbionts related to those found in Bathymodiolus (s.l.) aff.

boomerang (Duperron et al. 2005) have been identified in I. (s.l.) simpsoni (Ritt et al. 2012). This is despite high concentrations of methane at the study area [START_REF] Carlier | Heterogeneous energetic pathways and carbon sources on deep eastern Mediterranean cold seep communities[END_REF] . They also have at least two habitat associations in common (sunken wood and hydrocarbon seeps). Thus logically, their distributions ought to overlap geographically and bathymetrically, somewhere within the Mediterranean Sea. Such overlapping distributions are opportune for direct intra-generic 21 comparisons. In addition, as distantly related species only, a comparison of their anatomies may provide insight into hypothesised independent convergences in (or conservation of) morphoanatomy, suspected for the mytilids generally [START_REF] Distel | Phylogenetic relationships among Mytilidae (Bivalvia): 18S rRNA data suggest convergence in mytilid body plans[END_REF].

Although symbiont assemblages differ markedly in both species they are suspected to be extracellular in both, where in I. (s.s.) modiolaeformis at least, bacteria are located among a layer of microvilli which extends out from non-ciliated filament epithelia. Since both species represent less specialised species, some filter-feeding function is presumed to remain. Though frequently overlooked in regional ecosystem-based syntheses, organic falls are known to play host to several species which specialise in colonising organic material undergoing decomposition, making it a habitat type in its own right. Wooden debris is known to occur at a wide range of depths (e.g. in the Caribbean, [START_REF] Wolff | Magrofaunal utilization of plant remains in the deep sea[END_REF]). This appears to also be the case for organic debris occurring naturally on the seafloor both in the Mediterranean (e.g. wood, Pailleret et al. 2007b;Giusti et al. 2012 2.1) or while visiting submarine canyons (Lacaze-Duthiers Canyon). These were specifically designed to permit the qualitative collection of newly settled organisms upon various substrata (including two organic substrates), while reducing postsettlement mortality by constraining larger, mobile-predator access.

The housing of these devices consisted of a hollow PVC cylinder (14 cm diameter, 10 cm tall), in which many holes were drilled for water-circulation purposes. Fitted snugly inside this cylindrical casing was a matching basket-like insert constructed from plastic 2-mm garden mesh, into which one of three substratum types was placed. Substrata were either terrigenous carbonate, Douglas-fir pinewood or dried alfalfa grass, he e①the①fi st①t o①a ou ted①to①a①total①of①≈① ① u es①pe ①CHEMECOLI① u es① e e① ① ① ① ① ① ①a d①the① last was the volume of grass needed to fill the device entirely (with minimal compaction, standardised olu e①of①≈① . ①d 3 based on dimensions). Once filled, the basket was fitted with a lid made from the same mesh, so that the contents were sealed inside. In order to counter the buoyancy of the device, the lower edge of the device was weighted using stainless steel chain. Wood-filled devices were given time to soak in old① filte ed① sea ate ① i ① o de ① to① ou te ① the① d ① ood s① uo a .① De i es① e e① la elled① usi g① conspicuous syntactic foam floats, attached using thick nylon rope to make their recovery straight-forward by robotic arm (Figure 2 . These had previously been deployed attached to a sampling frame with considerable concrete ballast (Figure 2.2). Densities were extremely high on certain bones, and the subsample of bone mussels used for this research was selected to cover all sizes, thus size-frequency data from this subsample were meaningless.

2-5.2.2 Larger adults and live specimens

The largest adults from both species were collected in the Lacaze-Duthiers Canyon, from small pieces of oakwood (June 2012) and a large section of palm-tree trunk (April 2013) which had been deployed for 11

and 29 months respectively. The palm tree mussels were the individuals predominantly used in live-mussel observations in the Banyuls-sur-mer Marine Observatory (Chapter 3). Live observations were also performed on the limited number of juveniles and small adults collected from the CHEMECOLI from within the Lacaze-Duthiers Canyon (i.e. those mentioned in section 2-5.2.1, p. 122).

The work carried out on live organisms in Banyuls was to be repeated almost immediately, following the beginning of the mobility period in Aveiro, Portugal (starting July 2013), using live mussels from 24-month bone-deployments. However, rather unfortunately bones being recovered on my behalf were subject to excessive mechanical damage due to difficult sampling conditions, where bones were rolling during their transport to shore. The mussels did not survive and were fixed ahead of docking in order to preserve the samples before they spoiled completely. Compared to the preserved samples at 18 months (see section 2-5.2.1, p. 122), the populations densities at 24 months had also almost completely crashed (excessive canyon sedimentation and thus burial).

2-5.2.3 Additional DNA samples

Note that pre-extracted DNA originating from I. (s.s.) modiolaeformis mussels found in CHEMECOLI devices deployed at the Amsterdam MV (eastern Mediterranean), the Meknès and Darwin MVs and Gorringe ridge were also available which, along with the sampling described, were sent for high-throughput 454 sequencing to examine the biogeography of symbiont associations in these two species across the entire Mediterranean and East Atlantic. Unfortunately, delays in the subcontracted analysis mean that this data will not feature in the PhD (but will be processed as part of a 2-month post-doctoral extension).

Figure 2.3 Overview of sampling locations

Details are given in Table 2.1

3-1 Abstract

The survival of specialist deep-sea benthic species thriving in spatially and temporally finite reducing habitats necessitates long-ranging dispersal, suitably responsive reproductive strategies, and in numerous species also depends upon chemosynthetically derived nutrition from associated bacterial symbionts.

Ho e e ,① o t a ① to① the① elati el ① sustai ed① suppl ① of① sulphides① at① h d o a o ① old ① seeps① a d① hydrothermal vent fields, sulphide supply at the surface of sunken wood is only intermittent, raising doubts about whether chemosymbiotic organisms rely entirely on their bacterial symbionts for nutrition. To assess this, live observations of feeding and reproduction were examined in two divergent species of

3-2 Introduction

Since the discovery of hydrothermal vents (Lonsdale 1977) and the chemosynthetic basis of their productivity, other reducing habitats have subsequently been identified where each is home to their own array of specialist and visiting fauna (Levin 2007). Chemosynthetic bacteria occur as free-living chemotrophs, as symbionts, or as both, supporting communities by deriving metabolic energy from the electron donors and acceptors available at reduction-oxidation interfaces (Dubilier et al. 2008). In the deep sea, these reducing habitats shrouded in darkness, are typically ephemeral over decadal timescales or less and are highly discontinuous in their distribution (Duperron 2010). Thus, the survival of specialist deep-sea benthic species is favoured by long-ranging dispersal, suitably responsive reproductive strategies, and often depends upon taking advantage of the chemosynthetically derived nutrition available (Dubilier et al. 2008).

Such constraints in reducing habitats have resulted in a range of contemporary species that engage in chemosymbioses, availing novel metabolic capabilities to the host.

One of the most pervasive chemosymbiotic taxa to inhabit reducing habitat types in the oceans (Duperron 2010;Lorion et al. 2013;Thubaut et al. 2013b) is the mytilid bivalve subfamily Bathymodiolinae (used sensu lato hereafter, as with genera enclosed in quotation marks based on the phylogeny in Thubaut et al. 2013b). They colonise hydrocarbon seeps (e.g. Childress et al. 1986;Van Dover et al. 2003), hydrothermal vents (reviewed in [START_REF] Dover | The ecology of deep-sea hydrothermal vents[END_REF], the skeletons of decomposing megafauna (e.g. Deming et al. 1997;Tyler et al. 2009;Génio et al. in press), sunken wood (e.g. Dean 1993;Gaudron et al. 2010;Lorion et al. 2010;Bienhold et al. 2013;Cunha et al. 2013) and even oily drill-cuttings (Southward 2008).

Their success is attributed to suspected long-ranging larval dispersal capacities [START_REF] Tyler | Reproduction and dispersal at vents and cold seeps[END_REF]Mullineaux et al. 2010;Adams et al. 2012;Arellano et al. 2014), and to their capacity to host diverse chemotrophic bacterial associations (Vrijenhoek 2010). Extended larval dispersal has been proposed based on considerable differences between larval shell dimensions (prodissoconch I and II), indicating small hatching size and relatively large settling size respectively (e.g. Lutz et al. 1980;Arellano and Young 2009;Gaudron et al. 2012a). Such small hatching sizes, supported by documented mature oocyte diameters with limited energy reserves (<80 µm, Colaço et al. 2006;Arellano and Young 2009;Gaudron et al. 2012a), suggest that dispersal is planktotrophic (Lutz et al. 1980). Larvae of some small-sized bathymodiolins may be aposymbiotic, excluding the possibility of symbiont-derived nutrition during dispersal (Laming et al. 2014). Data on sex-ratios are scarce, however most bathymodiolins are believed to be gonochoristic, except

Bathymodiolus azoricus, which displays a mixed mode with limited protandry (Comtet et al. 1999;Dixon et al. 2006). Spawning frequency in the bathymodiolins appears to be predominantly seasonal (e.g. Van Dover et al. 2002;Van Dover et al. 2003;Colaço et al. 2006;Dixon et al. 2006;Tyler et al. 2007), though semicontinuous spawning has been proposed for B. thermophilus from the East-Pacific Rise based on gametogenesis [START_REF] Tyler | Reproduction and dispersal at vents and cold seeps[END_REF]) and smaller-sized Idas spp. based on settlement patterns (Dean 1993;Ritt et al. 2012;Laming et al. 2014).

When documented, bathymodiolin species host symbiotic bacteria within the abfrontal, non-ciliated epithelial surfaces of their hypertrophied gills, which can be extra-or intracellular (reviewed in Duperron 2010). In all cases, symbiont transmission is thought to be horizontal, acquired predominantly from the environment as free-living bacteria (Won et al. 2003;Duperron et al. 2008a;Gaudron et al. 2012a;Fontanez and Cavanaugh 2014;Laming et al. 2014). The timing of acquisition appears to vary between pre-and postsettlement, depending on the species (Streams et al. 1997;Won et al. 2003;Salerno et al. 2005;Wentrup et al. 2013;Laming et al. 2014). The retention of a functional, albeit-reduced digestive system (e.g. Gustafson et al. 1998) has fuelled speculation for mixotrophy, particularly during early benthic life [START_REF] Cowie | Plankton availability and retention efficiencies of cold-seep symbiotic mussels[END_REF][START_REF] Martins | Size-dependent variations on the nutritional pathway of Bathymodiolus azoricus demonstrated by a C-flux model[END_REF]Laming et al. 2014). For wood-associated mussels, a retained capacity for mixotrophy may represent one way to overcome fluctuations in sulphide supply, which appear to be highly intermittent on sunken wood [START_REF] Laurent | Sunken wood habitat for thiotrophic symbiosis in mangrove swamps[END_REF]Laurent et al. 2013;Yücel et al. 2013). Documented variability in symbiont densities within and across species of small-sized bathymodiolins strongly suggests a necessity for alternative nutrition (e.g. Duperron et al. 2008a;Duperron et al. 2008b;Ritt et al. 2012;Rodrigues et al. 2013;Laming et al. 2014), however mixotrophy remains hypothetical. Filter-feeding and assimilation have ee ①de o st ated①e pe i e tall ①i ①la ge① Bathy odiolus (sensu lato) childressi, B. thermophilus and B.

azoricus (Page et al. 1990;Page et al. 1991;Riou et al. 2010).

Several species and clades of small bathymodiolin mussels are deep-a hi g① ithi ①the①su fa il s① phylogeny (Jones et al. 2006;Kyuno et al. 2009;Vrijenhoek 2010;Duperron et al. 2013a;Lorion et al. 2013;Thubaut et al. 2013b). They might thus present more ancestral states for certain characters than the more intensively studied, highly specialised vent and seep species. Many of these mussels are loosely attributed to the genus Idas, with pronounced polyphyly in this genus as a consequence (e.g. Lorion et al. 2013;Thubaut et al. 2013b). However, the discovery and molecular analyses of new specimens of I. argenteus, the type species, has recently resolved this problem (Rodrigues et al. in press). The mostly extracellular symbioses of members this clade and other Idas-like mussels are less-integrated than those in vent and seep species, and this has prompted research into small-sized bathymodiolin biology in order to clarify the evolutionary history of the Bathymodiolinae as a whole (see Discussion, Duperron et al. 2008a;Tyler et al. 2009;Ockelmann and Dinesen 2011;Gaudron et al. 2012a;Thubaut et al. 2013a;Laming et al. 2014).

Two such small-sized bathymodiolin species are Idas modiolaeformis and I. simpsoni. While the species I. modiolaeformis truly belongs in its genus, I. simpsoni actually places within a deeper-branching clade that predates the later divergence of Idas (sensu stricto) and Bathymodiolus (sensu stricto) clades (Lorion et al. 2013;Thubaut et al. 2013b, Rodrigues et al. in press). Although Idas (s.s.) modiolaeformis and I. (s.l.) simpsoni belong to markedly divergent lineages, they display some similarities in morphoanatomy and habitat use with sunken wood and hydrocarbon seeps in common (Thubaut 2012). These two species are distributed over a wide variety of reducing environments and depth ranges both in the Mediterranean and the Atlantic (Thubaut 2012), which at their shallowest extremes in are likely to be subject to considerable vertical flux of particulate organic matter (Palanques et al. 2006). Despite overlap in their distributions, they are only known to co-occur on sunken wood in the Gulf of Lion (Thubaut 2012;Le Bris et al. in preparation).

Remarkable symbiont diversity has been demonstrated in I. modiolaeformis recovered from hydrocarbon seeps, with six bacterial types identified (Duperron et al. 2008a). Various subsets of these presumptive symbionts have been repeatedly described in other specimens and populations (e.g. Lorion et al. 2010;Lorion et al. 2012;Rodrigues et al. 2013;Laming et al. 2014). In Idas (s.l) simpsoni, only one dominant thiotroph bacterium has been observed in specimens from hydrocarbon seeps in the Marmara sea (Ritt et al. 2012;Thubaut et al. 2013b).

Although data on feeding and spawning behaviour are best collected during the examination of live organisms, only a handful of in vivo studies have been published for bathymodiolins, all from Bathymodiolus (Page et al. 1990;Page et al. 1991;Colaço et al. 2006;Kádár et al. 2006;Arellano and Young 2009;[START_REF] Mikkelsen | Speciation in Modern Marine Bivalves (Mollusca: Bivalvia): Insights from the Published Record*[END_REF].

This study sought to successfully maintain adult Idas-like mussels alive, in order to describe aspects of their Specimens used in live specimen work originated from these substrata (Figure 3.2). The previous year (LD4), some specimens were recovered from oakwood experiments deployed in 2011 (LD3, 11 month deployment). These were used in this study to support stable isotopes analyses and for FISH analysis.

3-3.1.3 Transport and sample sorting

The palm-tree trunk and CHEMECOLIs were transported intact in aerated, filtered seawater (13°C) to the LECOB laboratory facility at the Banyuls Marine Station (UPMC, CNRS), France. Mussels were suspected to be ready to spawn, since live dissections performed upon Idas sp. specimens collected the previous year (LD3-LD4, oakwood substrata) had resulted in the spontaneous release of gametes. The palm-tree individuals were obtained while sorting through subsamples, carefully torn from the trunk after transfer to the onshore laboratory 6-9 hours after sample recovery, while CHEMECOLIs were placed in the flowthrough container before sorting. Initially, small samples of substrate (one type per tank, split across two mesh baskets, Figure 3.1) were relocated to the experimental aquaria for acclimation, while samples were sorted. A subsample of Idas-like mussels from both substrates were then relocated to experimental aquaria for acclimation, gathered upon pieces of their corresponding recovered wood substrate to provide chemically reduced conditions. Aquaria were kept in the dark (mussels had clearly identifiable pigment spots, suggesting a level of photo-sensitivity), and examined twice daily for maintenance. Only mussels considered to be healthy were retained (i.e. normal resting behaviour: either shells were shut or marginally open with the mantle curled posteriorly forming two pseudo-siphons).

3-3.2. Induction of spawning and fertilisation

Before induction, aseptic seawater (ASW) was prepared by filtration (0.45 µm) and sterilisation (autoclave, 15 min at 121 °C) and then re-oxygenated (steam-sterilised air-sto e,① ≈ ① L① ai ① L and fixed for further analyses when fertilisation was first identified (presence of a thin fertilisation membrane), and when changes were apparent in fertilised oocytes (Table 3.1)

3-3.3. Anatomical dissections and fixation regime

Following the collection of gametes, mussels were returned to their substrate and relocated to the experimental aquaria to recover (1 wk). During the recovery period, one mussel (IF1) displayed atypical behaviour indicative of being moribund, and was removed and fixed immediately (Table 3.1). Mussels IF2 -IF14 were dissected microscopically to examine anatomy under a dissection microscope and fixed according to intended analyses (Table 3.1). Seeking to identify whether Idas spp. truly engage in filter-feeding and thus mixotrophy, detailed observations were made during anatomical examinations and live dissections.

Mussel IB1, not induced to spawn due to its impractical size, was also photographed under highmagnification using a digital SLR-mounted compound microscope (Canon EOS 650D, on a Leica Diaplan). Gill filtration activity in this mussel was then video-recorded at high magnification. This mussel and a second individual (species not determined) were fixed whole (Table 3.1). to identify whether differences in mean shell lengths were significant between the studied species. All analyses were performed in SPSS (17.0).

3-3.4.2 Molecular identification

Host DNA was extracted from frozen gill tissue using the QIAamp® DNeasy blood and tissue kit (QIAGEN, centrifuge protocol). A fragment of the mitochondrial Cytochrome Oxidase I-encoding gene (COI) was PCRamplified using primers LCO1490 (Folmer et al. 1994) and H691 (Duperron et al. 2008a). The program started at 94°C for 4 min, followed by 35 cycles of 94°C for 40 s, 50°C for 50 s, and 72°C for 1 min, with a final elongation step at 72°C for 10 min. PCR products were sequenced in both directions (GATC Biotech).

Sequences assembled and checked using BioEdit v. 7.2.5 [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. Only regions for which all residues had been identified were used (460-534 bp, depending on individual). Sequences were queried against the GenBank nucleotide sequence database using BLAST [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF]).

3-3.4.3 Stable Isotopes analysis

Foot tissue from mussels collected for this study (n = 14, after pooling individuals where necessary) and a subsample of Idas modiolaeformis collected from the canyon in 2012 (LD3-LD4, oakwood substrata) were prepared and analysed according to Gaudron et al. (2012b). C:N atomic ratios were calculated from the percentages of organic carbon and nitrogen.

3-3.4.4 Histological analysis

Viscera (comprising one half of the gut, gonad and mantle tissue, split medially) and gill tissue samples from each specimen were dehydrated in 100% alcohol, transferred incrementally to Histoclear, and embedded in paraffin wax. Slide-mounted (Superfrost plus), 7-µm thick sections cut using a microtome (Jung, Heidelberg)

e e① stai ed① ith① a idified① Ha is ① hae ato li ① L① gla ial① a eti ① a id① L -1 solution, 10 s), differentiated ith① . %① a eti ① a id① a . ,① lued① i ① " ott s① tap-water substitute (pH >8) and then counterstained with alcoholic Eosin-Y (pH 5, 3 s). Sections were dehydrated (increasing %-ethanol (aq.) series), cleared (Histoclear), and mounted immediately (Eukitt). ) to first identify whether unfertilised oocytes were in Prophase I or Metaphase I of meiosis, and second the stages of cell division following fertilisation, respectively. To establish whether previously identified bacterial phylotypes occur as symbionts in these mussels, fluorescence in situ hybridisations (FISH) were performed on a subsample of unstained 7-µm paraffin-sections of gill tissue, employing all dual-hybridisation combinations (Cy3 and Cy5labelled probes) of the general eubacterial probe EUB338 (Amann et al. 1990), the non-sense probe NON338 (Wallner et al. 1993), and specific oligonucleotide probes for type-M1 methanotrophs and type-T1

and -T2 thiotrophs (details in Duperron et al. 2008a), following the post-permeabilisation steps of the FISH protocol in Duperron et al. (2005).

3-4 Results and discussion

3-4.1. Species appearance

The genus Idas (s.l.) remains poorly resolved. However, recent multi-gene molecular analysis of type specimens for I. (s.l.) simpsoni have revealed that it actually belongs within a deep-branching clade of highly-related species in the Bathymodiolinae that predates the later divergence of Idas (s.s.) and Bathymodiolus (s.s.) spp. (Lorion et al. 2013;Thubaut et al. 2013b). This clade has been ascribed to a new but still not formally described genus provisionally named Nypamodiolus (Thubaut et al. 2013b); quotation marks are thus employed to emphasise the improper assignment of the Idas genus to this species.

Three species of mussel were identified in this study (Supplementary table 3 ).

Moderate differences existed between shell characteristics in the dominant species ( even in the largest specimen (IF1, Figure 3.3). However, some individuals deviated from these general trends (IF4, IF15, IF16), reaffirming the need for molecular validation when discriminating small-sized bathymodiolin species (Lorion et al. 2010).

3-4.2. Distribution and habitat use

Idas (s.s.) modiolaeformis and I. (s.l.) simpsoni represent the two of most widespread chemosymbiotic mussels documented in reducing environments within the Mediterranean Sea and eastern Atlantic (Thubaut 2012). Including data from the current study, I. modiolaeformis is now known to occur in the eastern Mediterranean (Nile Deep-sea Fan, NDSF, on authigenic carbonate crust and siboglinid tubes at methane-seeps, at several mud volcanoes and on sunken wood, Olu-Le Roy et al. 2004;Duperron et al. 2008a;Gaudron et al. 2012a;Lorion et al. 2012) comprises the north-eastern Atlantic (several independent cetacean bones at Rockall, Grand Fisher Bank, and off Faroe, Shetland and Orkney Islands, Marshall 1900;1901;Thubaut et al. 2013b) Nothi g① is① k o ① of① eithe ① spe ies ① eha iou ① i ① the① o te t① of① thei ① ha itats.① The① elati e① similarities (morphologically) and differences (phylogenetic divergence) between these two taxa are particularly intriguing, as their distribution is now reported to overlap marginally: sunken wood in the Gulf of Lion so far represents the only habitat and region in which both species cohabit. In both species, mussels were located beneath the utmost exterior of deployed substrate. In the palmwood experiment, they were e t e hed①alo g①the① i u fe e e①of①the①t u k s①sa ① oss-section between the cortex and the evidently sulphidic central cylinder (Supplementary figure 3.4). The I. modiolaeformis found in the CHEMECOLI

"upple e ta ①ta le① . ,① Pi e ① e e①su -peripheral, beneath the outermost structurally-degraded cubes (riddled with empty burrows of Xylophaga spp. ① ut① ot①i ①di e t① o ta t① ith①the①de i e s①sulphidi ① o e.①

Thus both micro-environments were at apparent REDOX transitions (organoleptic observations).

Both mussel species also displayed levels of mobility characteristic of mytilids [START_REF] Paine | Intertidal landscapes: disturbance and the dynamics of pattern[END_REF], where in shallow-water coastal species with deterred mobility, such movements reduce intraspecific crowding, mitigate the effects of wave action and can improve filter-feeding efficiency. They often adjusted their positions on aquarium substrata between maintenance checks (<8 hours). Pre-dissection observations revealed this process in more①detail.①The①foot①e te ded①a d① ea ed① a k①a d①fo th①i ①a① sea hi g ① otio .①

Contact with an attachment surface was identified with the tip but temporary attachment with the heal of the, while the tip of the foot continued searching. The tip contained a pit at the distal terminus of the pedal groove which is thought to have a sensory function. The tip itself subsequently attached to a proximal point on the substrate, allowing the heel to detach and the foot to contract, dragging the body. This was repeated process permitted the mussels to crawl using their foot.

3-4.3. Reproduction

The available data for Idas spp. generally, indicates probable planktotrophic larval development inferred from larval shell and gamete characteristics (Dean 1993;Ockelmann and Dinesen 2011;Gaudron et al. 2012a;Lorion et al. 2012;Ritt et al. 2012;Laming et al. 2014), a tendency towards protandry based on sizedependent patterns in sex ratios, (Tyler et al. 2009;Gaudron et al. 2012a;Laming et al. 2014) The smallest specimen (SL 5.09 mm) of I. (s.s.) modiolaeformis was male, the remainder (n = 5) being female. Idas (s.l.) simpsoni sex ratios approximated 50:50, the two smallest specimens being hermaphroditic, but functionally male (Supplementary table 3.1 Repeated induction procedures and (presumably) size-constrained fecundity (e.g. 3 rd induction: ≈ -400 oocytes ind.

-1

) may have limited the number of viable oocytes. Difficulties in discriminating between the co-occurring genera without real-time molecular confirmation (and fecundities too low for separate fertilisation treatments) probably led to numerous non-viable cross-fertilisations. Consequently, fertilisation rate was low despite a 1000:1 sperm-to-oocyte ratio. Communally spawned oocytes were also used in the fertilisation experiment, as no premature fertilisation was identified during counts made prior to mixing gametes. Oocytes were white and optically opaque (Figure 3.4A) and very slightly negatively buoyant at ambient pressure and 14 ±1°C. Hoechst staining revealed a subsample of the oocytes to be in also be responsible for the high phylogenetic similarity between I. modiolaeformis and its sister-species I.

macdonaldi (Lorion et al. 2012) found in the Gulf of Mexico, which also harbours chemoautotrophic symbionts (Won et al. 2008). However, the dispersal capacity of Idas spp. larvae will only be realised through improvements in the protocol for rearing larvae of this taxa. complete digestive anatomy with discernible contents in histological sections (Thubaut et al. 2013a).

Anatomical features observed in I. (s.s.) modiolaeformis in the current study, mirrored those previously identified in specimens from the NDSF (Laming et al. 2014). A fully-formed digestive system was apparent in both species (e.g.

3-4.4.2 Particle filtration and ingestion

The morphoanatomy of the gill filaments differed between the two species, where lateral regions of I. (s.l.) simpsoni gill filaments were not hypertrophied, as in I. modiolaeformis, instead being delicate and thin.

During live examinations under a dissection microscope, the convection of debris in the vicinity of the gills was unidirectional in I. modiolaeformis and in most I. (s.l.) simpsoni (not evident in the three largest specimens, IF12, IF7 and IF3). High-magnification observations of this process (Supplementary video 3.1), taken in the mid-to-anterior region of the ascending lamella for the smallest I. modiolaeformis mussel (IB1), confirmed that particle transport was being coordinated by ciliary action, where lateral cilia, latero-frontal cirri, and frontal cilia in the gill filaments could be identified (Figure 3.6D, labels 1, 2 and 3 respectively; Supplementary video 3.1, right inset). Initially, the labial palps -located anteriorly, inflexed posteriorlyappeared to be coupled with the most anterior gill filaments in all specimens (e.g. Figure 3.6A). This coupling disengaged somewhat during dissection, likely as a consequence of stress (Jørgensen 1990).

Initially, particles were captured and transported ventrally between interfilamental lateral cilia, and then anteriorly along the ventral particle groove towards the labial palps and mouth where particles mucous produced may have been abnormally abundant due to physiological stress incurred when opening the mussels (Jørgensen 1990). The ultimate fate of un-ingested mucous was unclear, but it was probably ejected as pseudofaeces and transported away from the mouth, as in other bivalves (Jørgensen 1990). To the① autho s① k o ledge,① these① ep ese t① the① fi st① di e t① i os opi ① o se atio s① of① particle ingestion in live bathymodiolin mussels providing evidence of filter-feeding behaviour in both species, demonstrated indirectly in some larger-sized members of this sub-family (e.g. tracking assimilation of radio-labelled waterborne bacteria Page et al. 1990;Page et al. 1991; naturally-occurring plankton removal rates, [START_REF] Cowie | Plankton availability and retention efficiencies of cold-seep symbiotic mussels[END_REF]; or based on a C-flux model, [START_REF] Martins | Size-dependent variations on the nutritional pathway of Bathymodiolus azoricus demonstrated by a C-flux model[END_REF]).

3-4.4.3 Evidence of symbiosis

All-but-one of the species of Idas previously examined for symbionts, have been shown to be chemosymbiotic (e.g. Deming et al. 1997;Duperron et al. 2008a;Southward 2008;Ritt et al. 2012;Rodrigues et al. 2013;Thubaut et al. 2013a), the exception being the type species I. argenteus, a larviphage (Ockelmann and Dinesen 2011). Remarkable symbiont diversity has been demonstrated in I.

modiolaeformis when recovered from hydrocarbon seeps (Duperron et al. 2008a), in stark contrast to young specimens of the same species in the same region living on wood (Laming et al. 2014). In I. simpsoni, a highly dominant thiotrophic bacterium was observed in specimens from hydrocarbon seeps in the Marmara sea (Ritt et al. 2012). FISH performed herein revealed markedly different symbiont patterns depending not only on species, but also on the year of collection. Although Idas modiolaeformis collected from oakwood deployments (2012) did not display the level of diversity seen in Duperron et al. (2008a), dense populations of bacteria were identified in the gills, mainly composing of highly dominant type-T1 thiotrophs and a much-reduced presence of type-M1 methanotrophs, established towards the interior regions of the gill (Figure 3.7). This sort of dual symbiosis has been seen before in this species, when settled upon wood (Lorion et al. 2012). General Eubacterial signals mirrored those of T1-thiotrophs, suggesting the thiotrophs accounted for almost all the Eubacteria present. In specimens of I. simpsoni from the same substrata and cruise (LD3-LD4, oakwood, recovered 2012), densities were lower (Figure 3.7). However, I.

simpsoni specimens were less than half the size of the I. modiolaeformis examined (SL 3.7 mm versus 9.0-9.2 mm). Attempts to elucidate phylotypes using specific oligonucleotide FISH probes were ultimately unsuccessful in I. simpsoni, though successful hybridisation with the EUB338 probe confirmed the signal to be eubacterial (Figure 3.7). Note that these mussels were not involved in live fertilisation experiments.

Symbionts could not be identified in mussels from the live observational experiments during postexperimental FISH. However, the FISH analyses presented above (specimens from previous year) and the stable isotopes data discussed below for each species (Figure 3.8; Supplementary table 3.2, both years), suggest that this absence reflects a short-term symbiont loss due to the conditions to which these specimens were subjected in the lab. Symbiont loss is a well-known phenomenon in the Bathymodiolinae (e.g. Kádár et al. 2006), and can be rapidly induced using experimental conditions (Duperron, unpublished data). In I. modiolaeformis, these comprised of thiotrophs (T1) and methanotrophs (M1). Eu-T1 overlaid image identified thiotrophs as highly dominant (Eu-M1 overlaid image has an abundance of eubacteria that were not methanotrophic). In I. simpsoni, eubacteria type could not be confirmed with the probes employed. ).

This was carried out prior to formal COI-confirmation of species, leading to a single instance where a pooled sample resulted in a mixed-species isotopic signature (IF4 and IF10 from palmwood, 0.3 and 0.4 mg contributions respectively). C/N atomic ratio values of foot tissues were similar in all groupings (3.3 -3.6).

Values① fo ① δ 13 C① a d① δ The scatter-plot① depi ts① the① δ 13 C① a d① δ 15 N values for each of the specimens analysed for stable isotopes ratios. All data are from ussels① used① i ① li e① o se atio s,① e ept① poi ts① la elled① .① The① half① i le/half① t ia gle① poi t① as① a① pooled① sa ple① of① the two species (due to initial misidentification of IF4 as I. modiolaeformis). Due to very low % nitrogen (0.18 -0.21 %) i ①Pi e ood,①o l ①δ 13 C value exists for the Pinewood (left-hand vertical line). No data are available for the oakwood substrate. (s.s.) modiolaeformis and I. (s.l.) simpsoni and those for organisms whose diets are linked to wood directly (e.g. wood boring bivalve species, wood-associated limpets and chitons, [START_REF] Zbinden | Bacterial o u ities① asso iated① ith① the① ood-feedi g① gast opod① Pe ti odo ta① sp. Patellogast opoda,① Mollusca)[END_REF]Duperron et al. 2013b). However these distinctions are not such that direct feeding on wood-derived nutrition can be ruled out (though not precedence for this exists in mussels). et al. 2004;[START_REF] Carlier | Heterogeneous energetic pathways and carbon sources on deep eastern Mediterranean cold seep communities[END_REF]. Since the incorporation of a diverse array of nutritional resources could also explain such signatures, the case for a mixotrophy diet cannot be discounted. Accordingly, the notablyde ia t① alues①fo ①δ 13 C①a d①δ 15 N for the 2013-pooled I. modiolaeformis found in the 10-month CHEMECOLI deployment (pinewood, Figure 3.8; Supplementary table 3.2), may reflect a diet dominated by photosynthetically-derived material, as might be expected based on the smaller sizes and presumably, younger ages [START_REF] Cowie | Plankton availability and retention efficiencies of cold-seep symbiotic mussels[END_REF][START_REF] Martins | Size-dependent variations on the nutritional pathway of Bathymodiolus azoricus demonstrated by a C-flux model[END_REF]Laming et al. 2014).

3-4.5. Species comparison and further considerations 3-4.5.1 Reproduction Spawning appears to be semi-continuous in both I. modiolaeformis and I. (s.l.) simpsoni. Oocyte diameters were typical of planktotrophic larval development (Arellano and Young 2009), and display slightly negative buoyancy only. This may affect ascension through the water column to shallower eutrophic waters, where the hatched, planktotrophic larvaeshown to be aposymbiotic (Laming et al. 2014)are thought to feed and develop, as in B. [s.l.] childressi (Arellano and Young 2011;Arellano et al. 2014). Oocyte properties for both species were so similar as not to be distinguishable, indicating that modes of dispersal in both I. (s.s.) modiolaeformis and I. (s.l.) simpsoni are likely identical, and may reflect a more general trend in Bathymodiolinae (see Introduction). Historically, I. modiolaeformis has displayed sex-ratios indicative of protandry (Gaudron et al. 2012;Laming et al. 2014), further corroborated by the size-specific prevalence for females in the current study at (smallest individual was male only, SL 5.09 mm).Idas (s.l.) simpsoni sex-ratios approximated 50:50 wherein the two smallest individuals at SLs < 6mm were hermaphroditic, but functionally male. While this hints at a similar pattern of protandry, the otherwise equal size-dependent sex ratios show that at these larger SLs in this habitat, both sexes co-occur. The biology behind these patterns remains difficult to decipher as a consequence.

3-4.5.2 Nutritional flexibility

The findings of this study, though preliminary, provide new insights into the nutritional and reproductive biology of two bathymodiolin species. Laming et al. (2014) collated evidence based on the developmental biology of Idas (s.s.) modiolaeformis which strongly suggested not only the capacity for mixotrophy (functional digestive system) but also a probable obligation to filter-feed, as symbiont densities were considered too low to solely support energetic requirements. In the current study, we provide further evidence that this capacity for mixotrophy is retained in adults of I. (s.s.) modiolaeformis, and also exists in I.

(s.l.) simpsoni, from the Gulf of Lion, on wood. This is despite the documented prevelance for dense populations of bacterial symbionts in the gills of these species, based on the current FISH analysis but also previous work (Duperron et al. 2008a;Duperron et al. 2008b;Lorion et al. 2009;Lorion et al. 2012;Ritt et al. 2012). Despite belonging to highly-divergent clades, which would ultimately warrant classification within distinct genera, commonality exists in the biology of I. (s.s.) modiolaeformis and I. (s.l.) simpsoni. This reflects either conservative or convergent features of their evolution, where such features appear to be widespread within the smaller-sized bathymodiolins.

Idas (s.s.) modiolaeformis and I. (s.l.) simpsoni evidently retain the capacity for filter-feeding and assimilating particulate organic matter (POM), based on live observations of active particle processing, on the presence of considerable volumes of digestive diverticula, and the preservation of excretory function with externally-visible gut contents. However, both species were also documented to engage in symbioses in the current study harbouring dense populations of bacteria in their gills, confirmed to be predominantly sulphur-oxidising in Idas (s.s.) modiolaeformis. Yet to be determined however are how relative contributions of heterotrophy and symbiont-based nutrition vary both ontogenetically, and whether habitat location and type influences nutritional dynamics, as seems likely. This is especially poignant given the welldocumented variability in symbiont composition and abundances in I. (s.s.) modiolaeformis at various localities and shell sizes (Duperron et al. 2008a;Lorion et al. 2012;Rodrigues et al. 2013). Disentangling these nutritional components remains a challenge in the absence of targeted nutritional-sources data for this dynamic canyon environment, as evidenced by the complex stable isotopes picture identified. A significant fraction of plume POM is terrestrial in origin within the Gulf of Lion [START_REF] Darnaude | Trophodynamic linkage between river runoff and coastal fishery yield elucidated by stable isotope data in the Gulf of Lions (NW Mediterranean)[END_REF]) due to unusually high terrestrial POM loading, originating from the Rhône delta (mean annual flow of 1,700 m N signatures (e.g. [START_REF] Mintenbeck | Depth-depe de e①i ①sta le①isotope① atio①δ 15 N of benthic POM consumers: The role of particle dynamics and organism trophic guild[END_REF]). However, the canyon ecosystem itself is subject to complex hydrodynamic and ecological features which prevent a simple assessment of POM origins. These factors, and the additional variables specific to the current study including deployment periods, seasonality, variable substrata type, and unresolved effects of symbiont metabolism (Robinson and Cavanaugh 1995), have rendered further interpretation impossible. The comparison of electron donor availability in different reducing habitats is difficult and is out of the scope of this study. However, as supply of sulphides (and by inference, biogenic methane) at the surface of sunken-wood is subject to considerable temporal variability [START_REF] Laurent | Sunken wood habitat for thiotrophic symbiosis in mangrove swamps[END_REF]Laurent et al. 2013;Yücel et al. 2013), sustenance derived solely from engaging in chemosymbioses may not meet the energetic demands of the organism and a degree of nutritional flexibility is advantageous. A capacity for facultative mixotrophy may also explain the multi-habitat distribution of both these species, by taking advantage of varying contributions from heterotrophy and chemosymbiosis.

This study is believed to be the first to report live observations concerning the nutrition and spawning behaviour of Idas-like mussels. Be it in situ (ideally) or ex situ as in the current study, the benefit of carrying out live-organism studies examining deep-sea organismal behaviour, and in particular larval development, has been continually reaffirmed within the deep-sea biology community, emphasising the need for long-term multidisciplinary experimental programmes which permit such work to be undertaken.

Aspects of the methodology presented here could benefit from further refinement. In particular, sulphidic conditions were not confirmed electrochemically, with possible consequences for bacterial symbioses in living organisms. Future attempts to keep organisms associated with sulphidic organic-falls, would benefit from a means of monitoring sulphide production (as in Yücel et al. 2013) and a deliberately reduced oxygen suppl ,① as① this① a ① ha e① i ad e te tl ① o ge ated① the① supplied① sulphidi ① su st ata① i ① the① u e t① stud ① (and thus induced symbiont loss). However, the current study has demonstrated that maintaining aquaria for smaller wood-based (and potentially bone-based) chemosymbiotic species need not be costly in terms of space or finances, following the collection of samples, which does remain logistically and financially challenging. It also aims to reopen discussion on larval development in deep-sea organisms, which remains poorly understood (one of the goals of INDEEP, The International Network for Scientific Investigations of Deep-Sea Ecosystems). If specimens are collected from the upper limits of their depth range in the deepsea, it is possible to keep them alive at ambient pressures [START_REF] Dixon | The effects of hydrostatic pressure change on DNA integrity in the hydrothermal-vent mussel Bathymodiolus azoricus: implications for future deep-sea mutagenicity studies[END_REF]Arellano and Young 2009).

Seasonal time-series sampling would be of great benefit in empirically demonstrating semi-continuous readiness to spawn, which remains a challenge for deeper habitats, while it is hoped that information presented herein on the nutritional requirements of these species, will aid future attempts to maintain adults, and ultimately grow larvae for further research.

IF07

4-2 Introduction

Repeated discoveries and descriptions of aphotic communities thriving upon hydrothermal vents (Lonsdale 1977), hydrocarbon seeps (Paull et al. 1984) and whale carcasses (Smith et al. 1989) initiated a new era in deep-sea biology, and altered conventional understanding of deep-sea ecosystems. It was realised that bacteria could metabolise reduced compounds and support whole communities either directly or indirectly through chemosymbioses, courtesy of simultaneous access to electron donors and electron acceptors at anaerobic-aerobic interfaces [START_REF] Stewart | Chemosynthetic endosymbioses: adaptations to oxic-anoxic interfaces[END_REF]Cavanaugh et al. 2006). Since then, other organic falls (e.g. other marine mammals, terrestrial vegetative debris) have been postulated as reducing habitats, being colonised by opportunistic chemosymbiotic organisms and sulphur-reducing bacteria (e.g. Bienhold et al. 2013), though anecdotal records of wood-fall communities (Challenger expedition), pre-date records of all known reducing habitats (Murray et al. 1891).

