Cédric Hubas

Bruno Jesus

Claire Passarelli

Christian Jeanthon

Tools providing new insight into coastal anoxygenic purple bacterial mats: review and perspectives

Keywords: microbial mats, anoxygenic phototrophs, sulfur purple bacteria, diversity, sediment stability, photosynthesis, spectral reflectance

Tools providing new insight into coastal anoxygenic purple bacterial mats: review and perspectives

Cédric Hubas, Bruno Jesus, Claire Passarelli, Christian

Introduction

Microorganisms have the ability to colonize different types of habitats and interact with each other, forming more or less complex communities. Microbial mats that develop in different geographical locations are a remarkable example of these associations. They are found for instance in coral reefs, hypersaline ponds and lakes, salterns, thermal springs, Antarctic lakes, and coastal sediments [START_REF] Stal | M icrobial mats: Structure and Environmental Significance[END_REF]. In the latter, they develop at the sedimentwater interface in shallow environments such as estuaries, intertidal areas, sandy beaches or hypersaline salt marshes [START_REF] Herbert | D evelopment of mass blooms of photosynthetic bacteria on sheltered beaches in Scapa Flow, Orkney Islands[END_REF], S tal and Caumette, 1994, V an Gemerden et al., 1989a, Van Gemerden et al., 1989b). Coastal microbial mats are principally inhabited by bacteria (heterotrophic, autotrophic and chemotrophic) as well as eukaryotic microalgae such as benthic diatoms. These consortia are often referred to as microbial mats, laminated microbial communities, microphytobenthos or simply biofilms, in the literature (or any combination) but describe ultimately the association of different microbial cells, embedded in an extracellular polymeric substance (EPS) matrix. These mats can exhibit different morphologies based on the physicochemical environments they experience. Most of the time, the cells are organized according to their physiologies in vertical laminated structure consisting of successive layers.

In the coastal zone, microbial mats are mostly photosynthetic and are composed of several functionally complementary groups of microorganisms whose composition can vary greatly depending on the energy and nutrient source from the top and bottom. Cyanobacteria are often the pioneer organisms and generally dominate the top layer. According to the chemical and light gradients available they can for instance be followed by aerobic or facultative heterotrophic bacteria, chemolithotrophic bacteria (among them colorless sulfur bacteria), anoxygenic phototrophs (purple and green) and sulfate-reducing bacteria, forming several laminated layers distributed within the EPS matrix principally produced by cyanobacteria.

Purple bacteria perform anaerobic anoxygenic (without release of oxygen) photosynthesis as, unlike cyanobacteria, they are unable to perform water photolysis due to the lack of the photosystem II. They mostly use, as electron donors, the intermediate products of organic matter degradation from primary producers and some compounds originated from fermentation and anaerobic respiration. In microbial mats, a large diversity in purple bacteria is generally observed and the genus Thiocapsa is often highly represented (Van Gemerden et al., 1989b).

In the last decade, community structure as well as physical-chemical environment of the microbial mats have been reviewed at several occasions [START_REF] Franks | F lat laminated microbial mat communities[END_REF][START_REF] Paerl | A mini-review of microbial consortia: Their roles in aquatic production and biogeochemical cycling[END_REF], S tal and Caumette, 1994[START_REF] Van Gemerden | Microbial mats -a joint venture[END_REF]. The introduction of molecular approaches has indeed provided new insight into the ecology of these mats by allowing the characterization of the community structure [START_REF] Ranchou-Peyruse | C omparison of cultivation-dependent and molecular methods for studying the diversity of anoxygenic purple phototrophs in sediments of an eutrophic brackish lagoon[END_REF][START_REF] Wieland | M icrobial mats on t he Orkney Islands revisited: Microenvironment and microbial community composition[END_REF]. Thanks to the development of high resolution microelectrodes, the physical and chemical environment of these mats was characterized at very small spatial scales (µm to mm, [START_REF] Revsbech | Photosynthesis of Benthic Microflora Measured with High Spatial-Resolution by the Oxygen Microprofile Method -Capabilities and Limitations of the Method[END_REF]Jørgensen, 1983, Visscher et al., 1991). Pigment diversity and in situ reflectance measurements revealed the spatial or temporal heterogeneity of microbial mat surfaces (e.g. Brotas andPlante-Cuny, 2003, P aterson et al., 1998). Finally, their role in sediment biostabilisation was revealed [START_REF] Paterson | B iological mediation of sediment erodibility: ecology and physical dynamics[END_REF]. This paper reports the main discoveries made through the introduction of these powerful techniques and points out the gap in current knowledge regarding anoxygenic phototrophic biofilms.