Chemosymbiotic bivalves represent a pervasive and diverse macrofaunal group in these environments. Fifty-three Atlantic and Mediterranean deep-sea species are already confirmed to engage in symbioses (Duperron et al. 2013), with hundreds more worldwide in diverse reducing environments, when all depth ranges are considered (Taylor and Glover 2010). Recent, intensified, ecological study of aphotic habitats driven by chemosynthetic primary production has yet to resolve gaps in our knowledge of basic developmental traits for even the dominant species (Young 2003). The life-histories of deep-sea chemosymbiotic bivalves thus remain speculative (Tyler et al. 2009). Studies have focused on the sex ratios, size and ultrastructure of gametes, and gametogenesis (reviewed by Le Pennec and Beninger 2000; more recently, Le [START_REF] Pennec | Spermatogenesis in the archaic hydrothermal vent bivalve, Bathypecten vulcani, and comparison of spermatozoon ultrastructure with littoral pectinids[END_REF]Colaço et al. 2006;Dixon et al. 2006;Kádár et al. 2006;Tyler et al. 2007a;Tyler et al. 2009;Gaudron et al. 2012), with one investigation based upon time-series sampling (Tyler et al. 2007a). Larval developmental studies in deep-sea chemosymbiotic bivalves are much rarer [START_REF] Gustafson | Development of the pericalymma larva of Solemya reidi (Bivalvia: Cryptodonta: Solemyidae) as revealed by light and electron microscopy[END_REF]Arellano and Young 2009), supplemented by inferred larval modes from shell morphometry, where the small prodissoconch I (indicative of hatching size) is compared with the larger prodissoconch II (indicative of settlement size, e.g. Lutz et al. 1980;Gaudron et al. 2012;Ritt et al. 2012). Observations on post-larval and juvenile development in chemosymbiotic deep-sea bivalves are conspicuously absent, other than incidental observations while examining immature symbiont associations (Streams et al. 1997;Salerno et al. 2005;Wentrup et al. 2013). This could be because size-related difficulties encountered during postlarval examination are many (microscopic sample-sorting, identification, dissecting, orienting tissue during embedding, and interpreting juvenile anatomy), while sample availability is limited (larval settlement might at times be supply-limited and sampling often favours larger, generally adult macrofauna). Prior to the sub-family has demonstrated however, that evolving the capacity to flourish in these habitats facilitated astounding levels of adaptive radiation (Lorion et al. 2013). This occurred over ca. 85 million years (Ma) with peak speciation rates at an estimated 41-32 Ma (0.17 speciation events Ma -1 ). Extant Bathymodiolinae currently display wide variability in habitat use, shell morphology, adaptive anatomy, developmental and reproductive mode and dependency upon their symbionts.

The bathymodiolin genus Idas, (polyphyletic at present, Lorion et al. 2010Lorion et al. , 2013;;Duperron et al. 2013;Thubaut et al. 2013b), colonises organic falls (Smith et al. 1989;Kiel and Goedert 2006;Samadi et al. 2007;Duperron et al. 2008a;Lorion et al. 2009Lorion et al. , 2012;;Tyler et al. 2009;Ockelmann and Dinesen 2011;[START_REF] Guisti | Il genere Idas (Mytilidae, Bathymodiolinae) in Mediterraneo con la descrizione di quattro nuove specie[END_REF]Bienhold et al. 2013;Cunha et al. 2013;Thubaut et al. 2013a), authigenic carbonate crusts at methane cold-seeps (Olu-Le Roy et al. 2004;Ritt et al. 2012) and sulphide crusts, bathed in diffuse hydrothermal vent fluids (Juniper et al. 1992;Southward 2008). A then undescribed species, Idas sp. Med, was first documented in the Eastern Mediterranean at various hydrocarbon seeps and mud volcano sites Once on board, random subsamples were fixed and preserved en masse using protocols specified in Gaudron et al. (2010). In the laboratory, the preserved substrata were sorted microscopically. Post-larval I. (s.s.) modiolaeformis from the two-week wood deployment were not used in this study. Mussels found in the 51-week wood and alfalfa CHEMECOLI deployments (no mussels were identified on carbonate) were fixed in either 4% buffered formaldehyde in twice-filtered seawater (TFSW) for 2-4 h at 4°C with a gradient transfer to 95% ethanol, or 95% ethanol directly (all stored at <5°C). The relative proportions of individuals fixed with each method and resulting analyses is summarised in a supplementary table (Supplementary table 4.1).

Individuals of I. (s.s.) modiolaeformis were identified based on morphoanatomy. Prior molecular studies had identified that all other mussels from these same deployments were of this species (Gaudron et al. 2010;Gaudron et al. 2012). The larval prodissoconch I and II were opaque pearly white and transparent red/orange respectively and, where present, the dissoconch was modioliform, colourless, and almost transparent in all juveniles or more opaque and off-white in individuals >4 mm. Periostracal byssus hairs were already numerous larger juveniles (> 1 mm), most concentrated on the postero-ventral margin flank.

Wood-and alfalfa-located mussels were pooled, providing a size-spectrum from post-settlement to putative adults. Thus, it was possible to document key developmental patterns in I. (s.s.) modiolaeformis from the Nile Deep-sea Fan, for the size-range examined.

4-3.2. Preliminary shell and tissue analysis

Individuals were measured where feasible (dissoconch: n = 41, prodissoconch II: n = 35) and photographed (Figure 4.1a-e) using a camera-mounted dissection microscope (Evolution VF camera, Media cybernetics;

Olympus S2X12 microscope, Japan; Image-pro v.5.1). Shell height (SH) was the greatest linear distance dorso-ventrally measured from the umbo, perpendicular to the hinge-line, while SL was the greatest linear distance antero-posteriorly measured parallel to the hinge line . Prodissoconch II dimensions e e① easu ed① usi g① the① sa e① app oa h,① ut① ith① sta da dised① la al ① hi ge-lines, to permit the aggregation of prodissoconch II data across all shell sizes (Figures 4.1f-g). Dissections were performed microscopically using custom-made tools (Supplementary figure 4.1). General anatomy was recorded and tissue was extracted, with minimal disruption. Where gills had remained intact (n = 35), the number of filaments comprising the descending lamella in either inner demibranch -the first to develop -was recorded, as a proxy for gill development. Microscopy Sciences, UK) filled with LR White resin (London Resin Company, UK), transferred to a fresh resin-filled gelatine capsule, orientated appropriately, capped, and polymerised at 55°C (20 hours minimum). Gelatine was then removed with hand-hot water.

Semi-thin sectioning maximised structural detail but retained staining contrast and optimised limited tissue availability. Resin blocks were wet-sectioned (glass knife) on a Leica EM Ultra Cut R Ultramicrotome (Germany). Supplementary figure 4.2 depicts the typical orientations. Where possible, 1 µm-thick sections on Superfrost plus slides were used for haematoxylin and eosin-Y staining (H&E), up to 1 µm-thick sections on Superfrost plus slides for fluorescence microscopy, and 350 nm-thick sections on carbon-film, 200-mesh, nickel grids for TEM. Periodic toluidine staining was performed to identify the cutting-a is①a d①lo atio ① ≈ -µm intervals).

Standard H & E staining procedures for thick sections were modified by extending the staining times and excluding ethanol, which compromises LR white resin. Semi-thin sections of select individuals Ta le①" ① e e① eh d ated① distilled① ate ,① ① i ,① lotted①d ①a d①stai ed① ①h ①i ①filte ed,①a idified①Ha is ① haematoxylin solution (5mL glacial acetic acid L -1 solutio .① "lides① e e① lued ① fo ① ① i ① u ning-water bath, pH >8). Differentiation was performed with 0.5% acetic acid (aqueous). Slides were rinsed (distilled water) to remove unknown-pH residues, air-dried, stained with 1% aqueous Eosin-Y (pH 5.0, 25 min), rinsed again (3 x separate 1% acetic acid baths, 1 x distilled water), and air-dried. Sections were cleared (Histoclear), and mounted immediately (Eukitt).

Slides were viewed under a camera-mounted compound microscope (Evolution VF camera, Media Cybernetics, USA; Olympus BX61, Japan) and micrographs were processed and measured, where necessary, using Image-pro plus (v.5.1).

4-3.4. Examination of symbiont patterns 4-3.4.1 Fluorescence

Select slide-mounted sections targeting the entire size-spectrum were equilibrated in phosphate buffered saline (PBS 1x), stained with the nucleic acid-specific stain 4',6-diamidino-2-phenylindole (DAPI, 300 nM in PBS for 5 min) and mounted in Slow-Fade Gold (Invitrogen). All body regions were examined. Gill tissue from Lorion et al. (2012) provided the positive control. Negative controls excluded DAPI altogether, to test for auto-fluorescence. Slides were photographed as in the H & E analysis, but with a monochrome filter.

4-3.4.2 Transmission Electron Microscopy (TEM)

A subsample of individuals (Supplementary table 4.1) which harboured putative bacteria, were examined using TEM (JEOL 2100 HC; GIF Tridiem Gatan Imaging Filter). Sections were contrasted with uranyl acetate (0.05 g mL -1 Milli-Q, 7 min in darkness) and lead (II) citrate alkaline solution (2 µg, and 0.01 mL NaOH [10 M] mL -1 Milli-Q, 7 min, CO 2 -free environment).

4-3.5. Statistical analyses

All graphs were created in Sigmaplot (v.11). Size-class frequency analyses were performed, employing 0.1mm intervals in shell length (informed by shell dimensions analysis). Differences in both whole-SL and la al,① p odisso o h① II① "L① e e① assessed① a oss① p esu pti e① oho ts ① usi g① ge e al① li ea ① odels① GLM,① unbalanced data: thus type III SS ① i ① "P""① . . ,① ha i g① o fi ed① the① dist i utio s① ho os edasti it ① Le e e s ① a d① o alit ① Kol ogo ov-Smirnov). Gill-filament counts (descending inner-demibranch lamellae only), were plotted against shell size; best-fit analysis was performed in Sigmaplot (v. 11) to assess gill①p olife atio ①as①a①fu tio ①of①"L.①②①sig ifi a e①le el① α ①of① . ① as①used.

4-4 Results

4-4.1. General specimen condition and classification

Of the 34 immature mussels found, five were plantigrades (i.e. post-larval mussels in which neither dissoconch deposition, nor gametogenesis was evident), all with shells intact (SL 0.38-0.42 mm). Tissue was damaged or already dehydrated in two cases. The remaining three were used for soft-tissue analysis regardless of fixation (Supplementary table 4.1), to maximise tissue availability. Despite their small size, much of the anatomy pertaining to later development was already evident. Small (<1 mm) and larger (>1 mm) juveniles, in which dissoconch growth was evident but gametogenesis was not, were most numerous (n = 29, SL 0.44-1.92 mm). The seven largest individuals (SL 2.35-6.54 mm) were adults with direct evidence of gametogenesis, discussed later. Formalin-fixed individuals were preferentially (but not exclusively) processed for tissue analyses (Supplementary table 4.1).

4-4.2. Shell-size patterns

SH ranged from 0.30-2.93 mm and SL from 0.38-6.54 mm in I. (s.s.) modiolaeformis, when they could be measured (n = 41, including plantigrades). Mean prodissoconch II heights ( ) and lengths ( ① ±σ① were 0.33 ± 0.017 mm and 0.40 ± 0.018 mm respectively (PdH: 0.30-0.36 mm, PdL: 0.38-0.42 mm, n = 35).

4-4.4. Reproductive development

At the post-settlement stage in development, gonad tissue was as yet unformed. In addition, at this size it proved impossible to identify primordial germ cells (PGCs), which migrate and differentiate to form germ cells (GCs) and then gametes, later in development. However, putative PGCs were observed in several juveniles and small adults, located between the posterior periphery of the digestive system, the ventral periphery of the pericardium and the dorsal periphery of the organ of Bojanus, the bivalve metanephridium (e.g. Figure 4.3e, encircled). In juveniles of I. (s.s.) modiolaeformis examined, this also placed the PGCs in proximity to the visceral ganglion and the posterior adductor muscle (e.g. Supplementary figure 4.9, upperright image). In juveniles < 1mm SL, these cells had already formed two small aggregatio s① ≈① ①µ ①lo g,① antero-posteriorly) located on either side of the mid-sagittal plane and embedded in vesicular connective tissue (smallest individual, Idas 10, SL = 0.850 mm, Supplementary figure 4.9). In larger specimens, cell proliferation appeared to facilitate PGCs branched migration, towards the utmost dorso-lateral regions of Only following migration to the predominantly posterior, dorso-lateral regions of the body, did PGCs differentiate into GCs and identifiable gametes. In terms of SL, this was first observed at 2.35 mm (Idas 13, male). Unambiguous spermatogenesis was observed in two dorso-ventrally flattened regions of acini (length postero-a te io l ①≈① ①µ ,①flush① ith①the① a tle①epitheliu ,①i ①do so-laterally symmetrical posterior positions ( The minimum size at which gonads develop is documented for the first time in I. (s.s.) modiolaeformis, from the migration of putative, medial, posterior germ-line cells in juveniles to the dorso-lateral regions of body.

In Gaudron et al. (2012), the smallest specimen examined at 3.6 mm (greatest postero-anterior length, regardless of hinge-line) had already reached sexual maturity. In the present study, two larger individuals and several smaller were sexually mature. The threshold of 2.35 mm identified here for size at first maturity is supported by the patterns of germ-line migration towards the dorso-lateral regions of smaller specimens, based upon classic histological descriptions [START_REF] Lubet | Recherches sur le cycle sexuel et remission des gametes chez les Mytilides et les Pectinides (Moll. Bivalves)[END_REF], which have since been corroborated by vasa-like gene-marker techniques where vasa-like gene expression is restricted to the germ cells and is undetectable in somatic tissues [START_REF] Fabioux | Oyster vasa-like gene as a marker of the germline cell development in Crassostrea gigas[END_REF]Obata et al. 2010).

Size at first maturity is not an ideal interspecies index for comparing developmental rates, on account of wildly different maximum sizes and growth rates. However, in deep-sea reducing environments su h① e a ples① i ① elatio ① to① a i u ① size① a e:① ≈ mm in Calyptogena magnifica (Berg 1985 The shallow-water genus Mytilus displa s① highl ① a ia le① sizes① at① fi st① atu it ① ≈ -7 mm SL), but less variable ages-at-first-maturity of 1-2 mo in M. trossolus, 4-8 mo in M. californianus a d①≈ -2 y in M. edulis, depending on habitat (Seed 1969;Suchanek 1981;Seed and Suchanek 1992). Additionally, B. thermophilus a ① atu e①at①≈ -13 mo, when documented size at first maturity (Berg 1985) and Von Bertalanffy growth analyses (Nedoncelle et al. 2013) Protandry is often argued as an adaptation to finite nutritional resources [START_REF] Ghiselin | The evolution of hermaphroditism among animals[END_REF], where the costs of reproductive activity as a female at a given size (or age) may be prohibitive in contrast to being male (or vice versa). In the case of protandry in I. (s.s.) modiolaeformis (Gaudron et al. 2012; this study) and I. washingtonius (Tyler et al. 2009), investment in oogenesis are evidently minimal below a threshold size or age (based on the absence of females < 7mm), where larger females would be more fecund. This would maximise somatic growth in smaller mature individuals, by partitioning a minimal amount of energy for spermatogenesis as males, and may also promote survival by reducing the period during which juveniles are most susceptible to predation. After a period of growth, and when nutritional resources allow, a switch in sex may then occur. With the staggering of sex-alternation, an array of small but numerous males, and larger fecund females would be reached. Tyler et al. (2009) The specificity and appearance of bacterial distributions (DAPI and TEM) on the non-ciliated lateral and abfrontal surfaces of gill-filament epithelia, were the same as in extracellular symbionts repeatedly identified in adult I. (s.s.) modiolaeformis (Duperron et al. 2008a, b;Rodrigues et al. 2013), its congeners (Gros et al. 2007;Southward 2008) and other bivalve species (e.g. Dufour 2005). In our TEM observations, their small size and the absence of concentrically-stacked intracellular membranes [START_REF] Cavanaugh | Symbiosis of methylotrophic bacteria and deep-sea mussels[END_REF] indicate that they were not methane oxidisers. Although preservation limitations do not permit the bacteria to be identified further, they are believed to be thiotrophic symbionts since I. (s.s.) modiolaeformis has previously been demonstrated to harbour multiple symbionts, where thiotrophs and methanotroph phylotypes dominate (Duperron et al. 2008a;Lorion et al. 2012;Rodrigues et al. 2013). Preliminary fluorescence in-situ hybridisation (Laming, unpubl. data), employed upon the same organisms using thiotroph-specific oligonucleotidic probes, appears to support this, along with the fact that sulphide emissions from these samples were detected using autonomous potentiometric sensors (Gaudron et al. 2010) and that free-living sulphur-reducing bacteria are now known to colonise CHEMECOLI substrata (Khelaifia et al. 2011). Duperron et al. (2008a), identified 6 bacterial phylotypes, where methanotroph-like and unspecified methylotroph-like bacteria collectively appeared to dominate other symbiont types. In the 2012 study, thiotroph-type bacteria were dominant, indicating a level of symbiont flexibility (Lorion et al. 2012). A fifth Gammaproteobacteria ph lot pe① s io t① G of unknown metabolism has been identified less predictably (Duperron et al. 2008a;Rodrigues et al. 2013). In the study by Gaudron et al. (2012), methanotroph-like bacteria were identified using FISH in the gill tissues of all 29 I. (s.s.) modiolaeformis collected. However tissue was not analysed for the remaining known symbionts. The absence of methanotroph-like bacteria in the TEMs of the mussels in this study was unexpected for the NDSF given that methane seepage was thought to occur in the area. Such exclusively-thiotrophic associations have previously been identified in I. (s.s.) modiolaeformis from the Gulf of Cadiz (Rodrigues et al. 2013).

Considering all the evidence, sulphide emissions due to anaerobic substrate degradation (e.g. (Wentrup et al. 2013). In that study the two symbiotic phylotypes infecting the tissues were found to be the only detectable bacteria present. However unlike the current study, size-at-first-acquisition could not be established, since post-larval specimens were not examined and aposymbiotic juveniles were never identified, which is not always the case (e.g. Streams et al. 1997). The increase in symbiont density with size probably reflects a developing association which has yet to attain stable densities, unlike the symbiont densities described for post-larvae onwards in B. heckerae and B. azoricus (Salerno et al. 2005), where symbiont morphotypes were observed in all specimens regardless of life-history stage and remained relatively unchanged in density regardless of size. Gaudron et al. (2012) proposed that symbionts in I. (s.s.) modiolaeformis are probably acquired horizontally (i.e. environmentally) rather than vertically (parentally), since bacterial symbionts were never detected in parental germ cells or unreleased oocytes. The current study provides additional evidence to further support this hypothesis, by isolating the probable period of acquisition to the post-settlement phase of development. The absence of bacteria in plantigrades, whose digestive system is well-developed, indicates that planktotrophic larvae and post-larval plantigrades are both exclusively heterotrophic.

4-5.4. Summary of findings

Although much of the anatomical developmental patterns in this species are parallel to those of shallow water mytilids, key developmental traits are viewed as adaptive. Idas (s.s.) modiolaeformis is capable of colonising and reaching maturity well within a year, at just 14% of its SL max . Whether this trait was selected for directly or is a by-product of selection for a reduced body size (e.g. though paedomorphism, Génio et al. 2012), the net effect is the same: it permits I. (s.s.) modiolaeformis to colonise ephemeral habitats and attai ① atu it ① ell① ithi ①the① o st ai ts①of①the①ha itat s①fi ite①e iste e① pa ti ula l ①fo ① ales .①E uall ,① the adaptive advantage of protandric, sequential hermaphroditism is in maximising early investments in growth (which are almost certainly underestimated in the current study), and ultimately attaining an optimised ratio of sexes across sizes [START_REF] Ghiselin | The evolution of hermaphroditism among animals[END_REF]. In larger wood falls, sulphide production is greater and the substratum persists①fo ①lo ge ① La i g,①u pu l.①data .①O e① olo ised,①the①populatio s①st atified①sizedistribution and sex-ratio structure would increase the likelihood of colonising nascent habitats in su se ue t①ge e atio s,① ① a i isi g①the①populatio s①pote tial① ep oductive output [START_REF] Ghiselin | The evolution of hermaphroditism among animals[END_REF].

The symbiotic partnership between I. (s.s.) modiolaeformis and its bacteria is perhaps the most e ide t① adaptatio ① i ① the① spe ies ① de elop e t,① i ① ha itats① that① ight① othe ise① e① su -optimal or intolerable physiologically. In the earliest stages (<1 mm) during filter feeding for nutritional requirements, hypertrophic gills would facilitate symbiont acquisition. Flexibility in nutritional mode would promote survival in juvenile individuals in which bacterial densities remain low during the early symbiotic period (qualitatively observed in the current study) and when infection is not mediated. The presence of bacteria on nearly all types of non-ciliated epithelia during the early symbiotic period is believed to augment the limited surface area available upon the gills at this size. As the gills increased in length and depth, the nonciliated regions soon represented a far greater surface area in cross section and longitudinal section. The linear relationship between descending lamellae filament counts and SL suggest that gill development was allometric (as in other Filibranchia), once increases in filament dorso-lateral length, fronto-abfrontal depth a d① the① i itiatio ① of① as e di g① la ella① de elop e t① at① sizes① ① . ① ① i ① le gth are considered. This allometric development would increase the symbiont carrying capacity considerably. It is unknown whether heterotrophic nutrition remains important at this stage, however the apparent increase in the relative volumes of gill tissue versus digestive tissue in larger specimens of the same species found upon carbonate crust (Gaudron et al. 2012), may indicate a switch in nutritional mode from heterotrophic to predominantlychemosymbiotic with increasing size.

The eventual isolation of bacteria to the gill regions only (presumably mediated by the host), places these symbionts in the best possible location to gain access to both electron donors such as H 2 S, and electron acceptors (O 2 in this case), thus maximising potential metabolic activity (Cavanaugh et al. 2006).

Assuming the host has a means to assimilate the resulting bacterial organic carbon, this proves mutuallybeneficial, particularly if the bacteria also indirectly confer a fluid detoxification role (Duperron 2010).

The larval colonisation devices (CHEMECOLI) employed permitted the collection of a comprehensive size-series from substrata providing habitat for the hosts and reduced compounds for their symbionts. The fact that I. (s.s.) modiolaeformis larvae can settle and grow to maturity successfully in these artificial environments validates their use. The findings of previous CHEMECOLI colonisation experiments (e.g. Gaudron et al. 2010;Cunha et al. 2013;Rodrigues et al. 2013) and wood deployments (e.g. Bienhold at al. 2013) all demonstrate that I. (s.s.) modiolaeformis is capable of rapidly and repeatedly colonising finite decaying organic debris in the Mediterranean and eastern Atlantic. The present study has identified that early post-larvae have a functional gut and no detectable symbionts, which suggests strict heterotrophy at the larvae and immediate post-settlement stages. Extracellular bacterial infection occurs shortly after metamorphosis, on various non-ciliated epithelia, but bacteria are only retained on non-ciliated gill epithelia following maturation, where densities increase. In much less than one year, juveniles reach maturity as males, which in addition to previous data (Gaudron et al. 2012), supports protandric hermaphrodism. Individuals in the present study were likely below the threshold size (or age) at which the first female alternation occurs. This study closes a previous gap of knowledge of post-settlement and juvenile development, a critical period for survival in the life-cycle of any species (Duperron et al. 2008a;Gaudron et al. 2012). Intuitively, selective pressures, early in the evolutionary adaptation of organic-fallassociated Bathymodiolinae are likely to have favoured species with a predisposition to grow rapidly and quickly reach maturity, in part by harnessing chemosynthetic symbionts. Together, these adaptive traits ould① ha e① fa ilitated① thei ① pe siste e① i ① pat h ① a d① ephe e al① ha itats.① This① stud s① fi di gs,① a d① the① evident success of I. (s.s.) modiolaeformis on organic falls and cold seeps, are testament to this fact.

and the elongation of staining steps to retain sufficient contrast in semi-thin sections. In addition, since the embedding media is not removed, initial steps involving a solvent to dissolve the embedding media and, if necessary, subsequent removal of solvent by replacement using alcohol (such as is the case in paraffin embedding), are not required. The sub-cellular detail which can be realised with semi-thin sections is superior to that of thicker preparations, singular-layer nature of the tissue under examination. Tissue loss should be minimised due to the retention of the embedding media on the slide (assuming the use of Superfrost slides) At this thickness (350 nm), expected contrasts associated with thicker paraffin-embedded specimens are unrealistic and difficult to achieve with aqueous Eosin-Y, which is less variable in intensity with variable tissue-types when compared to alcohol-based alternative, particular in the absence of Phloxine. The retention of the embedding media may be contributing factor to variable staining intensity, though trials are on-going.

5-2 Introduction

The discovery rate of new highly productive habitats in the deep sea has risen exponentially (Ramirez-Llodra et al. 2010) following the initial descriptions of dense megafaunal aggregations thriving in the vicinity of hydrothermal vents (Corliss and Ballard 1977;Lonsdale 1977 ① a d① old ① h d o a o ① seeps① Paull et al. 1984). Some of the most startling were the discoveries of specialized fauna found at large organic falls such as vegetative debris and megafaunal nektonic carcasses (whale falls being the best documented).

Inexplicably high species abundances on and around whale skeletons were initially surprising until the eside t①spe ies ①loose①affiliatio s①to① e t①fau a① e e① ealised,①suggesti g①that①o ga i ①falls①fo ed①si ila ① sulphide-based reducing habitats (Smith et al. 1989), but with elevated levels diversity in comparison with hydrothermal vents (Baco and Smith 2003 .①Follo i g①the①ha itat s①fo al①des iptio ① " ith①et①al.① ,①

the earliest studies carried out upon both natural-and experimental-whale-fall communities identified commonality with some vent and seep organisms, albeit as minor contributors to species richness (Bennett et al. 1994;Deming et al. 1997;[START_REF] Feldman | Vestimentiferan on a whale fall[END_REF]Smith et al. 2002;Baco and Smith 2003;Smith and Baco 2003;Schuller et al. 2004). Accordingly, extant or ancestral whale fauna have been implicated in the evolution of vent and seep fauna (Smith et al. 1989), based on the apparent similarities in taxa between the different types of reducing habitat; ancestral vent-and seep-species already developing symbiotic associations are believed to have inhabited large organic falls as dispersal pathways over their evolutionary history. Whilst most researchers in reducing habitats tend to agree that this hypothesis is plausible, the order of habitats remains contested due to somewhat conflicting patterns between fossil records and current multi-gene phylogenies, Kiel and Goedert 2006;Lorion et al. 2013). However, a similar hypothesis was subsequently put forward for sunken wood (Distel et al. 2000), suggesting that organic falls may have played a crucial role in the eventual adaptation of vent and seep 26 fauna to their respective habitat conditions.

Collectively, whale falls at various stages of degradation are estimated to cover 35 km2 of the ocean floor (Ramirez-Llodra et al. 2010). This is based on the knowledge that a 30t whale fall can enrich surrounding sediments up to 9 m from the site of the carcass (i.e. approximately 50 m 2 area of seafloor, Baco and Smith 2003;Treude et al. 2009), and an estimated 690 000 whale falls are decomposing on the seafloor at any one moment in time (from Ramirez-Llodra et al. 2010, using data from Baco and Smith 2003;Smith 2006;Treude et al. 2009). It follows that when averaged over the entire deep-sea①floo s①su fa e①a ea① (3.6 x 10 8 km

In the case of megafaunal nektonic carcasses, four stages of community succession have been recognised during decomposition, of which the first three are expedited by biological processes (Bennett et al. 1994), though the depth of the carcass appears to dictate to what extent each phase applies (Lundsten et al. 2010). These are the mobile scavenger stage, the enrichment-opportunist stage, the sulphidic stage and finally, when all organic sources of energy are depleted, a skeletal reef stage. Following the arrival of the carcass at the seafloor, mobile scavengers quickly consume the labile protein and lipid rich flesh.

Applying an estimated removal rate of 40-60 kg d -1 (Smith 2006), the very largest carcasses in the deep sea (e.g. 160 t Blue whale Balaenoptera musculus) could provide tissue in this way for up to a decade, though for an average-sized baleen whale (e.g. 35 t) this period is in the range of 1.5 years (Smith et al. 2002). Due to①settli g①pa ti les①of① a ass①flesh① ess ①feedi g ①a d① apid①i put①of①fae al① aste,①u de l i g①sedi e ts① typically become greatly enriched, supporting an array of organic-enrichment type opportunistic species (e.g. dorvellid polychaetes, cumaceans and oligochaetes, Smith and Baco 2003). Enrichment of this sort has been documented to last 0.3 -4.5 years, depending on carcass size (5 -35 t, n = 3, Smith et al. 2002).

Sediment enrichment is relatively rapid, occurring within days to weeks of the addition of whale biomass due to microbial sulphate reduction and methanogenesis (based on in vitro studies Treude et al. 2009). The levels of sulphide and methane found in sediments appear to depend on proximity of underlying sediments to the carcass, but at 0.5-m distance, values of 717 and 99 mmol m -2 d -1 of ex situ sulphate reduction and in vitro methanogenesis have been recorded (Treude et al. 2009). Approximately 10% of the total sulphides produced are as free sulphides, while sediments subject to direct whale biomass enrichment, can see rates of methanogenesis equivalent to 20 -30% of those for sulfate reduction (Treude et al. 2009). Decomposing whalebones are thus thought to create a habitat intermediate to that of vents and seeps (Treude et al. 2009).

Once bones are exposed, the resultant efflux of sulphides (Treude et al. 2009) can fuel the metabolism of chemoautotrophic bacteria, both free-living (e.g. microbial mats) and as symbionts in specialist chemosymbiotic metazoans that colonise the hard surfaces of organic falls. The semi-porous nature of bone permits limited release of sulphides from decomposing lipid-rich marrows internal to bones of whale skeletons (Higgs et al. 2011b). Sulphide reduction rates are considerably lower in bones than the su ou di g①sedi e ts① e.g.①≈① ① ol①total①sulphide①d -1 e sus①≈ . ①-2 mol d -1 in sediments). However, the① elease①of①sulphides① a ① e①a ele ated① ①the①e osi e①a tio ①of① i o ial① ats①o ①the① o e s①su fa e① and through①the①digestio ①of① o e①di e tl ① ①the① o e-eati g ①pol haete①spe ies① ge us① Osedax) in the course of accessing internal lipid-rich marrow (Higgs et al. 2011a). This is made possible by employing symbiotic heterotrophic bacteria housed in bacteriocytes located in the characteristic root tissue of this genus, typically buried into the bone (possibly aided by acidic mucopolysaccharide secretion through the carbonic anhydrase-catalysed hydration of CO 2 , [START_REF] Rouse | Osedax: Bone-eating marine worms with dwarf males[END_REF]Higgs et al. 2011a;[START_REF] Katz | The osedax trophosome: Organization and ultrastructure[END_REF][START_REF] Tresguerres | How to get into bones: Proton pump and carbonic anhydrase in osedax boneworms[END_REF]). In addition to the organic-fall siboglinid polychaetes specialists (Jones et al. 2008;[START_REF] Kiel | Osedax borings in fossil marine bird bones[END_REF]Rouse et al. 2011), small-sized chemosymbiotic bathymodiolins such as Adipicola (s.l.)

pacifica, Idas [s.s.] washingtonius and I. [s.l.] simpsoni are often present on bone surfaces during the sulphidic stage (e.g. Marshall 1900;1901;Southward 2008;Fujiwara et al. 2010), taking advantage of the release of free sulphides and methane (Treude et al. 2009) used by their bacterial symbionts. The first indication that members of the genus Idas (s.l.) might harbour bacteria in their gills, already known to be the case in larger bathymodiolin (s.l.) mussels at that time, was when Gram-negative bacteria were identified in the gills of I. (s.s.) washingtonius from whale bones (Deming et al. 1997). Evidence for the symbiotic nature of these bacteria was based upon gills testing positively for the presence of ribulose- (Smith and Baco 2003;McKiness et al. 2005;Duperron et al. 2008a;Duperron et al. 2008b;Southward 2008;Gaudron et al. 2012;Lorion et al. 2012;Rodrigues et al. 2013;Thubaut et al. 2013a;Laming et al. 2014; except the types species I. [d.f.] argenteus, Ockelmann and Dinesen 2011).

Considering the ca. 690000 whale carcasses distributed globally at a given time, only around 0.005% (approximately 30 carcasses) have been studied in detail. This reflects both the random way in which natural whale falls are discovered and the logistical (and ethical) problems associated with securing experimental carcasses. Thus, furthering our understanding of mammalian fall community dynamics is hindered by a lack of suitable organic substrate. One way in which this problem may be circumnavigated is by using analogous mammalian alternatives which are readily accessible to researchers. Precedence for this approach exists in a study by Jones et al. 2008, in which bovine femur bones stripped of meat were deployed in an area proximal to documented whale falls (Monteray Bay, USA). That study, which was focused upon collecting and identifying Osedax spp. found such deployments to be highly effective for this purpose, despite lower lipid contents in smaller mammalian bones compared to those of intact whalebone [START_REF] Evershed | Preliminary results for the analysis of lipids in ancient bone[END_REF]. The evident success of such deployments opens up a new avenue for colonisation experiments in the deep sea which can approximate natural nektonic falls.

Funded by Fundação para a Ciência e a Tecnologia (FCT), the CARCACE project was devised (project coordinator: Ana Hilário), bringing together a multidisciplinary international team of scientists with experience in various aspects of deep-sea chemosynthetic benthic ecology. Within the framework of the CARCACE project , entire bovine carcasses attached to concrete ballast, were deployed in Setúbal Canyon on the west Portuguese margin and at the Condor Seamount (near the Azores), with subsequent recoveries following 18-and 24-month durations (Génio et al. submitted). The principal aim was to study the community response to the arrival of large organic falls to deep-sea floor in the Atlantic Ocean and to gauge the importance of this localised enrichment as a sulphide-rich habitat. As part of this overall aim, a random size-st atified① su set① of① spe i e s① of① the① he os ioti ① ussel① Idas. ① s.l.) simpsoni, collected from bones recovered after 18 months i ①the①"etú al①Ca o ① ≈ ,① e e①sele ted①to①assess①post-larval to adult development in terms of general anatomy, rates of maturation and patterns of symbiont acquisition.

5-3 Methodology 5-3.1. Sampling and processing Details concerning the deployment of cow carcasses and their recovery may be found in Génio et al.

submitted. On board the ship, bones were sorted immediately and samples were fixed and preserved for a variety of analyses (Génio et al. submitted). Mussels for the current study were fixed in 4% formaldehyde (in filtered seawater) for 2-4 hours and then preserved in 96% ethanol, by serial transfer (70%, 80%, 96%).

Samples were stored at room temperature. In the laboratory, the preserved substrata were sorted microscopically.

Individuals of Idas (s.i.) simpsoni were identified based on morphoanatomy and partial Cytochrome Oxidase I data. The larval prodissoconchs I and II were opaque, pearly white and transparent, red/orange respectively and, where present, the dissoconch was modioliform, colourless (but iridescent), glossy, and almost transparent in all juveniles or translucent and fleshy-pink in individuals >2 mm (examples displayed in Figure 5.1). Bones recovered at 18 months were colonised by these mussels at prolific densities, along with at least one species of Osedax polychaete, such that the recovery resulted in 1000s of I. (s.l.) simpsoni individuals in total (Ana Hilário, personal observation). The subset of mussels used in this study was selected randomly from several bones, but with the requirement that the resulting size-spectrum was sufficiently wide to cover post-settlement to putative adults (in order to repeat procedures in Laming et al. 2014). Thus, it was possible to document key developmental patterns in I. (s.l.) simpsoni from the Setúbal canyon, western Portuguese margin, using the size-range selected.

5-3.1.1 Shell and preliminary soft tissue analysis Individuals were measured (dissoconch: n = 38, prodissoconch I: n = 24, II: n = 38) and photographed using a camera-mounted dissection microscope (Nikon Elements camera and software, Japan), as in Laming et al. (2014). Shell height (SH) was the greatest linear distance dorso-ventrally measured from the umbo, perpendicular to the hinge-line, while SL was the greatest linear distance antero-posteriorly measured parallel to the hinge line. Prodissoconch II dimensions were measured using the same approach, but with sta da dised① la al ① hi ge-lines, to permit the aggregation of prodissoconch II data across all shell sizes.

Prodissoconch I SLs, measured parallel to the vestigial provinculum, were estimated using their coloration to define margin limits. Prodissoconch II shells were oddly blistered on their surfaces, in contrast to I. (s.s.) modiolaeformis in Laming et al. (2014). Size-class frequency analyses were not performed here as the subset was an artificially size-stratified sample (such analysis will be included in Génio et al. submitted). for visual explanation) were achieved using a hair to roll the tissue into position, where plane orientation was alternated sequentially along the size spectrum, i.e. each aspect was equally represented in similarly sized organisms. Transverse orientations required double-polymerisation. The first was a catalysed polymerisation at room temperature, so that a flatbed mould could be used in the presence of oxygen. The mould was afloat on ice water to draw away exothermic heat generated during the 10-min reaction.

Surface resin was trimmed (removing resin polymerised imperfectly at the oxygenated surface), submerged vertically in a LR white-filled gelatine capsule, and polymerised as with sagittal and frontal orientation, above. Gelatine was removed with hand-hot water.

Semi-thin sectioning had previously been used in a similar developmental study with great success (Laming et al. 2014), so this approach was also employed in this species. Resin blocks were wet-sectioned (glass knife) on a Leica EM Ultracut UC6 Ultramicrotome (Germany). 1 µm-thick sections on Superfrost plus slides were used for haematoxylin and eosin-Y staining (H&E) and 2 µm-thick sections on Superfrost plus slides for fluorescence microscopy (to mitigate previously encountered poor signal to noise ratios, Laming et al. 2014). Periodic toluidine staining was performed to identify the cutting-axis a d①lo atio ① ≈ -µm intervals).

Standard H & E staining procedures for thick sections were modified for semi-thin LR-white sections (according to Laming et al. 2014;Chapter 4). In the rare instances where contrast remained poor and alcoholic Eosin-Y had to be used, section edges were clamped with a border Eukitt hard mounting medium, in order to mitigate resin distortion. Slides were viewed under a camera-mounted compound microscope (Evolution VF camera, Media Cybernetics, USA; Olympus BX61, Japan) and micrographs were processed and measured, where necessary, using Image-pro plus (v.5.1) or during post-processing in Photoshop CS3. soft-tissue analysis. Though small, much of the anatomy pertaining to later development was evident. Small (<1 mm) and larger (>1 mm) juveniles, in which dissoconch growth was evident but gametogenesis was not, were most numerous (n = 30, SL 0.43-2.45 mm). The three largest individuals (SL 2.61-3.74 mm) were adults with direct evidence of gametogenesis, discussed later. In serial sections, these three paired ganglia were elliptical, comprising of a peripheral layer of neuronal cell-bodies (axons being directed inwardly towards the nerve centre) with a point of exit for the nerve bundle (e.g. Figure 5.3B). It was not possible to identify connective nervous cords with the staining techniques used in this study, but cerebropleural commissures were identified in specimens sectioned along the dorso-ventral axis in the sagittal plane (not shown).

5-4.3.2 Digestive system

The digestive system was already well-developed in plantigrades with a scaled increase in volume and complexity with increasing shell sizes (Figure 5.3A, C). In plantigrades examined histologically (n = 3), ciliated labial palps were already well-developed (Sequential toluidine-stained sections in Supplementary figure 5.1), with a ciliated oesophagus, stomach and digestive gland. The oesophagus was practically flush to the utmost anterior region of the digestive system with most of its length orientated perpendicular to the hinge-line (Supplementary figure 5.1). Identifying whether palps were associated with the anterior region of the gill-basket directly, proved infeasible at this size with the techniques employed. The entire alimentary system typically occupied about a third of the area of the soft tissue in mid-sagittal section (Figure 5.3A).