Microbial communities in coastal purple phototrophic mats

Structure of coastal purple phototrophic mats

Photosynthetic microbial mats develop in many different habitats with salinities ranging from freshwater to hypersaline conditions [START_REF] Overmann | T he Phototrophic Way of Life[END_REF]Garcia-Pichel, 2006, Van Gemerden, 1993). Some prominent marine and hypersaline habitats where laminated microbial communities frequently develop in visible masses are represented by coastal sediments of the Great Sippewissett salt marsh (USA) [START_REF] Nicholson | Structure of a microbial mat at Great Sippewissett Marsh, Cape Cod, Massachussets[END_REF][START_REF] Rothermich | Characterization, seasonal occurrence, and diel fluctuation of poly(hydroxyalkanoate) in photosynthetic microbial mats[END_REF], coastal lagoons in the southern France [START_REF] Caumette | Phototrophic sulfur bacteria and sulfate-reducing bacteria causing red waters in a s hallow brackish coastal lagoon (Prévost lagoon, France)[END_REF][START_REF] Guyoneaud | Anoxygenic phototrophic bacteria in eutrophic coastal lagoons of the French Mediterranean and Atlantic coasts (Prevost Lagoon, Arcachon Bay, Certes fishponds)[END_REF], marine salterns in France [START_REF] Caumette | M icrobial mats in the hypersaline ponds of Mediterranean salterns (Salin-de-Giraud, France)[END_REF](Caumette et al., , G iani et al., 1989) ) and in Guerrero Negro (Baja California, Mexico, [START_REF] Canfield | Biogeochemical Cycles of Carbon, Sulfur, and Free Oxygen in a Microbial Mat[END_REF]Des Marais, 1993, Ley et al., 2006), sandy flats of the Ebro Delta [START_REF] Mir | Vertical stratification and microbial assemblage of a microbial mat in the Ebro Delta (Spain)[END_REF][START_REF] Navarrete | P hysiological status and community composition of microbial mats of the Ebro Delta, Spain, by signature lipid biomarkers[END_REF], and sheltered beaches on the Orkney islands (Van Gemerden et al., 1989a, Van Gemerden et al., 1989b, W ieland et al., 2003). In such ecosystems, the surface sediment layer covers a transition zone between oxic and anoxic conditions characterized by steep gradients of oxygen and sulfide. These gradients favor the maturation of vertically stratified, multicolored and cohesive layers of several functional groups of microorganisms. Although the uppermost layer, brown and green in color, may contain benthic diatoms, the dense top material is typically formed by unicellular and filamentous cells of cyanobacteria that are generally the driving force as they provide growth substrates for other organisms. For instance, newly colonized sands mostly comprised Oscillatoria sp. and Spirulina sp. [START_REF] Franks | F lat laminated microbial mat communities[END_REF]. The gliding cyanobacterium Microcoleus chthonoplastes often replaces these pioneer species and becomes dominant in mature intertidal mats [START_REF] Stal | Structure and development of a benthic marine microbial mat[END_REF], V an Gemerden, 1993). Below the cyanobacteria, a distinct layer of purple sulfur bacteria is often present, sometimes overlying a layer of green sulfur bacteria. Sometimes, a white layer or patches due to sulfide-oxidizing bacteria (including Beggiatoa spp.) are visible at the surface of marine sediments that have a sufficiently high production of sulfide from bacterial sulfate reduction [START_REF] Jorgensen | Distribution of colorless sulfur bacteria (Beggiatoa spp.) in a coastal marine sediment[END_REF].

They are followed vertically by sulfate-reducing bacteria whose activity leads to the precipitation of iron sulfides visible as black mud. Aerobic heterotrophic organisms are also functionally important as their activity leads to oxygen depletion, and fermentative organisms provide growth substrates for sulfate-reducers. Other, numerically less important groups are nitrifying and denitrifying bacteria and methanogens. Numerous examples have been described in the literature of colored blooms and mass accumulations of phototrophic bacteria in the coastal zones and lagoons [START_REF] Caumette | Phototrophic sulfur bacteria and sulfate-reducing bacteria causing red waters in a s hallow brackish coastal lagoon (Prévost lagoon, France)[END_REF]Baleux, 1980, Imhoff, 2001, andreferences herein). Mass developments of purple sulfur bacteria have been observed during warm summer months in the intertidal zone of sandy beaches [START_REF] Herbert | D evelopment of mass blooms of photosynthetic bacteria on sheltered beaches in Scapa Flow, Orkney Islands[END_REF], Van Gemerden et al., 1989a, Van Gemerden et al., 1989b). Three different laminated microbial mats were described, distinguished by the position of the cyanobacterial layer above or beneath the purple sulfur bacterial layer, or its complete absence and therefore exclusive development of purple sulfur bacteria in the top layer. On the Orkney islands [START_REF] Herbert | D evelopment of mass blooms of photosynthetic bacteria on sheltered beaches in Scapa Flow, Orkney Islands[END_REF], Van Gemerden et al., 1989a, Van Gemerden et al., 1989b) and in Roscoff Aber Bay (Fig. 1; Hubas, C., Jesus B. M., Jeanthon, C., unpublished data), the latter pattern occurs seasonally when beaches are supplied with a high load of organic matter due to decomposition of macroalgae. These purple sulfur bacteria are therefore almost permanently exposed to oxygen at the sediment surface (e.g. [START_REF] Herbert | E stablishment of phototrophic purple sulphur bacteria in microbial mat systems[END_REF]. Among the purple bacteria, the purple nonsulfur bacteria are also widely distributed in aquatic environments rich in organic matter (Guyoneaud et al., 1996, Hiraishi and[START_REF] Hiraishi | Isolation and characterization of Rhodovulum strictum sp. nov. and some other purple nonsulfur bacteria from colored blooms in tidal and seawater pools[END_REF].

Cultural and molecular diversity of purple sulfur phototrophic mats

Most ecological studies on the distribution of anoxygenic phototrophs in natural environments have been based on biochemical features such as photopigment composition (see section 3.1) and / or on e stimations of bacterial numbers, isolation and characterization of pure cultures [START_REF] Guyoneaud | Anoxygenic phototrophic bacteria in eutrophic coastal lagoons of the French Mediterranean and Atlantic coasts (Prevost Lagoon, Arcachon Bay, Certes fishponds)[END_REF][START_REF] Nicholson | Structure of a microbial mat at Great Sippewissett Marsh, Cape Cod, Massachussets[END_REF], Ranchou-Peyruse et al., 2006, Van Gemerden et al., 1989a). The most prominent purple sulfur bacteria, frequently observed and also isolated from marine coastal sediments, have been reviewed by [START_REF] Van Gemerden | Ecology of phototrophic sulfur bacteria[END_REF] and by [START_REF] Imhoff | True marine and halophilic anoxygenic phototrophic bacteria[END_REF]. They were assigned to Thiocapsa roseopersicina, Thiocystis violacea and Allochromatium vinosum. As an example, various organisms have been cultivated from microbial mat communities of the Ebro Delta, one site among the most intensively studied. Vacuolated bacteria, such as Thiocapsa rosea and Lamprobacter modestohalophilus as well as non vacuolated bacteria such as Marichromatium gracile, T. roseopersicina or Ectothiorhodospira sp. have been isolated [START_REF] Martinez-Alonso | Diversity of anoxygenic phototrophic sulfur bacteria in the microbial mats of the Ebro Delta: a combined morphological and molecular approach[END_REF][START_REF] Villanueva | Diversity and physiology of polyhydroxyalkanoate-producing and -degrading strains in microbial mats[END_REF]. T. roseopersicina , which is easily cultivated, is very common in marine coastal habitats and predominant in most systems where it can reach abundances of 10 6 to 10 7 cells.cm -3 (Van Gemerden et al., 1989a).. The adaptation to a wide range of salinities and the high metabolic versatility and flexibility of this organism (tolerance to oxygen and possible aerobic growth in the dark) are important competitive advantages that explain the success of its distribution (de Wit and [START_REF] De Wit | C hemolithotrophic growth of the phototrophic sulfur bacterium Thiocapsa roseopersicina[END_REF][START_REF] De Wit | Growth of the phototrophic purple sulfur bacterium Thiocapsa roseopersicina under oxic/anoxic regimens in the light[END_REF]. Allochromatium spp and Marichromatium spp are also often observed and may be locally dominant [START_REF] Imhoff | True marine and halophilic anoxygenic phototrophic bacteria[END_REF]. From red layers found in mats of hypersaline environments, other members of the family Chromatiaceae such as Halochromatium salexigens, H. glycolicum and Halothiocapsa halophila have also been isolated [START_REF] Caumette | Characterization of Chromatium salexigens sp. nov., a halophilic Chromatiaceae isolated from Mediterranean salinas[END_REF], 1991[START_REF] Caumette | Chromatium glycolicum sp nov, a moderately halophilic purple sulfur bacterium that uses glycolate as substrate[END_REF].

In the last decades, the species composition of microbial mats has mostly been described by dissecting cores into thin horizontal layers and extracting nucleic acids or other cell components for chemical and molecular analysis [START_REF] Martinez-Alonso | Diversity of anoxygenic phototrophic sulfur bacteria in the microbial mats of the Ebro Delta: a combined morphological and molecular approach[END_REF][START_REF] Mouné | Molecular sequence analysis of prokaryotic diversity in the anoxic sediments underlying cyanobacterial mats of two hypersaline ponds in Mediterranean salterns[END_REF][START_REF] Navarrete | P hysiological status and community composition of microbial mats of the Ebro Delta, Spain, by signature lipid biomarkers[END_REF], Ranchou-Peyruse et al., 2006[START_REF] Villanueva | Diversity and physiology of polyhydroxyalkanoate-producing and -degrading strains in microbial mats[END_REF]. With these techniques, a high degree of bacterial diversity was generally found. As an example, the microbial mats within hypersaline lagoons at Guerrero Negro generated more than 1500 16S rRNA sequences representing over 750 s pecies [START_REF] Ley | Unexpected diversity and complexity of the Guerrero Negro hypersaline microbial mat[END_REF]. Denaturing gradient gel electrophoresis separation of 16S rRNA gene amplification products obtained using specific primer combination for Chromatiaceae, the main family of purple sulfur bacteria, showed that the diversity of members of this family in microbial mats in the Ebro Delta was high and pointed out the presence of novel species not related to any known purple sulfur bacteria [START_REF] Martinez-Alonso | Diversity of anoxygenic phototrophic sulfur bacteria in the microbial mats of the Ebro Delta: a combined morphological and molecular approach[END_REF].

The pufM gene encodes for the medium (M) subunit of the photosynthetic reaction center of the anoxygenic photosynthetic bacteria of the Alpha-, Beta-, and Gammaproteobacteria and of the Chloroflexaceae. Molecular analyses using this functional gene have also been applied in order to specifically study the depth distribution of anoxygenic phototrophs in mat communities [START_REF] Fourçans | C haracterization of functional bacterial groups in a hypersaline microbial mat community (Salins-de-Giraud, Camargue, France)[END_REF](Fourçans et al., , W ieland et al., 2003)). Using this method, vertical diel migration of an anoxygenic phototrophic community in responses to oxygen concentrations and pH was detected at a m icroscale depth level [START_REF] Fourçans | Vertical migration of phototrophic bacterial populations in a hypersaline microbial mat from Salins-de-Giraud (Camargue, France)[END_REF]. Only few studies detailed the di versity of anaerobic purple bacteria by the analysis of pufM environmental libraries since the pioneering work of [START_REF] Achenbach | Photosynthetic and phylogenetic primers for detection of anoxygenic phototrophs in natural environments[END_REF] and [START_REF] Karr | Remarkable diversity of phototrophic purple bacteria in a permanently frozen Antarctic lake[END_REF] on Antarctic lake waters and mats. An environmental clone library of the pufM gene was obtained from a thin cyanobacterial mat developed at the top of black sediment samples from the Berre lagoon (France) [START_REF] Ranchou-Peyruse | C omparison of cultivation-dependent and molecular methods for studying the diversity of anoxygenic purple phototrophs in sediments of an eutrophic brackish lagoon[END_REF]. Surprisingly, most of clones were closely related to aerobic anoxygenic phototrophic bacteria related to the Roseobacter clade whereas only two Roseobacter strains were isolated. The culture-dependent approach performed in parallel revealed the dominance of anaerobic purple sufur bacteria in these samples. The coexistence of both aerobic and anaerobic anoxygenic phototrophic bacteria has also been demonstrated in sediments from Antarctic and saline lakes [START_REF] Karr | Remarkable diversity of phototrophic purple bacteria in a permanently frozen Antarctic lake[END_REF][START_REF] Thiel | Unique communities of anoxygenic phototrophic bacteria in saline lakes of Salar de Atacama (Chile): evidence for a new phylogenetic lineage of phototrophic Gammaproteobacteria from pufLM gene analyses[END_REF].