In juveniles, the moderate increase in shell capacity was occupied, in part, by an expanding digestive system. Sequential HE sections with detailed annotation from two similarly sized juvenile individuals are presented, cut in the transverse (Figure 5.4) and sagittal (Figure 5.5) planes. These Adults possessed all the attributes of juveniles, but scaled-up in line with shell capacities (not shown, for brevity). Alimentary organs and tissues were identified more easily on account of their characteristic structure, typical of descriptions available in the literature for related, shallow-water species (e.g. Bayne 1976). The stomach and digestive diverticula still represented a vast portion of the visceral mass in the dorsal region.

5-4.3.3 Attaining maturity

Maturation rate was estimated based on the size at first maturity. In I. (s.l.) simpsoni, the first individual to be identified as a mature male had a SL of 2.61 mm, based on clear evidence of spermatogenesis (Figure 5.3E). However, the gonad generally, and the structuring of the tissue particularly were already relatively developed, suggesting that this individual had been mature for a marked period of time prior to fixation.

The reproductive status of preceding mussel sizes was examined in as much detail as possible to assess whether earlier sizes showed signs of gametogenesis, but none was found of the gill lamellae, suggesting that these regions are the site of new filament formation (e.g. Figure 5.3D).

28 N.B. at the preceding specimen size, SL 2.45 mm, the tissue were bad damaged during a difficult dissection An unusual morphological trait in this species was the presence of an apparent thin connecting band of tissue along the antero-posterior length of the gill filaments (connecting band in Figure 5.3B and D). This band may also have been the dorsal bend of a developing ascending lamella, however no filaments of these secondary lamellae were identified with confidence, perhaps suggesting their relatively delayed development in this species. Gill filament numbers recorded during dissection increased linearly in I. (s.l.)

simpsoni with increasing SL (Figure 5.6; Linear regression, r 2 = 0.98, F 1,31 = 1236, P <0.0001). In post-larval plantigrades, these numbered between 5 and 7 simple, stout, gill bars, while 46 filaments were identified in one descending lamella of the largest specimen examined (SL 3.74 mm). Little or no mantle epithelia survived sample processing, so bacterial colonisation of these epithelia could not be confirmed.

Initial densities on gill epithelia were relatively low, however densities increased rather dramatically with increasing SL (Figure 5.7), so that some larger juveniles (>1 mm) had deep layers of bacteria between filaments (Figure 5.3,5.5,5.7 and 5.8). In some instances, the latero-abfrontal regions of gill filaments loaded with bacteria appeared to have undergone atrophy, based on their relatively thin sagittal cross-sectional area (e.g. Figure 5.3C). This may however be an early state in gill development before the effects of hypertrophy are truly witnessed (e.g. size series in Figure 5.7B).Despite employing a wide range of targeted oligonucleotidic probes during FISH analysis, only T1-thiotrophs were confirmed to reside on the filaments of I. (s.l.) simpsoni from the Setúbal Canyon. Superimposed signals from DAPI and the general Eubacterial probe EUB338 revealed complete overlap in bacteria (Figure 5.7A; Figure 5.8), although DAPI signal appeared more densely populated. A similar approach for the specific thiotroph probe Bthio-193, revealed high signal overlap also, but again extraneous signal in the DAPI could not be confirmed ith① the① ta geted① p o e s① sig al.① This① a ① efle t① the① fa t① that① D②PI① a d① the① oligo u leotide① p o es① a e① targeting DNA and ribosomal RNA respectively, meaning the latter will fail to hybridise with those bacteria that were not very active prior to fixation. Signal from the FISH probes in smaller mussels was quite weak but distinguishable (e.g. Figure 5.8B: Bthio-193 in particular), or non-existent, suggesting that some issues remained with an unknown aspect of the method. This may be partly related to focal depth of the frequency of light (this was noticeably shallower for Cy3 and Cy5).The least ambiguous signals (including those pictures in Figure 5.8) were from transverse-sectioned specimens that also happened to have been subjected to a different polymerisation protocol (see methodology).

5-5 Discussion

By examining size-specific trends in the developmental biology of Idas (s.l.) simpsoni, this study has considerably advanced our understanding of early post-settlement life all the way through to sexual maturation in a bathymodiolin species colonising bone remnants in the deep sea. This was made possible using relatively short term (for carcass falls) deployments: specimens, at previously undetermined key points along their developmental path to adulthood, were acquired and analysed. The results identify crucial developmental junctures which are believed to maximise this species survival in spatially and temporally finite environments: in this instance mammalian bones. 5-5.1. Nutrition Given that PdI SL roughly corresponds to the size of the D -veliger following initial shell formation, it is also thought to relate to oocyte diameter (Lutz et al. 1980). Diameters such as those above would thus be equivalent to oocytes at <96 µm, which is a small-sized oocyte and thus could only hold a small yolk reserve.

The PdII provides a proxy for pelagic larval duration, which in this case argues for an extended larval phase (M. edulis has a PdII SL of 120-252 µm by comparison, [START_REF] Sprung | Physiological energetics of mussel larvae (Mytilus edulis). I. Shell growth and biomass[END_REF]. As yolk reserves are likely to be low, researchers often cite these data together as evidence for planktotrophy, though this overlooks to the possibility of chemosymbiotic larval nutrition (e.g. as suggested for B. (s.s.) azoricus, Trask and Van Dover 1999). Fortunately, since bacterial signals were entirely absent from all epithelia in post-larval plantigrades regardless of means of analyses, no evidence exists to support symbiotic nutrition prior to settlement and metamorphosis. Taken with the larval shell data this argues for obligatory planktotrophy during larval dispersal, though the pre-acquisition of a very low seeding population of bacteria can not be entirely ruled out.

5-5.1.2 Acquisition of symbionts following settlement

It has been shown that, once settled and metamorphosed on bone material, I. (s.l.) simpsoni appears to spend minimal energy on growth before it acquires its first symbionts (8% increase in SL, from PdII SL 0.40 mm, to 0.43 mm with the deposition of a thin margin of dissoconch shell). A large proportion of these symbionts were certainly Eubacteria, and probably thiotrophic (T1-type), based on Bthio-193-specific bacterial FISH signals in host gill tissues (though bacteria were also identified on non-ciliated visceral epithelia). Following their initial appearance in very small juveniles, bacteria appeared to qualitatively increase in numbers unit -1 gill-space within a narrow range of shell lengths; in fact they rapidly achieved densities that seem to approach saturation, at least visually (e.g. Figure 5.5; Figure 5.7B even at SLs 846 and 974 µm). However, an increase in specificity towards the gill filament surfaces only, observed in other bathymodiolins (Wentrup et al. 2013;Laming et al. 2014), did not occur within the size range examined. In the largest individual, traces of bacteria could still be seen on the ventral surfaces of visceral epithelia. This probably reflects the fact that even the largest adult examined was less than 10% of the maximum size recorded for this species (SL max 45mm, from Warén and Carrozza 1990).

The only other chemosymbiotic Idas mussel to which this rate of acquisition can be compared directly is in I.

(s.s.) modiolaeformis, colonising wood and alfalfa substrata (Laming et al. 2014). When the two are spe ies ① a est al① adaptatio ① to① o ga i ① falls① spa e① li ited① i ① o pa iso ① ith① othe ① edu i g① ha itats,① Lorion et al. 2013). In fact, this appears to have taken place around the same time that Bathymodiolus (s.l.)

spp. evolved their contemporary levels of gigantism (Lorion et al. 2013).

5-5.1.5 Developmental anatomy

Finally, this study has described some general aspects of development in I. (s.l.) simpsoni in terms of organismal anatomy. In particular, it identified a functional digestive system in post-larvae of I. (s.l.)

simpsoni, with increasingly developed organisation at later stages nearing adulthood. The persistence of a fully-formed gut is intriguing in a species where symbionts seem abundant (at least for this habitat).

However, the retention of a fully-formed digestive system appears to be the norm in organic-fall-colonising small-sized bathymodiolins (s.l.), based on the limited data available (e.g. Gustafson et al. 1998;Thubaut et al. 2013a;Laming et al. 2014;Chapter 3, p. 127). This is despite evidence for intracellular chemoautotrophs in the bone-colonising species I. (s.s.) washingtonius (Smith et al. 1989;Deming et al. 1997)

2010).

In a group of mussels in which chemosymbiosis presents the most common nutritional state described in adults (except the carnivorous larviphage, I. argenteus, Ockelmann and Dinesen 2011), the retention of a functional gut could be the result of ongoing selective pressures (i.e. a gut remains a requirement for survival) or the presence of the gut is neutral to survival, and thus is simply an echo of evolutionary history.

However, in the large bathymodiolins, obligatory chemosymbiosis (i.e. at most deeper hydrothermal vents and hydrocarbon seeps) typically coincides with a moderate reduction in the gut, now known to have occurred independently multiple times (Gustafson et al. 1998;Lorion et al. 2013;Thubaut et al. 2013b). In all bathymodiolin species, the chemosymbiotic mode is believed to have evolved relatively recently (Lorion et al. 2013), and thus varying degrees of symbiont dependency in the subfamily may represent several stages in an evolutionary transition towards further holobiont integration, as in the pliocardiin vesicomyid clams. If this were so, Bathymodiolus spp.① ould① u e tl ① e① the① ost① de i ed ① state① o e① edu ed① digestive system, intracellularas opposed to extracellularbacteria, and stable isotopes data supporting a high dependency on chemosynthetically derived nutrition in some species, [START_REF] Cavanaugh | Evidence for methylotrophic symbionts in a hydrothermal vent mussel (Bivalvia: Mytilidae) from the Mid-Atlantic Ridge[END_REF]Dubilier et al. 2008). However, there are no arguments per se, to suspe t①that① ath odioli s①a e t①al ead ①suita l ① adapted to their habitats, i.e. no evidence for active selection towards a more integrated holobiont state. In fact, there is growing evidence in the literature that the retention of a partially or fully formed digestive system in bathymodiolins (including many Bathymodiolus [s.l.] spp.) reflects a real need; a retained capacity forand active engagement infilter-feeding is postulated for several species (e.g. Page et al. 1990;Page et al. 1991;Chapter 3) modiolaeformis has settled in Laming et al (2014). An alternative explanation could be a①te de ①fo ① leak ① vertical transmission in I. (s.l.) simpsoni,① he e①a① seedi g ①populatio ①of① a te ia,①at①de sities①too①lo ①to① detect, were transported from the natal site and thus were already present on arrival as larvae to the cow bones. In other words, larvae were pre-equipped with a very low density starter population of bacteria, removing the need for environmental acquisition and possibly accelerating the patterns of symbiont expansion in the gills. This could explain site-specific bacteria assemblages in I. (s.s.) modiolaeformis (Rodrigues et al. 2013), which suggest acquisition is truly after settlement. However, both scenarios remain plausible, and the argument is rooted in relative rather than absolute rates of acquisition. A future hypothesis to be tested would be whether the symbiont assemblages in I. (s.l.) simpsoni hosts display phylogenetic affinities with bacteria living at the host s natal site, rather than its source.

6-2 Introduction

Symbiosis between bacteria and metazoans is key to the productivity of deep-sea cold seeps and hydrothermal vents, in which most of the primary production is ensured by chemoautotrophic symbionts (Dubilier et al. 2008). Symbiosis is usually facultative for the bacteria and several shifts between free-living and symbiotic lifestyles during the life of a bacterium or during evolution of bacterial groups are reported (Dubilier et al. 2008;Duperron et al. 2013;Wentrup et al. 2013). Taxa within the annelids and bivalves on the other hand live in obligate symbiosis with bacteria which contribute to their nutrition, and symbiosis is considered the key synapomorphy that allowed efficient colonization of reducing habitats (Won et al. 2008;Duperron 2010). Habitat and depth shifts (and reversals) can occur (Won et al. 2008;Thubaut et al. 2013) (Ockelmann and Dinesen 2011). This mytilid is of particular taxonomic importance, as it is the type species of its genus (Lorion et al. 2013;Thubaut et al. 2013). Whether I. argenteus is a relict species belonging to a deep-branching lineage emerging before the acquisition of symbiosis in Bathymodiolinae or belongs to a recently emerging lineage in which symbiosis was secondarily lost, has not been investigated due to the lack of material for molecular studies.

We

6-3.2. Molecular characterization

DNA was extracted from one specimen from each site using the QIAamp DNA MicroKit (QIAGEN).

Fragments of three mitochondrial loci (encoding COI, NADH4 and 16S rRNA) and three nuclear loci encoding ",① "① 'N②① a d① Histo e① ① e e① a plified① fo ① ph loge eti ① a al sis.① ② out① ① p① of① COI,① ① p① of① NADH4, 480 bp of 16S, 1650 bp of 18S, 1000 bp of "①a d① ① p①of①H ① e e①a plified①usi g①p i e s①a d① PCR amplification profile summarized in Table 1. PCR products were purified using QIAquick Gel Extraction Kit (QIAGEN) and send to sequence in both directions at GATC Biotech (United Kingdom). DNA sequences obtained during this study were complemented with data from GenBank and following available datasets (Lorion et al., 2013;Thubaut et al., 2013) (Table S1) and aligned using ClustalW [START_REF] Thompson | The Clustal X-Windows interface: flexible strategies for multiple sequence alignments aided by quality analysis tools[END_REF].

Phylogenetic reconstructions were performed for single and concatenated genes using maximum likelihood (ML) and a General Time Reversible model with Gamma distribution of rates and a fraction of invariant sites using MEGA 6 [START_REF] Tamura | MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods[END_REF].

The presence of bacteria was tested by PCR of fragments of genes encoding 16S rRNA, 23S rRNA and APS reductase using previously described protocols (Table 1; Rodrigues et al., 2013). Obtained by maximum-likelihood (ML) analysis of the multigene data set (2 mitochondrial and 3 nuclear genes), details in Table S2 6-4.2. Phylogenetic relationships Sequences from LD and MAR specimens displayed similarities between 99.4 to 99.9% for the six studied genes. Phylogenies based on COI (Table S2, Figure S2), on concatenated datasets of 5 genes (including closest relatives, Figure 2) and on all 6 genes (excluding I. washingtonius and I. macdonaldi, not shown)

presented similar topologies and well-supported nodes using ML methods. PCRs on all 4 bacterial genes using various DNA concentrations and PCR conditions including nested PCR failed to yield any product despite success with similar-sized symbiotic Idas spp. from the same experiment.

6-4.3. Live observations and anatomy

The largest LD individual liberated sperm during dissection, confirming it to be male. Ctenidial ciliary action was evident and a ventral particle groove was present. Rotating mucous-bound debris was identified within the ciliated labial palps, which were not obviously plicate, but enlarged (Figure S1). The adult male was highl ① a ti e① du i g① o se atio s,① o i g① a ou d① the① Pet i① dish① usi g① it s① e ifo ① foot① Figu e① .① The① pericardial vale could clearly be seen during these observations, where beat rate increased during movement, in comparison to resting entirely still.

6-5 Discussion

Idas argenteus were found associated with sunken wood colonization experiments on the Rainbow MAR vent field and the LD canyon. Although Bathymodiolus azoricus occurs at Rainbow and I. simpsoni and I.

modiolaeformis are reported at LD (Thubaut 2012;this research, Section 3-4.2, p. 136), this is the first report of I. argenteus at these two sites.

This study provides the first phylogenetic analysis of I. argenteus. Its closest relative is I. Ockelmann & Dinensen 2011 reported no evidence of bacteria in the gills of I. argenteus. However, this could be due to a site-specific rarity or absence of symbionts. Without exception, symbiont acquisition in other bathymodiolin mussels occurs long before sexual maturation (Laming et al., 2014;Wentrup et al., 2013). The fact that symbiont genes were not detected by PCR tests in the specimens studied here is thus not size-or age-related. Even if present in small numbers, below the detection limit of PCR approaches used herein, symbiont rarity would imply a negligible contribution to host nutrition.

From the 13 Idas species belonging to the I. argenteus clade in the COI tree (Figure S2, Table S1), symbiosis is documented in only five. I. macdonaldi, I. washingtonius, Idas sp. C, and Idas sp. D have sulfuroxidizing symbionts, meanwhile I. modiolaeformis has up to 6 different phylotypes with relative dominance of methanotroph or thiotrophs depending on time of sampling or habitat (Duperron et al., 2008;Lorion et al., 2012;Rodrigues et al., 2013;Thubaut et al., 2013). Symbiosis in other species is not yet documented. At this stage, it can be assumed that the Idas clade had a common ancestor with thiotrophic symbionts, most likely extracellular as in all species but I. washingtonius (Deming et al., 1997). Extracellular thiotrophic The history of nomenclature for wood-and bone-associated mussels has been complex, with members of the genus (s.l.) as it currently stands having been assigned one or more of the following genera over time ; Idasola; Myridas,; Myrinopsis; Habepegris; Myrina, Adipicola Idas, Terua. For scientists working on these small-sized mussels this has been highly frustrating, since making assertions about the evolutionary origins of the various genera is, by definition, confounded by ongoing debate concerning their taxonomy and more recently, phylogeny. Some researchers have however contributed considerable advances in the cladistics of the subfamily (e.g. Jones et al. 2006;Miyazaki et al. 2010;Lorion et al. 2013;Thubaut et al. 2013b;Rodrigues et al. in press, Chapter 6, p. 232).

Adula

7-1.1.1 Observations on the type species, Idas argenteus

As part of the research carried out within the framework of this PhD, (Rodrigues et al. in press,Chapter 6,p. 232), key characteristics of the adult-shell sculpture, umbo placement, larval prodissoconch size and hingeplate denticulation were used to provisionally identify specimens collected as the species I. (d.f.) argenteus (Figure 7.1). Digitate labial palps characteristic of the species (Ockelmann and Dinesen 2011), were discernible from live video observations of the small male from the Lacaze-Duthiers Canyon, though grabbed still images were not of sufficient quality to demonstrate this feature formally in publication (see Figure 6.1). Unfortunately, prior to the realisation that this specimen was I. argenteus, the soft-tissue had been used in its entirety for DNA extraction and thus a very detailed anatomical investigation was never carried out on this adult specimen. Ockelman and Dinesen (2011) predicted that the inner muscular folds of the mantle edge could probably extend a considerable degree beyond the ventral margins of the shell valves, based on the musculature of preserved specimens. During live observations while the mussel was resting, it was indeed possible to see a curtain of mantle extending along most of the length of the ventral margin, and in particular where pseudo-siphons formed at the posterior region of the specimen. The entire inner fold then briefly extended further still, prior to complete valve-closure when the specimen was accidentally disturbed (vibrations of microscope). This behaviour may flood the mantle cavity with oxygenated water in preparation for closure. However, during periods when the foot was extended during movement, the mantle curtain was retracted within the shell. The absence of the incomplete and modified outer demibranchs (Ockelmann and Dinesen 2011) was probably a question of size. The adult specimen from the Lacaze-Duthiers Canyon (SL 2.13 mm) fell well below the minimum size identified for outer demibranch formation in this species (> 4 mm, Ockelmann and Dinesen 2011). Based on all these observations, the evidence available indicated that these specimens were indeed the type species for the genus. This was subsequently confirmed by Graham Oliver of the National Museum of Wales. Concerted efforts have been made to establish more robust concatenated trees for the Bathymodiolinae (s.l.) subfamily (e.g. Jones et al. 2006;Miyazaki et al. 2010;Lorion et al. 2013;Thubaut et al. 2013b;Rodrigues et al. in press, Chapter 6, p. 232). The use of multigene concatenated tree construction has resolved some of the incongruence typical of single-gene trees (seen most recently in Fontanez and

Cavanaugh 2013), greatly increasing bootstrap support for many the more recently emerging nodes (Lorion et al. 2013). One example is the study by Thubaut et al. (2013b), in which some of the most drastic alterations to the current phylogeny are made. A multi-gene concatenated Bayesian tree, identified 10 distinct clades with robust support for 8 based on the posterior probabilities and bootstraps values cited (two clades terminate in genera for which only one species is known). The analysis examined 51 taxa/ESUs and 5912 base pairs (bp) from two mitochondrial and five nuclear genes. These were fragment sequences for mitochondrial genes which code for cytochrome oxydase I (mtCOI) and the 16S region of ribosomal RNA (16S), the nuclear genes 18S rDNA (18S) and 28S rDNA (28S), which code for the 18S and 28S region of ribosomal RNA respectively, the nuclear gene histone H3 (H3), the nuclear gene that codes for the 70-kDa heat-shock protein (HSP70), and the gene that codes for adenosine nucleotide (ADP/ATP) translocase (Ant).

However, these studies rarely make concerted efforts to acknowledge any divergence, or conservativeness/convergence in morphoanatomy, where the erection of new genera in particular, appears to be based upon genetics alone (Thubaut et al. 2013b;but Lorion et al. 2010;Thubaut et al. 2013a).

Additional criticism might be levelled at the revision of the nomenclature proposed in Thubaut et al. emerged much earlier, prior to all Bathymodiolus (s.l.) and Idas (s.s.), as in Thubaut et al. (2013b). This discrepancy exists in a region of both trees where low posterior probabilities and boot-strap support are common place. Although fewer species (and a slightly different array of genes) were used, the concatenated tree presented in Chapter 6 (Rodrigues et al. in press) appears for the most part to agree with the structure of the tree from Lorion (2013). Despite these discrepancies, in all instances the lineage which terminates in the Bathymodiolus (s.s.) and Idas (s.s.) clades, is highly divergent from the clade to which I.

(s.l.) simpsoni appears to belong. Thus, an opportunity presented itself to assess the corresponding degree of divergence in the morphoanatomy of these species, to examine whether such differences might also be apparent in their biology.

7-2 Synthesis of current research

The research results presented in the previous chapters (excluding Chapter 6) are summarised in Table 7.1, which also provides some data on habitats occupied by I. (s.s.) modiolaeformis and I. (s.l.) simpsoni that

were not examined during this study (i.e. seeps). Note that this table effectively represents a walkthrough guide for the sections below.

7-2.1. General species information

An updated version of known distributions of the two species is presented (Figure 7.

Chemosymbiosis?

Heterotrophy?

Chemosymbiosis

7-2.2.2 Gamete characteristics and gametogenesis

Due to the constraints of the live-mussel experiment undertaken at the Banyuls-sur-mer Marine Observatory (co-occurrence of two species, low fecundity, no sure means of species ID in real time), released oocytes from both species were inevitably pooled for fertilisation experiments. This, and a rather ad-hoc means of temperature control (use of tap-water baths), evidently presented some problems for maintaining larvae alive. Consequently much of the work that was envisioned, in relation to embryonic and larval development (see section 2-1, p. 107), was not realised fully. However, as only one oocyte type was obvious during oocyte collection and examination, this suggests that either the oocytes of both species are impossible to discriminate, or only one species spawned oocytes in significant numbers. The latter seems implausible since I was witness to several of the individuals spawning directly (although diameters of unreleased oocytes varied significantly between species). The net result is that gamete characteristics and estimates of fecundity had to be pooled across both species (Table 7.1). Gamete sizes were similar to those previously cited for I. (s.s.) modiolaeformis (Gaudron et al. 2012, having accounted for fixation effects) and are in keeping with recorded values for the prodissoconch I (representative of larval hatching size; see Table 7.1). The mean, released oocyte diameter of 59 µm, suggests that a minimal reserve of energy is invested in these oocytes (Young 2003), and goes some way to explain how relatively small specimens can spawn epeatedl ① a d① p odu e① a ① a e age① of① ≈① ① oo tes① i di idual -1 (Table 7.1). Measurements of gametes were also made upon fixed gonad tissue in the current research, in both preserved mussels of each species from the live experiments and on the limited I. (s.s.) modiolaeformis adults available from the eastern Mediterranean and I. (s.l.) simpsoni adults from the Setúbal Canyon. Results from these measurements overlapped for oocytes and spermatids/spermatozoids within each species, consequently intraspecific gamete values for wood-associated mussels were pooled (regardless of site or embedding protocol, Table 7.1). As no I. (s.l.) simpsoni females from the Setúbal Canyon were analysed within the framework of this PhD, no comparable oocyte diameters exist at this location. Preserved gamete sizes for I. (s.s.)

modiolaeformis measured within the current research consistently fell below those recorded for this species at seeps (Gaudron et al. 2012). However, this is suspected to be an artefact, either due to slight differences in sample processing or marginal variance in microscope calibrations. Spermatozoid acrosome le gths① e e① asi all ① ide ti al① i ① oth① spe ies,① hile① u eleased① atu e ① oo te① dia ete s① fo ① ea h① species differed significantly (Table 7.1). Given that this conflicts somewhat with the inferred homogeneity in released oocyte diameters across both species, it may indicate that the oocytes deemed mature in I. (s.s.) modiolaeformis specimens, may not have been developed fully (these were however, the largest oocytes retained subsequent to spawning). The resulting tanks included holding aquaria, which were used successfully during the observational experiments (Section 3-3.1.1, p. 130) and larval post-hatching tanks, which though built and tested, were ultimately not employed. Unfortunately due to the constraints imposed by the facilities, the species under examination and of working alone when employing the fertilisation protocol, larval development observations remained unattainable. During this process however, a great deal was learnt about other behavioural aspects of these species which proved highly informative, principally in relation to nutrition (section 7-2. 5.3, p.264). This first attempt though not wholly successful, had provided insight and impetus to try to repeat the fertilisation protocol based on what had been learnt. This was scheduled to be repeated in dedicated, temperature-controlled, filtered, closed-aquarium systems in the University of Aveiro and employing a monospeciate assemblage (together removing several of the constraints previously encountered). However, the necessary coincidence of suitable facilities and test organisms stipulated in the objectives of this research was not realised (see Methodology section 5-3, p. 207). However, a limited amount of data concerning adult reproduction (see previous section), fertilisation and early embryonic development were gathering during the course of the live spawning experiments performed in Banyuls-surmer (Table 7.1).

7-2.3.1 Fertilisation rates and embryogenesis

Fertilisation rates were low, despite a 1:1000 oocyte-to-sperm mixing ratio. A combination of working alone (gametes remained on ice for an overly long period of time) and the pooling of oocytes from two species are likely to be the principal causes for this, rather than any natural biological phenomenon. In addition, a large proportion of fertilisation incidences will have been hybridisations, and thus doomed from the start.

However, the formation of a fertilisation membrane, a vitellin jelly coat, and subsequently the first polar body were identified following to gamete mixing. As far as it went, embryogenesis was relatively slow, with the 4-cell stage being first identified at around 11 hours (about double the time taken in shallow-water Mytilus spp.), but typical of deep-sea Bathymodiolins (Arellano and Young 2009). Cleavage patterns habitat types (seeps and bones), though admittedly this is in comparison to a single shell measured by Ritt et al. (2012). In both cases, the small prodissoconch I (and of course, gamete size, Table 7.1) along with the large disparity between hatching and settling size, suggests low maternal energy investment oocyte -1 and a long dispersal period in the plankton. This has previously been cited as evidence for a planktotrophic larval phase (Lutz et al. 1980;Arellano and Young 2009). Having the flexibility of feeding in the plankton has both positive and negative consequences, given the energy that must be spent searching and catching prey, and the elevated risks of predation, associated with an extended planktonic duration. However, a precedence for planktotrophic dispersal, within the top 100 m of the water column has been demonstrated rather conclusively (both in terms of physiological feasibly and by direct capture for the larger seep species Bathymodiolus (s.l.) childressi, (Arellano and Young 2009;Arellano and Young 2011;Arellano et al. 2014). In fact the arguments used by Lutz et al. (1980), have since been applied to multiple examples (see 1-9.3.2, p. 70 for details) from the Bathymodiolinae (s.l.), suggesting that this trait is a ubiquitous one in the subfamily.

7-2.4.2 Post-settlement growth

Although derived estimates for time-averaged minimum growth rates cited herein make some assumptions a out①ea h①spe ies ①g o th,①the ①appea ①to① e①i ①g oss①agreement with the closest related species available for comparison within the bivalves (see section 4-5.1.2, p. 177). The assumptions of the methodology in (s.l.) simpsoni in the current research was not the very largest recovered (Thubaut 2012), comparable growth rates for both species were identified, with those in I. (s.l.) simpsoni being marginally greater and separated by sex (Table 7.1); estimates for each sex used the largest male and female respectively. By way of validation, the growth rates calculated independently for I. (s.s.) modiolaeformis from wood deployments in the NDSF and Lacaze-Duthiers Canyon, were found to be in close agreement. The relatively similar averaged growth rates across quite divergent shell sizes suggests that, much like I. argenteus (Dean 1993), growth in these species is unusually consistent with increasing size, a trait that is probably either a function of their lineage, habitat or both. Discerning the contribution from habitat effects however, would require growth data from these environments, which are currently unavailable. edulis (Cannuel et al. 2009), particularly the emergence of new gill filaments from the distally located budding zone, and the order of development; the descending lamellae of the inner demibranchs developed first, and grew in length and filament number (Table 7.1), before the initial development of the ascending lamella of the inner demibranch. The onset of ascending-lamella development was at 0.77 mm in I.

modiolaeformis, similar to that of the same lamellae in M. edulis hi h①appea ① at①<① ① Cannuel et al. 2009). Although the outer demibranch is known to be fully developed in larger I. (s.s.) modiolaeformis adults (5 mm +), the moment at which this begins developing appears to exceed SL 4.37 mm 31 , in the mussels examined from the eastern Mediterranean. In I. (s.l.) simpsoni, the initiation of ascending lamellae

was not identified at all in the samples from bone deployments in the Setúbal canyon; that said, these observations were based on histology alone, as specimen soft tissues were not examined prior to embedding. By the time the mussels had matured, 31 -33 filaments in each inner descending lamella had developed, where filament number and SL were directly proportional (4-4.5, p. 173).

7-2.4.5 Acquisition of (or infection by) symbionts

Part and partite to measuring gill development was the examination of associated symbiont densities and if possible, the moment that symbionts were first acquired or first infected the host: the relative degree of mediation of each partner remains unknown. A principal result common to both species was the initial absence of symbionts in post-larval plantigrades. The most parsimonious conclusion is therefore that larvae arriving at settlement sites as pediveligers were probably also aposymbiotic. This suggests strict heterotrophy during larval dispersal, which further supports the likelihood of planktotrophic development, since this remains the only means by which nutritional needs could be met (in the absence of both a yolk reserve and symbionts). Despite being the larger-sized species, I. (s.l.) simpsoni actually acquired symbionts at a slightly smaller size both in absolute and relative terms: SLs were 0.43-0.51 mm as opposed to 0.60 mm in I. (s.s.) modiolaeformis, representing quite different proportional changes in shell sizes following settlement (i.e. % increase in SL when compared with the Pd II size; Table 7.1).

7-2.5.2 Symbiont diversity

Using oligonucleotide probes previously designed to target the six known phylotypes for I. (s.s.)

modiolaeformis (Duperron et al. 2008) as well as an additional Bathymodiolus spp. thiotroph phylotype, BangT-642 (Duperron et al. 2005), symbiont diversity was assessed in a subset of the mussels collected from the NDSF (wood and alfalfa-filled CHEMECOLI), Lacaze-Duthiers Canyon (oakwood, 2012;palmwood and wood-filled CHEMECOLI, 2013) and Setúbal Canyon (on cow bones). The level of diversity documented in I. modiolaeformis recovered from oakwood deployments in the Lacaze-Duthiers canyon in 2012 32 were found to house at least two phylotypes, T1-thiotrophs and M1-methanotrophs. Of the two the T1-thitrophs were present at very high densities, raising some questions about how efficiently water can flow through filaments under bacterial saturation. Methanotrophs were at low to moderate densities located at interior regions of the gill, thought to be close to the dorsal gill axis (orientation of dissected gill tissue was difficult to confirm). The presence of such high symbiont levels suggests that the supply of sulphides (and presumably significant traces of biogenic methane) were considerable under the specific conditions experienced by those mussels tested for bacteria (3 of 30 I. (s.s.) modiolaeformis collected that year).

Oakwood samples were contained with netting and placed on the seafloor, so it is thought that sulphides and methane production were through sulphate-reducing bacteria and methanogens internal to the wood [START_REF] Fagervold | Microbial communities associated with the degradation of oak wood in the Blanes submarine canyon and its adjacent open slope (NW Mediterranean)[END_REF]Yücel et al. 2013) and possibly in the underlying sediment and faecal matter of Xylophaga spp. (Fagervold et al. 2014).

Curiously, the two specimens of I. (s.l.) simpsoni collected from the same oakwood substrata experiments (though not on identical pieces of wood) were found to contain moderate levels of putative bacteria, however the phylotypes could not be confirmed with FISH, despite identical fixation, probes and hybridisation conditions. Since the general eubacterial probe employed only weakly hybridised with bacteria, this suggests that issues persisted with the specificity of hybridisation rather than the presence of novel bacterial phylotypes. FISH and DAPI were inconclusive (but suggested a surprisingly low number of symbionts) for the larger palmwood specimens used in the live fertilisation experiments. These specimens had been kept alive in aquaria for several weeks during spawning induction trials p. 144) with access to unfiltered seawater (due to the constraints of the facilities). Access to particulate organic matter from shelf seas (inlet supply), rather than more refractory material within the canyon, may have 32 the year prior to samples collected for live experiments previously). The adaptability of I. (s.s.) modiolaeformis and I. (s.l.) simpsoni to their various environments is astounding and unquestionable. It helps to explain their collectively wide geographical and bathyal distribution, their capacity to take advantage of multiple reducing habitat types, and their ability to adapt to the heterogeneity of these environments. In the latter case, this seems to be in part, through a retained level of motility (a trait common to loosely analogous shallow-water counterparts) which is thought to facilitate access to reduced compounds for chemosymbiosis. In particular however, it is their capacity to supplement chemosymbiotic nutrition with facultative filter feeding that permits such extraordinary flexibility.

7-3.1.1

The lifecycle of Idas (s.s.) modiolaeformis

In terms of what we can now state definitively or infer with confidence, the post-larval to adult period of these mussels lifecycle has benefited the most form this research. To help visualise what has been established for Idas (s.s.) modiolaeformis (as an example), I return to the lifecycle of the bathymodiolins presented in the introduction (Figure 7. 3). In this case the main details summarised in Table 7.1 are included as annotations and as two anatomical schematics, based on multiple dissections. In effect, within a short period of time after settling a d① eta o phosi g①o ① ood① ≈① ①da s ,① ussels①ha e①al ead ①a ui ed① their symbionts. Mounting symbiont densities are compounded by an exponential increase in filament surface area as the gills develop allometrically (Chapter 4, p. 163), and the ascending lamellae begin to de elop① ≈① ① da s .① With① the① e e g ① p o ided① ① he os iosis① a d① filte ① feedi g① the① a e① to a ds① maturity as males is at a relatively fast rate of growth, in comparison to analogous species. So it is that within 4 months, mussels have matured as males. By 8.5 months, bacteria have become isolated to gill filaments only, presumably mediated by the host. As a protandric species, a period of time passes as the mussels grow, before an eventual switch in sex occurs (seemingly after at least 12 months), which requires a brief period as a hermaphrodite. As yet, the exact timing of this switch in sex is not known for either species on wood, however at seeps all functional I. (s.s.) modiolaeformis females were above SL 7 mm while the largest hermaphrodite had a SL 11.6 mm. If such sizes are typical of the species generally (i.e.

independent of growth, then the first females could likely have appeared after 13.5 months (using the growth rate 0.017 mm d -1

), while the most delayed switches in sex would be expected to occur at around 22 months (employing a SL 11.6 mm), assuming immediate settlement in both cases.

7-3.2. Gigantism, Miniaturisation, nutrition and maturation

The①geologi al①age①of①ha itats①is①p o a l ① e ts→seeps→ ood→ hale.①Ho e e ①i ①te s①of①the①e olutio ① of the Bathymodiolinae, fossil records suggest that the mussels appear on seeps first, then wood habitats very closely followed by whale associations when whales became large enough or lipid-rich enough to support sulphophilic organisms, and finally, at vents. It is not hard to imagine an Idas-like ancestor with a single bacterial association taking one of two paths in terms of evolution from the Early to Middle Miocene onwards (Lorion et al. 2013); either down the avenue of gigantism with a dependence on reducing fluids and a reduction in gut functionality, or down the avenue of paedomorphism, retention of full filter feeding capacity and the evolution of superior symbiont flexibility in order to cope with more unpredictable highly ephemeral organic fall conditions. These trends are supported by remarkably consistent patterns of size within the fossil record. These show that seep-colonising mytilids were less than 50 mm in length until the Early Miocene (Amano and Little 2005;[START_REF] Kiel | New and little known mollusks from ancient chemosynthetic environments[END_REF][START_REF] Saether | New fossil mussels (Bivalvia: Mytilidae) from Miocene hydrocarbon seep deposits, North Island, New Zealand, with general remarks on vent and seep mussels[END_REF][START_REF] Amano | New fossil Bathymodiolus (sensu lato)(Bivalvia: Mytilidae) from Oligocene seep-carbonates in eastern Hokkaido, Japan, with remarks on the evolution of the genus[END_REF], and then their dimensions abruptly increased from SL 10 cm in the Mid-Miocene to more than 30 cm in extant species. This flexibility in the subfamily (s.l.) as a whole has resulted in members being represented at nearly all types of reducing habitat. Even in the larger-sized bathymodiolins that have sacrificed some complexity in their gut for a greater reliance on symbionts, filter feeding still persists (Page et al. 1990;Page et al. 1991). This behaviour may be a widespread trait in the subfamily, but one which has gone out of regular use in some species in the absence of a strict need for it.

Vents and seeps appear to provide higher energy environments with a greater amount of colonisable space, in comparison to typically spatially limited and ephemeral organic falls. These factors may have dictated the extremes that exist in terms of size at these habitats [START_REF] Saether | New fossil mussels (Bivalvia: Mytilidae) from Miocene hydrocarbon seep deposits, North Island, New Zealand, with general remarks on vent and seep mussels[END_REF]. Mussels inhabiting relatively sparse organic debris might have benefited from heightened paedomorphism which would constrict the growth of the species, reduce spatial competition and permit the allocation of energy resources towards reaching maturity sooner (Lorion et al. 2013). The current research (and other studies) demonstrate that mussels from organic falls reach sexual maturity quickly and at much reduced sizes (Tyler et al. 2009;Ockelmann and Dinesen 2011;Gaudron et al. 2012;Laming et al. 2014).

7-3.3. Convergence versus divergence

Developmental patterns of germ-line cell migration, size-at-first-maturity, gill growth and symbiont acquisition and localisation are presented for the post-larva to adult transition. Evidence for protandry supports previous findings (Gaudron et al. 2012) while data on gametes, larval shells and the likelihood of feeding, being aposymbiotic, fit with the assumptions made for many of the members of the subfamily.

These aspects are what have made them such an intriguing group to study. Biological traits aside from symbiosis appear to reflect conserved ancestral phylogenetic traits, particularly in aspects of development communities (Duperron 2010;Duperron et al. 2013). This suggests that for these mussels, interspersed softsediment environments do not provide refuges and must be traversed in order to populate new reducing habitat sites. This either reinforces the hypothesis that larval dispersal must be considerable in order to maintain the apparent levels of connectivity between distant sites (e.g. Bathymodiolus sp., Schultz et al. 2011) or of course, it suggests that reducing-habitat frequency may be greatly underestimated.

Larval development takes many forms, with many species adopting a planktonic phase (over 70% in the Atlantic: [START_REF] Thorson | Reproductive and larval ecology of marine bottom invertebrates[END_REF][START_REF] Connell | The influence of interspecific competition and other factors on the distribution of the barnacle Chthamalus stellatus[END_REF]Milekovsky, 1971;Vance, 1973). Of the larval phases that are planktonic, planktotrophy is the most prevalent mode in marine invertebrates, particularly in the tropics [START_REF] Thorson | Reproductive and larval ecology of marine bottom invertebrates[END_REF]Pechenik, 1999), but also in deep sea environments (Young 2003). This larval mode requires minimal oocyte investment, as the oocytes hatch quickly into heterotrophic, planktonic larvae with a minimal or absent yolk reserve. Large numbers of young may be produced per unit of energy expended (high fecundity), and the capacity to feed permits an extended period of larval transport, which may facilitate dispersal (Cowen and Sponaugle 2009). However their planktonic prey is dependent on fluctuating environmental conditions, so successful larval development is constrained to periods when food availability is high (Pechenik, 1999). Moreover, the cost of prolonged planktotrophy is in increased mortality from predation and abiotic pressures (e.g. over-dispersal or dispersal into unsuitable environment). Mortality is typically very high at up to 99% before settling [START_REF] Thorson | Reproductive and larval ecology of marine bottom invertebrates[END_REF]Milekovsky, 1971).

Evidently, with data being accumulated on the distribution and phylogeny of deep-sea metazoan species in chemosynthetic habitats from many of these underrepresented regions, there is an immediate need to resolve how dispersal is acting to influence connectivity within and perhaps between these environments to better understand their resilience to disturbance, particularly in light of the gathering interest in the deepsea for mining minerals (Van Dover 2011a).

7-3.5.3 Resilience to and recovery from disturbances

Despite the fragmented knowledge available for habitat connectivity and stability in the deep sea, some indications of resistance to disturbance can be surmised based on habitat characteristics and some oppo tu isti ①studies①of① e olo isatio ①of① as e t ①ha itat.①The① esista e of a community to disturbances imposed by human activities will depend on its predisposition to similar levels of natural disturbance.

Accordingly, much emphasis has been placed on the need to better describe the patterns of distribution and connectivity, which exist courtesy of larval dispersal in the deep-sea, particularly in spatially isolated reducing habitats. This is an expanding and evolving field, evident for example, from the changeable nature of accepted biogeographic provinces described for vent species globally (Bachraty et al. 2009;Rogers et al. 2012). Clearly, as more sites are discovered and explored, the resolution and thus reliability of the global biogeography for vent species will likely increase. Contrastingly, in hydrocarbon seeps, or at least those located upon the Atlantic Equatorial Belt, isobathyal distance appears to be of less importance than that of depth in determining species composition and similarity between regions, particularly between 1000m and consequences of stress, tolerance and parental effects on larval development. Indeed the next decades of deep-sea larval biology can only be improved by such advances.

Deep-sea marine science is now at a critical juncture in its history. The advent of technology for scientific exploration was only made possible by the investment of telecommunications companies and those interested in offshore hydrocarbon fuel extraction. Ironically, it is through these avenues of development that ROVs have become so accessible to scientific ocean exploration. Deep-sea environments are subject to multiple levels of environmental impact, including deep-sea fisheries and imminent mineral-extraction exploration using huge underwater drilling apparatus. This comes at a time when we still have so much to learn and gain from describing these habitats more definitively ( Van Dover 2011b).

At present, a niche exists for new researchers to fill: that of the deep-sea larval developmental biologist.

Such a research niche is rare in biological studies and whilst the challenges it represents are not insignificant, the importance of the potential results of such research cannot be overstated. Studies examining coastal systems, which have acted to shape the way in which we approach the discipline of larval biology, reproductive kinetics, supply, recruitment into the adult population and thus the sustainability of community compositions remain seminal pieces of marine scientific literature (e.g. [START_REF] Hatton | Essais de bionomie explicative sur quelques espèces intercotidales d'algues et d'animaux[END_REF][START_REF] Coe | Resurgent populations of littoral marine invertebrates and their dependence on ocean currents and tidal currents[END_REF][START_REF] Connell | The influence of interspecific competition and other factors on the distribution of the barnacle Chthamalus stellatus[END_REF][START_REF] Thorson | Some factors influencing the recruitment and establishment of marine benthic communities[END_REF][START_REF] Woodin | Polychaete abundance patterns in a marine soft-sediment environment: the importance of biological interactions[END_REF][START_REF] Gaines | Larval settlement rate: a leading determinant of structure in an ecological community of the marine intertidal zone[END_REF][START_REF] Roughgarden | Recruitment dynamics in complex life cycles[END_REF][START_REF] Connolly | A latitudinal gradient in recruitment of intertidal invertebrates in the northeast Pacific Ocean[END_REF] ...①Ge eti s① a ot①a s e ①every uestio

Figure 1 . 1

 11 Figure 1.1 Various equipment used in deep-sea research ........................................................................................................

Figure 1 . 2

 12 Figure 1.2 The evolution of manned submersible technology ..................................................................................................

Figure 1 . 3

 13 Figure 1.3 Rates of habitat discovery in the deep sea ...............................................................................................................

Figure 1 . 4

 14 Figure 1.4 Impacts in the deep sea ..........................................................................................................................................

Figure 1 . 5

 15 Figure 1.5 Map of Marine Protected Areas generated by UNEP World Conservation Monitoring Centre .............................

Figure 1 . 6

 16 Figure 1.6 Distribution of deep-sea habitats within an ocean bordered by a passive and an active margin ..........................

Figure 1 . 7

 17 Figure 1.7 Data and predictions for hydrothermal vents, hydrocarbon seeps, gas hydrates, and organic falls ......................

Figure 1 . 8

 18 Figure 1.8 Methane production in a hydrocarbon seep environment ....................................................................................

Figure 1 . 9

 19 Figure 1.9 Chemosymbioses in different marine habitats .......................................................................................................

Figure 1 .

 1 Figure 1.10 The biogeochemistry of hydrothermal-vent fluid formation................................................................................

Figure 1 .

 1 Figure 1.11 Eleven-provinces biogeography for vent species .................................................................................................

Figure 1 .

 1 Figure 1.12 Flow-diagram depicting those principal themes and factors in the marine environment that affect the lifecycle of a chemosymbiotic invertebrate species ................................................................................................................

Figure 1 .

 1 Figure 1.13 Critical junctures in the lifecycle of chemosymbiotic organism ...........................................................................

Figure 1 .

 1 Figure 1.14 Theoretical relationships between the spatial and temporal aspects of larval transport, dispersal, and (reproductive) population connectivity for a sedentary species .............................................................................................

Figure 1 .

 1 Figure 1.15 The combined processes contributing to larval dispersal and survival and thus demographic connectivity .......

Figure 1 .

 1 Figure 1.16 The cost of reciprocal benefit between two organisms (A and B). .......................................................................

Figure 1 .

 1 Figure 1.17 Endosymbiotic theory...........................................................................................................................................

Figure 1 .

 1 Figure 1.18 Dispersal, and links with symbiont transmission .................................................................................................

Figure 1 .

 1 Figure 1.19 Multigene tree for Bathymodiolinae ....................................................................................................................

Figure 1 .

 1 Figure 1.20 Global distribution of members of the Bathymodiolinae: genus Idas ..................................................................

Figure 1 .

 1 Figure 1.21 Global distribution of members of the Bathymodiolinae: genus Bathymodiolus ................................................

Figure 1 .

 1 Figure 1.22 Habitat use in chemosymbiotic bivalves ..............................................................................................................

Figure 1 .

 1 Figure 1.23 Hypothetical lifecycle of a Bathymodiolin ............................................................................................................

Figure 1 .

 1 Figure 1.24 A diagrammatic comparison of pediveliger to plantigrade morphoanatomy ......................................................

Figure 1 .

 1 Figure 1.25 Patterns of gill ontogeny in Mytilus galloprovincialis ...........................................................................................

Figure 2 . 1

 21 Figure 2.1 Idas (s.l.) simpsoni and Idas (s.s.) modiolaeformis from Ritt et al. 2012 ..............................................................

Figure 2 . 2

 22 Figure 2.2 Methods employed to collect specimens .............................................................................................................

Figure 2 . 3

 23 Figure 2.3 Overview of sampling locations ............................................................................................................................

Figure 3 . 1

 31 Figure 3.1 Acclimation aquarium for the maintenance of Idas spp. ......................................................................................

Figure 3 . 2

 32 Figure 3.2 Wood-olo isatio ①e pe i e t s①lo atio ①i ①the①La aze-Duthiers Canyon, western Mediterranean ..................

Figure 3 . 3

 33 Figure 3.3 Shell morphology of specimens in this study separated by halpotype and species .............................................

Figure 3 . 4

 34 Figure 3.4 Images of liberated ooctes during spawning and following fertilisation (pooled from both species) ..................

Figure 3 . 5

 35 Figure 3.5 Haematoxylin and Eosin Y staining in 7mm histological sections from both species ...........................................

Figure 3 . 6

 36 Figure 3.6 Detail of the dissection performed on IB1, a small I. (s.s.) modiolaeformis male ................................................

Figure 3 . 7

 37 Figure 3.7 Fluorescence in situ hybridisations performed on gill tissue of I. (s.s.) modiolaeformis and I. (s.l.) simpsoni .....

Figure 3 . 8

 38 Figure 3.8 Stable Isotopes plot for I. (s.s.) modiolaeformis and I. (s.l.) simpsoni from various wood substrata ...................

Figure 4 . 1

 41 Figure 4.1 Micrographs of Idas (s.s.) modiolaeformis specimens examined .........................................................................

Figure 4 . 2

 42 Figure 4.2 Plot of SL against SH of Idas modiolaeformis .......................................................................................................

Figure 4 . 3

 43 Figure 4.3 Micrographs of Haematoxylin & Eosin-stained mid-sections from Idas (s.s.) modiolaeformis of various sizes ...

Figure 4 . 4

 44 Figure 4.4 High magnification micrographs of Haematoxylin & Eosin-stained gonad and gill tissue from Idas (s.s.) modiolaeformis of various sizes .............................................................................................................................................

Figure 4 . 5

 45 Figure 4.5 Putative bacteria lining the lateral-to-abfrontal regions of gill filaments in Idas (s.s.) modiolaeformis ...............

Figure 4 .

 4 Figure 4.6 Gill-fila e t① ou ts①i ①the①i e ①de i a h s①des e di g①la ella①of①Idas① odiolaefo is ...............................

Figure 4 . 7

 47 Figure 4.7 Summary of pivotal periods in the development of Idas (s.s.) modiolaeformis as a function of size ...................

Figure 5 . 1

 51 Figure 5.1 Micrographs of Idas (s.l.) simpsoni specimens examined ..................................................................................... Figu e① . ①Plot①of①"L①agai st①"H①of① I. ① s.l. ①si pso i .............................................................................................................

Figure 5 . 3

 53 Figure 5.3 Overview of Haematoxylin and Eosin-Y developmental histology in Idas (s.l.) simpsoni ......................................

Figure 5 . 4 Figure 5 . 5

 5455 Figure 5.4 Serial Haematoxylin and Eosin-Y stained transverse sections of a juvenile Idas (s.l.) simpsoni (SH 0.59, SL 0.92)213 Figure 5.5 Serial Haematoxylin and Eosin-Y stained sagittal sections of a juvenile Idas (s.l.) simpsoni (SH 0.59, SL 1.05) ....

Figure 5 .

 5 Figure 5.6 Gill-fila e t① ou ts①i ①the①i e ①de i a h s①des e di g①la ella①of①Idas① odiolaefo is ...............................

Figure 5 . 7

 57 Figure 5.7 Analysis of symbiont localisation and the period of acquisition using fluorescence microscopy .........................

Figure 5 . 8

 58 Figure 5.8 Fluorescence in situ hybridisations of one of the largest specimens not confirmed as an adult, and a small juvenile ...................................................................................................................................................................................

Figure 6 . 1

 61 Figure 6.1 Live observations, shell morphology and anatomical analysis of Idas argenteus .................................................

Figure 7 . 1

 71 Figure 7.1 Idas argenteus morphometry with increasing size (multiple studies and sample sites), and its anatomy ...........

Figure 7 .

 7 Figure 7.2 distribution of Idas (s.s.) modiolaeformis and Idas (s.l.) simpsoni based on literature and current research ......

Figure 7 . 3

 73 Figure 7.3 The lifecycle of Idas (s.s.) modiolaeformis ............................................................................................................

Figure 1 . 1

 11 Figure 1.1 Various equipment used in deep-sea research A) Little Hercules ROV exploring the Mid-Cayman Rise. Image courtesy of NOAA Okeanos Explorer Program, MCR Expedition 2011. B) Alvin submersible: plan for the 2013 refurbishment. Illustration by E. Paul Oberlander, for WHOI. C) 3-D multibeam sonar image of Norfolk Canyon, eastern United States, looking down the axis toward the canyon mouth (Steve Ross: Deepwater Mid-Atlantic Canyons cruise 2011). D) A benthic rover a mobile physiology lab assessing carbon supply and demand at the seafloor, being tested at the cabled observatory MARS. Image from MBARI website. E) Multicorer with 6 polycarbonate coring tubes. KC Denmark research equipment. F) Calcein fluorescence-labelling chamber for marking organisms after which growth can be tracked. Inset is view of coral inside. 2014 Nautilus Exploration Program. G) A Raman spectrometer, which uses laser excitation and measurement of molecular vibration, specific to the chemical bonds and symmetry of molecules. Image from MBARI website

Figure 1 .

 1 Figure 1.1B). The most recently constructed submersible to be used for scientific exploration is the DEEP Challenger submersible (Figure 1.2B). However, exploration of the deepest parts of the ocean remains challenging exemplified by the p o a le①i plosio ①of①the①'OV① Ne eus ①du i g①e ploration of the Kermadec trench (WHOI Media-Relations_Office 2014). ROVs today can be mounted with all the equipment necessary

Figure 1 . 2

 12 Figure 1.2 The evolution of manned submersible technology A) The Bathyscaph Trieste [i], made the first voyages to the Marianas Trench. View of pilot sphere from front [ii]. B) The CHALLENGER DEEP submersible having just reached a predetermined depth (during mid-depth test dive). All bodywork except the pilot sphere is made from advanced syntactic foam to reduce operating weight. Scuba divers in support during pre-dive checks [ii]. C) The various incarnations of Alvin, the figurehead submersible for the Woods Hole Oceanographic Institute, whose debut dive was in 1964.

  floor >500m depth, for a predicted ca. 201,000 seamounts from 100-6700 m in height, Hillier and Watts 2007). The oceanic trenches normally located at sites of o ea i ①su du tio ,① ep ese t①≈ %①of①the①ho izo tal① o e age,①though① o side a l ① o e①i ① e ti al① elief① with depths of nearly 11 km in the Marianas Trench (10908 m, DEEPSEA CHELLENGER, 2013). Reducing environments and the habitats therein currently represent incidental features by comparison, with hydrothermal vents on spreading ridges, back-arc basins and volcanic seamounts, hydrocarbon seeps, which include methane seeps, gas hydrates, asphalt flows and oil seeps, cold-water coral reefs, whale falls and oxygen minimum zones (OMZ) representing <1% of the ocean floor, based on current knowledge.

Figure 1 . 3

 13 Figure 1.3 Rates of habitat discovery in the deep sea Depicted is a plot of key moments in the last 160 years at which various aspects of novel habitats have been recognised by the scientific community. Highlighted, are those habitats most relevant to the current research presented in this thesis. Adapted from Ramirez-Llodra et al. 2010, with annotation from data therein.

1- 4

 4 Deep-sea conservation: research and challenges 1-4.1. The paucity of baseline data ②lthough①the① e e t①ad e t①a d① apid①de elop e t①of①e uip e t① apa le①of①sa pli g①the①o ea s①depths① has enabled the exploration of habitats under pressure, technical and habitat-specific biases persist in our understanding of deep-sea biology and ecology. While global patterns in bathymetry, marine hydrography and geochemical processes are being resolved, to date, many deep-sea community-based studies are systematically hindered by low sample sizes and a lack of replicate sampling both temporally and spatially.

1- 4 . 1 .

 41 Identified challenges of deep-sea conservation 1-4.1.1 An impact assessment of the deep-sea In the first assessment of its kind for the deep sea, Ramirez-Llodra et al. (2011) collated the documented reports of anthropogenic impacts on deep-sea environments. The themes (Figure 1.4 ① e e① disposal ① (human-o igi ① aste① a d① litte ,① li ate ① o ea ① a idifi atio ① a d① li ate① ha ge,① e a e ated① ① a th opoge i ① a ti ities ① a d① e ploitatio ① the① i pa ts① of① deep-sea resource prospecting and exploitation). These were considered for 12 deep-sea habitats: mid-ocean ridges (MOR), sedimentary slope (SLO, background fauna), canyons (CA), seamounts (SM), cold-water coral reefs including associated species (CO), active hydrothermal vents (VENT), cold seeps (SEEP), oxygen minimum zones (OMZ), abyssal plains (AP), manganese nodule provinces (MnAP), trenches (HT), and bathypelagic water column (BP; see Ramirez-Llodra et al. 2011 for more detailed habitat descriptions). Based on their collective judgement each of the impacts was assessed in detail as far as data would allow. Mea ① disposal ①i pa t①fo ①all①ha itats① as①highest①i ①the①past, on account of a recent worldwide a ① o ① offsho e① aste① disposal.① Mea ① i pa t① i di es① fo ① e ploitatio ① i eased① fo ① those habitats in which current or predicted commercially valuable resources occur (particularly mineral extraction and fish sto ks .①It① as①i ① ea ① li ate ①i pa ts①ho e e ①that①the①g eatest①i eases① e e①p edi ted① efle ti g①the① hitherto-underestimated impact that climate change, and in particular, ocean acidification is expected to have upon the deep sea in the foreseeable future (Smith et al. 2008). 1-4.1.2 Areas of focus for conservation Ramirez-Llodra et al. (2011) identified four deep-sea habitats that are at greatest risk in the near future. These were upper-slope sedimentary benthos (accumulative effects of climate change-related shifts in nutrient input, ocean acidification and spreading hypoxia; global fishing effort), cold-water corals (for identical reasons), seamount communities (fishing effort, fishing gear damage to the benthos, climate change-driven changes in circulation and increased stratification), and finally the canyon-based benthos (innovative fishing activity, increases in refuse, chemical pollution and nutrient loading, climate changedriven changes in circulation and increased stratification).

Figure 1 . 4

 14 Figure 1.4 Impacts in the deep seaSchematic of all impacts on the deep sea considered in[START_REF] Ramirez-Llodra | Man and the Last Great Wilderness: Human Impact on the Deep Sea[END_REF]. Each impact was initially scored from 0-5 based on the collective expertise of 23 deep-sea researchers (0 = no impact,① ①=① Major anthropogenic impact including death of all life at the point of impact. Likely to have subsequent regional effects ,① ith①a①spe t u ①of①i pa ts①i ① et ee .①②① at i ① as①the ① o piled①to① identify the presence or absence of antagonistic interactions between impacts. The cross-links displayed are the results of this analysis. CFCs = Chlorofluorocarbons; PAHs = Polycyclic Aromatic Hydrocarbons. From[START_REF] Ramirez-Llodra | Man and the Last Great Wilderness: Human Impact on the Deep Sea[END_REF]

Figure 1 . 5

 15 Figure 1.5 Map of Marine Protected Areas generated by UNEP World Conservation Monitoring Centre This map is the most recent official map of MPAs available, generated by UNEP World Conservation Monitoring Centre (UNEP WCMC) using data from the World Database on Protected Areas (WDPA) potentially destructive nature of commercial deep-sea exploration and resource extraction deserves a better notion of what we have to lose. Such threats to a domain for which so much remains unknown or poorly studied, have created the impetus to gather foundational data that can reliably inform decisions regarding conservation. The sustainable and ethical use of deep-sea resources can only be realised through ongoing exploration of poorly surveyed regions of the oceans, routinely assessing biodiversity and ecosystem function in known habitats and initiating measures to optimise conservation efforts (German et al. 2011; Ramirez-Llodra et al. 2011).

1 .

 1 Figure1.7) and regions of elevated wood and macroalgal offshore transport. Historic sighting and catch data for whales suggest that certain regions of the ocean are likely to see disproportionately high numbers of natural whale deaths. As part of the Census for Marine Life such data has been compiled for the most frequently sighted whale species[START_REF] Smith | Spatial and Seasonal Distribution of American Whaling and Whales in the Age of Sail[END_REF]; redrawn in Figure1.7B). Available whale-fall data appears to agree with whale sightings and catch data (Figure1.7B). By layering global remote sensing data of the present-day distribution of forests worldwide and the main (i.e. navigable) global network of rivers, their catchment areas and their associated mean monthly discharge, it is possible to identify, semiobjectively, regions of the planet that are most likely see the greatest proportion of coastal efflux of woody material (Figure1.7C). Together these maps suggest that open abyssal expanses are dominated by soft sediments, but punctuated with volcanic and geothermal features, which include hydrothermal vents.

Figure 1 . 6

 16 Figure 1.6 Distribution of deep-sea habitats within an ocean bordered by a passive and an active marginThe schematic displayed attempts to summarise the principal processes and resulting habitats which are known in deep-sea environments. Not marked but occupying the vast majority of the deep sea, are soft-sediment environments. See text for description of each habitat. Habitats which relate directly to this research are highlighted in blue (wood falls) and pink (whale falls). Conceptual schematic compiled by S.R. Laming.

Figure 1 . 7

 17 Figure 1.7 Data and predictions for hydrothermal vents, hydrocarbon seeps, gas hydrates, and organic falls A) Map compiled from vent, seep and hydrate distribution data from InterRidge Vents and ChEss Databases and the MARUM website. B) Reproduction of data on caught and sighted whale species since records began from Smith et al. 2012, overlaid with whale fall locations from the ChEss Database. C) Objective predictions (by S.R. Laming) for wood falls accumulations based on the locations of forested areas on land (2009 global forest/non-forest map from the Japan Aerospace Exploration Company) and river distribution/discharge data (navigable rivers from the CIA World DataBank II/ Major river discharge data from polylines of 687 rivers, classified by discharge from the Global River Discharge Centre, inset map).

  September 2010, over 150 confirmed deep-sea vent sites and over 130 cold seep sites had already been studied with respect to the fauna communities associated with them[START_REF] Baker | Map of Hydrothermal Vent Sites [Online[END_REF][START_REF] German | Diverse styles of submarine venting on the ultraslow spreading Mid-Cayman Rise[END_REF] Van Dover 2011b), illustrated in Figure1.7. Approximately 70 additional unconfirmed vent-site locations have been postulated, extrapolated from water-chemistry anomalies recorded during oceanic surveys (Van Dover 2011b; Figure1.7), as well as just less than 80 known methane hydrate sites, though not all in marine environments.The geothermal processes that form vent fluids are quite well established for basalt-hosted hydrothermal activity[START_REF] Tivey | Deducing patterns of fluid flow and mixing within the TAG active hydrothermal mound using mineralogical and geochemical data[END_REF] Figure 1.10). Initially, seawater containing Mg 2+ and SO 4 2-ions among others (both notable by their removal or reduction in vent end-member fluids), must permeate faulted and fissured igneous crust often rich in metal ores and minerals. If the crust is subject to proximal volcanism, infiltrating water can be chemically altered by a number of ion exchanges with the rock as it pe et ates①the①deepe ① e ha ge ①zo e① Ti e ① .①B ①the①ti e①the①deepest① HT① ea tio ①zo e①is① ea hed① (Figure 1.10) under elevated temperatures and pressures, evolved fluids are notably stripped of Mg 2+ increased fluid acidity and caused the precipitation of CaSO 4 anhydrite. Water and Fe-bearing minerals reacting together create conditions favouring the reduction of SO 4 2-to a mixture of H + , HS -and H 2 S. Thus, reactions occurring in the recharge zone result in an evolved fluid that is somewhat acidic, anoxic, rich in alkalis and Mg-poor compared with entering seawater. The reaction zone sees the solubilisation of sulphur and metals (e.g., Cu, Fe, Mn, Zn, Li, Pb) from the crust, usually a period of phase separation into brines and vapour (the critical point between both being 407 °C at 298 bar, Tivey 2007) and varying degrees of magmatic volatiles degassing into the fluid (CO 2 , CH 4 , H 2 ). As a o se ue e,① e d-e e ①fluids①a e① uo a t①a d①as e d① apidl ①to①the① ust-sea interface via the path of least resistance (Figure 1.10). On contact with seawater and with a fall in temperature and pressure, some minerals are precipitated to form chimneys and deposits (e.g. CuFeS, FeS 2 , ZnS, Tivey et al. 1995). Endmember fluids from on-axis peridotite-hosted (ultramafic) hydrothermal activity differs by being marginally less acidic and H 2 and CH 4 -e i hed① ①se pe tisatio ,①e ept① 'ai o ①M②',① hi h①is①H 2 and CH 4 -enriched but more acidic and Fe-saturated). The single known off-axis ultramafic vent site exhibits atypically alkaline end-e e ① fluids① Lost① Cit ,① off-MAR, H 2 and CH 4 -enriched, pH 9-10, Kelley et al. 2001; Kelley et al. 2005). Andesite-hosted hydrothermal activity at back-arc basins in contrast are enriched in Ca, Mn, Si, andFe and even more acidic (due to magmatic off-gassing of SO 4 ), relative to basalt-hosting.

Figure 1 . 8

 18 Figure 1.8 Methane production in a hydrocarbon seep environment The < and > signs indicate if fluxes are likely to be smaller or larger in reality, respectively, based on the underlying uncertainties. Figures are derived from literature reviewed by Boetius and Wenzhofer 2013. Brown arrows indicate fluxes in diffusion driven systems, blue in advective (seep) systems. POC particulate organic matter, AOM anaerobic oxidation of methane in the sulphate-methane transition zone. From Boetius and Wenzhofer 2013

m 2 ,

 2 . When averaged over the entire deep-sea①floo s① surface area (3.6 x 10 8 km 2 ,① hale① falls① o ld ide① p o a l ① o l ① ep ese t① ≈ . %① of① the① a kg ou d① particulate organic carbon (POC) flux to the deep-sea even under the most oligotrophic central gyre waters (Baco and Smith 2003, details and assumptions therein). However the reality at the habitat scale is that whale falls provide massive influxes of labile organic matter to localised areas of the seabed (typically around 50 Baco and Smith 2003). These can be in the region of 2000 years worth of background POC flu ①at①a ssal①depths①i ①a①si gle①pulse① Ba o①a d①" ith① .①What s① o e,①if① o se ati e①p edi tio s①fo ① whale-fall frequencies based on natural surface aggregations are accurate (e.g. nearest neighbour distance of <16 km for gray whales within their known North Pacific range, Baco and Smith 2003) then whale-fall

  generations. Experiments employing coincident reducing habitats which display similarities in fluid emissions generated by different chemical processes, have identified little overlap in between-habitat communities (e.g. whale-fall community in comparison with nearby seeps and vents, Japan,[START_REF] Fujiwara | Three-year investigations into sperm whale-fall ecosystems in Japan[END_REF]; organic falls in relation to mud volcano [MV] seeps, Gulf of Cadiz,Cunha et al. 2013).1-5.4. Biogeography of deep-water ecosystemsSeveral projects within the CoML initiative (e.g. ChEss, COMARGE, MAR-ECO, and CenSeam) set out to define the biogeographic distribution of deep-water chemosynthetic and hotspot provinces worldwide, while gaining a better understanding of the processes maintaining these ecosystems. The biogeography of at least one of the deep-sea habitats concerned, hydrothermal vents has been investigated in some detail, however disagreement persists over differing interpretations. Based on a biogeographic model using phylogenetic data available at the time,Bachraty et al. in 2009 suggested that at least six major hydrothermal provi es①i ①the① o ld s①o ea s①e ist① i ①ag ee e t① ith①Va ①Do e ①et①al.① ,① ith①se e ① paths for dispersal wherein the Northern East Pacific Rise (NEPR) was identified as a likely dispersion centre.

Figure 1 .

 1 Figure 1.11 Eleven-provinces biogeography for vent speciesBased on Vent provinces are resolved comprising the Mid-Atlantic Ridge, the ESR, the northern, central, and southern East Pacific Rise, a further province located south of the Easter Microplate, four provincesin the western Pacific, and a further Indian Ocean province. FromRogers et al. 2012

Figure 1 .

 1 Figure 1.12 Flow-diagram depicting those principal themes and factors in the marine environment that affect the lifecycle of a chemosymbiotic invertebrate species The diagram displays factors within each of 4 themes that influence the lifecycle of a generic deep-sea invertebrate with chemoautotrophic symbionts. Interactions between components of themes are indicated. The diagram, though not exhaustive, helps to convey the complexities that face marine organisms (and scientists studying their lifecycles). The highlighted components (*) are those areas into which this PhD investigates directly or for which it provides additional insight indirectly. Arrow widths indicate relative perceived importance/impact of relationships. Bi-directional coloured links are highlighted with broken transitions. Compiled by S.R. Laming

Figure 1 .

 1 Figure 1.14 Theoretical relationships between the spatial and temporal aspects of larval transport, dispersal, and (reproductive) population connectivity for a sedentary speciesSurvivorship is not represented for the sake of clarity. In both figures the sum of larval transport distances is assumed to be greater than the ultimate (vectoral) dispersal distance. White circles are locations in space with coordinates x-y at times t in figure A, and x-y-z at times t in figure B (a 3D interpretation of the theory behind A, accounting for depth), though the white circles are not intended to be directly comparable across the figures. All locations are pelagic except x0,y0 and x4,y4 (in A), which are benthic. N.B. between settlement and sexual maturity there is only a passage of time. Figure A taken from Pineda et al Pineda et al.., 2007, figure B was drawn by S.R. Laming.

Figure 1 .

 1 Figure 1.15 The combined processes contributing to larval dispersal and survival and thus demographic connectivityThe spawning output (abundance) for each of the populations (represented by a single bands on the left) is spatially variable due to variability in the adult reproductive capacity of each. Advection and diffusion causes mixing and dilution of larvae: crossing lines and increases larval distribution spatially (expanded size, lighter colour). Larval behaviour (e.g., vertical migration), acts to mitigate diffusion through biophysical interactions; spatial extents decrease and densities increase. Spatially dependent and therefore variable larval loss (mortality: predator/prey interactions, availability of settlement habitat, and post-settlement survivorship with carryover effects) result in ongoing depletion of survivors (progressively smaller bands). Evidently the source (left) and sink locations (right) have markedly differing spatial distributions and densities providing a picture of the scale of successful dispersal and demographic patterns (from Cowen and Sponaugle 2009).

Figure 1 .

 1 Figure 1.18 Dispersal, and links with symbiont transmissionDepicted, are the various proposed scenarios for transmission of symbionts, in the context of dispersal in bivalves. A) Displayed are the various dispersal strategies of bivalve molluscs.[1] Teleplanic, with (typically) planktotrophic larvae dispersing either in the euphotic zone[1] or in deeper water layer(2). Larvae can grow during dispersal and can settle far from the natal site. Shorter-distance dispersal (3) can be achieved by lecithotrophic larvae, which live on maternal reserves and settle closer to their natal site in theory. B) Bacterial symbionts can be maternally inherited (1), acquired from free-living populations of bacteria at site of origin (2a, green bacterium) or at site of settlement (2b, yellow bacterium), or laterally acquired from another host either at site of origin (3a, blue bacterium), or at site of settlement (3b, red bacterium). Situations 1, 2a and 3a lead to host-symbiont co-dispersion, as emphasized by the colour arrows. Different transmission modes can co-exist within a given species. Redrawn from Duperron et al. 20131-7.3.4 Symbiont localisation and compartmentalisation

seeps,

  Bathymodiolus boomerang von Cosel and Olu 1998), Vesicomyidae (e.g. Calyptogena spp. Boss and Turner 1980) and the Solemyidae (deep-sea genus Acharax, shell lengths up to 200 mm (A. bartschii, Taylor and Glover 2010). The highly productive environment at vents (and some seeps) are thought to permit rapid growth rates (e.g. the mytilid, B. thermophilus, Nedoncelle et al. 2013; the vesicomyid Calyptogena magnifica,

1 ) 3 ) 4 )

 134 marisindicus and Gigantidas gladius, respectively), and from 80-360 mm at hydrocarbon seeps(B. hirtus and B. boomerang, respectively). Paedomorphism has been posed as one means by which this has occurred i ①the①s alle ① ath odiolifo ① ussels① G io①et①al.① ,①though①it① ould①e uall ① e① atte ①of①suppl ① versus demand, where reducing environments that sustain the largest species within this (and other) symbiotic bivalve families are those where energy supply can meet the demands of rapid or extended growth. This is supported generally by the differences in size between bathymodiolin species from vents and seeps and those found at organic falls, being considerably smaller, though exceptions exist in I.macdonaldi at the Garden Banks seeps, Gulf of Mexico(Gustafson et al. 1998) and Adipicola sp. and I. washingtonius at vents on the Juan de Fuca ridge (McKiness et al. 2005; Southward 2008of atypically massive bivalves (e.g. the genera Bathymodiolus and Calyptogena) at vents and seeps, bivalves engaging in chemosynthetic and heterotrophic bacterial symbioses have been found in a comprehensive array of reducing environments ranging from organically enriched sands and muds, mangroves, seagrass beds, accumulations of sunken vegetative debris, offshore sewage sites and nektonic megafaunal carcasses (Taylor and Glover 2010). In the deep sea, bivalve assemblages regularly figure as dominant macrofaunal components of reducing habitats (Duperron 2010).Understanding the lifecycle biology of model species from this group can provide insight into the different, habitat-specific processes that allow communities to persist (their suitability for lifecycle studies is considered in more detail as part of the following chapter on the rationale for the research undertaken during the PhD) Bivalves engaging in chemosymbiotic associations have been reported or postulated for seven distinct families in the deep sea that vary markedly in their basic anatomy and life habits (Taylor and Glover 2010), based on information from several reviews and extraneous incidental records (Tunnicliffe et al. 1998; Taylor and Glover 2010; Brissac et al. 2011; Roeselers and Newton 2012). These are the Solemyidae, Mytilidae, Lucinidae, Thyasiridae and Vesicomyidae, with more speculative accounts in the Nucinellidae (synonym = Manzanellidae) and Montacutidae, where the first five are by far the most dominant in reducing environments, when all habitats are considered. At present, these proven (or hypothesised) symbiotic bivalves are recorded across deep-sea reducing habitats as follows: Hydrothermal vents (all types): a. the Mytilidae, genera Bathymodiolus, Gigantidas, Idas, Adipicola, Benthomodiolus, Vulcanidas 4 ; b. the Solemyidae, genera Acarax, Solemya; c. the Vesicomyidae, genera Abyssogena, Calyptogena, Vesicomya, Waisiuconcha; d. the Thyasiridae, genus Thyasira e. the Lucinidae, genus Bathyaustriella 2) Hydrocarbon seeps (all types): a. the Mytilidae, genera Bathymodiolus, Gigantidas, Idas, Tamu; b. the Solemyidae, genera Acarax, Solemya; c. the Thyasiridae, genera Adontorhina, Conchocele, Parethyasira, Spinaxinus, Thyasira, Axinulus, Maorithyas; d. the Vesicomyidae, genera Calyptogena, Laubiericoncha, Vesicomya, Isorropodon, Chrisinteconcha, Pliocardia, Elenaconcha; e. the Lucinidae, genera Myrtea, Lucinoma, Mesolinga 5 Wood-and whale-falls a. the Mytilidae, genera Idas, Adipicola, Benthomodiolus; b. the Solemyidae, genus Solemya; c. the Thyasiridae; genus Thyasira (juveniles) d. the Vesicomyidae; genera Archivesica, genus unknown (juveniles) e. the Nucinellidae, genus Nucinella 6 Deep-sea soft sediments with REDOX layers and oxygen-Solemyidae (Protobranchia, with putative Paleozoic representatives, Taviani et al. 2011,

  alongside some taxonomic misidentifications(Krylova and Sahling 2010;[START_REF] Krylova | A new genus of large Vesicomyidae (Mollusca, Bivalvia, Vesicomyidae, Pliocardiinae) from the Congo margin, with the first record of the subfamily Pliocardiinae in the Bay of Biscay (northeastern Atlantic)[END_REF]; Decker et al.① ,① pa ti ula l ① the① i a u ate① lassifi atio ① of① se e al① s all ① esi o id① la s① as① the① ge us① Vesicomya (Krylova and Sahling 2010). Consequently, several species assigned at some time or other to the Cal ptoge a ①ge us① ased①o ①g oss① o pholog ,①ha e① ee ① elo ated①to①othe ①e isti g①o ① e e tl ①erected genera (e.g. Abyssogena, Christineconcha), based both on molecular analyses and more detailed morphoanatomies. 1-8.2.4 The Pteriomorpha: Mytilidae: Bathymodiolinae (sensu lato) Current phylogeny and morphology-based taxonomy for the Bathymodiolinae (s.s.) are not yet concurrent and the status of this subfa il ① e ai s①a iguous.①It s①e ide t①f o ①a① ealth①of① ole ula ① o k① a ied① out on chemosynthetic mytilids that the Bathymodiolinae (s.s.) fails to account for several genera of smaller mussels which fall within this clade and for which symbiosis has been demonstrated in several members.

  described based upon malacological characteristics alone, sometimes by default, being the only material available. Examples are: the type species I. (d.f.) argenteus, I. (s.l.) lamellosus, I. (s.l.) coppingeri, I. (s.l.) dalli, I. (s.l.) simpsoni, I. (s.l.) indicus, and I. (s.s.) modiolaeformis. Two species, (I. [s.l.] simpsoni and I. [s.s.] modiolaeformis) have recently been sequenced for fragments of mitochondrial and nuclear DNA from paratypes that were confirmed taxonomically by Thubaut et al. (2013b), and by both Olu-Le Roy et al. (2004) and Lorion et al. (2012), respectively. These two species, along with the three other species that have been sequenced, I. (s.s.) macdonaldi, I. (s.s.) washingtonius and I. (s.l.) japonicus, frequently feature in phylogenetic analyses, wherein marked levels of divergence warrant their classification in at least two distinct genera (e.g.Thubaut et al. 2013b). An additional species, A. (s.l.) iwaotakii, was cited as I. (s.s.)

Figure 1 .Figure 1 .

 11 Figure 1.19 Multigene tree for Bathymodiolinae Adapted from Thubaut et al. 2013b with genera as they currently stand included for comparison

Figure 1 .

 1 Figure 1.21 Global distribution of members of the Bathymodiolinae: genus Bathymodiolus Data from OBIS principally, created by S.R. Laming

  . (s.l.) anteumbonatus, B. (s.l.) elongatus and the most westerly record for B. (s.s.) thermophilus (based on OBIS data), though a peer-reviewed account of this last record could not be identified (in fact, obviously erroneous data occurred quite frequently on OBIS during distribution searches). In contrast the East Pacific plays host to only two species: B. (s.s.) thermophilus and B. (s.s.) antarcticus. Hybridisation between these two species has been identified (Johnson et al. 2013). The type species for Gigantidas, G. gladius forms de se① eds① ea ① e t①sites①o ①'u le①III①sea ou t,① o th est①of①Ne ①)eala d s①No th①Isla d, in the South-West Pacific. In the Atlantic, a total of seven species occur; three in the West Atlantic at hydrocarbon seeps in the Gulf of Mexico (B. (s.l.) childressi, B. (s.l.) heckerae , and B. (s.l.) brooksi), two at hydrothermal vent fields in the Mid-Atlantic Ridge (B. azoricus, B. puteoserpentis) and two which are trans-Atlantic seep species (B. mauritanicus, B. boomerang 11 ). Similar to B. thermophilus and B. antarcticus in the Pacific, a hybridisation zone exists between B. azoricus and B. puteoserpentis from 37°N and 14°N on the Mid-Atlantic Ridge, (O'Mullan et al. 2001). Curiously, shell morphometry and variability in mitochondrial sequences indicate

(

  Taylor and Glover 2010; Duperron et al. 2013), for which sulphur-oxidising symbionts have been identified without exception in species examined in detail, suggesting that symbiosis is a pan-pliocardiin characteristic. One record of gill bacteria, not yet confirmed to be chemoautotrophic, has been published fo ① the① spe ies① Vesicomya sergeevi, however current taxonomic opinion places this species in a sister family, the Kelliellidae (i.e. Kelliella sergeevi13). The principal features that characterise the Pliocardiinae are medium-to-large white shells (though they are smaller in Isorropodon), well-developed siphons, labial palps that are reduced to simple plicated ridges around the mouth, a basic gut and a small stomach[START_REF] Mortensen | The functional morphology of the organs of feeding and digestion of the hydrothermal vent bivalve Calyptogena magnifica (Vesicomyidae)[END_REF][START_REF] Fiala-Médioni | Structural adaptations in the gill of the japanese subduction zone bivalves (vesicomyidae) Calyptogena phaseoliformis and Calyptogena laubieri[END_REF][START_REF] Krylova | A new genus of large Vesicomyidae (Mollusca, Bivalvia, Vesicomyidae, Pliocardiinae) from the Congo margin, with the first record of the subfamily Pliocardiinae in the Bay of Biscay (northeastern Atlantic)[END_REF]) and sub-filamental tissue in the gills(Krylova and Sahling 2010) which are thickened. The Vesicomyinae by contrast possess a fully retained gut, relatively short siphons and an absence of sub-filamental tissue in the gills. In adult Pliocardiinae, domed, microvilli-covered gill epithelial bacteriocytespacked with intracellular sulphur-oxidising autotrophic Gammaproteobacteriaare aligned in layers within the abfrontal region of the engorged gill filaments, separated by a central blood lacuna and interspersed with intercalary cells[START_REF] Fiala-Médioni | Structural adaptations in the gill of the japanese subduction zone bivalves (vesicomyidae) Calyptogena phaseoliformis and Calyptogena laubieri[END_REF] Taylor and Glover 2010). These pliocardiin symbionts, which form a compact clade with highly homogeneous 16S rRNA sequences, are related to those within mytilid and poriferan hosts, and various free-living bacteria(Duperron et al. 2012).