Pigment diversity and reflectance measurements

Pigment diversity of microbial mats

The microenvironment within a mat is characterized by physical-chemical gradients (e.g. light, pH, nutrients), leading to high variability in the distribution of phototrophic microorganisms, both vertically within the top mm of the sediment (taxonomic stratification) and horizontally (high patchiness). Frequently, there is also significant temporal variability on biofilms that colonize intertidal areas as a result of the large physicochemical variations caused by the tide. The high variability exhibited by biofilms in such small scales cause significant sampling problems, e.g. to fully capture biofilm variability, many samples have to be taken, often more than it is logistically possible. Also, until recently, most of the available techniques for assessing microorganisms abundance or pigment diversity in biofilms were destructive (e.g. pigment extraction with organic solvents and quantification by HPLC). The destruction of the biofilm removes the existing physical-chemical gradients, changing significantly the environmental conditions of the biofilm under investigation. Thus, there is growing interest in developing remote sensing techniques that allow the non-destructive and non-invasive study of phototrophic microbial biofilms. S uch techniques include: spectral reflectance, O 2 micro-electrodes, optodes, Pulse-Amplitude-Modulation (PAM) fluorometry, Fast Repetition Rate Fluorometry (FRRF) fluorometry and Infra Red CO 2 Gas Analyzer (IRGA) benthic chambers (e.g. [START_REF] Kühl | O ptical microsensors for analysis of microbial communities[END_REF], K uhl and Polerecky, 2008[START_REF] Migné | A closed-chamber CO2-flux method for estimating intertidal primary production and respiration under emersed conditions[END_REF][START_REF] Stephens | Effects of microalgal communities on reflectance spectra of carbonate sediments in subtidal optically shallow marine environments[END_REF], T har et al., 2001, V opel and Hawes, 2006[START_REF] Wiggli | Reflectance spectroscopy and laser confocal microscopy as tools in an ecophysiological study of microbial mats in an alpine bog pond[END_REF]. All these techniques allow the repetition of measurements in the same biofilm area and some can be used to infer about biofilm biomass or taxonomic composition. In this section we focused on the use of spectral reflectance in the study of photosynthetic microbial mats.

Microbial biofilm taxonomic diversity is reflected in the presence of different pigments. Some of these pigments can be used as "signatures" of the presence of specific groups in the biofilm, e.g. diatom dominated biofilms will show the abundant presence of fucoxanthin and chlorophyll c; cyanobacteria dominated biofilms will show a variety of cyanobacterial specific pigments (e.g. mixoxanthophyll, equinenone, etc.); and an anoxygenic bacterial biofilm will mainly show bacteriochlorophylls and carotenoids (Table 1). If for diatom and cyanobacterial biofilms numerous studies exist showing their pigment composition (e.g. [START_REF] Andréfouët | Airborne hyperspectral detection of microbial mat pigmentation in Rangiroa atoll (French Polynesia)[END_REF], B rotas and Plante-Cuny, 2003, S tephens et al., 2003), only few papers focus on anoxygenic bacterial biofilms (e.g. [START_REF] Masse | Experimental study of interactions between purple and green sulfur bacteria in sandy sediments exposed to illumination deprived of nearinfrared wavelengths[END_REF]. Spectral reflectance can be used to identify and quantify the presence of different pigments in the biofilms, but it is first necessary to determine the spectral signatures of these pigments. It is thus useful to have good "ground truth" studies, i.e. spectral measurements taken together with measurements of the pigments present in the biofilm. Currently there are few studies that have attempted to establish the pigment spectral signatures of anoxygenic phototrophic biofilms.

Although they are not consensual about which wavelengths should be used to detect bacteriochlorophyll, there are 3 m ain absorbance peaks attributed to bacteriochlorophyll a: around 800 nm , around 850 nm and around 870 nm. The exact wavelengths depend on t he type of bacteria present (Table 1).

Spectral reflectance of microbial phototrophic mats

Spectral reflectance measurements have been used often in the estimation of biofilm microalgal biomass, using chlorophyll a as a biomass proxy (e.g. [START_REF] Carrère | Comparison of simple techniques for estimating chlorophyll a concentration in the intertidal zone using high spectral-resolution fieldspectrometer data[END_REF].

Chlorophyll a strongly absorbs red light and reflects most of the infrared light. Using this information, a wide variety of chlorophyll a based reflectance studies were developed, e.g. Normalized Difference Vegetation Index (NDVI), modified soil-adjusted vegetation index (MSAVI). To our knowledge no s imilar index exists to estimate anoxygenic phototrophic biofilms; although bacteriochlorophyll a is frequently used to infer about the presence of anoxygenic phototrophic bacteria (e.g. [START_REF] Gitelson | Optical characteristics of the phototroph Thiocapsa roseoparsicina and implications for real-time monitoring of the bacteriochlorophyll concentration[END_REF][START_REF] Kühl | Spectral light measurements in microbenthic phototrophic communities with a fiber-optic microprobe coupled to a sensitive diode array detector[END_REF][START_REF] Stal | T he Simultaneous Assay of Chlorophyll and Bacteriochlorophyll in Natural Microbial Communities[END_REF], S teenbergen and Korthals, 1982), it is not common to use the pigment content to quantify anoxygenic phototrophic bacterial biomass. Bacteriochlorophyll a dominated biofilms typically show absorption features in the infrared region, whereas chlorophyll a dominated biofilms do not (e.g. [START_REF] Stal | T he Simultaneous Assay of Chlorophyll and Bacteriochlorophyll in Natural Microbial Communities[END_REF]. In the Roscoff Aber Bay where anoxygenic photosynthetic biofilms seasonally developed at the sediment surface, reflectance spectra recorded from different sediment areas allowed the determination of bacteriochlorophyll absorption features in the infrared region, with absorption maxima at 792 and 850 nm (Fig. 2).