2 . 2

 22 Spawning behaviour (Figure 1.23a) The most thoroughly studied species in the Bathymodiolinae are B. (s.l.) childressi, a mixotrophic species (i.e. employing filter feeding and chemosymbiosis, Page et al. 1990) which harbours methanotrophs exclusively (Childress et al. 1986), residing at hydrocarbon seeps occurring on the Louisiana slope in the Gulf of Mexico, and B. (s.s.) azoricus, another mixotrophic (Riou et al. 2010a) species harbouring a dual thiomethanotrophic symbioses (Fiala-Médioni et al. 2002), on the MAR (e.g. Menez Gwen and Lucky Strike vent fields). Gametogenesis, spawning behaviour, and aspects of larval development have been examined in B. (s.l.)

  g. B. (s.l.) satsopensis and other examples,[START_REF] Kiel | The earliest bathymodiolin mussels: an evaluation of Eocene and Oligocene taxa from deep-sea methane seep deposits in Western Washington State, USA[END_REF]. The apparent ancestral origins of planktotrophic larval development is of potential importance for the evolutionary history of bathymodiolins[START_REF] Kiel | The earliest bathymodiolin mussels: an evaluation of Eocene and Oligocene taxa from deep-sea methane seep deposits in Western Washington State, USA[END_REF]) since this trait is restricted for the most part to this group of chemosymbiotic bivalves (accepting certain tropical lucinids,[START_REF] Glover | Diversity of chemosymbiotic bivalves on coral reefs: Lucinidae (Mollusca, Bivalvia) of New Caledonia and Lifou[END_REF], wherein lecithotrophic development is the most ubiquitous larval mode in the Solemyidae, Nucinellidae, Thyasiridae, Lucinidae, and Vesicomyidae[START_REF] Gustafson | Larval and post-larval morphogenesis in the gutless protobranch bivalve Solemya reidi (Cryptodonta: Solemyidae)[END_REF][START_REF] Von Cosel | Vesicomyidae (Mollusca: Bivalvia) of the genera Vesicomya, Waisiuconcha, Isorropodon and Callogonia in the eastern Atlantic and the Mediterranean[END_REF][START_REF] Oliver | The Thyasiridae (Mollusca: Bivalvia) of the British continental shelf and North Sea oil fields: an identification manual. National Museums & Galleries of Wales, Cardiff, Oliver PG, Holmes AM[END_REF] Krylova et al. 2010

Figure 1 .

 1 Figure 1.24 A diagrammatic comparison of pediveliger to plantigrade morphoanatomy A) Diagrammatic reconstruction of pediveliger of Mytilus edulis. an anus, ant.ad anterior adductor muscle, a.p apical plate, a.pit apical pit, by.d byssus duct, by.g byssus gland, c.gan, cerebral ganglion, d.d. didestive diverticulum, f foot, ga.s gastric shield, gill.f gill filaments, int. Intestine, kid. kidney, m. mouth, man. mantle, mid.gut midgut, oe. oesophagus, o.p oral palp, os. osphradium, p.ch posterior chamber of mid-gut, p.gan pedal ganglion, p.g.s. secretions of the phenol (or purple) gland, pig.s pigment spot, post.ad posterior adductor muscle, p.r.m pedal retractor muscle, s.sac style sac, st. stomach, stat. statocyst, v. velum, v.gan visceral ganglion, v.r.m. velar retractor muscle, wh.g white gland. B) Diagrammatic reconstruction of plantigrade (post-larva) of Mytilus edulis before dissoconch deposition. c.gan cerebral ganglion, gill.f gill filaments, l.l.p lower labial palp , upper labial palp u.l.p . From Bayne (1971).

  g. B. (s.l.) childressi), suggesting other as-yet-undetermined factors play a role in overcoming the physiological constraints observed development from post-larva to adult has received little attention in deep-sea mytilids, while in shallow water species, contemporary studies have examined more detailed aspects of tissue development (e.g. in situ hybridisation with myvlg to identify expression patterns in primordial germ-line cells,Obata et al. 2010; or gill development using SEM,Cannuel et al. 2009). Basic developmental biology in shallow-water species has identified that even as plantigrades, the critical organs necessary to function are in place, with the exception of gonads (Figure1.24b). Organs then increase in size and complexity as the animal develops, such that in adult heterotrophic mussels, the stomach, the mass of digestive diverticula (forming the digestive gland), gills, and gonads represent the majority of soft tissues.

1 )

 1 g. B. (s.s.) thermophilus,[START_REF] Rhoads | Growth and predation activity at deep-sea hydrothermal vents along the Galapagos Rift[END_REF] Nedoncelle et al. 2013). A posteriori backcasting from von Bertalanffy growth curves plotted for adult shell measurements of the vent-based B. (s.s.) thermophilus(Nedoncelle et al. 2013) identified an average growth rate of 4.2 cm year -1 for post-larval plantigrades immediately after settlement, assuming a larval settling size of 0.04 cm in B. (s.s.) thermophilus. This rate of growth precipitously decreased so that by 10 years old (growth rate of 1.1 cm year -, mussels had already attained a length approaching 95% of their theoretical asymptotic maximum size (L∞ = . ① ,①at①≈ ① ea s①old). These rates of growth were validated in newly established populations of B. (s.s.) thermophilus settling on nascent basaltic substrata following an eruption (unpublished results fromNedoncelle et al. 2013). Interestingly, microvariability in daily shell increments appear to relate to the circalunidian rhythm of the tidal signal for that part of the Pacific Ocean, perhaps as a consequence of near-bottom tidal current variability(Nedoncelle et al. 2013). This is despite the experiment being performed on mussel beds 2,500+ m deep. Such circalunidian timing in growth rates has also been identified for B. (s.s.) brevior[START_REF] Schöne | Growth increments and stable isotope variation in shells of the deep-sea hydrothermal vent bivalve mollusk Bathymodiolus brevior from the North Fiji Basin, Pacific Ocean[END_REF] from the North Fiji Basin.

(

  maintaining growth rates in later years), or a reduced investment in other energetically demanding processes (such as gametogenesis). This follows a pattern established by earlier work (e.g.[START_REF] Grassle | Macrofaunal colonization of disturbed deep-sea environments and the structure of deep-sea benthic communities[END_REF] which suggests that congeneric trends in developmental rates in organically enriched environments differ between shallow-water and deep-sea species. Examination of gonads revealed that, following maturity, gamete production was relatively high. 1-9.4.4 Organ development: gills and gonads Two of the organs in greatest need of rapid development in deep-sea chemosymbiotic mytilids are arguably; a) the gills, since it is within the gills that symbionts will be housed in adults, and; b) the gonads, whose speed of① de elop e t① ill① defi e① the① populatio s① ep odu ti e① esilie e① i ① the① fa e① of① ha itat① ephemerality. Regrettably, these processes remain shrouded in mystery in the Bathymodiolinae, where even age or size at first maturity remains poorly defined (one known example: 40-60 mm in B. (s.s.) thermophilus depending on sex, Berg Jr 1985). These aspects of growth have been examined in shallow water Mytilidae however. Mytilid gills follow a particular developmental template (e.g. M. edulis, Cannuel et al. 2009). A number of gill bars initially develop at the pediveliger stage (Figure 1.24a). Consequently, when larvae first metamorphose into plantigrade post-la ae①a① e ①s all① u e ①of①fila e ts①a e①al ead ①p ese t① ≈① -,①fo i g①a①gill① asket ① (Bayne 1971; Cannuel et al. 2009; Figure 1.24b, Figure 1.25a). As the mussel grows, gill complexity increases, both structurally and functionally. Structurally, the number of filaments and lamellae increases and each of the elements become bound together as a single organ by the formation of various ciliary junctions

1- 10

 10 Overall summary Despite being bathed in the scientific limelight, deep ocean ecology beyond the continental shelf margins still remains poorly understood (hydrothermal vent systems accepted, Adams et al. 2012). Much has been learned courtesy of the endeavour of early research (e.g. the Challenger expedition, Murray et al. 1891) and the recent advent and rapid development of deep-sea equipment capable of cataloguing the environmental ha a te isti s① a d① e olog ① of① the① o ea s① depths.① Ho e e ,① hile① these① te h i ues① i fo ① us about community-wide biology, distribution and heterogeneity and habitat conditions, they cannot account for

  the research project was to characterise critical junctures in the lifecycle of less-specialised15 deep-sea chemosymbiotic benthic macrofauna associated with organic falls.Particular periods in the lifecycle of such organisms are severely lacking across a variety of phyla. The most ubiquitous macrofaunal taxa that routinely settle on organic substratesdistinct from the fauna that colonise the proximal sedimentsinclude several examples of small-sized bathymodiolin (s.l.) mussels, and the siboglinid polychaetes. Of these, the bathymodiolins were considered the more suitable model group (discussed below). A priori review of the literature identified a dearth of data regarding planktonic and postlarval development, including a near absence of basic biological parameters such as modes of planktonic development, settlement patterns, growth rates, post-larval and juvenile developmental morphoanatomy, and size or age at which juveniles recruit into the adult population (excludingThubaut et al. 2013a). Given the ephemeral nature of organic falls in the deep sea, these parameters were considered fundamental in describing adaptations of organic-fall chemosymbiotic mytilids to the constraints of their habitat. As nearly all mytilid species from organic-fall communities that have been examined in detail have been shown to e gage① i ① s ioses① as① adults,① ide tif i g① the① ole① s io ts① pla ① du i g① the① ou se① of① these① spe ies ① development was equally crucial.

  the larval, post-larval and juvenile period of the lifecycle, with emphasis on reproductive (linking to Theme 1) and nutritional development (linking to Theme 3) from the moment of gamete fertilisation, up to size/age at first maturity in settled juveniles. It consequently formed the largest part of the envisioned research and was split into larval and post-larval/juvenile subthemes, for increased clarity.

  -larval and juvenile development: To meet the above aims, organic larval colonisation devices were either already awaiting sorting, having been deployed for a period in a deep-sea environment, or were deployed during the course of the PhD research and subsequently examined. N.B. The sorting of preexisting substrates at the beginning of the PhD ultimately had considerable bearing on the choice of model organism, given that sample availability was an obvious criterion for the research (detailed in the following section 2-2.1 p. 110 onwards). Specimens of the model organism at various sizes, amassed during substrate sorting (with three predetermined methods for preservation), were first to be measured to detail sizefrequency distributions and thus possible patterns in larval supply, based on the presence of surviving postsettlement individuals.

  (s.s.) thermophilus group (e.g. Idas [s.s.]), while several clades emerged much further back in evolutionary time (e.g. the Benthomodiolus and Vulcanidas clades, and the polyphyletic clade that currently contains the species I. [s.l.] simpsoni, I. [s.l.] japonicus and A. [s.l.] longissima, Thubaut et al. 2013b).

  relation to what was known of I. (s.s.) modiolaeformis from the eastern Mediterranean. In that study, similar differences in sizes were recorded (Figure2.1). In fact, Idas-like nov. sp. was assigned a putative new species status, based on comparative malacology against seven other species known to occur in the Mediterranean Sea including I. (s.l.) simpsoni type specimens. This demonstrates the sort of difficulties typically encountered during species description, which plague this highly plastic group of mussels.

Figure 2 . 1

 21 Figure 2.1 Idas (s.l.) simpsoni and Idas (s.s.) modiolaeformis from Ritt et al. 2012 A,C, E are Idas (s.l.) simpsoni , B,D, F are I. (s.s.) modiolaeformis. Views from top: ventral, lateral-right and dorsal

2- 5

 5 Overall study area and sampling regime 2-5.1. The Mediterranean and eastern Atlantic I ①the①past,①the①Medite a ea ①"ea①has① ee ①du ed①a① hotspot ① egio ①i ①te ms of land-and coastal-based biodiversity. The deep-sea has often been dismissed under the presumption that biodiversity at greater depths is very low in Mediterranean waters, but theoretical extrapolations from rarefaction curves provide an estimate (excluding prokaryotes) of approximately 2805 species in the overall deep-sea Mediterranean (in that study, considered from 200 -4000+ m in depth), where 66% remain undiscovered[START_REF] Danovaro | Deep-sea biodiversity in the Mediterranean Sea: The known, the unknown, and the unknowable[END_REF]. According to this review, these exist in a wide variety of habitats including: continental slopes; deepsea basins; submarine canyons and seamounts with deep-water stony corals at both; some isolated hydrothermal vents and; hydrocarbon seeps and deep-hypersaline anoxic basins, particularly in the eastern Mediterranean[START_REF] Danovaro | Deep-sea biodiversity in the Mediterranean Sea: The known, the unknown, and the unknowable[END_REF]. Within and around the area of study in the northeast Atlantic Ocean, synonomous habitats exist to those in the Mediterranean Sea, with numerous methane-seeping MVs in the Gulf of Cadiz (e.g.[START_REF] León | Classification of sea-floor features associated with methane seeps along the Gulf of Cádiz continental margin[END_REF], an extensive series of vents along the MAR (e.g.[START_REF] Sudarikov | Geochemistry of the Snake Pit vent field and its implications for vent and non-vent fauna[END_REF][START_REF] Douville | The rainbow e t① fluids① ° ′N,① M②' :① the① i flue e① of① ult a afi ① o ks① a d① phase① sepa atio ① o ① t a e① etal① content in Mid-Atlantic Ridge hydrothermal fluids[END_REF][START_REF] Silantyev | Peridotite-gabbrotrondhjemite association of the Mid-②tla ti ①'idge① et ee ① ° ′①a d① ° ′N:①②shadze①a d①Loga he ① hydrothermal vent fields[END_REF], and seamounts (e.g. seamounts Josephine, Erik, Gettysburg (Gorringe ridge), Ampere and Seine,[START_REF] Morato | Seamount physiography and biology in the north-east Atlantic and Mediterranean Sea[END_REF]). In addition the continental margins are densely populated by submarine canyons, carved into the continental slope along both the Portuguese and North African coasts (majority are blind canyons, though some cut the shelf break, Harris and Whiteway 2011).

Figure 2 . 2

 22 Figure 2.2 Methods employed to collect specimens Bottom left: CARCACE project deployment in the Setúbal Canyon, Portugal. Middle-left: samples from the Lacaze-Duthiers Canyon, near Banyuls (2012). Centre and mid-right samples collected from following cruise in Lacaze-Duthiers Canyon on wood, 2013. Top row concerns the deployment and recovery of CHEMECOLI in the Eastern Mediterranean (Nile Deep-sea Fan, 2007).

  chemosymbiotic mussel, Idas (sensu stricto) modiolaeformis and I. (sensu lato) simpsoni, collected from wood-colonisation experiments deployed in the Lacaze-Duthiers Canyon, Western Mediterranean. Both species are probable semi-continuous spawners based on multiple, induced dribble spawning events, and post-spawning retention of mature oocytes. Oocytes display practically neutral buoyancy at atmospheric pressure and 14°C with diameters indicative of planktotrophy in both species. Although both species harboured bacteria in their gills, each retains a capacity for active particle processing based on live observations. The presence of fully-equipped alimentary, digestive and excretory systems, and mixed diet of host tissues support facultative mixotrophy in both species.

  nutritional and reproductive biology based upon live observations and histological analyses following dissection and fixation. Specimens were collected from a variety of wood-colonisation experiments, deployed in the Lacaze-Duthiers (LD) Canyon at 525 m depth (Gulf of Lion, France, LeBris et al. in preparation). Live studies were performed at the Banyuls Marine Station (UPMC, CNRS), France. experimental aquaria (Figure3.1) and an additional flow-through container for holding unsorted material, were installed and then acclimated under deep-sea Mediterranean conditions (dark, 13°C ± 1°C) for five days prior to receiving samples. Both were supplied continuously with unfiltered seawater imported from shallower depths around the Banyuls Marine Station. In the aquaria, seawater circulated above an elevated, mesh-covered sample platform, driven by gentle downward inflow from the inlet manifold with corresponding upwelling from aeration at the opposing end (Figure3.1). The manifold introduced seawater equally across the width of the tank. The sub-platform outflow, equipped with an antisiphon vent, removed sinking wastewater and material. Aquaria were emptied and cleaned on

Figure 3 . 1

 31 Figure 3.1 Acclimation aquarium for the maintenance of Idas spp. Aquarium (20 x 14 x 14 cm) design was based loosely on Høeg(1984) and Utting and Spencer(1991). Access was via the viewing window [A]. The sample platform [B] was raised from the floor of the tank which along with the manifold design [C], the anti-siphon vent [D] and bubbles from aeration [E], collectively created a gentle circulatory flow [F]. Waster material sank to the base of the tank [G] while dissolved waste was removed by the outflow (dashed arrow). Samples were held in plastic 1-mm mesh cages [H]. 3-3.1.2 Substrate experiments A multi-annual sunken-wood deployment-recovery experimental programme was established in the Lacaze-Duthiers canyon site at 525 m in October 2010 (42°32.728'N, 3°25.267'E, 5 LD-cruises in total, Le Bris et al. in preparation). Using the ROV SuperAchille (COMEX) recoveries and new deployments have since been performed over a series of 4 additional cruises (LD1-LD5, chief scientist: N. Le Bris, R/V Minibex), the last being in April 2013. As part of the LD5 cruise in April 2013, various pre-deployed wood experiments were recovered, including a section of palm-tree trunk (~0.4 m girth) deployed on the seabed during the LD1 cruise (2.5 year deployment), and two pinewood cube-filled larval colonisation devices (CHEMECOLI, Gaudron et al. 2010) deployed on an sampling frame during the LD4 cruise (10 month deployment).

Figure 3 . 2

 32 Figure 3.2 Wood-olo isatio e peri e t's lo atio i the La aze-Duthiers Canyon, western Mediterranean Depicted is the Gulf of Lion with the Banyuls Marine Station and Lacaze-Duthiers Canyon in red, where the remaining canyons are in yellow. The deployment/recovery location was near the head of the canyon (42°32.728'N, 3°25.267'E) at a depth of 525 m.

  oocytes collected during the fertilisation experiment, were stained using the fluorescent dye Hoechst 33258 (Herzog and Schütze 1968, 5 μg ml -1

Figure 3 . 3 ,Figure 3 . 3 .

 3333 Figure 3.3 .① Pe iost a al① ssus① hai s ① Choo et al. 2014) were absent on I. (s.l.) simpsoni (except IF4 and IF15) in contrast to I. modiolaeformis, (hairs concentrated along the postero-ventral margin). The periostraca were golden-brown in both species, but glossier and more tanned in I. (s.l.) simpsoni. The inner nacreous layer was pearly and off-white in I. (s.l.) simpsoni shells but barely discernible in I. modiolaeformis,

Figure 3 . 3

 33 Figure 3.3 Shell morphology of specimens in this study separated by halpotype and speciesHaplotypes are based on fragment COI sequence similarities (single haplotypes are 100% similar). Exterior and interior views of the left valve are shown in each case (upper and lower respectively). Note that IF2 is placed with I. (s.l.) simpsoni provisionally as DNA was not successfully sequenced.

  ). Mean diameter for released oocytes (pooled across all mussels) was 59.1 ± 2.31 µm (n = 50). Mean diameters for unreleased and potentiallyimmature, paraffin-embedded oocytes in I. (s.l.) simpsoni (IF12 and IF7, n = 75) and I. (s.s.) modiolaeformis (IF14, n = 10) were 33.5 ± 3.65 µm and 25.3 ± 2.98 µm respectively. In the latter, the number of available eggs to measure was limited by the volume of the gonad, which was also almost spent. These diameters are comparable to previously-documented vitellogenic oocyte diameters for I. (s.s.) modiolaeformis (32.5 µm), though smaller than sizes for oocytes in fully-ripe gonads(Gaudron et al. 2012a); this is attributed to the release of all fully-formed oocytes during repeated spawning events though diameters may also underestimate true sizes, as oocytes showed signs of heat-shrinkage. Two inductions had been attempted in a subsample of the individuals prior to the success of the third. Initial attempts to induce spawning with 2 mmol l -1 serotonin microinjections (Femtojet, Eppendorf) into the anterior adductor muscle (Arellano and Young 2009) proved ineffective, while the batch of oocytes from the second more successful attempt was damaged while being sieved. Successful induction was by mechanical shock (10 min, Sprung and Bayne 1984), saline shock (size-calibrated injections of 0.55M KCl into mantle cavity, Young and Tyler 1993) and the ①the al① sho k① °C① ought① to① °C①o e ① ≈ ① i ,① Sprung and Bayne 1984). All but one (IF1) of the adults appeared to recover from these inductions, based on behaviour and visually-assessed body condition.

Figure 3 . 4

 34 Figure 3.4 Images of liberated ooctes during spawning and following fertilisation (pooled from both species) A) Oocytes realised by the largest I. (s.l.) simpsoni female IF7 during dissection (thus, not used in fertilisation experiment). B) Hoechst Staining identifying i) released, but unfertilised, oocyte in Metaphase I; ii) 4-cell pre-morula division stage. Arrow identifies detachment of membrane/possible polar body. C) Phase contrast microscope images of fertilised oocytes, i) arrowheads delimit fertilisation membrane; ii) with jelly coat and; iii) following formation of polar body

Figure 3 . 5

 35 Figure 3.5 Haematoxylin and Eosin Y staining in 7mm histological sections from both species Note that A, G, H are from IF3, male I. (s.l.) simpsoni; B, C, D, F are from IF7, female I. (s.l.) simpsoni. E is from IF10, female, I. (s.s.) modiolaeformis. A) Sagittal view in region where digestive gland and gonad coincide. Large white arrows = spermatogenesis. B) Oesophagus in sagittal section, surrounded by digestive diverticula. C) Kidney-like nephridia and edge of spent acini in gonad (bottom right of image). D) Similar region to C, but focused over gonad. Follicles are spent, though putatively mature oocytes persist (black arrows). E) Gonad, with some immature oogonia indicated with black arrows. G) View of tubules, some full, some partially depleted (region H). H) Magnified view of box in G. Empty region is where spermatozoa formerly were, prior to release. c.ep cuboid epithelial cells, d.d digestive diverticula, ep epithelium, e.c secretory epithelial cells, go gonad, in intestine, ne nephridium, oc mature oocyte, oe oesophagus, sc spermatocytes, sp spermatozoa

3- 4 . 4 .

 44 Evidence for mixotrophy in adult Idas (s.s.) modiolaeformis and I. (s.l.) simpsoni 3-4.4.1 Alimentary, digestive and excretory system Laming et al. (2014) previously examined post-larval to adult anatomical development in I. (s.s.) modiolaeformis from the NDSF, in the eastern Mediterranean (CHAPTER). In that study, many components for a funtioning digestive system were already in place in post-larval plantigrades (SL 0.38-0.42 mm), prior to the acquisition of their symbionts (at SL 0.59 mm). The low symbiont densities prior to maturation, and the presence of an almost fully-formed gut, prompted Laming et al. to suggest a necessity and potential for mixotrophy in I. modiolaeformis, respectively. In that study, qualitative increases in symbiont densities and gill-specificity with increasing shell-length (SL) suggested that, at the very least, adults were shifting towards increasingly-chemosymbiotic mixotrophy. Mixotrophy had also been postulated recently for a Pacific congeneric, A.(s.l.) iwaotakii based on the simulataneous presence of thiotrophic symbionts and relatively

Figure 3 .

 3 6, yellow-pigmented digestive diverticula beneath foot of I. (s.s.) modiolaeformis) with a dorsal mid-and hind-gut, visible on account of dark sediment-like contents. In I. (s.s.) modiolaeformis one intestinal loop was typically identifiable; I. (s.l.) simpsoni sometimes had two (not shown). Histological examination of stained 7-µm sections of viscera from I. (s.s.) modiolaeformis and I. (s.l.) simpsoni, revealed the alimentary route from the mouth (Figure 3.6A: I. [s.s.] modiolaeformis) and heavily-ciliated oesophagus (Figure 3.6B; I. [s.l.] simpsoni) to the stomach and ultimately the mid-gut (partially sectioned, Figure 3.6A; I. [s.l.] simpsoni). All dorsal regions of the mantle cavity unoccupied by other organs, were flush with extensive digestive diverticula in both species (Figure 3.6A), which normally absorb soluble compounds in the stomach. In I. (s.l.) simpsoni, metanephridia were well-developed (Figure 3.5C), and structurally comparable to Mytilus edulis (Pirie and George 1979), but in I. (s.s.) modiolaeformis metanephridia were not evident.

Figure 3 . 6

 36 Figure 3.6 Detail of the dissection performed on IB1, a small I. (s.s.) modiolaeformis maleNote that this mussel is the same as that in Supplementary video 1, and was not induced to spawn. A) Ventral view of the buccal region during the accumulation of mucus, actively rotating between labial palps (dotted white perimeter), delivered by the feeding groove (yellow path). The foot is curled posteriorly (far left). B) Ventral view of the medial region. The pedal groove extends from the byssus gland to the sub-distal pit (left and right stop-marks on dotted line). Note coloration of viscera in both A and B. C) Sagittal view of left lateral region. Mucus string is depicted with a white perimeter, delivered along the ventral particle groove (yellow path). D) a high magnification view of the frontal region of filaments, with the lateral cilia (1), laterofrontal cirri (2) and frontal cilia (3), involved in trafficking particles ventrally (yellow path). a.a anterior adductor, a.r anterior retractor, by byssus gland, d.g digestive gland, ft foot, g gill, l.p lower labial palp, m mouth, m.f mantle fold, p.a posterior adductor, pd prodissoconch, p.g pedal groove, p.r posterior retractor, u.p upper labial palp. Scale bars 1 mm (inset D: 50 µm)

Figure 3 . 7

 37 Figure 3.7 Fluorescence in situ hybridisations performed on gill tissue of I. (s.s.) modiolaeformis and I. (s.l.) simpsoni All micrographs from specimens collected from oakwood in 2012. FISH failed to identify symbionts in IF# mussels (2013) used in live observations (see main text). Eubaterial probes confirmed DAPI signals associated with lateral-abfrontal filament surfaces to be Eubacteria in both species (images labelled Eu).In I. modiolaeformis, these comprised of thiotrophs (T1) and methanotrophs (M1). Eu-T1 overlaid image identified thiotrophs as highly dominant (Eu-M1 overlaid image has an abundance of eubacteria that were not methanotrophic). In I. simpsoni, eubacteria type could not be confirmed with the probes employed.

15NFigure 3 . 8

 38 figure 3.5). However stable isotope signatures are highly variable depending on the processes that generate the sources of sulphide and methane, and the relative proportions of each when both are available for dual s ioses.①O e① eed①o l ①e a i e①δ 13 C-values for mussel tissues from seeps in the Gulf of Mexico (B. [s.s.]

  heckerae,Nelson and Fisher 1995) and the Japan trench(B. [s.l.] platifrons,[START_REF] Goffredi | Species-specific variation in sulfide physiology between closely related Vesicomyid clams[END_REF], which range from -76.4 --62. ① ‰① eth lot oph① do i ated① s ioses ,① a d① o pa e① the ① ith① those① at① ult a afi ① MAR vent sites (e.g. B. [s.s.] puteoserpentis -37.3 --32.5, thio-/methanotroph dual symbiosis, Robinson et al. 1998). In Bathymodiolus (s.l.) spp. where mixotrophy has been identified (B. [s.s.] azoricus, B. [s.s.] thermophilus, B. [s.l.] childressi, Page et al. 1990; Page et al. 1991; Riou et al. 2010) values are approximately comparable to those of I. (s.l.) simpsoni (palmwood) and I. (s.s.) modiolaeformis (oakwood) in this study (B. [s.s.] azoricus δ 13 C: -32.6 --21.3 ‰,①δ 15 N: -10.5 -+ . ①‰,①Trask and Van Dover 1999; Fiala-Médioni et al. 2002; and B. [s.s.] thermophilus δ 13 C: -37.1 --30.5 ‰,①δ 15 N: -8.1 -+9.6 ‰ .①The①e eptio ①is① B. [s.l.] childressi collected from seeps in the Gulf of Mexico, which hosts methanotrophs only (-67.1 --37.5 ‰,① Nelson and Fisher 1995 .① Ho e e ,① i ① the① Gulf① of① Me i o,① δ 13 C signatures for methane hydrates are highly variable (Supplementary figure 3.5), depending on the thermogenic or biogenic origins (or a mixture of the two, Brooks et al. 1985 .① Negati e① o ① e ① slightl ① positi e① δ 15 N values typical of larger chemosymbiotic mussels, are not comparable with those found in this study (except B. [s.s.] thermophilus, Supplementary figure 3.5), however this may reflect an alternative nitrogen source at seeps and vents, or another unknown factor attributable to the larger-sized Bathymodiolus (s.l.). In small-sized bathymodiolin mussels for which such data exists, values for both carbon and nitrogen isotopes are much more akin to the current study. In the small undescribed vent mussel (probably 24 Benthomodiolus sp., Thubaut et al. 2013b) at the Juan de Fuca ridge Mid-Valle ① e t①fields,① alues①fo ①δ 13 C①a d①δ 15 N vary from -30.7 --. ①‰①a d① . ① -. ①‰① espe ti el ① McKiness et al. 2005; Bergquist et al. 2007). Isotope values for the two species in the current study are only known from seep environments. Idas (s.s.) modiolaeformis found at several mud volcanoes in the Olimpi and Anaximander areas of the NDSF (Olu-Le Roy et al. 2004; Carlier et al. 2010) in the eastern Mediterranean displayed 14 C-depleted① δ 13 C signatures of -41.6 --. ①‰.①This① a ①ho e e ① efle t①a①p edo i a e①of① eth lot ophi ①s io ts①usi g① methane as an energy source, such as in B. [s.l.] childressi, B. [s.s.] heckerae and B. [s.l.] platifrons (Nelson and Fisher 1995; Barry et al. 2002; Olu-Le Roy et al. 2004). Such methylotroph symbionts were either at low abundances (as M1-methanotrophs, I. (s.s.) modiolaeformis), or not identified at all in the current study. Values①fo ①δ 15 N in I. (s.s.) modiolaeformis cited in Olu-Le Roy et al. (2004), were also slightly 15 N-depleted, ranging from 0.2 -. ①‰,①a d①lo e ①tha ①those①i ①this①stud .① Notably for I. (s.l.) simpsoni,①δ 13 C①a d①δ 15 N values approach those documented for this species in the①Ma a a①"ea,①at①sites①of① etha e①seepage;①the e,①δ 13 C varied between -. ①‰①--. ①‰① hile①δ 15 N alues① a ged①f o ① alues① et ee ① . ‰①-. ‰① this study:①δ 13 C -. ①±① . ①‰,①δ 15 N①① . ①±① . ①‰ .①The① 24 this①spe ies①is①so eti es①assig ed①to① Idas washingtonia , probably inaccurately ele ated① δ 15 N values in that study were already unusually 15N-enriched for a chemosymbiotic mussel (perhaps suggesting that nitrogen sources were not strictly chemosyntheticRitt et al. 2012). In the current again, reflecting a probable input from photosynthetically derived nitrogen, based on the filter feeding behaviour identified in both species.Mixotrophic diets are thus supported for both species (albeit more biased towards chemosymbiosis in I. (s.l.) simpsoni), although an undetected dual symbiosis cannot be ruled out (as these a ① ha e① si ila ① δ 13 C-signatures, e.g.Trask and Van Dover 1999 .① "u tle① disti tio s① e ist① et ee ① δ (more negative or equivalent) identified in the current study for I.

  15N-enriched values suggest that the diets of both species, but particularly I. (s.l.) simpsoni, are not exclusively of symbiotic or free-li i g① a te ial① o igi ,① ith① δ 15 N values approximating those of primary heterotrophs[START_REF] Carlier | Trophic relationships in a deep Mediterranean cold-water coral bank (Santa Maria di Leuca, Ionian Sea)[END_REF][START_REF] Carlier | Heterogeneous energetic pathways and carbon sources on deep eastern Mediterranean cold seep communities[END_REF] ,① athe ①tha ①othe ① he os ioti ①spe ies①at①≈①-1 --11‰① Cavanaugh et al. 1992; Trask and Van Dover 1999; Fiala-Médioni et al. 2002), the exception being values for small-sized mussels (McKiness et al. 2005; Ritt et al. 2012) other than I. (s.s.) modiolaeformis (Olu-Le Roy

2 ,

 2 [START_REF] Darnaude | Trophodynamic linkage between river runoff and coastal fishery yield elucidated by stable isotope data in the Gulf of Lions (NW Mediterranean)[END_REF][START_REF] Bourgeois | Distribution and lability of land-derived organic matter in the surface sediments of the Rhône prodelta and the adjacent shelf (Mediterranean Sea, France): a multi proxy study[END_REF]. Recent studies have also identified depth-depe de t① effe ts① o ① POM① δ 15 N (varying across plankton size categories) as POM degradation increases exponentially with water-olu ① depth,① esulti g① i ① fu the ① alte atio s① i ① δ

  Supplementary video 1 may be found on the CD Archive***4-1 AbstractThe Bathymodiolinae are pervasive in reducing environments in the deep sea, yet data on post-larval and juvenile development and on the process of symbiont acquisition remain elusive. To understand how these opportunistic metazoans survive in ephemeral reducing habitats, individuals of the small bathymodiolin, Idas (s.s.) modiolaeformis, were examined histologically to trace their reproductive development, and with fluorescence and transmission electron microscopy to identify patterns of infection by their environmentally-acquired bacterial symbionts. A size series of these mussels was retrieved from larval colonisation devices containing vegetative substrates, deployed for 51 weeks(November 2006(November -2007) ) in the e t al① Po k a ks ① egio ① site① ② ①of①the①Nile①deep-sea fan in the eastern Mediterranean (NDSF), a zone where methane seepage can occur (N 32° 31.97, E 30° 21.18, 1693 m deep). Developmental patterns of germ-line cell migration, size-at-first-maturity, and symbiont acquisition and localisation are presented for the post-larva to adult transition. The smallest mature adult was a male with shell length (SL) 2.35 mm. All larger individuals in the series were male (maximum SL 6.54 mm). Based on the absence of bacterial signals, plantigrades were aposymbiotic, indicating strict heterotrophy in larvae and early post-larvae. During the early stages of dissoconch deposition, extracellular symbiont infection was non-specific. This was followed by increasing specificity on non-ciliated gill epithelia in adults. These observations on early development in I. (s.s.) modiolaeformis represent evolutionary adaptations to their ephemeral, reducing habitats.

  i o po atio ① of① s io ts,① h d o a o ① old ① seeps① a d① o ga i ① de is probably embodied physiologically-suboptimal habitats during deep-sea mussel evolution. Evolutionary adaptations believed to have facilitated their exploitation, such as the well-documented bacterial symbioses of large deep-sea chemosymbiotic Bathymodiolinae (reviewed in Dubilier et al. 2008; Duperron et al. 2013), now restrict bathymodiolin distributions to these reducing environments. This is evidenced by their conspicuous a se e①f o ① a kg ou d ① ath al①fau a① Duperron 2010). A time-calibrated molecular phylogeny of this

(

  Olu-Le Roy et al. 2004). Several molecular-based analyses of Idas-like mussels have revealed Idas sp. Med to be one of the most pervasive chemosymbiotic bivalves known in the Mediterranean and eastern Atlantic(Gaudron et al. 2010(Gaudron et al. , 2012;; Lorion et al 2010; Cunha et al. 2013; Rodrigues et al. 2013; S.R. Laming, unpubl data from Lacaze-Duthiers Canyon, Gulf of Lyon, western Mediterranean).Lorion et al. (2012) tentatively ascribed this species to Idas (s.s.) modiolaeformis[START_REF] Sturany | Zoologische Ergebnisse VII. Mollusken I (Prosobranchier und Opisthobranchier; Scaphopoden; Lamellibranchier) gesammelt von S.M. Schiff "Pola" 1890-1894[END_REF] based on morphological characters in the absence of type-specimen organic tissue for molecular analysis (I. [s.s.] modiolaeformis and Idas sp. Med are thus considered synonymous herein). This species is presently documented upon authigenic carbonate crust and tubes of Lamellibrachia sp. bathed in hydrocarbon seep fluids (Nile Deep Sea Fan (NDSF),Gaudron et al. 2012; Lorion et al. 2012), and upon various types of sunken vegetation. The latter was either 1) deployed as small colonisation experiments, such as alfalfa-or pinewood-filled colonisation devices called CHEMECOLI(Gaudron et al. 2010) placed on mud volcanoes in the Gulf of Cadiz(Cunha et al. 2013; Rodrigues et al. 2013) and at cold seeps in the eastern Mediterranean(Gaudron et al. 2010; Rodrigues et al. 2013, present study) and at the base of submarine canyons in the western Mediterranean (Lacaze-Duthers Canyon, Gulf of Lyon; S.R. Laming, unpubl data); 2) larger wood deployments (NDSF, Lorion et al. 2012; Bienhold et al. 2013; Lacaze-Duthiers Canyon, Gulf of Lyon, S.R. Laming, pers obs, J. Thubaut, pers comm) or; 3) naturally-occurring sunken wood (Gorringe Bank, eastern Atlantic, Rodrigues et al. 2013). The species has a wide depth range, 350-3015 m (Mercator MV, Gulf of Cadiz to Cheops Mud volcano, NDSF respectively). In a genus associated almost exclusively with thiotrophic bacterial symbionts (Duperron 2010, excludes the asymbiotic I. [d.f.] argenteus), atypically-diverse associations have been documented in I. (s.s.)modiolaeformis, particularly in the eastern Mediterranean. Six bacterial phylotypes have been documented(Duperron et al. 2008a) in adults from authigenic carbonate crust associated with fluid seepage (2130 m modiolaeformis post-larvae (seeGaudron et al. 2010). At the same site duri g①this①e pe i e t s① e o e ,① one of each substrate (i.e. three devices in total) was deployed by remotely operated vehicle (ROV Quest 4000, MARUM, Germany in 2006) for 51 weeks from November 2006 (recovery by ROV Victor, Ifremer, France in 2007). The identified individuals ranged from post-larvae to mussels that exceeded 3.6 mm in shell①le gth① "L ,①the①s allest① e o ded①"L①at① hi h①this①spe ies ① atu it ①had① ee ①e a i ed①Gaudron et al. 2012).

Figure 4 . 1

 41 Figure 4.1 Micrographs of Idas (s.s.) modiolaeformis specimens examined Micrographs are all but two of examined specimens. Grouping based upon morphometric analysis: SL (a) 0.38-0.85 mm ; (b) 1.22-1.92 mm; (c) 2.35-2.94 mm; (d) 4.37 mm and; (e) 6.54 mm (empty left valve). Figures (f) and (g) concern measurement protocol. SH dorsoventrally perpendicular to hinge-line (f: right-angles), SL antero-posteriorly parallel to hinge line. Prodissoconch hinge line always considered as post-larval (PdII). Dissoconch hinge line was individual-specific (Diss.). Growth patterns based on overlaid micrographs, with prodissoconchs approximately aligned. Scale bars 500 µm

Figure 4 .

 4 Figure 4.2 displays a scatter-plot of both measurements.

4. 2 ,

 2 for explanatory schematic), ranging from plantigrade-sized to the smallest identified adult, are provided as supplementary material (Supplementary figures 4.4-4.11). Identifiable across the entire size spectrum were a functional digestive system including an oesophagus with a stratified ciliated epithelium and extensive digestive diverticula around a stomach(Figures 4.3c, d and e, Supplementary figures 4.4-4.11), while hypertrophied gills (Figure 4.3e, Figures 4-4a-e, Supplementary figures 4.4-4.11), a style sac (e.g.

Figure 4 .

 4 3e) and a recurrent loop (identified during dissection) in the microvilli-lined intestinal tract were first recorded with certainty in Idas 4 (0.57 mm) and then in all larger individuals examined with histology (preservation permitting).

Figure 4 . 2

 42 Figure 4.2 Plot of SL against SH of Idas modiolaeformis SL and SH of intact prodissoconch and whole shells (n = 35, black dots and n = 36, grey dots, respectively). Prodissoconch measurements from plantigrades and juveniles/adults. Whole shells exclude plantigrades.

Figure 4 . 3

 43 Figure 4.3 Micrographs of Haematoxylin & Eosin-stained mid-sections from Idas (s.s.) modiolaeformis of various sizes Sections along or parallel to mid-sagittal plane as in (a), arrows = direction. A plantigrade (b), two juveniles (c), (d) and the smallest adult (e). a.a.: anterior adductor muscle a.d: ascending inner demibranch, a.r: anterior (byssus or pedal) retractors, Boj: organ of Bojanus, by: byssus gland, c.g: cerebropleural ganglion, co: neural commissures, d.d: descending inner demibranch, d.g: digestive gland, ft: foot tissue, g.ax: gill axis, i.j: interfilamentous ciliary junctions, l.p: lower labial palps, m: mantle, oe: oesophagus, p.a: posterior adductor muscle, p.by.r: posterior byssus-retractor, p.c: pericardial cavity, p.g: pedal ganglion, p.p.r: posterior pedal-retractor, st: stomach, s.s: style-sac, u.p: upper labial palps, v.g: visceral ganglion, * = gill filaments. Dashed arrow, observed developmental path with increasing SL of germline cells (encircled) towards gonads (location = arrowhead, see also Figure S11).

Figure 4 -

 4 4b and f, lateral-left dorsal region; Supplementary figure4.11, lateral-right dorsal region). All but two of the individuals > 2.35 mm SL were confirmed to be male (e.g. Figures4-4c and g). The two remaining unsexed individuals still harboured gonad-like interstitial cells and an additional sequence of cells which, based on cell membrane profiles (they lacked contents due to incomplete ethanolpreservation), were undergoing spermatogenesis-type patterns of differentiation.4-4.5. Gill proliferation and growthAt the post-la al①stage,①a① gill-asket ①of①fou ①to①si ①si ple①fila e ts①had①formed: the earliest incarnation of the① i e ① de i a h s① des e di g① la ellae① "upple e ta ① figu e① . ① uppe -far-left). The gill axesthe dorsally-located rigid tissue supporting each gill lamellawere not parallel to the hinge-line in post-larvae,

Figure 4 . 4

 44 Figure 4.4 High magnification micrographs of Haematoxylin & Eosin-stained gonad and gill tissue from Idas (s.s.) modiolaeformis of various sizes One juvenile (Idas 12: a, d) and two adults (Idas 13: b, e, f and Idas C5-2: c, g). For orientation terms, see Supplementary figure4.2. Images (a) to (c), full-organism sections. d: digestive system, g: gill, ft: foot. (a) Dorso-medial region in frontal section. (b) Left-lateral region in sagittal section. (c) Anterior region in transverse section. Images (d) and (e) gill filaments. Solid arrowheads = putative bacteria, open arrowheads = lateral cilia. (d) magnification of box 1 in (a). Gill filaments in frontal-section (x100) with putative bacterial distributions (left) and gill nomenclature (right). (e) magnification of box 1 in image (b). Gill filaments in sagittal-section with gill nomenclature. Note interfilamentous ciliary junction (broken), encircled. Images (f) and (g), gonad regions undergoing spermatogenesis. (f) magnification of box 2 in image (b). (g) magnification of box 1 in image (c). Spermatozoid development is labelled sequentially: spermatogonia, spermatocytes, spermatids and spermatozoids (1, 2, 3 and 4 respectively). Germ-line cell proliferation zones are indicated (gcpz). d.g. digestive gland. Scale bars 100 µm (a) to (c), and 10 µm (d) to (g).

  ut①ele ated①f o ①it① ≈ ° ①at①the①poste io ①e d,①to a ds①the① e t al① a gi ① ide tified①du i g①disse tio .① Gill filaments in plantigrades and the smallest juveniles were the least numerous, the shortest dorsoventrally from the gill axis to the ciliated terminal ends (approximately congruent) and the shallowest along the frontal-to-abfrontal axis(Figures 4.3c & 5g; Supplementary figure 4.4 upper-far-left). Consequently, filaments appeared comparatively stout.

Figure 4 . 5

 45 Figure 4.5 Putative bacteria lining the lateral-to-abfrontal regions of gill filaments in Idas (s.s.) modiolaeformis Images (a) to (k): sagittal (a) to (f ) and frontal (g) to (k) gill-filament sections at various sizes, stained with DAPI. Bordering arrows indicate increasing SL (boxes). White arrowheads = putative bacteria (tiny dots). Large nuclei belong to filament epithelia (also mantle epithelia in (a), Idas 42). Pictured; (a) to (f) Idas 42, Idas 28, Idas 3, Idas 8, Idas 13 and Idas C5-2 respectively, along sagittal plane in lateral region of gill filaments; (g) to (k) Idas 28, Idas 9, Idas 10, Idas 12 and Idas C5-2 respectively, frontal gill-filament sections (abfrontal region-bottom of micrograph, frontal-top). Images (l) and (m): TEM images of gill epithelia, Idas 7; (l) bacteria (white arrowhead) in association with gill tissue, with bacteriocyte visible (black arrow); (m) magnified view of similar bacteria, with gram negative-like double-membrane encapsulation (black arrowheads). Scale bars: 1 µm (left), 200 nm (right). I ① ju e iles① a d① adults,① as① the① i e ① de i a h s① des e di g① la ellae① elo gated① poste oanteriorly and filaments became more numerous with increasing specimen-size (Figures 4-4a-c), dorsoventral filament length (Figure 4-4e) and frontal-abfrontal filament depths (Figures 4.5h-k) increased. The shortest filaments were always found at the posterior end of lamellae, terminating in bud-like filaments (Idas 7, Supplementary figure 4.6, lower-left). New filaments are thus thought to develop from the posterior ends of the gill axes. In larger individuals, the posterior gill-egio ① floated ① ithi ①the① a tle① a it ①e ept① distally, being attached to the inner mantle fold by a band of elastic tissue (repeated observations during dissections). Using all measured individuals, the relationship between filament number in the lamellae and SL was linear in the size range examined (Figure 4.6; Least-squares linear regression, r 2 = 0.99, F 1,32 = 4213, P <0.001). Standard curved-line fits (e.g. power-function) resulted in lower r 2 values. Counts ranged from 4-6 gill filaments in post-larvae to 88 filaments in the largest individual.

Figure 4 . 7

 47 Figure 4.7 Summary of pivotal periods in the development of Idas (s.s.) modiolaeformis as a function of size Distribution of individuals is based on length-frequency analysis.

  cite variability in microhabitat as a potential determinant for the diverse range of sizes over which sex-switching occurred, a pattern also seen in I. (s.s.) modiolaeformis . Microhabitat heterogeneity within the CHEMECOLI may therefore have contributed to the sex-switching patterns inGaudron et al. (2012). elongation appeared to follow patterns documented in other mussel species (e.g. Mytilus edulis post-larvae,Bayne 1971; post-larvae-to-adults, Cannuel et al. 2009). The long, developed profile of frontal and lateral cilia and the latero-frontal cirri on gill filaments for example is more typical of heterotrophic mytilids than larger Bathymodiolinae(Southward 2008, figure1 therein).

4- 9 . 3 .

 93 List of equipment and consumables  Extraction Hood  Oven or hot-plate at <60 °C  Compound Microscope (20x to 40x objective lens as a minimum)  Silicone pen  Superfrost slides  Adjustable micropipettes  Filter paper  20ml syringe with 0.2 µm filter  Glacial Acetic acid  Distilled water-source  Running tap-water (ideally with a pH above 7)  Suitable bath with slide-rack insert  Harris' Hae ato li (worki g-stock): Mix well. Pour off a given volume of standing-stock into a clean vessel and filter (0.2 µm) to remove particulates. Add 2-4ml glacial acetic acid per 100ml of stain immediately prior to use, to increase the precision of nuclear staining. Use immediately.  S ott's tap-water (bluing) solution: Magnesium sulphate (MgSO 4 )-30.0 g; Sodium bicarbonate-2.0 g; Tap water-3 litres  Aqueous Eosin-Y (working-stock): Mix well. Pour off a given volume of standing-stock into a clean vessel. Add glacial acetic acid dropwise (using a 10 or 20 µl micropipette) to attain a pH of between of 5.0, monitored using a pHmeter. Be conservative! Too acidic (< pH 4), and the Haematoxylin stain contrast is reduced (as this stain turns increasingly pi k with loweri g pH a d Eosi stai i g is loudy . Too alkali e > pH 5 a d the Eosi o trast will be drastically reduced.

Figure 5 . 1

 51 Figure 5.1 Micrographs of Idas (s.l.) simpsoni specimens examined Micrographs are representative subsample of the total number of specimens examined for this study (n = 38). The top 8 specimens are the smallest individuals examined at < 500 µm. The smallest mature individual and largest specimen examined are also pictures (small and large ♂ respectively).

1 .

 1 mounted sections targeting the entire size-spectrum were equilibrated in phosphate buffered saline (PBS 1x), stained with the nucleic acid-specific stain 4',6-diamidino-2-phenylindole (DAPI, 300 nM in Milli Q for 3 min) and mounted in Slow-Fade Gold (Invitrogen). All body regions were examined. Gill tissue fromLorion et al. (2012) provided the positive control. Negative controls excluded DAPI altogether, to test for auto-fluorescence. To establish whether previously identified bacterial Idas-symbiont phylotypes occur as symbionts in these mussels, fluorescence in situ hybridisations (FISH) were performed on a subsample of unstained 1-2-µm whole-specimen LR white sections, employing dual-hybridisation combinations (Cy3 and Cy5-labelled probes) of the general eubacterial probe EUB338(Amann et al. 1990), the non-sense probe NON338(Wallner et al. 1993), and specific oligonucleotide probes for type-M1 methanotrophs and type-T1, -T2 and Bang-T 27 thiotrophs (details inDuperron et al. 2008a) following the post-permeabilisation steps of the FISH protocol inDuperron et al. (2005). Slides were photographed as in the H & E analysis, but with a General specimen condition and classificationOf the 38 mussels, 35 were still immature. Five of these were plantigrades (i.e. post-larval and immature, no dissoconch deposition evident: SL 0.405-0.479 mm). Two of these were unfortunately lost during the dissection-embedding-i filt atio ① p o ess① tissue① as① ≈① µ ① lo g .① The① e ai i g① th ee① e e① used① fo ①

Figure 5 .

 5 2 displays a scatter-plot of both measurements.27 Previously identified in this species(Ritt et al. 2012) easily identified in all individuals across all size ranges, on account of their distinctive appearance and conservative size.① Ga glia① a ged① f o ① ≈ ① µ ① i ① dia ete ① i ① post-la al① pla tig ades,① to① ≈ ① µ ① i ① atu e① specimens. The respective locations of cerebropleural-, pedal-, and visceral pairedganglia were, immediately dorso-lateral to the mouth, centrally within the proximal region of the foot, and in proximity to the posterior adductor muscle (Figures 5.3-5.5). Prior to dissection, pigment spots were actually visible within the translucent tissue under the microscope as two pairs of tiny black dots. These were subsequently identified in Haematoxylin and Eosin (HE) stained sections, coupled directly with the cerebropleural ganglia, either side of the mouth and very close to the anterior-most gill filament.

Figure 5 . 2 Figure 5 . 3

 5253 Figure 5.2 Plot of SL against SH of I. s.l. si pso i SL and SH of prodissoconch and whole shells (n = 38, black and grey dots, respectively). Prodissoconch measurements from plantigrades and juveniles/adults. Whole shell measures exclude plantigrades. Prodissoconch SL range marked horizontally

Figure 5 . 4

 54 Figure 5.4 Serial Haematoxylin and Eosin-Y stained transverse sections of a juvenile Idas (s.l.) simpsoni (SH 0.59, SL 0.92) A) Series of sections taken at sequential locations along the antero-poste io ①a is①[❶=① id-a te io ,①❽①=①Fa -posterior]. B) ②①s he ati ①that①seg egates①the① a ious①o ga s①a d①tissues①ide tified①i ① .①I set:① ag ifi atio ①of① egio ①i side① box, highlighting the location of non-gill bacteria (arrowhead). An enlarged version is available in Supplementary figure 5.2. C) A generic Idas-like mussel drawing, with region of sections displayed (approximate). by.g byssus gland, b.t byssus thread, d.g digestive gland, ft foot, in.l intestinal loop, l-h.g lower hindgut, m.g midgut, n.b nerve bundle, p.g pedal ganglion, p.r posterior retractor, r rectum, st stomach, u-h.g upper hindgut, v.g visceral ganglion. * = gill filament

Figure 5 . 5

 55 Figure 5.5 Serial Haematoxylin and Eosin-Y stained sagittal sections of a juvenile Idas (s.l.) simpsoni (SH 0.59, SL 1.05) A) Series of sections taken at sequential locations along the latero-late al①a is①[❶=①Left-late al①side,① =①'ight-lateral side]. B) ②①s he ati ①that①seg egates①the① a ious①o ga s①a d①tissues①ide tified①i ① ,①highlighti g①the①lo atio ①of① ogill bacteria (arrowhead). An enlarged version is available in Supplementary figure 5.3. C) A generic Idas-like mussel drawing, with region of sections displayed (approximate). a.r anterior retractor, by.g byssus gland, c.g cerebropleural ganglion, d.g digestive gland, ft foot, g.a gill axis, h.g hindgut, l.p lower labial palp, m.g midgut, m mouth, p.a posterior adductor, p.c pericardial cavity, p.g pedal ganglion, p.r posterior retractor, r rectum, st stomach, u.p upper labial palp, v.g visceral ganglion. * = gill filament

28.

  Thus a cautious size at first atu it ①of① . ① ①as①a① ale,①is①gi e .①The①t o①la ge ①spe i e s① e e①also①ide tified①to① e① ale. 5-4.4. Development of the gills in the context of symbiosis 5-4.4.1 Gill development Gill development followed similar trends to those in Laming et al. (2004) for I. (s.s.) modiolaeformis and more generally for Mytilus edulis (Cannuel et al. 2009) and are consequently discussed briefly herein. Gill filaments in plantigrades and the smallest juveniles were the least numerous, the shortest dorso-ventrally and densely ciliated, with exception to the minimal abfrontal region present, on the interior face of filaments (Figure 5.3A, B; Supplementary figure 5.1). Filaments became more numerous with increasing specimen-size, were more elongate dorsoventrally, and of greater depth fronto-abfrontally. The extension in depth was mainly through the hypertrophic proliferation of the abfrontal zone, when comparing filaments in plantigrades (Figure 5.4A, Supplementary figure 5.1) to juveniles of increasing size (from Figure 5.3B to D and from Figure 5.4 to Figure 5.5). Shortest filaments were located distally in the posterior regions

5- 4 . 4 . 2 Figure 5 .

 4425 Figure 5.5B: regions indicated with arrowheads).

Figure 5 .Figure 5 . 7

 557 Figure 5.6 Gill-fila e t ou ts i the i er de i ra h's descending lamella of Idas modiolaeformisFilament counts in one inner-demibranch descending lamella (n = 33) as function of SL. Grey dots: not analysed for bacteria. Black: bacteria not identified during fluorescence microscopy. White: bacteria present on non-ciliated epithelia, but most dense on abfrontal and lateral non-ciliated gill filament surfaces(DAPI and FISH, n = 15)

  (s.l.) simpsoni, versus the still-unrivalled symbiont diversity of I. (s.s.) modiolaeformis, though the localisation of signal during FISH in both suggest that they are both extracellular. Oligonucleotide probes that are designed based on bacterial sequences identified from the gill tissue of I. (s.l.) simpsoni (and other Idas-like species), may reveal hitherto unknown levels of symbiont diversity, and thus flexibility. The almost immediate appearance of bacteria in the gills of I. (s.l.) simpsoni raises questions about the origins of these bacteria. Such rates of acquisition suggest one of two immediately obvious hypotheses. The first is that the relative speed with which I. (s.l.) simpsoni became symbiotic, reflects a greater abundance of free-living symbiont candidates in the vicinity of bones versus those around the wood habitat on which I. (s.s.)

1 .

 1 recently collected specimens resembling I. argenteus during colonization experiments deployed in the Lacaze-Duthiers (LD) Canyon (Gulf of Lion, Mediterranean) and at the Rainbow hydrothermal vent site in the Mid-Atlantic Ridge (MAR). Multi-locus sequencing is used to ascertain the phylogenetic position of these specimens. Assignation to I. argenteus is based on the comparison of shell and anatomy with original and subsequent descriptions (Jeffreys 1876; Dean 1993; Oliver and Holmes 2009; Ockelmann and Dinesen 2011). Tissue histology and PCR amplification of bacterial genes are employed to test for the presence of symbionts and the presence of a functional digestive tract. Sampling and histology Mussels were collected from CHEMECOLI colonization devices deployed for 414 days (n=2) at the Rainbow h d othe al① e t① M②',① º . N,① º . W,① ,①Gaud o ①et①al.① ①a d① ①da s① = ①i ①the① LD① a o ① ° . N,① ° . E,① .① The① LD① specimen was observed alive under compound microscope. Specimens were preserved in 4% formaldehyde (2-4hrs, gradient transfer to 96% ethanol).

Figure 2 .

 2 Figure 2. Phylogenetic relationships among Bathymodiolinae

  I. argenteus was most closely related to I. washingtonius (K2P COI distance: 19.7 to 20.0%) and belonged to a clade that included Idas ESU O, Idas sp. P, Idas SAL1 (Figure 2) but also Idas ESU K and Idas ESU N (COI tree, 2S).

  washingtonius found in organic falls and cold seeps in the Eastern Pacific. All other Idas species in the clade were found in the Western Pacific, mainly associated with wood. The only two Atlantic species (Idas macdonaldi and I. modiolaeformis) displayed COI K2P distances with I. argenteus between 19.3 and 20.4%, in the upper range of distances measured among members of the clade (9.3 to 23.1%). Because I. argenteus is the type species of the genus Idas, this clade is actually the true Idas clade, and our results confirm that this genus is polyphyletic.

Figure 1

 1 Figure S3 ML tree obtained from the analysis of the Cytochrome Oxidase I (COI) mtDNA data set (table S2)Only bootstraps higher than 50% are given

Figure 7 . 1

 71 Figure 7.1 Idas argenteus morphometry with increasing size (multiple studies and sample sites), and its anatomy Depicted is the full record of shells used to assess the species, based on the description in Ockelmann and Dinesen (2011), and on the original holotype[4]. Note that the valves from source[3] bear a strong resemblance to those of Idas lamellosus. Scale bars are 500 µm (when above image); and 1 mm (when below image). Image sources are:[1] this PhD research; [2] Dean 1993; [3] Oliver et al. 2010; [4] Jeffreys 1881; Oliver et al. 2010. 7-1.1.2Consequences for the phylogeny of the Bathymodiolinae (s.l.)

(

  2013b), for the re-introduction of the genus TeruaBartsch & Rehder (1938), previously synonymised with Adipicola Dautzenberg, (1927), since Adipicola takes precedence chronologically. Equally, the amalgamation of① e e s①of①the① childressi ①g oup① ith①A. crypta and the limited number of Gigantidas sequences (one described species only) to form a single clade ranked at the genus level (Gigantidas) is also questionable in their arrangement between the two papers. The clade containing the othe ① Nypamodiolus ① e e s① I. japonicus, ESU I, and ESU J and A. longissima), is not as deep branching inLorion (2013) as the equivalent clade inThubaut (2013), which also includes I. (s.l.) simpsoni. Consequently, depending on which analyses one trusts, I. (s.l.) simpsoni would either be in a clade that is most closely related to the Idas (s.s.) and Bathymodiolus (s.s. ① lades① ut① a hi g①late ①tha ①the① B. (s.l.) childressi ①g oup①Lorion et al. 2013) or it

  2). Occasional records cited in the literature (e.g. some withinPelorce and Poutiers 2009) could not be confirmed directly (in particular, the location of the largest recorded specimen of I. (s.l.) simpsoni, fromWarén and Carrozza 1990). When viewed in conjunction with Table7.1, habitat associations can be placed in the context of geographical location. Notably, through the course of the current research, new records of I. (s.s.) modiolaeformis include specimens found associated with various deployed wood experiments both in the Lacaze-Duthiers Canyon and at the Rainbow vent field (I. aff. modiolaeformis/macdonaldi), while new records of I. (s.l.) simpsoni are from deployed wood experiments in the Lacaze-Duthiers Canyon. Rather surprisingly, the juvenile I. aff. modiolaeformis/macdonaldi at the Rainbow Vent Field appeared to be possess few or no symbiotic bacteria, based on histological evidence (toluidine blue, DAPI, FISH) and a failure to amplify bacterial DNA in soft tissues (PCR). The distribution clearly demonstrates a considerable overlap in habitat distribution and yet co-occurrence of the two species is apparently restricted to the wood recovered from the Lacaze-Duthiers Canyon in the Gulf of Lion, western Mediterranean. This is probably due to the unavoidably sporadic nature both of deep-sea sampling and reducing habitat distributions (based on current knowledge). It seems likely however that with a greater sampling effortfocussed

  Gametogenic and spawning periodicity were both thought to be semi-continuous based on the minimal amounts of connective acinal or inter-acinal tissue, which suggests active gametogenesis as opposed to the storage of gametes (LePennec and Beninger 2000), and on the capacity to spawn multiple times (live experiments, possibly indicative of dribble spawning). Certainly mussels were ready to spawn in June of 2012 and May 2013 in the Gulf of Lion. However, this assessment remains speculative in the absence of time series sampling that might identify relative changes in gametogenic patterns (e.g. such as inTyler et al. 2007a), or changes in the amount of inter-acinal tissue or the thickness of the acinal walls, indicative of seasonal gametogenesis (LePennec and Beninger 2000).7-2.3. Theme 2a: Larval developmental biology subthemeThe objectives of both the larval and post-larval development subthemes were the most ambitious of the current research. Of the two, the latter was met most fully by the research presented herein. The objectives of the larval developmental work were well met at the design stage but fell short in terms of useable results. Preparations for live observations were considerable. As a part of this, aquaria were designed and constructed based on several previously validated systems both academically and in commercial hatcheries.

Chapter 4 (

 4 p. 163 onwards;Laming et al. 2014), rendered estimates of growth in I. (s.l.) simpsoni on cow bones from the Setúbal canyon impossible however, since this represented a stratified random subsample of mussels only (i.e. largest individuals recorded were not available). However, following the approach employed for eastern Mediterranean I. (s.s.) modiolaeformis specimens (section4-5.1.2, p. 177; Laming et al. 2014), rough estimates of growth could be inferred for I. (s.s.) modiolaeformis and I. (s.l.) simpsoni from the Lacaze-Duthiers Canyon wood deployments as well. These were based on SL in the relatively large male and female adults, but over longer deployment periods. Despite the fact that the largest specimens of I.

  Identifying the rate of maturation in juvenile mussels represented the central aim as far as developmental biology was concerned. Size at first maturity was assessed based on the smallest size at which all stages of 7-2.4.4Gill development and growth Symbionts play a vital role in the lifecycle of Idas spp. Of particular interest for this research was the initiation of this symbiosis, in terms of acquisition and localisation. Thus, it was of prime importance that the structures within which these bacteria are housed were investigated. The development and proliferation of gills in I. (s.s.) modiolaeformis and I. (s.l.) simpsoni was thus studied in detail. In some respects the observed patterns of development echoed those identified in the shallow-water mussel M.

  (s.s.) modiolaeformis associated directly with methane-seeping carbonate crusts in Pockmarks Central 2A region of the NDSF, eastern Mediterranean (up to 6 symbiont phylotypes, Duperron et al. 2008) was never identified in I. (s.s.) modiolaeformis mussels in this research, including those from the same region, but colonising wood. Despite the fact that these individuals occurred in a similar region, only a single T1thiotroph phylotype was identified (not presented, signals very low). In the western Mediterranean, I. (s.s.)

Figure 7 . 3

 73 Figure 7.3 The lifecycle of Idas (s.s.) modiolaeformis

  . Prof. Craig Young, one of the most experienced and influential researchers to have invested several decades of his academic career in the study of larval and reproductive biology, alongside many up and coming deep-sea researchers whose field of expertise resides in reproductive biology, have recently reiterated the need for research into the larval biology of deep-sea organisms. The dearth of such researchers was palpable at the last Deep-sea Biology Symposium in December 2012, where only one researcher, Prof. Craig Young himself, presented any data which actually incorporated empirical larval biology (within the context of a modelling approach,[START_REF] Young | Dispersal of deep-sea larvae from the intra-American seas: simulations of trajectories using ocean models[END_REF]. Addressing the lack of investment in this research discipline in deep-sea organisms, could significantly contribute to our understanding of the dynamics of deep sea benthic environments, as they has in shallow water systems. Prof. Craig Young put it quite succinctly, when he was heard to say:

Table 1 .

 1 1 Depth and geographic distributions of the Bathymodiolinae (s.l.)...........................................................................

Table 2 .

 2 1 Site and sampling details ........................................................................................................................................

Table 3 .

 3 1 Fixation and preservation regime .......................................................................................................................... Table 7.1 Synthesis of the results from this research integrated with those from other habitats ........................................

		ABBREVIATIONS
	±	"either side of"
	≈	approximately equal to
	≡	equivalent to
	µm	micrometre
	mm	millimetre
	‰	parts per thousand
	σ	standard deviation
	♀	female
	♂	male
		hermaphrodite

  Bowie seamount, Canada; Tasmanian seamounts, Australia and; fisheries closures over 19 seamounts spread throughout the New Zealand, Pitcher et al. 2007), no-fishing zones in cold-water coral reef habitats (e.g. Bering Sea-Aleutian Islands ecosystem, Ruckelshaus et al. 2008, assessed globally with generally positive results, Selig and Bruno 2010), and MPAs prohibiting the disturbance, damage, destruction or removal of any living marine organism from submarine canyons (The Gully, Canada; Soquel Canyon State Marine Conservation Area, Monteray Bay, US, pelagic fin-fishing accepted).

	Future human activities could place additional habitats at threat through the extraction of lucrative
	mineral resources such as polymetallic sulphide deposits at	hydrothermal vents, cobalt-laden
	ferromanganese concretions on seamounts, and hydrocarbon reserves (e.g. methane, gas hydrates, oil)
	buried at hydrocarbon seeps (Ramirez-Llodra et al. 2011; Van Dover 2011a). This was the argument behind

). MPAs for the four high-risk habitats previously listed include the Northeast Atlantic Fisheries Commission Regulatory Area for upper-slope sedimentary benthic communities, numerous MPAs which restrict or prohibit bottom fishing on seamounts communities (e.g. Formigas-Dollabarat Bank, Azores, Portugal; Sitka Pinnacles Marine Reserve, Alaska; the establishment of the International Seabed Authority (ISA): an intergovernmental body which seeks to orchestrate and control all mineral-related commercial activities expected to take place beyond the limits of national jurisdiction, i.e. in the High Seas. The ISA itself was a product of UNCLOS (United Nations Convention on the Law of the Sea) which delineates the entitlements and responsibilities of ramified nations concerning global oceanic ecosystem services, by creating guidelines for enterprise, conservation, and the ethical, sustainable management of natural marine resources.

  -8.4. Organic-fall mytilidsIn the Bathymodiolinae, although knowledge on aspects of adaptive biology and physiology in the large Bathymodiolinae (s.s.) is growing, data are wholly deficient regarding the biology of small-sized genera that typically colonise decomposing wooden debris and megafaunal carcasses. This includes timing of symbiont acquisition in the context of other aspects of development, but also basic metrics of ontogeny, settlement, rates of growth, reproductive development and spawning habits, lifelong nutritional needs and anatomical or physiological adaptations (if any) to reducing habitats. Gaining a handle on these issues seems particularly pertinent considering some members of the small-sized Bathymodiolinae appear to branch basally within the subfamily (e.g. Benthomodiolus, Vulcanidas, Lignomodiolus and Nypamodiolus,Thubaut et al. 2013b), with obvious evolutionary implications. The suggestion that both Idas and Adipicola represent analogous genera to a Bathymodiolus ancest al① state① i ① te s① of① ha itat① use① oode ① steps ①

. This pattern of general-to-specific infection appears to occur in two other Bathymodiolus (s.l.), B. (s.s.) azoricus and B. (s.s.) puteoserpentis, confirmed by employing FISH upon host tissues using targeted oligonucleotide probes for the expected bacterial symbionts

(Wentrup et al. 2013)

. Symbionts were present in all specimens, the smallest being juveniles of 4mm shell length, however, infection in the smallest individuals was specific in terms of phylotype but generalist in terms of tissue type. In adults, increased specificity resulted in the disappearance of non-gill epithelial symbionts. In B. (s.s.) azoricus at the Lucky Strike hydrothermal vents and the seep species B. (s.l.) heckerae at Blake Ridge cold seeps, patterns of symbiont infection appeared to vary independently of size (ranged down to post-larval specimens with no evidence of post-settlement growth,

Salerno et al. 2005)

, instead reflecting variations in habitat fluid composition between the seep (predominance of methanotrophic symbionts) and vent environments (being chemoautotrophic bacteria dominated). Stable isotopes signatures supported chemosymbiotic life habits at all stages of benthic development.

1hypothesis,

Distel et al. 2000)

, has since been tested more rigorously as sequence data amasses. For the most part, such a hypothesis remains plausible from a genetic point of view, with only a few examples hi h① do t① fit① the① p oposed① ood-whale-vent evolutionary progression, considered atypical habitat reversals (e.g. I. macdonaldi, A. [s.l]. pacifica). Their appropriateness as analogous species is sometimes called into question, based on the equivalent evolutionary position they adopt in most phylogenetic trees for the subfamily

(Dubilier et al. 2008)

. Despite this, they present less-integrated, but more diverse symbioses and conversely, a greater flexibility in habitat suitability, which suggests that they represent lessspecialised lineages. Developmental traits that permit the efficient colonisation of organic falls and their exploitation remain mysterious. What is more, the reproductive kinetics and ecology underpinning organic fall① ha itats① a e① still① e t apolated① f o ① eithe ① shallo ① ate ① e ui ale ts① o ① e d-state ① spe ies① of① Bathymodiolus, restricted to highly specific vent habitats. 1-9 The hypothesised bathymodiolin lifecycle 1-9.1. What is known Lorion et al. 2013 identified that bathymodiolin mussels (s.l.) at deep vents (particularly the Bathymodiolus sensu stricto clade) are end-state species with regard to habitat use. In other words they have reached an e olutio a ①dead①e d .①I ①light①of①this,①it s①see s① ou te -intuitive to back-cast biological assertions about

  Ockelmann and Dinesen 2011 and; the bone colonising mussel A. (s.l.) pacifica,[START_REF] Kinoshita | Protandric hermaphroditism in the whale-fall mussel Adipicola pacifica[END_REF]. Sex determination appears to be one area within which the genera differ, since larger Bathymodiolus species are often gonochoristic, including B. azoricus; B. childressi; B. mauritanicus and B. puteoserpentis(Duperron et al. 2013). Reports can be conflicting however, asComtet et al. 1999 suggest that B. azoricus might exhibit hermaphroditism, while in B. (s.s.) thermophilus, which was initially reported as protandric on the MAR (at 11°N to 13°N) as part of the species description

	1-9.2. Adult reproductive biology
	1-9.2.1	Sex determination and ratios (Figure 1.23a)
	One of the attributes which has been better documented in small-sized chemosymbiotic is their tendency

towards protandric hermaphroditism, identified in all four species examined to date, of which three were Idas spp. (the seep and wood-based mussel I. modiolaeformis,

Gaudron et al. 2012

; the bone-colonising mussel I. washingtonius,

Tyler et al. 2009; the wood-colonising, asymbiotic, larviphage I. argenteus,

  . Spawning patterns remain unresolved for most other species on account of a lack of intra-annual temporal sampling, however there is strong evidence for discontinuous spermatogenesis in B. (s.s.) puteoserpentis and B. (s.s.) elongatus[START_REF] Pennec | Ultrastructural characteristics of spermatogenesis in three species of deepsea hydrothermal vent mytilids[END_REF]) and a readiness to spawn in November in Idas modiolaeformis (only mature gametes were identifiable regardless of sex), from the central pockmarks methane-seepage region of the NDSF, eastern Mediterranean.

  Arellano and Young 2009), where it was only after 40 h that embryos had reached the hatched blastula stage (Figure1.23c) in B. (s.l.) childressi.

	1-9.3.2	Late embryonic and larval development
	Subsequent to hatching, B. (s.l.) childressi (Arellano and Young 2009) took a period of about 7 days at 7-8 °C
	1-9.3. Larval development
	1-9.3.1	Fertilisation and early embryonic development (Figure 1.23b-c)
	Following gamete release, fertilisation is presumed to be external (Figure 1.23b, Seed 1969) as in shallow
	water relatives; however no data confirming this one way or another is published. Cleavage is spiral in other
	Mytilidae, mirrored in those bathymodiolin species in which cleavage has been witnessed (e.g. Arellano and

Young 2009)

. The rate of cell division and embryonic growth appears to be retarded in Bathymodiolinae (taking approximately twice as long) based on the limited data available (B. (s.l.) childressi,

  ; Figure1.23g-i, minus the filamentous substratum). Such behavioural features in deep-sea mytilid species settling within spatially constrained reducing habitats might prove pertinent since intraspecific larviphagi in adult bathymodiolins has been reported (unpublished observations in[START_REF] Comtet | Population structure and recruitment in mytilid bivalves from the Lucky Strike and Menez Gwen[END_REF], and is known to influence settlement mortality rates in shallow water mytilids when it occurs[START_REF] Porri | Does cannibalism of larvae by adults affect settlement and connectivity of mussel populations? Estuarine[END_REF] but Tamburri et al.

2007

). The propensity for this behaviour in deep-sea mytilids is however, unknown, though some juvenile traits, such as settlement on adult shells (e.g. Bathymodiolus (s.s.) spp. at Lucky Strike vent field, MAR) are mirrored in M. edulis.

  [START_REF] Gustafson | Molluscan Life History Traits at Deep-Sea Hydrothermal Vents and Cold Metha e/"ulfide①"eeps.①Reproduction, larval biology, and recruitment of the deep-sea benthos[END_REF] Salerno et al. 2005) and is suggested for B. mauritanicus byArellano et al. (2014).Studied species in the genus Idas (s.l.) display similar trends, such as in the holotype Idas (de facto) argenteus, Ockelmann and Dinesen 2011; the seep and wood-fall mussel Idas (s.s.) modiolaeformis,(Gaudron et al. 2012); the seep, whale and wood-fall mussel Idas (s.l.) simpsoni Idas o .①sp.,①Ma a a ,①

including the aforementioned B. (s.l.) childressi

(Arellano and Young 2009)

, B. (s.s.) thermophilus

[START_REF] Berg | Reproductive strategies of mollusks from abyssal hydrothermal vent communities[END_REF] Craddock et al. 1995a

) and B. (s.s.) antarcticus by inference

[START_REF] Johnson | A hybrid zone between Bathymodiolus mussel lineages from eastern Pacific hydrothermal vents[END_REF]

; B. (s.s.) azoricus,

(Colaço et al. 2006)

; B. (s.l.) securiformis and B. (s.l.) hirtus

[START_REF] Okutani | Two new species of Bathymodiolus (Bivalvia: Mytilidae) from methane seeps on the Kuroshima Knoll off the Yaeyama Islands, southwestern Japan[END_REF]

); B. (s.s.) heckerae,

  Adult host biology is reasonably well-documented in select end-state Bathymodiolus spp. in the subfamily. With each newly published study, the dynamics of symbiont assemblages in relation to alterations in their chemical environment are becoming better-esta lished.①Ho e e ,①as① a ① e①see ①f o ①the①a o e①se tio s,①the①i teg ati e① holo io t ① biology requires renewed focus, particularly regarding timing of acquisition, which is thought to be horizontal. Although studies exist which identify the presence of symbionts at various stages of the life cycle, no studies exist that actually identify precisely when it is that bathymodiolin species first become symbiotic. Symbiont acquisition remains unresolved in nearly all chemosymbiotic species, except in isolated species employing horizontal transmission (e.g. the siboglinid, Riftia pachyptila[START_REF] Marsh | Larval dispersal potential of the tubeworm Riftia pachyptila at deep-sea hydrothermal vents[END_REF] B. (s.l.) childressi,Streams et al. 1997; B. (s.s.) azoricus,Salerno et al. 2005; Wentrup et al. 2013; B. (s.s.) heckerae,Salerno et al. 2005; B. puteoserpentis,

  . Two sister species are known to occur at methane seeps (I. [s.s.] modiolaeformis and I. [s.s.] macdonaldi, Gustafson et al. 1998; Olu-Le Roy et al. 2004), with one species even occurring at vents (I. [s.s.] washingtonius, Southward 2008). Other deepbranching clades, in which one includes species assigned to Adipicola (s.l.) and Idas (s.l.) spp., and the other Benthomodiolus, represent additional examples of basal clades frequently with organic-fall associations.

Vulcanidas, in contrast, may have a quite different evolutionary history; contemporary species occur at shallow vents but fossil records originate from much deeper vent sites. This suggests deep-to-shallow migration, perhaps driven by large-scale deep-sea anoxia during the Cretaceous-Paleogene extinction event

(Jones et al. 2006)

or competitive exclusion by the emerging Bathymodiolus (s.l.) spp.

[START_REF] Kiel | The earliest bathymodiolin mussels: an evaluation of Eocene and Oligocene taxa from deep-sea methane seep deposits in Western Washington State, USA[END_REF]

.

Co side i g①the①e ti e①su fa il s①ph loge ① he e① o -Bathymodiolus spp. typically branch deeper in the trees), and the tendency both for less-integrated extracellular symbioses (s.l.) and less-hypertrophied gills in the small-sized Bathymodiolinae (s.l. ,① a① steppi g① sto es ① h pothesis① fo ① ha itat① use① Distel① et① al.① ) remains plausible. This could explain contemporary phylogenetic trends in the Bathymodiolinae (s.l.), with isolated habitat reversals in a few species

(Miyazaki et al. 2010)

. The order of habitat transition in terms of type (and depth, Lorion et al. 2013) over evolutionary time remains contentious, since discrepancies exist between evolutionary clock estimates and the appearance of bathymodioliform mussels in fossil records (being rather seeps first, and then almost parallel appearances on wood and whale falls, Amano and Little ;① Kiel① ;① Kiel① a d① Goede t① ;① Kiel① a d① ② a o① .① Esse tiall ,① although① it s① ot① lea ① u de ① which circumstances the rapid radiation of the Bathymodiolinae (s.l.) occurred (Lorion et al. 2013), contemporary species which fall① outside① the① u e t① lades① o tai i g① Bathymodiolus ① s.l.) spp. (which includes Gigantidas [s.s], Miyazaki et al. 2010; Lorion et al. 2013; Thubaut et al. 2013b), seem to be of a less-specialised state.

  2-3.2. Known distribution and habitat use Idas (s.l.) is one of the most ubiquitous bathymodiolin genera (s.l.) found in habitats subject to reducing conditions worldwide, both in terms of locality and depth. Current records of members of the genus Idasi.e. described Idas [s.l.] spp. and Idas-like ESUsindicate a frequent habitat association with sunken, de o posi g① ood① oth① i ① elati el ① shallo ,① a d① deepe ① offsho e① ate s,① the① g eatest① k o ① spe ies①

	One species, Idas washingtonius, is even reported from the Mid-Valley vent fields, Juan de Fuca Ridge,
	northeast Pacific (Juniper et al. 1992; Southward 2008).
	The genus Idas (s.l.) thus displays an intriguing array of habitats for investigation with some species capable
	of living in association with multiple reducing habitats, including multiple examples wood and bone (Lorion

di e sit ① ei g①f o ① ate s①<① ①deep①

Samadi et al. 2007; Duperron et al. 2008b; Lorion et al. 2009; Pelorce and Poutiers 2009; Lorion et al. 2010; Giusti et al. 2012)

. Of course, this may in part be due to sampling bias. Over 20 ESUs have been collected from the South Pacific alone

(Lorion et al. 2013)

, while several putative species from sunken wood and dredged sediments, rich in organic matter, are recorded in the Atlantic (e.g.