A spectral reflectance index is currently being developed by the authors to estimate bacteriochlorophyll content of this biofilm using bacteriochlorophyll absorption features. Spectral reflectance has also been widely used with benthic phototrophic biofilms to follow diatom vertical migration within the sediment matrix (e.g. [START_REF] Serôdio | Effects of chlorophyll fluorescence on the estimation of microphytobenthos biomass using spectral reflectance indices[END_REF], to follow photo-regulatory vertical movements (e.g. [START_REF] Perkins | Vertical cell movement is a primary response of intertidal benthic biofilms to increasing light dose[END_REF], to follow photo-physiological mechanisms [START_REF] Jesus | Detection of diatom xanthophyll cycle using spectral reflectance[END_REF], and to a lesser extent to identify the presence of different taxonomic groups, e.g. microalgae, cyanobacteria, green and purple bacteria (e.g. [START_REF] Bachar | Two-dimensional mapping of photopigment distribution and activity of Chloroflexus-like bacteria in a hypersaline microbial mat[END_REF][START_REF] Prášil | Emission spectroscopy and kinetic fluorometry studies of phototrophic microbial communities along a salinity gradient in solar saltern evaporation ponds of Eilat, Israel[END_REF][START_REF] Wiggli | Reflectance spectroscopy and laser confocal microscopy as tools in an ecophysiological study of microbial mats in an alpine bog pond[END_REF]. P resently, most of the research done with spectral reflectance on anoxygenic biofilms seems to have been focused on the identification of the presence of different taxonomic groups in the biofilm. With the introduction of hyperspectral (HS) imaging technology it b ecame possible to map sediment biofilms with high spectral and spatial resolution (e.g. [START_REF] Bachar | Two-dimensional mapping of photopigment distribution and activity of Chloroflexus-like bacteria in a hypersaline microbial mat[END_REF]. HS imaging is a very sensitive and minimally invasive tool that can be used in the investigation of biofilm spatial organization role in mat ecosystem functions, providing the possibility of imaging microbial identity and activity at high spatio-temporal resolution. Presently, the majority of the work involving HS imaging seems to address mainly questions relating spatial distribution of the different taxonomic groups that colonize the sediment, vertically and horizontally (Kuhl andPolerecky, 2008, Polerecky et al., 2009). However, some research has started to emerge using combinations of imaging techniques to infer about the relationships between the different microalgal groups and their environment. For instance, [START_REF] Bachar | Two-dimensional mapping of photopigment distribution and activity of Chloroflexus-like bacteria in a hypersaline microbial mat[END_REF] used hyperspectral imaging of reflectance spectra (4 th derivative of spectral images with 460-913 nm spectral resolution at 30 × 30 µm spatial resolution) and of emission spectra to map the distribution of different pigments (chlorophyll a, phycocyanin, bacteriochlorophyll a and bacteriochlorophyll c). Both spectral methods were sensitive enough to detect biofilm stratification within the sediment, showing the spectral signatures of chlorophyll a and zeaxanthin closer to the sediment surface, a m id layer 3-4 mm of bacteriochlorophyll c and bacteriochlorophyll a at deeper layers (5.5-7 mm). Using HS imagery these authors rejected their original hypothesis that Chloroflexaceae would be closely associated with the distribution of oxygenic phototrophs and proposed an alternative hypothesis that Chloroflexaceae is maximal in locations where both photosynthate excretion and sulfate reduction occur during a light / dark cycle.

In conclusion, although considerable research on microalgae phototrophic biofilms using spectral reflectance tools already exists, there is a gap in current knowledge regarding the use of these techniques for quantification and study of anoxygenic phototrophs.

Role of microbial mats in the functioning of coastal ecosystems

Role of microbial mats in sediment stability

Although the cohesive strength of one sediment may depend on its physicochemical properties, such as water content, density, mineralogy, plasticity, salinity and pH [START_REF] Dade | Predicting erosion resistance of muds[END_REF], its stability may correlate better with biological parameter than with nonbiological ones [START_REF] Paterson | Variations in sediment properties, Skeffling mudflat, Humber Estuary, UK[END_REF]. Microbial exopolymeric secretions are increasingly recognized as a major stabilising factor [START_REF] Stal | M icrophytobenthos as a biogeomorphological force in intertidal sediment stabilization[END_REF]. Extracellular Polymeric Substances (EPS) are a ubiquitous component of marine ecosystems primarily composed of carbohydrates, proteins and lesser amounts of other components. They have multiple roles in aquatic systems: attachment to substrata, flotation and locomotion, feeding, protection against desiccation / UV / pollution, development of biofilms, communication [START_REF] Decho | Microbial exopolymer secretions in ocean environments: their role(s) in food webs and marine processes[END_REF]. These molecules, mostly produced by diatoms and bacteria, compose a highly hydrated matrix more or less associated with cells. Tightly-wound capsules are secreted during exponential growth phase and allegedly serve protective effects to the cell, whereas loose slimes allow microorganisms to attach each other and to sediment [START_REF] Decho | Microbial exopolymer secretions in ocean environments: their role(s) in food webs and marine processes[END_REF]. The high amounts of EPS present in the sediment glue the grains together, thus enhancing the resistance of sediment to erosion and making it more stable [START_REF] Paterson | Variations in sediment properties, Skeffling mudflat, Humber Estuary, UK[END_REF][START_REF] Stal | M icrophytobenthos as a biogeomorphological force in intertidal sediment stabilization[END_REF]. If the resistance to erosion generally correlates well with carbohydrate and protein concentrations, variations in EPS quality influence as well sediment stability [START_REF] Sutherland | The effect of carbohydrate production by the diatom Nitzschia curvilineata on t he erodibility of sediment[END_REF][START_REF] Van Duyl | C onsequences of diatom mat erosion for carbohydrate concentrations and heterotrophic bacterial activities in intertidal sediments of the Ems-Dollard estuary[END_REF]. Moreover, cyanobacterial filaments trap sediment particles and reinforce cohesion [START_REF] Stal | M icrophytobenthos as a biogeomorphological force in intertidal sediment stabilization[END_REF]. Figure 3 summarised the potential influence of microbial mats on sediment stability. Given the importance of sediment stability in coastal ecosystems (which are typically constrained by strong physical and geochemical gradients), microorganisms are increasingly recognized as ecosystem engineers.