Southward 2008; Rodrigues et al. 2013

) and the Mediterranean

(Gaudron et al. 2010; Giusti et al. 2012; Duperron et al. 2013

).

To a lesser extent associations with whale skeletons are known, often as a second habitat in species otherwise known for colonising wood

(Lorion et al. 2009

). These associations appear to occur independent of geography, in the Mediterranean (e.g. I. [s.l.] cylindricus), Atlantic (I. [s.l.] simpsoni, I. [s.s.] washingtonius, Tyler et al. 2009), and Pacific (I. washingtonius, Smith et al. 1989). Bone-associations do not appear to be cetacean-specific, but applicable to megafaunal nektonic carcasses in general (based on the dense colonisation of bovine skeletons in experimental carcass deployments within Portuguese canyons, A. Hilário, personal communication). A few Idas spp. are recorded in moderate densities at hydrocarbon seeps (e.g. I. (s.s.) macdonaldi, exclusively endemic to Garden Banks, Gulf of Mexico, Gustafson et al. 1998; I. (s.s.) modiolaeformis found on MVs in the Gulf of Cadiz, and at the Nile deep Sea Fan, East Mediterranean, Olu-Le Roy et al. 2004; Rodrigues et al. 2013 and; I. (s.l.) simpsoni in the Marmara Sea, North-east Mediterranean, Ritt et al. 2012). et al. 2009); and then I. [s.s.] modiolaeformis on wood and seeps (Sturany 1896; Olu-Le Roy et al. 2004; Gaudron et al. 2010; Rodrigues et al. 2013; the current research); I. [s.l.] simpsoni on wood, bones and at seeps (Ritt et al. 2012; Thubaut et al. 2013b; the current research) and finally; I. [s.s.] washingtonius on wood, bones and at vents

  Two of the most prevalent Idas (s.l.) species in the Mediterranean and Atlantic are I. (s.s.) modiolaeformis

	2-4.2.1	Co t asti g①aspe ts①i ①the①stud ①spe ies ①k o ① iolog
	u k o ① ea s,①i ①s	io t① G ① Dupe o ①et①al.①	a .①The①p ese e①of①all①these①s	io ts①i ①a①si gle①

and I. (s.l.) simpsoni. These two species differ in three fundamental regards: phylogenetically, morphologically and in terms of their respective symbioses

(Ritt et al. 2012)

. Based on what we know of these two species, they are not actually congeneric but instead are distantly related species only, where the latter emerges much earlier in the evolutionary history of the Bathymodiolinae, within a less-derived clade

(Thubaut et al. 2013b

, Figure

1

.19,① a otated① as① simpsoni .① I ① fa t,① the① t o① spe ies① displa ① ge eti ① dissimilarity to such an extent that they likely diverged long before colonising one of their habitats, the Mediterranean-Sea seeps

(Ritt et al. 2012

).

Idas (s.s.) modiolaeformis has repeatedly been shown to host multiple symbionts (e.g. thio-, methano-and methylotrophic symbionts;

Duperron et al. 2008a; Duperron et al. 2008b; Southward 2008;[START_REF] Halary | Direct image-based correlative microscopy technique for coupling identification and structural investigation of bacterial symbionts associated with metazoans[END_REF]

), the highest number, six, being in specimens from carbonate crust at methane seeps in the central pockmarks region of the eastern Mediterranean

(Duperron et al. 2008a)

. Such uncommonly diverse symbiont assemblages presumably provide I. (s.s.) modiolaeformis with access to highly adaptable sources of nutrition. Although the metabolic role of a sixth phylotype, Bacteroidetes, remains unknown

(Rodrigues et al. 2013)

, the remaining bacterial assemblage meet their metabolic demands by oxidising sulphur (2 phylotypes), methane (1 phylotype), an undefined methylate substrate (1 phylotype) and via an additional

  modiolaeformis (i.e. using all probes fromDuperron et al. 2008a available bar the type-T2 thiotroph probe) and Bathymodiolus (s.l.) aff. boomerang (methanotroph and thiotroph phylotypes,Duperron et al. 2005). Thus, while the symbiotic association seen in I. (s.l.) simpsoni resembles that of many other Idas spp., it differs considerably from that of I. (s.s.) modiolaeformis(Ritt et al. 2012).

	Data available on shell sizes suggest that I. (s.s.) modiolaeformis attains a maximum size (shell length 17mm,
	Olu-Le Roy et al. 2004) less than half that of I. (s.l.) simpsoni, recorded at 45 mm (cited within species
	description of I. [s.l.] ghisottii, Warén and Carrozza 1990). This is supported by a recent (unwitting)
	o pa ati e①stud ① et ee ①the①t o①spe ies① 'itt①et①al.①	,①i ① hi h① Idas-like nov. sp. ①-now known to
	be I. [s.l.] simpsoni, based on new COI data for this species (Thubaut et al. 2013b) -was examined in

, suggesting that I. (s.l.) simpsoni has either not developed active associations with methanotrophs, or that the methanotrophs in question were overlooked during FISH. The latter seems improbable since FISH upon gill tissue incorporated a comprehensive array of oligonucleotidic probes, targeting bacteria known to occur in both I. (s.s.)

  ; whale bones,Pelorce and Poutiers 2009) and the Atlantic (e.g. woodRodrigues et al. 2013). The frequency of occurrence of larger, natural wood falls and whale falls in these regions is unknown, but they most certainly occur; artificial organic-fall experiments repeatedly identify rapid colonisation rates by specialist organic-fall species, suggesting relatively local larval sources (typically plant material e.g.Gaudron et al. 2010; Cunha et al. 2013;[START_REF] Romano | Submarine canyons as the preferred habitat for wood-boring species of Xylophaga (Mollusca, Bivalvia)[END_REF] Fagervold et al. 2014; but also bovine deployments, CARCACE, project leader-Ana Hilário, preliminary results akin toJones et al. 2008). It was through such deployments (e.g. as part of the European projects CHEMECO and CARCACE), that many of the samples for this study were collected,

	summarised below.
	2-5.2. Sampling approach
	2-5.2.1	Plantigrades, juveniles and smaller adults

Wood-and alfalfa-associated specimens that ranged from newly settled plantigrades to recently matured adults were collected using standardised colonisation devices called CHEMECOLI

(Gaudron et al. 2010

, Figure 2.2), typically deployed and recovered by remotely operated vehicle during scientific cruises either to vent and seep regions (MAR and NDSF respectively, Figure 2.3; Table

  .2). As a minimum, at each experiment site one CHEMECOLI with each substratum was deployed. These were at the NDSF (eastern Mediterranean, I. (s.s) modiolaeformis collected only), Rainbow vent-field (MAR, I. (d.f.) argenteus only) and Lacaze-Duthiers Canyon (I. (s.s) modiolaeformis and I.

	(d.f.) argenteus). Once recovered, substrata were normally spilt and fixed as subsamples immediately. The
	exception to this was the Lacaze-Duthiers Canyon CHEMECOLI deployed in June 2012 and recovered in April
	2013, which were kept intact in aquaria and the ①so ted① li e ,① he e①fi atio ① as①o ①a① ase① ① ase① asis①
	rather than en masse.
	Bone-associated plantigrades, juveniles and small adults of I. (s.l.) simpsoni, came from remnant bones
	recovered from 18-month bovine carcass deployments in the Sétubal Canyon (SW Portugal, Figure 2.2;
	Figure 2.3; Table 2.1)	22

Table 2 .1 Site and sampling details

 2

	Devices	Substrate	Deployment	Cruise	Collection	Cruise	Site name	Site details	Immersion time (wks)
	CHEMECOLI C7	Douglas Pine cubes	14/07/2007	Momardream	31/08/2008	MOMAR08	MAR, Rainbow, EXO2	36°13.7454N, 33°54.0513W 2279 m deep	59
	CHEMECOLI C9	Packed alfalfa grass	14/07/2007	Momardream	31/08/2008	MOMAR08	MAR, Rainbow, EXO2	36°13.7454N, 33°54.0513W 2279 m deep	59
	Carcass (bovine)	Bone remains	?	CARCACE	?	?	Setúbal Canyon	exact location unknown (1000m)	78
	Wood deployment (small and large)	Oak	04/09/2011	LD3	03/07/2012	LD4	Lacaze-Duthiers Canyon	42°32.728'N, 3°25.267'E (525m)	43
	CHEMECOLIs x 2	Douglas Pine cubes	06/07/2012	LD4	24/04/2013	LD5	Lacaze-Duthiers Canyon	42°32.728'N, 3°25.267'E (525m)	42
	CHEMECOLIs x 3	Packed alfalfa grass	06/07/2012	LD4	24/04/2013	LD5	Lacaze-Duthiers Canyon	42°32.728'N, 3°25.267'E (1000m)	42
	Wood deployment (V. large)	Palm incl. cortex	Nov-10	LD1	25/04/2013	LD5	Lacaze-Duthiers Canyon	42°32.728'N, 3°25.267'E (525m)	129
	CHEMECOLI C13	Douglas Pine cubes	18/11/2006	Bionil	10/11/2007	Medeco	Pockmark area (centre)	32 31.9772N, 30 21.1779E 1693 m deep	51
	CHEMECOLI C11	Packed alfalfa grass	18/11/2006	Bionil	10/11/2007	Medeco	Pockmark area (centre)	32 31.9772N, 30 21.1779E 1693 m deep	51

3 Sorbonne Universités, UPMC Banyuls, UMR8222 Laboratoire d'Ecogéochimie des Environnements Benthiques (UPMC CNRS), Station marine de Banyuls, Observatoire Océanologique 4 Institut Universitaire de France, Paris, France Corresponding author: svenlaming@googlemail.com N.B. This manuscript is written for submission to Deep Sea Research (pending)

  Musselshells were scrubbed and then rinsed in ASW. The most effective methods for induction were shock treatments (mechanical, thermal and saline, discussed in results). Initially, mussels were kept together (to improve induction success,Arellano and Young 2009) in a flat-bottomed container with a black background These were collected separately by filtering (60 µm Nitex mesh) and extracting the oocytes by pipette from the more concentrated ASW eluate.Sperm concentrations were calculated using single-use haemocytometers (Euromedex Ltd.).Oocyte numbers were calculated from triplicate 1 ml subsample counts. Gametes were kept on ice until fertilisation. Due to apparent dribble-spa i g① a d① lo ① esulti g① fe u dit ① total① ≈ ① oo tes ,① all① oocytes were pooled, while sperm from each individual were kept separate. Subsamples of unfertilised oocytes and sperm were photographed. Fertilisation was carried out in sterile Pyrex Petri dishes, at a density of 20 oocytes ml

	for contrast. Mussels believed to have begun spawning were relocated to individual compartments in sterile
	6-well cell-culture plates (Evergreen Scientific) with renewed ASW, for the collection of gametes separately
	by pipette (Supplementary figure 3.1E-F). However, a notable number of buoyant waterborne oocytes
	were found during a post-isolatio ①e a i atio ①of①the① o	u al① i du tio ①②"W①afte ① ussel①isolatio .①
		-1 ASW: Supplementary
	figure 3.1A). After five days, sixteen healthy putatively-adult mussels (i.e. > 2.35 mm in I. modiolaeformis,

Laming et al. 2014)

were selected for spawning (sample code IF#). Those awaiting induction were kept immersed in unfiltered seawater (15-18 °C, running tap-water bath, Supplementary figure 3.1B-C). -1 ASW (15mleach, 7 dishes, from 7 males, 1000 spermatozoa : 1 oocyte). After 30 min, each mixture was sieved through a 45-, 60-then 100-µm Nitex mesh to remove organic debris (mainly body fluids and excess sperm, Loosanoff and Davis 1963; Arellano and Young 2009), during which time the ASW was replaced. Air was introduced into each dish via parallel air-lines fitted with a sterile micropipette tip, to aerate the oocytes and reduce sedimentation (Supplementary figure 3.1G). ASW changes were made every 6 h approximately. Oocytes were examined before fertilisation and every 30 min following, for the first 4 hours, with increasing gaps in time thereafter (ranged from 2 up to 8 hours) over the course of the next 3 days, when the experiment was terminated due to halted development. Oocytes were photographed

Table 3 .1 Fixation and preservation regime

 3

	Tissue	Fixation / storage	Purpose
		IF1
	Shells	96% ethanol	Shell description
	Gill (x1)	96% ethanol	DNA extraction
	Remaining tissue	4% Formaldehyde (ASW), 2 hrs (RT) / gradient transfer 96% ethanol (4°C)	FISH / Histology
		IF2-IF14
	Shells	96% ethanol	Shell description
	Foot	96% ethanol	Isotopes analysis
		Liquid Nitrogen / Frozen (-80°C)	Host DNA
	Gill (x2)	4% Formaldehyde (ASW), 2 hrs (RT) / gradient transfer 96% ethanol (4°C)	FISH / Histology
	Mantle	Liquid Nitrogen / Frozen (-80°C)	Host DNA (backup)
	Gut Gonad	4% Formaldehyde (ASW), 2 hrs (RT) / gradient transfer 96% ethanol (4°C)	H & E staining
	Oocytes	4% Formaldehyde (ASW), 2 hrs (RT) / gradient transfer 96% ethanol (4°C)	Hoestch staining
		IF15, IF16, IB1, IB2
	Whole	4% Formaldehyde (ASW), 2 hrs (RT) / gradient transfer 96% ethanol (4°C)	Shell description / Histology / (extended) DNA extraction / FISH
	3-3.4. Material analysis
	3-3.4.1	Shell morphology

Shells were photographed and shell heights (SH) and lengths (SL) were measured using a standardised approach

(Laming et al. 2014

) under a camera-mounted dissection microscope (Olympus S2X12 microscope, Japan). Image-stacks of all focal-planes in the Z-axis were aligned in FIJI (Image-J 1.48r, StackReg, scaled rotation) and extended depths of field were achieved using the EDOF ①plug-in (easy mode) in FIJI (Image-J 1.48r; Supplementary figure 3.2). Tiled fully-focused images were merged in Photoshop CS3 (without transformation) for the three largest shells. Relationships between SL and SH with increasing size in both species were examined using Pea so s① o elatio ① oeffi ie t,① ith①a①t o-tailed test for significance (homoscedasticity and normality confirmed). A type III sums of squares (SS) ANOVA was employed (to accommodate unbalanced data and low samples sizes, homogeneity of variance and normality confirmed)

  .1). Idas (s.l.) simpsoni

	dominated numerically (n = 11), identified based on shell morphology and 99-100 % COI sequence
	similarity). All-but-one of the remaining mussels (including IB1) in this study matched Idas sp. Med (n = 6,
	99%; Idas (s.s.) modiolaeformis, Lorion et al. 2012). Mussels recovered from the previous year from
	oakwood substrata, were dominated numerically by I. (s.s.) modiolaeformis in contrast (n = 25, versus I.
	(s.l.) simpsoni n = 2, Thubaut 2012, note different wood and deployment conditions). The haplotypes of the
	two dominant species differed by 86 polymorphic sites in 497 bp (excluding gaps/missing data). This level of
	divergence warrants classification in two distinct genera, as indicated by Thubaut et al. (2013b). The
	remaining mussel (IB2), was a third unidentified species (Supplementary table 3.1, not considered further in
	this paper	23

  , the eastern Atlantic (on a dolphin skull, Bay of Biscay, Quero 1973; on bone experiments, Iberian margin, Setúbal canyon, Génio et al. submitted), the western Mediterranean (on wood experiments, Gulf of Lion, Thubaut 2012, this study), the central Mediterranean (northern Tyrrhenian Sea and the Adriatic Sea, Bolotin et al. 2005; Pelorce and Poutiers 2009; Giusti et al. 2012) and the north-eastern Mediterranean (at methane seeps as Idas sp.① o ,① Ma a a① "ea,① Ritt et al. 2012). It is unclear which combination of factors determines the geographic locations of these species, as both exhibit wide depth ranges and utilise diverse chemicallyreduced habitats (I. modiolaeformis wood and seeps, 350-3015 m; I. simpsoni bone, wood and seeps, <100m -1120 m).

  , and an age at first maturation (for males) identified at approximately 4 months following settlement, for I. (s.s.) modiolaeformis in the eastern Mediterranean (51-week wood deployment, no female sex-switching,Laming et al. 2014).

table 3 .2 A summary of the stable Isotopes data used to plot Figure 3.8

 3

	IF12 IF11 IF08 IF15 IF16 IF02 IB02	Palm Palm Palm Palm Palm Palm Palm Pine	♀ ♀ ♂ ♂ ⚥ ⚥ ♂ ♂	I. (s.l.) simpsoni ? ≈ Idas sp. ESU D	507/508(99%) 529/532(99%) 528/530(99%) 509/510(99%) 492/493(99%) 493/494(99%) COI 374/436(86%)	"I."simp C "I."simp D "I."simp E "I."simp F "I."simp G "I."simp H Unconfirmed Oblong Idas sp.
	Supplementary Sample ID				δ 15 NAIR (‰)	δ 13 CV-PDB (‰)
				Banyuls 2012 (Oakwood)
	CO28D a					1.4	-27.9
	CO28F a & CO28C a "alcohol"				1.6	-28.1
	CO28A a					2.1	-27.9
	CO24F a & H a				2.2	-27.7
	CO24E a					2.2	-25.6
				Mean	1.9	-27.4
				St. Dev.	0.38	1.05
				Banyuls 2013 (Pinewood)
	IF09 a & 14 a					4.8	-20.7
				Banyuls 2013 (Palmwood)
	IF04 b & 10 a					9.6	-29.7
	IF07 b					8.1	-31.7
	IF13 b					8.2	-31.5
	IF5 b					8.4	-32.1
	IF8 b					8.4	-31.2
	IF12 b					8.7	-31.1
	IF02 (b) & 06 b					9.0	-31.1
	IF03 b					9.6	-31.0
				Mean	8.7	-31.3
				St. Dev.	0.50	0.40
				Substrata 2013
	Palmwood					10.7	-24.

  Age at first maturity is generally considered a more robust measure of comparative reproductive developmental rate, being less sensitive to variable growth rates (though not always, e.g.[START_REF] Nakaoka | Size-dependent reproductive traits of Yoldia notabilis (Bivalvia: Protobranchia)[END_REF]).

	4-5.2.2	Approximate age at first maturity
		,
	ep ese ti g①≈ %①of①la gest① e o ded①"L,①"L max = 263 mm, from Boss and Turner 1980) and; 40-60 mm in B.
	thermophilus depending on sex (Berg 1985 ,① ep ese ti g①≈ . -29% of SL max (205 mm, from Nedoncelle et
	al. 2013). I. (s.s.) modiolaeformis falls① elo ①these①t o①at①≈ %①of①"L max (17 mm, Olu-Le Roy et al. 2004).

  are integrated. The time-averaged growth rate of 0.017 mm d-1 in the current study, may be cautiously applied to the size-at-first-maturation for comparison, where a SL of 2.35 mm, 36% of the largest SL (6.54 mm), represents an age of① ≈ ① o.① This① o e sio ① assu es① that① ea l ① growth rates in I. (s.s.) modiolaeformis are less variable than in coastal species. The lack of obvious growth discontinuities in the shell may reflect a reduced dependency on an external food supply(Seed and Suchanek 1992). The second is that compound size-scaling effects upon daily increments of growth are limited; since the size range examined here covers <40% of the range recorded for the species, this is considered only a moderate source of error. This rough① esti ate① of① ≈ ① o① fo ① age-at-first-maturity, is half the age of the relatively massive Bathymodiolinae example available, younger than most heterotrophic mussels of the genus Mytilus (except M. trossulus), and considerably younger than many other benthic invertebrate species (listed in Gosselin

	and Qian 1997, appendix 1). This, and the fact that the associated size at first maturity accounts for only
	14% of the known L max , are thought to represent evolutionary adaptations maximising reproductive
	developmental rate.
	4-5.2.3	Protandric hermaphroditism in reducing habitats
	All individuals >2.35 mm were confirmed male. Based on a larger number of adults, Gaudron et al. (2012)
	demonstrated that sex ratios are size-dependent in I. (s.s.) modiolaeformis from authigenic carbonate crust
	in the same region. I ①se uall ①fu tio al① t a sitio ① ales① . -11.6 mm SL, female acini were frequently
	present but non-functional (disintegrated oocytes). Females were exclusively > 7 mm in length, with a single
	size-intermediate hermaphrodite. This information provided compelling evidence for protandric
	hermaphrodism with a proposed intermediate period of abrupt sex-switching, similar to I. washingtonia
	(Tyler et al. 2009, since redescribed as I. washingtonius, Coan and Valentich-Scott 2012), where 7 mm also
	represented the lower female size threshold. Neither mature females nor female-sized empty shells, were
	identified during sample-sorting in the current study, providing evidence that maturation in this species is
	towards becoming predominantly male, and that the first sex alternation may not occur within the first 51
	weeks (or <6.54 mm). For the local population to reach size/age at first reproduction, a period in excess of
	① eeks①is①thus① e essa ,①u de ①the①e pe i e t s①e i o ental conditions.

  Laurent et al. 2013) likely predominated in the immediate environment of the organic substrata provided, which would have permitted thiotrophic symbionts to dominate. However the possibility that different symbionts are acquired at different stages of development cannot be ruled out.Specimens in this study <0.595 mm appeared to be either devoid of bacteria, or bacterial signals were undetectable. When infected, putative bacteria identified upon non-ciliated epithelia in individuals from 0.595-<4.37 mm in SL (confirmed with TEM, except in the smallest infected individuals) were comparable to gill-associated bacteria. This infection became more gill-spe ifi ① i ① adults① spe ifi all ① . ① .①Although this pattern of general-to-specific tissue infection is based on signals of putative symbionts only, it has already been shown in juveniles (4-21 mm) of two considerably larger bathymodiolin species,

	4-5.3.2	If not symbiotic, then what?
	Bathymodiolus azoricus and B. puteoserpentis

  13) Examine slide under microscope again. If satisfied, proceed to Eosin staining.

	4-9.4.3	Eosin-Y staining (aqueous)
	14) Blow-dry slides rapidly (this step was included to prevent alteration of Eosin stain pH by dilution from
	precious rinse step
	15) Working-stock Eosin-Y stain (see above) is applied by aliquots (30-50µl) to each section under
	examination.
	16) Leave for 20 mins (prolonged staining step)
	17) Rinse slides for 10 seconds with aqueous, glacial acetic acid (pH adjusted to same range as eosin, pH
	4.3 -5).
	N.B. Originally a rinse of distilled water (and on one occasion, running tap-water) was used, but this may
	have been removing or altering Eosin staining as results were poor
	4-9.4.4	Dehydration
	IMPORTANT: this step is compounded by methyacrylate resins such as LR White being sensitive to alcohol-
	it compromises the resin integrity and can lift sections as a consequence
	Dehydration is necessary to ensure that all water is removed prior to application of a suitable mounting
	medium (if not, the Eosin stain tends to be leached). Options consist of:
	18) Air-dry only at <60 °C
	19) N.B. Vacuum-assisted dessication diminished eosin staining
	20) Reduced-length alcohol series step (this seems to cause irrevocable damage to sections (shrinkage)
	but results in better contrast in staining (probably due to complete removal of water)
	4-9.4.5	Clearing and mounting of sections
	N.B. Clearing of slides using Histoclear II: this clearing medium is not compatible with many xylene-based
	permanent mounting fluids. If such a mounting medium is being used, replacing the last histoclear step with
	Xylene will removed problems associated with mixing and bubbles. An alternative is the use of Omnimount©
	(Electron Microscopy Sciences RT 17997-01), formulated to be used in conjunction with Histoclear II. If using
	xylene to clear slides, most permanent mounting fluids are suitable, however xylene (and toluene) are both
	highly-toxic.
	21) Place slides in holder with Histoclear for 30 seconds.
	22) Transfer to second bath of Histoclear for a further 30 seconds.
	23) If necessary (see note above), an additional bath of Xylene for 1 minute.
	24) Without drying slides, mount in a sufficient volume of suitable permanent mounding medium (see
	note above) to just cover samples upon addition of a cover-slip
	25) Place cover-slip and seal with nail varnish.
	26) View under compound microscope (plan-fluorite objective-lens as a minimum).

  However, SLs and SHs were plotted against one another to examine relative changes in dimensions with increasing size (Sigmaplot v. 11). Dissections were performed microscopically using custom-made tools (see supplementary figure 4.1 of Chapter 4). General anatomy was recorded and tissue was extracted, with

	minimal disruption. Where gills had remained intact (n = 31), the number of filaments comprising the
	descending lamella in either inner demibranch was recorded, as a proxy for gill development, as in Laming
	et al. (2014). Counts were plotted against shell size (Sigmaplot v. 11); best-fit analysis was performed in
	Sigmaplot (v. 11) to assess gill proliferation as a function of SL.
	5-3.2. Embedding, sectioning and histology
	Tissue was blotted dry and infiltrated (8 x 30-min infiltrations) in a gelatine capsule (size 00, Electron
	Microscopy Sciences, UK) filled with LR White resin (London Resin Company, UK), transferred to a fresh
	resin-filled gelatine capsule (orientated appropriately), the capsule was capped, and polymerised at 55°C
	(20 hours minimum). Orientations (sagittal, frontal and transverse, see Supplementary figure 4.2, Chapter 4,

  Morphometric analysis of larval shells revealed the prodissoconch I to be around 96 ± 6.3 µm. A study byRitt et al., (2012) described the phylogeny and symbiosis of a putative new species of Idas (s.l. ,① Idas nov sp. ① hi h① as①ulti atel ①ide tified①as①I. (s.l.) simpsoni, based on partial sequences released subsequently for the gene that codes for Cytochrome Oxidase I in this species(Thubaut et al. 2013b). Examination of the scanning electron micrograph of the prodissoconch for I. (s.l.) simpsoni presented inRitt et al. (2012) reveals the prodissoconch I to be approximately equivalent, at almost 100 µm. Values for related species are typically slightly smaller than this (Adipicola (s.wood/alfalfa (n = 20), measured after submission of Chapter 4 as a manuscript). Prodissoconch II①di e sio s① PdII①"H① ①"L①[±σ],① ①[±① . ]① ①403 [± 27.9] µm, n = 38) were also very similar to the single value available in the literature for this species (PdII SH x SL, 450 µm x 404 µm,Ritt et al. 2012). In descending order, values for related①spe ies① e e①PdII①"L① ±σ 544.33 ± 37.58 µm, n = 15,(A. [s.l.] iwaotakii:

	Thubaut et al. 2013a), ≈①	①µ ①i I. (d.f.) argenteus (Ockelmann and Dinesen 2011), 379 [± 11.7] x 344 [±
	5.0], n = 14, n = 27 in Idas [s.s.] modiolaeformis from seeps (Gaudron et al. 2012) and 398 [± 17.5] x 322 [±
	17.4], n = 35 in Idas [s.s.] modiolaeformis from NDSF samples on wood/alfalfa (Laming et al. 2014).
	5-5.1.1	Pre-settlement inferences

l.) iwaotakii:①PdI①"L①[±σ],① . ①±① . ①µ ,①n = 15, named Idas iwaotakii , Thubaut et al. 2013a; I. (d.f.) argenteus:①≈① ①µ ,①Ockelmann and Dinesen 2011 and Idas (s.s.) modiolaeformis: 78.6 ± 3.4 µm, n = 6 from seeps, Gaudron et al. 2012; and 80.3 ± 5.14 µm NDSF samples on

  . Species that colonise whale falls may similarly rely on retaining a level of mixotrophy. Idas (s.s.) washingtonius i ① pa ti ula ① displa s① ildl ① a ia le① δ 13 C① a d① δ 15 N isotope ratios, depending on the habitat it occurs on, ranging from values that may be considering reliably indicative of chemosymbiosis (at vents on the Juan de Fuca Ridge and larger whale falls in Monteray Bay Smith and Baco 2003; McKiness et al. 2005; Bergquist et al. 2007), to those which mirror signatures of benthic secondary consumers (e.g. small whale carcasses Smith and Baco 2003) with specimens of I. (s.s.) washingtonius on wood falling between the two extremes (Smith and Baco 2003). The rather divergent phylogeny of I. (s.l.) simpsoni (Thubaut et al. 2013b) in comparison with I. (s.s.) modiolaeformis, I. (s.s.) washingtonius, I. (s.s.) macdonaldi and the various Idas (s.l.) ESUs that colonise sunken wood in the Idas (s.s.) clade (as in Thubaut et al. 2013b) is reflected somewhat by morphological characteristics and digestive anatomy. No style-sac could be identified in the stomach (the region in coastal mytilids where enzymatic and mechanical digestion is most active, putatively identified in I. (s.s.)

modiolaeformis,

Laming et al. 2014)

. However, I. (s.l.) simpsoni has a slightly more convoluted intestinal tract when compared with I. (s.s.) modiolaeformis. In terms of patterns of symbiont acquisition and diversity, principal differences include the rate and the inherent abundance of symbiont acquisition in I.

  . It is① thus① easo a le① to① p opose① that① s iosis① a ① disappea ,① o ① e o e① less① sig ifi a t① to① the① host s① metabolism under particular circumstances. Recent examples within the bathymodiolin mussels (Mytilidae: Bathymodiolinae) indicate the presence of extracellular symbionts, sometimes in low abundances and possibly, intra-specific variability in symbiont types depending on sites (Duperron 2010; Rodrigues et al. 2013; Thubaut et al. 2013). In at least one case, Idas argenteus Jeffreys, 1876 from sunken wood in the north Atlantic, microscopy-based evidence supported the absence of bacterial symbionts and a larviphagous regime

Table 1 -Primers and PCR parameters used for amplifications and sequencing reactions

 1

		Gene		Primer	Primer sequences (5'3')	Reference
				names
	Host	COI mtDNA	50ºC	H691	GTRTTAAARTGRCGATCAAAAAT	Duperron et al. 2008b
			(35)	LCO1490	GGTCAACAAATCATAAAGATATTGG01	Folmer et al. 1994
		NADH4	50ºC	NADP2H	TGGAGCTTCTACGTGRGCTTT	Arevalo et al. 1994
		mtDNA	(35)
				ArgBl	CAAGACCCTTGATTTCGGCTCA	Bielawski and Gold 1996
		16S rRNA	55ºC	16SA	GGARGTASGCCCTGCCCWATGC	Baco-Taylor 2002
			(35)	LRJ	CTCCGGTTTGAACTCAGATCA	Ratnasingham and Hebert
						2007
		18S rRNA	55ºC	1F	ACCTGGTTGATCCTGCCAGTAG	Giribet et al. 1996
			(35)	5R	CTTGGCAAATGCTTTCGC	Giribet et al. 1996
				3F	GTTCGATTCCGGAGAGGG	Giribet et al. 1996
				9R	ATCCTTCCGCAGGTTCACCTAC	Giribet et al. 1996
				Bi	GAGTCTCGTTCGTTATCGGA	Okuzu et al. 2003
				A2	ATGGTTGCAAAGCTGAAAC	Giribet et al. 1996
		28S rRNA	55ºC	C1prime	ACCCGCTGAATTTAAGCAT	Hassouna et al. 1984
			(35)	C4	TCGGAGGGAACCAGCTACTA	Hassouna et al. 1984
		Histone 3	55ºC	F1	ATGGCTCGTACCAAGCAGACVGC	Colgan et al. 1998
			(35)	R1	ATATCCTTRGGCATRATRGTGAC	Colgan et al. 1998
	Bacteria 16S	45°C	27F	AGAGTTTGATCATGGCTCAG	Lane 1991
			(27)	1492R	GTTACCTTGTTACGACTT	Lane 1991
		23S	53°C	3505F	GACCGTCAGCTAAGGTCCCAA	Stewart and Cavanaugh 2009
			(35)	4761R	CCAGTCAAACTACCCACCATG	Stewart and Cavanaugh 2009
		APS	58°C	APS1-FW	TGGCAGATCATGATYMAYGG	Meyer and Kuever 2007
		reductase	(25)
				APS4-RV	GCGCCAACYGGRCCRTA	Meyer and Kuever 2007
		V5-V6			CAAACAGGATTAGATACCCTG	Wang and Qian 2009
					TGTTGGGTTAAGTCCCGRAACG	Wang and Qian 2009

Table 7 .1 Synthesis of the results from this research integrated with those from other habitats

 7 In red, are the data that have been elucidated from the work undertaken within the framework of this PhD. Themes, broken down into multiple aspects of the research are listed in the first columns. Data contributed by the PhD research are in red. Known distribution notation refers to the distribution map presented in Figure7.2.

	I. modiolaeformis	"I." simpsoni

A larval duration which is extensive enough to serve as a means for long range pelagic dispersal ~ s① k ① o ① o e and which originates from continental shelf benthic species

Unfortunately, neither bacteria nor their hosts have developed the ability (or inclination) to fill out scientific questionnaires.

N.B. deeper end of depth range is ba el ①deep①at①≈ ①fo ① oth①Vulcanidas and Gigantidas

Putatively assigned to this genus based on shell morphology[START_REF] Okutani | A new species of lucinid bivalve (Heterodonta: Lucinidae) from Kanesu-no-Se Bank near the Mouth of Suruga Bay, with a review of the recent species of the chemosynthetic genus Lucinoma from Japan[END_REF].

This species was collected in a JAMSTEC cruise, but it is uncertain whether it is actually associated with a chemosynthetic environment[START_REF] Okutani | Noteworthy abyssal mollusks (excluding vesicomyid bivalves) collected from the Nankai Trough off Shikoku by the ROV Kaiko of the Japan Marine Science and Technology Center[END_REF]. The genus is generally known from normal deep-sea environments.

NOT EXHAUSTIVE, Likely to be several other families, given the lack of sampling effort in both environments, which frequently overlap

Valves only, thus chemosymbiosis is inferred only

Oxygenated deep-sea soft sediments, but as a commensal on the deep-sea echinoid Pourtalesia sp. (possibly utilising commensal organic waste products)

"o e①autho s①a gue①agai st①this①le el①of①spe iatio :①o se ed① o phologi al① a iatio ①a o g①the① brevior ① tilid-complex may be due to adaptive radiation, but equally it could be a product of developmental plasticity in shell growth patterns[START_REF] Vrijenhoek | Cryptic species, phenotypic plasticity, and complex life histories: Assessing deep-sea faunal diversity with molecular markers[END_REF].

B. (s.l.) mauritanicus is considered by many to be synonymous with B. (s.l.) childressi and similarly, B. (s.l.) boomerang with B. (s.l.) heckerae: they are sister species at their most distant

This may be influenced by sampling bias given their pervasive presence in shallow-water environments

Huber, M. (2014). Kelliella sergeevi(Filatova, 1971). Accessed through: World Register of Marine Species at http://www.marinespecies.org/aphia.php?p=taxdetails&id=505370 on 2014-07-02

currently considered A. [s.l.] iwaotakii on the WORMS database (accessed 20/07/2014)

In terms of the state of their anatomy and the degree to which symbionts are integrated within the host

Includes Idasola, Iredale

[START_REF] Dell | Mollusca of the family Mytilidae (Bivalvia) associated with organic remains from deep water off New Zealand, with revisions of the genera Adipicola Dautzenberg, 1927 and Idasola Iredale[END_REF] Myridas, Iredale 1939; Myrinopsis Nordsieck, 1969; Habepegris Bernard, 1978; and indirectly Myrina H.Adams & A. Adams, 1854: which was changed to Adipicola, since the former was a junior homonym

Depths of mussels found at the Mercator MV, Gulf of Cadiz(Cunha et al.

2013) and at the Cheops Mud volcano, NDSF,[START_REF] Ritt | Diversity and distribution of coldseep fauna associated with different geological and environmental settings at mud volcanoes and pockmarks of the Nile Deep-Sea Fan[END_REF], respectively20 Depth of mussels found on a whale skull, Great Fisher Bank, NE Atlantic (40-50 fathoms, Marshall 1900) and at methane seeps in the northeast Central Basin of the Marmara Sea(Ritt et al. 2012), respectively[START_REF] Jones | Coral reef fish larvae settle close to home[END_REF] The two are sufficiently divergent to assume, as a minimum, placement in two separate genera(Thubaut et al. 2013)

Courtesy of Ana Hilário as part of the CARCACE project, Portugal

This specimen was later identified as a mature male specimen of I. (de facto) argenteus, the type species for the genus and the subject of Chapter 7, p. )

Micrographs of 600 nm semi-thin sections stained with toluidine blue at incremental locations along the lateral-lateral (left-right) axis in the small juvenile, Idas 8. Shell dimensions are cited in the grey box. a.d: ascending demibranch, a.r: anterior (byssus or pedal) retractors, by: byssus gland, c.g: cerebropleural ganglion, d.b: dorsal bend (along arrow), d.d: descending demibranch, d.g: digestive gland, ft: foot, g.ax.: gill axis (along arrow), l.p: lower labial palps, m: mantle, oe: oesophagus, p.by.r: posterior byssus-retractor, p.c: pericardial cavity, p.g: pedal ganglion, st: stomach, s.s: style-sac, u.p: upper labial palps, v.g: visceral ganglion, * = gill filaments.

This step may not always be necessary for semi-thin sections

), whale falls worldwide probably onl ① ep ese t①≈ . %①of①the① a kg ou d①pa ti ulate①o ga i ① carbon (POC) flux to the deep-sea even under the most oligotrophic central gyre waters(Baco and Smith 2003, details and assumptions therein). However the reality at the habitat scale is that whale falls provide massive influxes of labile organic matter to localised areas of the seabed(Baco and Smith 2003).26 This is under debate, given the fossil records for seep associated bathymodiolins predate other habitats

the largest individual at 6.54 mm was not entirely sectioned due to time constraints, and may have possessed the beginnings of outer demibranchs at its most posterior region

ACKNOWLEDGEMENTS

Whe e① to① sta t...① Well① that s① o ious.① Wi e.① Tha k① ou① f o ① the① otto ① of① ① sto a h.① The① usual①

3-5 Acknowledgments

Our thanks go to the pilots and crew (COMEX) and the Banyuls Marine Station LECOB research team for use of facilities (Erwan Peru, Franck Lartaud, Dimitri Kalenitchenko, Mustafa Yücel, Pierre Galand, Jadwiga Orignac) and to Marie-Catherine Boisselier, Justine Thubaut and Laure Corbari for help with sample sorting.