In the future, more studies are required in order to understand how EPS composition and diversity modify sediment properties. Particularly, little is known about stabilisation in mats of anoxygenic phototrophic bacteria. Yet abundance of purple sulphur bacteria may correlate with erosion threshold of sediment, and these bacteria appear to produce far more EPS than diatoms [START_REF] Grant | Prediction of coastal sediment stability from photopigment content of mats of purple sulfur bacteria[END_REF]. Thus the erosion of sediment is lower when purple phototrophic mats are present (Van Gemerden et al., 1989a). Recent measurements of sediment adhesion in Roscoff Aber Bay showed that sediment cohesion was enhanced and that sediment was stabilised by purple phototrophic bacteria, particularly under high bacterial abundance (Fig 4). Further investigations are now required to link stabilisation with the quantity or quality of the EPS produced by purple sulphur bacteria. 4.2. Production and respiration of microbial mats organic matter and its fate into the coastal food web Microbial mats are very productive ecosystems (e.g. about 200 gC.m -2 .y -1 in the Ebro Delta, [START_REF] Urmeneta | Oxygenic photosynthesis and respiratory activity in microbial mats of the Ebro Delta, Spain, by oxygen exchange method[END_REF]. The Winkler titration method [START_REF] Winkler | Die Bestimmung des im Wasser gelösten Sauerstoffen[END_REF], the incorporation of 14 C labeled bicarbonate, the fast-responding CO 2 microelectrodes [START_REF] De Beer | A fast-responding CO2 microelectrode for profiling sediments, microbial mats, and biofilms[END_REF] or the measurements of total DIC fluxes (e.g. [START_REF] Wieland | B iogeochemistry of an iron-rich hypersaline microbial mat (Camargue, France)[END_REF] have been used extensively to measure primary production, but most of the estimates in benthic photosynthetic mats were performed to date with oxygen microelectrodes [START_REF] Oren | Saltern evaporation ponds as model systems for the study of primary production processes under hypersaline conditions[END_REF], by measuring rates of oxygen depletion at different depths during light-dark shifts [START_REF] Revsbech | Photosynthesis of Benthic Microflora Measured with High Spatial-Resolution by the Oxygen Microprofile Method -Capabilities and Limitations of the Method[END_REF]. This method allows accurate estimation of gross primary production rates across the mat-water interface from the profiles providing that irradiance, temperature as well as porosity of the substrates are known [START_REF] Wieland | Irradiance and temperature regulation of photosynthesis and O2 consuption in a hypersaline cyanobacterial mat (Solar Lake, Egypt)[END_REF]. Oxygen measurements can provide information about both gross primary production and respiration of the microbial mats at millimetre scale. They revealed that microorganisms thrive in such a closeness that they mutually influence each other [START_REF] Van Gemerden | Microbial mats -a joint venture[END_REF]. Biological processes usually metabolically incompatible are found to occur simultaneously within the mats, which imply a tight coupling between them. The different members of the community are thus mutually dependent so that the entire ecosystem is often considered as self-sustaining [START_REF] Des Marais | Biogeochemistry of hypersaline microbial mats illustrates the dynamics of modern microbial ecosystems and the early evolution of the biosphere[END_REF]. The development of electrochemical and optical microprobes has attracted many microbiologists during the past decades probably because their resolution is particularly suitable for the study of microbial environments. But, ironically, whilst we have to date a good understanding of the chemical and physical conditions that microorganisms experience at millimeter scales in microbial mats, we still do not know precisely which role microbes play in biogeochemical cycles at larger scales.

In addition, microbial mats can represent a significant source of fixed carbon and nitrogen to the surroundings and they may serve as an important food source to higher trophic levels [START_REF] Joye | Benthic microbial mats: Important sources of fixed Nitrogen ans Carbon to the Twin Cays, Belize ecosystem[END_REF]. Recently, it has been shown that anoxygenic microbial mats may support the diet of inhabiting mud snails [START_REF] Riera | T rophic plasticity of the gastropod Hydrobia ulvae within an intertidal bay (Roscoff, France): A stable isotope evidence[END_REF]. In addition, microbial mats of the intertidal area which are dominated by diatoms generally serve as a f ood source for many invertebrates of the macrofauna and meiofauna (e.g. Hagerthey et al., 2002, Riera and[START_REF] Riera | Trophic ecology of nematodes from various microhabitats of the Roscoff Aber Bay (France) : importance of stranded macroalgae evidenced through ä13C and ä15[END_REF], including many commercial species such as penaeid shrimp postlarvae [START_REF] Al-Maslamani | Feeding on i ntertidal microbial mats by postlarval tiger shrimp, Penaeus semisulcatus De Haan[END_REF] or the oyster Crassostrea gigas [START_REF] Riera | Isotopic determination od f ood sources of Crassostrea gigas along a trophic gradient in the estuarine bay of Marénnes-Oléron[END_REF]. But despite the marked role they play into the coastal food web, the fate of microbial mats organic matter has seldom been addressed. Bacterial production has been proved to be a significant food source to benthic grazers and a sink of organic carbon in the food web of intertidal sediments [START_REF] Van Oevelen | T he fate of bacterial carbon in an intertidal sediment: Modeling an in situ isotope tracer experiment[END_REF] but further studies are still needed particularly concerning anoxygenic microbial mats. Indeed, mass bloom of anoxygenic phototrophic bacteria can develop at the sediment surface if the organic matter input is strong enough [START_REF] Herbert | E stablishment of phototrophic purple sulphur bacteria in microbial mat systems[END_REF] forming purple microbial mats which are characterised by the absence of oxygenic photosynthesis. The accumulation of organic matter at the sediment surface stimulates respiration and, below 2 to 3 mm depth, sediment becomes totally anoxic and characterised by very high sulfate-reducing rates [START_REF] Bolam | Effects of macroalgal mats on intertidal sandflats: an experimental study[END_REF][START_REF] Nedergaad | T he importance of sulfate reduction associated with Ulva lactuca thalli during decomposition: a mesocosm experiment[END_REF], allowing the exclusive growth of anoxygenic purple bacteria. Primary production and respiration rates measurements are still scarce on these types of mats. Recently, the high contribution of Chloroflexus-like anoxygenic phototrophs (green non-sulphur bacteria) to the gross primary production and community respiration of a microbial mat was found to be strongly dependent upon the light availability in the near infrared region [START_REF] Polerecky | C ontribution of Chloroflexus respiration to oxygen cycling in a hypersaline microbial mat from Lake Chiprana, Spain[END_REF]. This highlights the fact that understanding the contribution of anoxygenic phototrophs to total primary production and respiration is more complex than previously thought and that more studies on anoxygenic microbial mats are required.

Microbial mats are a remarkable example of the various forms of respiration that co-exist in aquatic habitats. Anaerobic respiration as well as aerobic respiration and re-oxydation processes have been relatively well studied in these systems. From the surface to the depth, the redox potential decreases, which influence the distribution of the different respiration pathways. Along the sediment depth, the chemical reactions involve different terminal electron acceptors and display apparent free energy yields which decrease with increasing depth [START_REF] Hoehler | Biological energy requirements as quantitative boundary conditions for life in the subsurface[END_REF]. Carbon fluxes across the mat-water interface are generally deduced from oxygen measurements by applying known respiratory quotients (RQ). However, most of the RQ apply to conventional aerobic respiration and have no useful meaning in case total respiration mainly occurs via anaerobic pathways [START_REF] Williams | Respiration in aquatic ecosystems: history and background[END_REF]. Total DIC fluxes in benthic chamber enclosures or chambers equipped with a CO 2 infra-red gas analysers [START_REF] Migné | A closed-chamber CO2-flux method for estimating intertidal primary production and respiration under emersed conditions[END_REF] are an efficient way to measure C fluxes across the mat-water or mat-air interface. They have been used extensively on emersed diatom biofilms to estimate their annual carbon budgets [START_REF] Hubas | D oes seasonal proliferation of Enteromorpha sp. affect the annual benthic metabolism of a small macrotidal estuary? (Roscoff Aber Bay, France)[END_REF], M igné et al., 2004, S pilmont et al., 2006) but rarely on other microbial mats to our knowledge.

Conclusions and future directions

The current knowledge of ecology, ecophysiology and role of anoxygenic purple microbial mats is far less documented than those dominated by cyanobacteria and / or diatoms.

Molecular approaches have provided fresh insight into the diversity of microbial mats but the role of microbial species diversity in sustaining ecosystem processes like primary production has seldom been addressed (but see [START_REF] Forster | B iodiversityecosystem function relationship in microphytobenthic diatoms of the Westerschelde estuary[END_REF]. As revealed by spectral reflectance measurements, the distribution of phototrophic microorganisms in coastal ecosystems is highly variable. Remote sensing methods are probably an efficient way to perform a real integration of these systems at larger scales. Indeed, coastal habitats are amongst the most productive ecosystems on earth and the contribution of anoxygenic bacteria to the coastal C and N cycles and their role in sustaining local food webs are probably underestimated.

Techniques such as HS imaging combined with state of the art optical sensors (e.g. optodes, Imaging PAM) will surely help to elucidate the distribution, trophic, geochemical roles of anoxygenic phototrophic biofilms as well as investigate in depth their photobiology in intact samples. In addition, new generations of ion microprobes based on m ass spectrometry of secondary ions (SIMS) are now available and particularly appropriate to the study of microbial mats. They allow the analysis of any isotopic composition of a given sample surface and the distribution of labelled molecules (e.g. H 13 CO 3 -) at a sub-cellular scales. It is thus now possible to determine the rate of carbon and nitrogen fixation at the cellular level [START_REF] Musat | A single-cell view on t he ecophysiology of anaerobic phototrophic bacteria[END_REF] which would give valuable information about the functioning of the microbial mats in the future. Bchla (805,860,880),Spi (480,520,550) Absorbance and reflectance Thiocapsa roseopersicina [START_REF] Gitelson | Optical characteristics of the phototroph Thiocapsa roseoparsicina and implications for real-time monitoring of the bacteriochlorophyll concentration[END_REF] BChl (800,801,804,806,808,835,837,862,865,867,868,870,879) Absorbance Hyperspectral imaging Bacterial mat under didemnid ascidian Kühl & Polerecky (2008)

Figures

Figure 1 :

 1 Figure 1: pictures of purple phototrophic mats, underwater (top panel) and emerged (bottom panel), in Roscoff Aber Bay.

Figure 2 :

 2 Figure 2: left panel: pictures of sites with low (A), medium (B) and high (C) biomass of purple sulfur bacteria, in Roscoff Aber Bay. Right panel : spectral reflectance of the sediment, in sites with low (dashed line), medium (solid line) and high (dotted line) biomass of these bacteria. The arrows point the bacteriochlorophyll a absorption peaks.

Figure 3 :

 3 Figure 3: biotic and abiotic parameters influencing sediment stability. Arrow with black solid line: stimulates. Arrow with gray solid line: inhibits. Arrow with dashed line: has an influence on.

Figure 4 :

 4 Figure 4: sediment cohesion (mTa; mean ± SD) in sites with low, medium and high biomass of purple sulfur bacteria, in Roscoff Aber Bay (see fig 2). Measurements were performed using a Magnetic Particle Inducer (MagPI), a d evice recently developed by Larson et al. (2009).

  in vivo pigment spectral "signatures" collected from available references. Numbers between brackets refer to the absorption features of each pigment. Emphasis was given to references where bacteriochlorophyll samples were found. Alo-alloxanthin, BChl-bacteriochlorophyll, ß-car-ß-carotene, BChlabacteriochlorophyll a, BChlc -bacteriochlorophyll c, BChld -bacteriochlorophyll d, BChle -bacteriochlorophyll e, Car-carotenoides, Chla-chlorophyll a, Chlb-chlorophyll b, Chlc-chlorophyll c, Chld-chlorophyll d, DD-diadinoxanthin, Fuco-fucoxanthin, Lut-lutein, Myxo-myxoxantophyll, Oke -Okenone, PC-phycocianin, PB-phycobilin pigments, PE-phycoerythrin, Per-peridinin, Spi -Spirilloxanthin, Zea-zeaxanthin

  photosynthetic bacteria

Acknowledgement: We would like to thank three anonymous reviewers for their constructive remarks as well as Nicolas Spilmont and Dominique Davoult for their help with field work. This work is a contribution to the French national CNRS program EC2CO-MicroBien (Microbiologie Environnementale). Thanks are due to Ecole Normale Supérieure (ENS) Paris for supporting Claire Passarelli PhD grant. Bruno Jesus was funded by a FCT postdoctoral grant (POCI ⁄ BPD ⁄ 20993 ⁄ 2004), the project SEAPhoB (PDTC/MAR/101410/2008) and the program ASSEMBLE (nº227799).