We thank Michael Manuel and Muriel Umbhauer (UPMC Paris 6) for the trial use of an Eppendorf Femtojet. Sample collection was through the Lacaze-Duthiers wood experiment programme (Chair Biodiversity, Extreme Marine Environment & Global Change, UPMC-Fondation Total). This research was supported by Diwood (CNRS) and by European Funds (COMPETE) and National Funds (Portuguese Science Foundation, FCT) within project PEst-C/MAR/LA0017/2013. S. Laming was co-funded by UPMC, HERMIONE EC (FP7/2007-2013-n° 226354) and a MARES PhD Grant. MARES is a Joint Doctorate programme selected under Erasmus Mundus, coordinated by Ghent University (FPA 2011-0016). See www.mares-eu.org for extra information. Funding sources had no involvement in the choice of research, the analyses, the interpretation of results and the writing or publication of the manuscript. . 4-6 Acknowledgments We thank Antje Boetius and Catherine Pierre, for samples collected during the BIONIL (M70/2b) and MEDECO cruises with RVs Meteor and RV Pourquoi Pas? funded by the ESF EUROCORES projects: CHEMECO and EuroDEEP and by the European Commission: EU HERMES and DIWOOD programs. Additional funding and logistics were met by IFREMER, CNRS and the Max-Planck-Institut für Mikrobiologie. We thank the captains and crews of RVs Meteor and RVs Pourquoi Pas? and those teams operating Quest 4000 (MARUM, Bremen, Germany) and ROVs Victor 6000 (Ifremer, France). At UPMC, our thanks go to Ghislaine Frébourg and Géraldine Toutirais (IFR 83) for assistance with TEM analysis. This work was co-funded by UPMC, HERMIONE EC (FP7/2007-2013-n° 226354) and a MARES Grant. MARES is a Joint Doctorate programme selected under Erasmus Mundus coordinated by Ghent University (FPA 2011-0016). See www.mares-eu.org for extra information. 5-6 Acknowledgements We are indebted to the pilots and crew of the Deep-sea Benthic cruise which was involved in the recovery of cow bone samples (chief scientists Marina Cunha and Ann Vanreusel). Our thanks go to Sandra and Antonío Calado for the gracious use of their ultramicrotome, and to Jörg Christian Frommlet for the use of laboratory facilities. This research was supported by ESF EUROCORES projects: CHEMECO and EuroDEEP and by the European Commission: EU HERMES and the CNRS research group Diwood, a European Joint-research programme on Wood and associated fauna. In Portugal work was supported by European funds (COMPETE) and by national funds through the Portuguese Science Foundation (FCT-EURODEEP/0001/2007 and FCT-PEst-C/MAR/LA0017/2013). Finally, S. Laming was co-funded by UPMC, HERMIONE EC (FP7/2007-2013-n° 226354) and a MARES Grant (1298/2008/EC, candidate no. 20100174). MARES is a Joint Doctorate programme selected under Erasmus Mundus coordinated by Ghent University (FPA 2011-0016). See www.mares-eu.org for extra information. . Bathymodiolus tangaroa (AY608439) Bathymodiolus securiformis (HF545109) Bathymodiolus mauritanicus (FJ890502) Bathymodiolus platifrons (HF545106) Bathymodiolus japonicus (HF545108) Bathymodiolus sp Kikaijama (HF545112) Bathymodiolus sp. Sissano 2 (HF545122) Gigantidas sp. 1 Broken Bay (KF611693) Bathymodiolus sp. Sissano 1 (HF545125) Gigantidas taiwanensis (GU966638) Gigantidas gladius (AY649802) Gigantidas horikoshii (HF545113) Gigantidas sp. Ashzuri (HF545120) Gigantidas sp. 2 Broken Bay (KF611692) Gigantidas sp. Aitape (HF545119) Bathymodiolus brooksi (AY649798) Bathymodiolus aff. thermophilus (AF456317) Bathymodiolus thermophilus (GU966639) Bathymodiolus brevior (AY649799) Bathymodiolus septemdierum (HF545111) Bathymodiolus heckerae (AY649794) Bathymodiolus boomerang (FJ890503) Bathymodiolus azoricus (AY649795) Bathymodiolus puteoserpensis (AY649796) Adipicola arcuatilis (FJ937033) Adipicola crypta B'' (EU702315) Adipicola crypta B' (EU702319) Bathymodiolus manusensis (GU966637) Bathymodiolus aduloides (HF545118) Tamu fisheri (AY649803) Idas macdonaldi (AY649804) Idas modiolaeformis (FJ158585) Idas ESU L (FJ937193) Idas s. D (EU702357) Idas sp. C (EU702376) Idas ESU M (FJ937202) Idas ESU K (FJ937192) Idas ESU O (FJ937211) Idas argenteus MAR Idas argenteus LD Idas washingtonius (AY275546) Idas ESU N (FJ937205) Idas SAL1 (DQ340775) Idas ESU P (FJ937222) Adipicola longissima (DQ340773) Idas ESU H (FJ937073) Idas ESU (FJ937188) Idas ESU J (FJ937189) Idas japonicus (FJ937190) Idas ESU G (FJ937161) Adipicola iwaotakii (A'') (EU702322) Adipicola iwaotakii (A') (EU702333) Idas simpsoni (KF611695) Idas ESU T (FJ937283) Adipicola pacifica (HF545115) Vulcanidas insolatus (FJ767936) Idas ESU Q (FJ937230) Idas SAL4 (DQ340776) Idas ESU R (FJ937239) Idas sp. S' (FJ937240) Idas sp. S'' (FJ937258) Idas SAL3 (DQ340772) Idas ESU E (FJ937079) Idas ESU F (FJ937127)

This work was (co-)funded through a MARES Grant. MARES is a Joint Doctorate programme selected under Erasmus Mundus coordinated by Ghent University (FPA 2011-0016). Check www.mares-eu.org for extra information.

Tunnicliffe V, Embley RW, Holden JF, Butterfield DA, Massoth GJ, Juniper SK (1997). Biological colonization of new hydrothermal vents following an eruption on Juan de Fuca Ridge. Deep Sea Research Part I: Oceanographic Research Papers, 44(9): p. 1627-44 Tunnicliffe V, McArthur AG, McHugh D (1998). A biogeographical perspective of the deep-sea hydrothermal vent fauna. Advances in Marine Biology, 34: p. 353-442 Turner JT (2002). Zooplankton fecal pellets, marine snow and sinking phytoplankton blooms. Aquatic Microbial Ecology, 27(1): p. 57-102 Tyler P (1988). Seasonality in the deep sea. Oceanography and marine biology: an annual review, 26: p. 227-58 Tyler P, Young C, Billett DSM, Giles L (1992). Pairing behaviour, reproduction and diet in the deep-sea holothurian genus Paroriza (Holothuroidea: Synallactidae). J Mar Biol Assoc UK, 72, : p. 447-62 Tyler P, Young C (1999). Reproduction and dispersal at vents and cold seeps. Journal of the Marine Biological Association of the UK, 79(2): p. 193-208 Tyler P, Young CM, Dolan E, Arellano SM, Brooke SD, Baker MC (2007). Gametogenic periodicity in the chemosynthetic cold-seep① ussel① Bathymodiolus ①childressi. Marine Biology, 150(5): p. 829-40 Tyler PA, Marsh L, Baco-Taylor A, Smith CR (2009). Protandric hermaphroditism in the whale-fall bivalve mollusc Idas washingtonia. Deep Sea Research (Part II, Topical Studies in Oceanography), 56(19-20): p. 1689-99 Van Den Avyle MJ, Maynard MA (1994). Effects of saltwater intrusion and flow diversion on reproductive success of striped bass in the Savannah river estuary. Transactions of the American Fisheries Society, 123(6): p. 886-903 Van Dover CL (2000). The ecology of deep-sea hydrothermal vents. Princeton University Press, Van Dover CL, German CR, Speer KG, Parson LM, Vrijenhoek RC (2002). Evolution and Biogeography of Deep-Sea Vent and Seep Invertebrates. Science, 295(5558): p. 1253-57 Van Dover CL, Aharon P, Bernhard JM, Caylor E, Doerries M, Flickinger W, . . . Vrijenhoek R (2003). Blake Ridge methane seeps: characterization of a soft-sediment, chemosynthetically based ecosystem. Deep Sea Research (Part I, Oceanographic Research Papers), 50(2): p. 281-300 Van Dover CL (2011a). Mining seafloor massive sulphides and biodiversity: what is at risk? ICES Journal of Marine Science, 68(2): p. 341-48 Van Dover CL (2011b). Tighten regulations on deep-sea mining. Nature, 470, : p. 31-33 Vetter E, Dayton P (1998). Macrofaunal communities within and adjacent to a detritus-rich submarine canyon system. Deep-Sea Research Part II, 45(1-3): p. 25-54 Vetter EW (1994). Hotspots of benthic production. Nature, 372: p. 47 Vetter RD (1991). Symbiosis and the evolution of novel trophic strategies: thiotrophic organisms at hydrothermal vents. Symbiosis as a source of evolutionary innovation: p. 219-45 Villanueva R (1992). Continuous spawning in the cirrate octopods Opisthoteuthis agassizii and O. vossi: features of sexual maturation defining a reproductive strategy in cephalopods. Marine Biology, 114(2): p. 265-75 Vine FJ, Matthews DH (1963). Magnetic Anomalies Over Oceanic Ridges. Nature, 199(4897): p. 947-49 Vogt P, Cherkashev A, Ginsburg G, Ivanov G, Crane K, Lein A, . . . Egorov A (1997). Haakon Mosby mud volcano: a warm methane seep with seafloor hydrates and chemosynthesis-based ecosystem in late Quantemary Slide Valley, Bear Island Fan, Barents Sea passive margin. EOS Transactions of the American Geophysical Union Supplement, 78(17): p. 187-89 Voight JR (2009). Diversity and reproduction of near-shore vs offshore wood-boring bivalves (Pholadidae:

 [START_REF] Von Cosel | Bathymodioline mussels of the Bathymodiolus (s. l.) childressi clade from methane seeps near Edison Seamount, New Ireland, Papua New Guinea: (Bivalvia: Mytilidae)[END_REF]Methane seeps near Edison Seamount ? B. elongatus (Cosel, Métivier & Hashimoto, 1994) ?

. heckerae (Turner, Gustafson, Lutz & Vrijenhoek, 1998) Blake Ridge 2 155 S West Florida Escarpment 3 314 S B. hirtus [START_REF] Okutani | Two new species of Bathymodiolus (Bivalvia: Mytilidae) from methane seeps on the Kuroshima Knoll off the Yaeyama Islands, southwestern Japan[END_REF] Kuroshima Knoll, Off Yaeyama Islands 637 S B. japonicus (Hashimoto & Okutani, 1994) Off Hatsushima, Sagami Bay 1170-1180 S B. manusensis (Hashimoto & Furuta, 2007) PACKMANUS Field E, Manus Basin 1627-1629 V B. marisindicus (= B. brevior?) (Hashimoto, 2001) Kairei Field, Southern Central Indian Ridge 2443-2454 V Kairei Field, Southern Central Indian Ridge 2415-2460 V B. mauritanicus (Cosel, 2002) West Africa 1000-1267 S B. platifrons (Hashimoto & Okutani, 1994) Off Hatsushima, Sagami Bay B. puteoserpentis (Cosel, Métivier & Hashimoto, 1994) Snake Pit, Mid-Atlantic Ridge 3023-3510 V Snake Pit, Mid-Atlantic Ridge 3023-3510 V B. securiformis [START_REF] Okutani | Two new species of Bathymodiolus (Bivalvia: Mytilidae) from methane seeps on the Kuroshima Knoll off the Yaeyama Islands, southwestern Japan[END_REF] Kuroshima Knoll, Off Yaeyama Islands

S

could indicate alternative means of sustenance other than chemosymbiotically derived nutrition were to be collated in both immature and mature organisms (in collaboration with Themes 2 and 1, respectively).

2-1.3.1 A priori research objectives

To meet these aims, the research would have to combine existing and newly acquired evidence for symbioses at various stages of development and in adulthood, with evidence that implied some level of heterotrophic nutrition (integrating data from Themes 1 and 2). The focus of research would depend upon the model group, since siboglinids are lecithotrophic and chemosymbiotic mytilids are suspected to be planktotrophic (e.g. Lutz et al. 1980;[START_REF] Nussbaumer | Horizontal endosymbiont transmission in hydrothermal vent tubeworms[END_REF]Arellano and Young 2009;Arellano et al. 2014). However, essentially, the principal objectives were to use histological and targeted in situ hybridisation techniques to identify the presence and distribution of symbionts within the host tissues and infer patterns of phylotype succession, localisation andusing electron microscopythe degree of integration of symbionts in the host tissues (compartmentalisation), based on how these distributions changed with size of organism. This would require access to a size series of the model organism, both above and below the size at first maturity. Much like Theme 2, samples were to be acquired in the course of sorting preserved colonised substrata from colonisation experiments (small post-settlement specimens) but also longer deployments for larger adult specimens. Availability of post-embryonic and larval samples would depend upon the success of live experiments from Theme 1.

Heterotrophy was either to be witnessed directly in live individuals (i.e. feeding behaviour in larvae, postlarvae and juveniles, andin mytilidsadults) or inferred. Nutritional inferences would principally be from the ontogenetic or size-specific organisation and state of organs involved in nutrition, such as feeding apparatus and digestive/endocrine tissues, based mainly on observations during specimen dissections and subsequent histological analyses. If specimen availability allowed for it, evidence to support these approaches was to come from chemical analyses of soft tissue, such as wax-esters, fatty-acids and stableisotopes analysis.

2-2 Studying deep-sea chemosymbiotic lifecycles

2-2.1. Choosing a model taxon

Studying the lifecycle of a species, whose distribution is restricted to deeper depths, continues to be fraught with difficulty. However, advances must be made if deep-sea metazoan developmental biology is to be better understood (Adams et al. 2012). There are two main obstacles which provide the greatest hindrance.

The first is the logistical challenge of working under deep-sea conditions characteristic of the larval-source,transport and -sink environments and the collection and recovery of organisms from these environments at various stages of development. For example, the collection of larvae is severely constrained by the dilution of the larval pool during dispersal, while the collection of benthic lifecycle stages are constrained by high operating pressures, where live recovery often results in sub-lethal to lethal consequences for the organisms retrieved. The second constraint is the recreation of suitable conditions in aquaria (assuming live 3-7 Chapter 3 Annex 1

3-7.1. Supplementary Material

Supplementary figure 3.1 An overview of the fertilisation experiment conditions

A) The use of a tap-water bath to control temperature conditions of the aseptic filtered seawater (blue-lidded flasks, one being aerated in preparation for use) and the animals waiting induction (white baskets in suspended tub). B-C) A plan-view of the selected substrates for holding tanks: B) pinewood cubes from CHEMECOLI and C) palmwood. E) Individuals were separated when spawning began F) IF7 I. (s.l.) simpsoni in the process of spawning small white eggs. G) The conduit for aeration of embryonic development dishes.

Supplementary figure 3.2 Achieving an extended depth of field in micrographs

A) A stack of images are taken at incremental levels of focus in the vertical z axis (the lit area of shell is in focus). B) The images are then imported into FIJI (Image-J) either as a stack or as separate images-the stack can be formed form these within the FIJI software. The image stack is then subjected to an alignment (STEP 1) using the turbo-reg/StackReg add-on for FIJI. Scaled rotation worked best.

The EDOF algorithm available for FIJI is then used to identify regions of highest contrast density to define focussed slices and a composite image entirely in focus is generated. If necessary, these images were then tiled in Photoshop CS3 (if the focus stacks were of parts of the shell only)

Supplementary figure 3.3 Shell dimensions depending on species and sex

Scatter-plot of shell height as a function of shell length in I. (s.s.) modiolaeformis and I. (s.l.) simpsoni.

Supplementary figure 3.4 Location of mussels in the palmwood experiment

Mussels (asterisks) of both species were located in the space between the bark (cortex) and the core water and food transport system, the central cylinder.

Supplementary figure 3.5 Isotopes plot for study species in the context of other bathymodiolins

The scatter-plot① depi ts① the① δ 13 C① a d① δ 15 N values for each of the specimens plotted in figure 8, in the context of values from the literature for both the study species (from methane seeps) and other bathymodiolin mussels from hydrothermal vents and hydrocarbon seeps (see values in the appended and two distinct thiotrophs were subsequently confirmed again 4 years later, in mussels from various substrata (Lorion et al. 2012). Relative symbiont dominance shifted from the methanotroph to the two thiotrophs between these two studies, demonstrating a predisposition for diverse, adaptable symbiont assemblages. Considered the prevalent symbionts in I. (s.s.) modiolaeformis, these bacteria can co-occur in mussels from the Nile Deep Sea Fan region, but in the eastern Atlantic only one or two phylotypes associate with this species (e.g. Rodrigues et al. 2013, including strains related to the 5 th phylotype, symbiont G, Duperron et al. 2008a). Symbiont acquisition is considered environmental since bacterial symbionts, though present in gills, are absent from gonad tissue in adults (Gaudron et al. 2012). The proliferation of this symbiosis remains undocumented. The larvae of I. (s.s.) modiolaeformis are believed to be planktotrophic based on prodissoconch shell records, and adults are thought to be protandric hermaphrodites (Gaudron et al. 2012). Idas (s.s.) modiolaeformis is prevalent in reducing environments in the Mediterranean suggesting that it has evolved life-history traits to optimise survival. These mussels thus present a fascinating case study for the evaluation of life-history adaptations in highly ephemeral habitats under reducing conditions.

4-2.1. Research aims

The aim of the present study was to describe elements of the life-cycle of I. (s.s.) modiolaeformis likely to be adaptive in its particular habitat. A size series of I. (s.s.) modiolaeformis was collected from larval colonization experiments in a cold seep region of the eastern Mediterranean.

Based on this size-series, this study sought to answer several questions by recording tissue development from metamorphosis to adulthood. absent in heavily-ciliated regions such as the entire ventral ciliated zone and the frontal-to-lateral ciliated zones along the length of the filaments (e.g. . Putative DAPI-stained bacteria, identical in appearance, were identified on other non-ciliated epithelial surfaces, such as the mantle itself, the dorsal side of the foot, epithelial retractor-muscle sheaths and the visceral epithelium (e.g. Supplementary figure 12). Bacterial densities identified in the gills appeared to increase qualitatively in abundance with increasing SL (Figure 4.5), while densities on other epithelia did not. In the two largest mussels examined, Idas 27 and Idas C5-2, bacteria were only identified on non-ciliated gill epithelium (e.g. Presumptive juveniles based on their size only. 2 Sex could not be determined but poorly preserved reproductive tissue was identified. Fixation and preservation (i.e. no transfer to 96% Ethanol after 4 hr of fixation). 4 DAPI analysis was performed during which bacteria were not identified, however the sample was ethanol-fixed so epithelial preservation was not complete.

Supplementary figure 4.1 Brief description of the methodology for constructing micro-dissection tools and their subsequent use

Micro-dissection pins and pliable metal scalpels (< 10 µm point/blade) were manufactured by electrolytic sharpening of either steel entomological minuten pins (A) or feather-blade razors (B) respectively (0.5M NaCl solution, 4.5V DC current), wherein the blade or pin which needed to be reduced in size formed the anode, and a 1-mm thick electrical copper wire (immersed length stripped of plastic oati g ① fo ed① the① athode① ot① depi ted .① Pi s① e e① sha pe ed ① di e tl ① usi g① this① set-up (with resulting silhouette profile). However, blade-edges were first snapped off the razor along their length in a vice (B1), to create shallow blades but maintain their straight edge-profiles (B2), and then the razor edge was dipped evenly into the electrolytic solution (not depicted). Blades and pins, which were often bent into specific forms using forceps (C), were mounted on applicator sticks (or toothpicks). Valve-margin o ie tatio ① as①fa ilitated① ①pla i g①the① ussels①i ① disse tio ① ells ①of① a i g①sizes①a d①depths that had been melted into the upper surface of sterile, plastic Petri-dish bases prior to dissection (D). Using the highly pliable fine blade, it was possible to part the valves of even the smallest plantigrades, open shells, with minimal damage to the organism, having cut the anterior and posterior adductor muscles.

Supplementary figure 4.2 Summary schematic to help visualise the orientation of organisms during sectioning

Pictured, is an axonometric drawing of a Idas modiolaeformis, based upon the animals dissected for this study. Axes of orientation are the dashed lines. Arrowheads indicate the direction of view for each region of the animal. Planes are labelled in white print. Serial sections cut from the animal (in either direction) along the lateral (left)-lateral (right) axis, would be in the sagittal plane (royal blue), they would be in the transverse plane (green) if cuts were made along the posteroanterior axis, and along the dorso-ventral axis, they would be in the frontal plane (turquoise).

Supplementary figure 4.3 Length-class frequency histogram of Idas (s.s.) modiolaeformis

The histogram displays the number of individuals that occurred in consecutive 100 µm length-class intervals over the entire dataset (sample sizes are encircled). Dark grey counts are individuals from wood substrate, while light grey counts are those from alfalfa, in which counts are stacked e.g. the highest frequency for a size class at 7 individuals (400 -<500 µm) is comprised of 1 individual from alfalfa and 6 from wood.

Supplementary figure 4.12 Presence of putative bacteria on visceral epithelia in Idas 12

Micrographs of 350 nm semi-thin section stained with DAPI cut in the frontal plane along the dorso-ventral axis, in the large juvenile, Idas 12 (between the upper-central-right and far-right sections in Supplementary figure 10). General region is depicted in inset image (white box is magnified). Bacteria = white arrowheads. d.g: digestive gland, v.e: visceral epithelium. Scale (inset) = 50 µm. have been aimed at Marine Ecology, as part of a special issue.

5-1 Abstract

On the scale of the habitat, the arrival of whale falls to the seafloor provides massive influxes of labile organic matter to localised areas of the seabed. Sulphidic conditions can last from a year or two in small shallow-water whale falls to many decades in deep large carcasses. The benthic chemosymbiotic fauna that specialise in colonising these organic substrates must be adapted to dynamic variability in their habitat which simultaneously provides their principal source of food. This study thus investigates two such adaptations, rate of maturation and the patterns of developmental symbiosis within the context of general development towards adulthood. A size series of mussels that spanned post-larvae to confirmed adults was used. Aposymbiosis in post-larval plantigrades provides evidence for entirely heterotrophic larvae.

Acquisition of symbionts was found to be rapid and in abundance, almost immediately following settlement: shells had only increased in size by less than 10 % when bacteria were first detected.

Maturation was at less than 2.6mm shell length, or the equivalent of < 6% of this species maximum known size. These results are discussed in relation to its habitat and in relation with other chemosymbiotic species for which data is available. boomerang Bang-T-like symbiont (Ritt et al. 2012). However this bacterium was not present in sufficient densities to elicit a signal during FISH using the Bang-T probe in the current samples from bones I. (s.l.)

simpsoni. In fact, with the exception of the T1 thiotroph (and EUB338), no other phylotype combinations revealed signal characteristic of bacteria in Idas-like mussels (these included ImedM-138, ImedT2-193, BhecM2-822, and Bang-T, for details see Duperron et al. 2005;Duperron et al. 2008a).

5-5.1.4 Reproductive energetics

Symbiont associations deliver novel metabolic capabilities to the host (Dubilier et al. 2008). Such patterns of symbiont acquisition and proliferation can ultimately provide the host with a plentiful food supply which might be harnessed to fuel augmented growth rates or drive the development of new host tissues (e.g.

early pre-maturation acini formation). Perhaps reflecting the metabolic advantages of acquiring symbionts earlier and at higher densities as young juveniles, size at first sexual maturity as a male, was ator likely preceded -SL 2.61 mm, which represents < 6 % of the current maximum size recorded for I. (s.l.) simpsoni. symbionts were reported as an early acquisition dating back to the stem of the group, almost 30 Myr (Lorion et al., 2013). Since I. washingtonius still harbors thiotrophic symbionts, it can be assumed that the loss (or considerable reduction) of symbiosis is a derived state and occurred within the branch leading to I.

argenteus. A clock analysis using known calibrations (Lorion et al., 2013) estimated that I. argenteus diverged from I. washigtonius between 11.1 and 13 Myr. Loss or reduction of symbiosis is not unexpected given the predisposition of Idas mussels for diverse, adaptable symbiont assemblages (Lorion et al., 2012;Rodrigues et al., 2013). Absence of symbionts in an otherwise symbiotic group has been observed in the Thyasiridae where some species have symbionts while others do not. A recent study even demonstrated that a single (potentially two cryptic) species may either harbor or lack symbionts in two distinct populations [START_REF] Batstone | Divergent chemosymbiosis-related characters in Thyasira cf. gouldi (Bivalvia: Thyasiridae)[END_REF], a flexibility that could potentially also exist in deep-sea mussels.

The results described here concur with previous studies suggesting the absence of symbionts in Idas argenteus (Ockelmann & Dinesen, 2011). This is to date the only reported case of symbiosis loss or strong reduction within the Bathymodiolinae. This shift to strict heterotrophy could be linked with the organic-enriched habitats, where larviphagy or filter-feeding may alone sustain the animal nutrition.

Whether this will remain a unique example, or a convergent trend in different mussel clades will require further investigation of symbiosis in smaller mussels.

6-6 Acknowledgments

We thank the chief scientists, captains, crew and ROV pilots of the two cruises.

6-7 Data accessibility

The sequences were deposited in GenBank (accession numbers: XXXXXX-XXXXX). Table S1 Specimen collection sites and GenBank acession numbers for Mytilidae included in the phylogenetic analyses. that are presumed to determine sex alternations (Tyler et al. 2009;Gaudron et al. 2012). Although such variability is also known to occur in association with sunken wood (Laurent et al. 2013;Yücel et al. 2013), a large component of the mussels diet could be supported by particulate organic matter (POM), reflected in the complex stable isotopes results presented for mussels recovered from the Lacaze-Duthiers Canyon, particularly for nitrogen p. 135). The vertical flux of particulate organic matter in the Lacaze-Duthiers Canyon is likely to be relatively high, due to coastal proximity, cascading effects (Palanques et al. 2006) and shallower water depths in comparison to seeps in the eastern Mediterranean (525 m, in comparison to 1158-3015 m respectively). Thus, periods of high, less-refractory, POM input may trigger rather abrupt changes in sex within the submarine canyon, in order to take advantage of an episodic abundance in food. The remaining I. (s.s.) modiolaeformis mussels used in this research were those from the developmental size series collected from the eastern Mediterranean from wood and alfalfa substrates, deployed in an area known for methane seepage (Central Pockmarks 2A, NDSF; Chapter 4, p. 163).

6-8 Funding statement

Conditions at this site effectively represent some sort of intermediate habitat state between the two other I. (s.s.) modiolaeformis sites. However, the largest individual collected from these experiments was only SL 6.54 mm, so data from this experiment can only support protandry generally (all mature individuals were male).

Analysis of sex-ratio patterns in I. (s.l.) simpsoni are somewhat more difficult to interpret.

Contrasting patterns existed between the data available from wood associations in the Lacaze-Duthiers Canyon, and those from bone-colonising I. (s.l.) simpsoni in the Sétubal Canyon off the coast of Portugal (Table 7.1). In the former, only adults were collected, where the two smallest individuals were hermaphroditic, but functionally male (SLs 5.6 and 5.8 mm). No obvious size-dependent trend in sex could be identified statistically or even qualitatively (Section 3-4.3, p. 138), at least within the rather limited sample size available (n = 12). In contrast, trends in the developmental size series of I. (s.l.) simpsoni recovered from cow bones in the Sétubal Canyon, were comparable to patterns from the aforementioned developmental size series of I. (s.s.) modiolaeformis. All mature individuals (n = 3, of 38), analysed in the current research, were male. In addition, colleagues at the University of Aveiro working upon a larger subset of the total number of mussels (in the 100s) recovered from these experiments found that for the 18-month carcass deployment, only one (relatively giant) female was found (Ana Hilário, personal communication), putatively identified based on alcohol fixed histology (preservation was poor). The resulting sex ratio approached 1:1000 (♀:♂). However, considering the presence of two hermaphrodites of almost identical size at the Lacaze-Duthiers Canyons and the exclusively small males found at the Setúbal Canyon, sex-switching remains plausible. As adults however, instead of exclusively large females prevailing, it would seem that both sexes coexist. These contrasting trends in sex ratios evidently warrant further investigation, since the results herein are preliminary and derived from two quite different habitats with non-overlapping size ranges.

appeared to be spiral based on Hoechst staining. Embryonic development beyond the 4-cell stage did not occur, as the embryos died following these observations. 7-2.4. Theme 2b: Post-larval developmental biology

The objectives set out for this subtheme were met almost entirely in full. In only two instances were objectives not met. The first was in attempts to use immuno-labelling to track the migration of the primordial germ-line on its course to forming gonads in mature individuals. This was trialled at the University of Caen, in collaboration with Anne-Sophie Martinez. However issues concerning either the methacrylate embedding medium or a vasa-like gene mismatch (the oyster vasa-like gene marker was employed, based on its availability at that lab), could not be overcome. This was instead inferred from cellclumps which could not be assigned to another tissue type (see Chapter 4,p. 163). The second, which was to use larval shells for possible chemical analysis (e.g. trace-elements or stable-isotopes analysis) was limited technically and by sample availability (insufficient volumes of shell). However, a considerable quantity of new data is now available regarding the developmental biology of both I. (s.s.) modiolaeformis and I. (s.l.) simpsoni (Table 7.1). These were based on size-frequency distributions (I. (s.s.) modiolaeformis), larval-shell measurements (prodissoconch I and II) and descriptions of developmental patterns in key anatomical features with increasing size, considered prudent to the survival of these mussels in reducing habitats (Table 7.1).

7-2.4.1 Larval biology, inferred from larval shell characteristics

The examination of vestigial larval features in post-larval, juvenile and adult shells revealed a consistent trend in both I. (s.s.) modiolaeformis and I. (s.l.) simpsoni, regarding the relative dimensions of the prodissoconch I (a proxy for larval hatching size, Lutz et al. 1980) and the prodissoconch II (a proxy for size at settlement and metamorphosis, Lutz et al. 1980). In both species, the prodissoconch I was easily identifiable as a result of its pearly white appearance, while the prodissoconch II could be discerned due to its red/orange coloration. Thus their measurement was possible under a high magnification using a dissection microscope only. Despite this potential technical limitation, values recorded were in close agreement with those found for these species in the literature (Gaudron et al. 2012;Ritt et al. 2012), in individuals from quite different habitats (Table 7.1) although the ratios of prodissoconch II SL and SH in I.

(s.s.) modiolaeformis collected from wood (those cited in table two are from the eastern Mediterranean only), appeared to be greater than from equivalent measures in specimens from seeps (Gaudron et al. 2012), resulting in a slightly more elliptical larval shell shape. The overall approximate area (and thus volume) of the shells were comparable however. Assuming similar growth rates in the plankton, this suggests similar transport times for I. (s.s.) modiolaeformis larvae settling in the east Mediterranean, ega dless①of①su st ate① hoi e .①La al①shell①di e sio s①i ① I. (s.l.) simpsoni were similarly consistent across gametogenesis were identifiable. Despite their markedly different maximum sizes, both I. (s.s.) modiolaeformis and I. (s.l.) simpsoni matured as males at SL a little over 2 mm (NDSF on wood, and Setúbal Canyon on bones, respectively). In the case of I. (s.s.) modiolaeformis, the smallest mature individual identified was 2.35 mm long (approximately 12% of its SL max , Table 7.1), where the gonad tissue was barely discernible and poorly structured, i.e. in its earliest, least-developed incarnation. At this size reproductive output is likely to be low, however as a male this presents less of an obstacle, given the investment per spermatozoid is also minimised. All individuals above this size were both mature and male (Laming et al. 2014;section 4-4.4, p. 171). For I. (s.l.) simpsoni, although a size at first maturity was identified at 2.6 mm (bone-associated Setúbal specimens, approximately 6% of its SL max , Table 7.1), the gonad was considerably more developed, which suggests that a notable period of time has passed since the initial establishment of functional gonad tissue in this individual. As no evidence for gametogenesis was observed in the next smallest individual (2.2 mm), size at first maturity was constrained simply to the size difference between these specimens. By combining growth rate estimates and the size at first maturity, estimates for age at first maturity in I. (s.s.) modiolaeformis were around 4 months following settlement. In the absence of growth estimates for the Setúbal Canyon I. (s.l.) simpsoni mussels, no such value could be estimated for this site. However, given all individuals of this species were mature at the Lacaze-Duthiers Canyon, this suggests a maturation of less than 6 months based on growth rate estimates and the smallest individual found for this species (5.6 mm functional male; Table 7.1), which was already in the early stages of a switch in sex (likely male to female). Collectively these rates of maturation are well within the longevity of larger wood and bone accumulations, based on the literature available (Smith et al. 2002;Smith 2006;Bernardino et al. 2010;Cunha et al. 2013;Yücel et al. 2013), making these mussels well adapted to these temporally finite habitats. However, the estimate for size and age at first maturity in females, and thus first reproduction in the population (i.e. both gametes are available for fertilisation), was not possible for I. (s.s.) modiolaeformis.

In I. (s.l.) simpsoni, since the smallest female was already quite large (SL 18.1 mm), size and age at first maturity as a female are both large and late at <18.1 mm (by definition) and <23 months respectively. Given that these mussels represented a subset of the total mussels found on the palmwood deployment, it is quite likely that smaller females not used in the current research existed. This fact, and the likely underestimate of growth (for similar sub-sampling reasons), suggest that these values are likely to be conservative. In effect, this fact holds true for all growth (and thus age at first maturity) estimates cited.

Given the dearth of data available for maturation rates of wood-associated reproductive development in the literature, such rough estimates though based on certain assumptions can still provide direct evidence to support what has otherwise been well-understood in theory, but assumed in practice:

like the wood-boring bivalves before them (Tyler et al. 2007b), which establish the apparently necessary preconditions for sulphophilic species to settle, chemosymbiotic mussels reach sexual maturity in an impressively short passage of time.

7-2.5. Theme 3: Dynamics of nutrition

In terms of the initial objectives, much of what was decided at the outset was achieved, with the exception of some of the more refined aspects of FISH analyses. Using basic histological and targeted in situ hybridisation techniques symbionts were localised and patterns in the symbiotic association with size were documented.

7-2.5.1 Qualitative symbiont densities with size Following symbiont acquisition which was in very small juveniles (Table 7.1), the number of bacteria to be found on the abfrontal and lateral non-ciliated regions of gill filaments depended strongly on species and/or habitat (Table 7. contributed to a certain degree of symbiont loss and a switch in trophic dominance from chemosymbiosis to heterotrophic filter-feeding. Issues were also initially encountered when attempting to identify the phylotypes of highly abundant putative bacteria in I. (s.l.) simpsoni from cow bones in the Setúbal Canyon.

The level of auto-fluorescence in gill sections suggested that formalin fixation had been overly long (performed remotely by a colleague). However, following an extended stint in the freezer, slides responded better during examination. Generic eubacterial-probe signal was high enough to overcome backgrounds levels, demonstrating that at the very least, the observed accumulations on non-ciliated regions of the gill filaments noted in histological sections, were eubacterial in origin. In some larger individuals TI-thiotrophs hybridisation also took place and to a lesser extent in juveniles too, though signals were of lower intensity, which may reflect a reduced number of or activity in bacteria present. In adults of both I. (s.s.) modiolaeformis and I. (s.l.) simpsoni, filter feeding behaviour was observed, where particles were actively transported to the buccal region by the gills, along the ventral particle groove.

Mucous entrainment was not evident in the frontal regions of the gill, suggesting that particle entrainment relied on fluid dynamics, as in shallow-water mytilids (Jørgensen 1981;1990). At the mouth, particles were ingested in advance of mucous accumulation which was seemingly not ingested (the degree of mucous is likely to have been stress related, Jørgensen 1990). Observations made of the extent of the digestive system in both species, but particularly I. (s.l.) simpsoni in which the intestinal tract appeared to loop more than once, indicate that the capacity to assimilate food, within the stomach (style-sac was putatively identified but the style was not preserved) and in the digestive diverticula. Equally in most cases, contents in the hind-gut were identified either histologically as heavily stained, optically fractal material, or as dark coloured regions during dissection (material compaction made identification unambiguous). Nephridia were also putatively identified in some instances, and certainly in the larger I. (s.l.) simpsoni where their organisation rendered them conspicuous among other tissues. No gastric shields were seen in any specimens. Thus anatomically andcruciallybehaviourally, both species are capable of, and engage in facultative heterotrophy ( (s.s.) modiolaeformis on wood are highly variable, from the dense thiotroph associations found in specimens recovered from oakwood in the Lacaze-Duthiers Canyon, to the low densities on pinewood and alfalfa from the NDSF, eastern Mediterranean and in small adult I. (s.s.) modiolaeformis found in the Lacaze-Duthiers Canyon, the latter being known to engage in filter-feeding. In I. (s.l.) simpsoni found on wood (Lacaze-Duthiers canyon), stable isotopes results, moderate densities of putatively identified symbionts in adults (excluding those used in the live experiments), and the filter-feeding behaviour observed in this species below a certain size, all point towards a mixotrophic diet. Finally, while there is likely to be a heterotrophic component to the diet of I. (s.l.) simpsoni on bone falls, the evident abundance of symbiotic bacteria even at early stages of development, suggest that chemosymbiosis forms the primary source of nutrition within this habitat (Table 7.1)

7-3 The flexibility of mixotrophy

Prior to the research undertaken in this thesis, little or nothing was known of several aspects of the lifecycle biology of small-sized mussel species, which colonise a variety of reducing habitats. Through this research several key attributes can now be described for the first time (though some have been inferred in literature (e.g. cleavage patterns, embryology, gross adult anatomy). Equally, some traits have probably evolved subsequently, such as the disappearance of abfrontal cilia and the hypertrophy of gills, both of which are likely to have facilitated an increase in bacterial symbiont densities historically, permitting host organisms to place greater dependence upon their symbionts. The former may be a by-product of neoteny, thought to be particularly prevalent in small-sized Bathymodiolinae (s.l., Génio et al. 2012).

7-3.4. Concluding remarks

Based on the absence of bacterial signals, plantigrades were aposymbiotic, indicating strict heterotrophy in larvae and early post-larvae. This information is of particular utility in dispersal modelling, though currently it is not certain where in the water column larvae principally disperse. Symbiont acquisition may be a critical juncture in the lifecycle of small-sized multi-habitat mussels, but the retention of a heterotrophic condition eleases① the ① f o ① a ① o ligatio ① to① a ui e,① o ① die .① ① ②s① a① o se ue e① of① thei ① t ophi ① fle i ilit ,① the① distribution of these mussels is astoundingly widespread. The only apparent constraint is the need for a hard substratum to attach to (a trait that select bathymodiolin species have even dispensed with (e.g. B.

[s.l.] boomerang). These observations on early development in I. (s.s.) modiolaeformis represent evolutionary adaptations to their ephemeral, reducing habitats. Extraordinary rates of adaptive radiation in the last 100 Ma suggest some event(s) took place which accelerated speciation, hypothesised to be the initial (repeated and independent) incorporation of free-living bacteria as symbionts in host species. Host speciation is suspected to have been facilitated by the incorporation of symbionts with diverse metabolic capabilities (e.g sulphur-and methane-oxidisers). The flexibility of symbiont associations and retention of filter-feeding and assimilation capabilities certainly furnish both I. (s.s.) modiolaeformis and I. (s.l.) simpsoni with an unrivalled level of flexibility within the Bathymodiolinae studied to date.

7-3.5. Future considerations and perspectives 7-3.5.1 Phylogeny

Family-to-subfamily hierarchy and generic cladistics within the Bathymodiolinae remain contradictory, as a consequence of conflict between classical constraints of taxonomic nomenclature for species described prior to the discovery of reducing environments, and data emerging from state of the art molecular techniques and analyses. Accordingly, the finer details concerning the systematics of the Bathymodiolinae must continue to be elucidated as part of an overhaul to reconcile polyphyletic (e.g. Idas, Adipicola) and paraphyletic taxa (i.e. the Bathymodiolinae sensu stricto) that contain many of the most thoroughly studied species in terms of deep-sea biology and ecology (Jones et al. 2006;Miyazaki et al. 2010;Fontanez and Cavanaugh 2013;Lorion et al. 2013;Thubaut et al. 2013b).

7-3.5.2

The larval/post-larval black box

The bathymodiolins are probably one of the most widespread chemosymbiotic bivalve groups in reducing environments worldwide, and yet they have never been recorded in background abyssal faunal 2000m (Olu et al. 2010), suggesting a degree of connectivity between seep habitats within a given depth range. The level of connectivity across organic falls remains difficult to assess since predicting organic fall distributions and locating them is not as straight-forward as for ventsusing known and predicted regions of geothermal activity and then water-chemistry anomalies locally, and seepsidentifying regions with a geologically stable history for organic sedimentation, that are now subjected to some form of sediment destabilisation.

However In the framework of these initiatives, the expansion of our understanding of larval biology in deep-water environments ought to be possible and indeed encouraged. Greatest gain in this area would include characterisation of dispersal potential (e.g. the pelagic larval duration coupled with oceanographic models) in conjunction with realised dispersal of keystone species (derived from genetic connectivity and elemental fingerprinting). This would not only provide two independent measures of connectivity, which can be co-validated against one another, but it also would help determine the degree of export or retention at natal sites. In addition, through the application of elemental marker techniques it may be possible to begin mapping population connectivity within the deep-ocean and help inform networks for marine protected areas (Christie et al. 2010;Van Dover 2011a). Finally the characterisation of larval nutrition during its pelagic phase, using isotopic and nutritional analysis may yield exactly the information required to better define the conditions needed for successful in-vitro larval rearing to characterise development. Once this is possible, many of the other larval traits for which empirical data are now emerging in shallow-water species, may eventually be categorised in deep-water descendents, such as swimming behaviour, behavioural responses to environmental stimuli in terms of orientation both during dispersal and at competency and the