Ian Probert
email: probert@sb-roscoff.fr

Raffaele Siano

Camille Poirier

Johan Decelle

Tristan Biard

Akihiro Tuji

Noritoshi Suzuki

Fabrice Not

(Dinophyceae), a dinoflagellate commonly found in symbiosis with polycystine radiolarians

Keywords: dinoflagellate, polycystines, Peridiniales, Radiolaria, Scrippsiella, symbiosis, taxonomy, Zooxanthella

come   L'archive ouverte pluridisciplinaire

Introduction

Mutualistic associations involving photosynthetic microalgae are common in both benthic and pelagic ecosystems and are essential for establishing and maintaining the structure of marine communities [START_REF] Caron | Symbiosis and mixotrophy among pelagic microorganisms[END_REF]. Symbiosis between corals and the dinoflagellate genus Symbiodinium Freudenthal is fundamental for the survival and ecological success of coral reef ecosystems. Members of the genus Symbiodinium have been intensively studied with respect to their morphology and life cycle [START_REF] Freudenthal | Symbiodinium gen. nov. and Symbiodinium microadriaticum sp. nov., a zooxanthella: Taxonomy, life cycle and morphology[END_REF][START_REF] Fitt | The relation of diel patterns of cell division to diel patterns of motility in the symbiotic dinoflagellate Symbiodinium microadriaticum Freudenthal in culture[END_REF][START_REF] Trench | Symbiodinium microadriaticum Freudenthal[END_REF], and genetic diversity [START_REF] Coffroth | Invited Review: Genetic diversity of symbiotic dinoflagellates in the genus Symbiodinium[END_REF][START_REF] Sampayo | Cohesive molecular genetic data delineate species diversity in the dinoflagellate genus Symbiodinium[END_REF][START_REF] Lajeunesse | Improved resolution of reef-coral endosymbiont (Symbiodinium) species diversity, ecology, and evolution through psbA non-coding region genotyping[END_REF][START_REF] Stat | Variation in Symbiodinium ITS2 Sequence Assemblages among Coral Colonies[END_REF]. Studies on this coastal benthic symbiotic relationship significantly increased when the coral-bleaching phenomenon was brought to global attention and associated to increases in sea surface temperature, enhanced light intensity, and ocean acidification [START_REF] Hoegh-Guldberg | Coral reefs under rapid climate change and ocean acidification[END_REF].

Symbiotic interactions between pelagic hosts and microalgae have received less attention, despite the fact that they are widespread in the photic layer of the world ocean where they play a fundamental role in the ecology of the planktonic ecosystem [START_REF] Stoecker | Acquired phototrophy in aquatic protists[END_REF]Decelle et al. 2012). Recent studies have demonstrated that dinoflagellate symbionts of Foraminifera belong to Pelagodinium Siano, Montresor, Probert et de Vargas, a genus that is related to Symbiodinium within the order Suessiales [START_REF] Siano | Pelagodinium gen. nov. and P. beii comb. nov., a dinoflagellate symbiont of planktonic foraminifera[END_REF], and that Acantharia typically associate with members of the prymnesiophyte genus Phaeocystsis Lagerheim (Decelle et al. 2012), although one taxon, Acanthochiasma sp., can contain multiple symbiotic partners, including distantly related dinoflagellates (from the genera Pelagodinium, Heterocapsa Stein, Azadinium Elbrächter et Tillmann and Scrippsiella Balech ex Loeblich III) as well as a haptophyte (Decelle et al. 2012b). Nassellaria) are single-celled, heterotrophic, biomineralizing planktonic protists from the Rhizaria lineage that are widely distributed in the ocean and are found throughout the entire water column [START_REF] Boltovskoy | World Atlas of Distribution of Recent Polycystina (Radiolaria)[END_REF]. Many polycystines host microalgae within their cytoplasm [START_REF] Anderson | Radiolaria[END_REF]. Cells containing photosynthetic microalgae have been shown to survive for longer periods in nutrient-poor water than those that do not have microalgal partners and the microalgae are therefore assumed to be symbionts that play a nutritive role for the hosts [START_REF] Anderson | Radiolaria[END_REF].

Polycystines form associations with various dinoflagellate, prymnesiophyte and prasinophyte partners (usually not at the same time), with dinoflagellates being the most common symbiotic partners [START_REF] Anderson | Ultrastructure of a colonial radiolarian Collozoum inerme and a cytochemical determination of the role of its zooxanthellae[END_REF][START_REF] Anderson | Radiolaria[END_REF][START_REF] Anderson | Assimilation of symbiont-derived photosynthates in some solitary and colonial radiolaria[END_REF]). In the late nineteenth century, Karl Brandt was the first to recognize that the "yellow cells" within polycystines, actinian corals and hydrozoans were microalgae, which he collectively described in the new genus Zooxanthella Brandt [START_REF] Brandt | Ueber das Zusammenleben von Thieren und Algen[END_REF]), although they were not immediately recognized as dinoflagellates. Soon afterwards, the species Z. nutricula Brandt was proposed for the symbiont of the collodarian polycystine Collozoum inerme collected from the western Mediterranean Sea and it was stated in the description that this species was presumably identical to the yellow cells of other polycystines [START_REF] Brandt | Ueber die morphologische und physiologische Bedeutung des Chlorophylls bei Thieren[END_REF]. The subsequent taxonomic history of this genus and species have been very confused (see review by [START_REF] Blank | Nomenclature of endosymbiotic dinoflagellates[END_REF], and the plural noun 'zooxanthellae' has persisted as a colloquialism used to describe marine microalgal endosymbionts in general.

The symbionts of the 'by-the-wind sailor' hydrozoan jellyfish Velella velella were reported to be similar to those of polycystines initially by [START_REF] Hovasse | Endodinium chattoni (nov. gen. et sp.). Son cycle de multiplication endogène. Variation du nombre de ses chromosomes[END_REF] [START_REF] Taylor | Ultrastructure of the "zooxanthellae" Endodinium chattonii in situ[END_REF] and [START_REF] Hollande | Les Xanthelles des Radiolaires Sphaerocollides, des Acanthaires et de Velella velella: infrastructure -cytochimie -taxonomie[END_REF] nutricula (Brandt) Banaszak, Iglesias-Prieto et Trench, respectively (Banaszak et al. 1993), but these names remain technically invalid because reference was not made to the exact page of the basionym.

Using molecular methods, [START_REF] Gast | Molecular phylogeny of symbiotic dinoflagellates from planktonic foraminifera and radiolaria[END_REF] found that the dinoflagellate symbionts in six different polycystine species from the Sargasso Sea (the collodarians Collozoum caudatum and Thalassicolla nucleata, three unidentified collodarian species and the spumellarian Spongostaurus sp.) had identical SSU rDNA sequences that they assigned to Scrippsiella nutricula. These molecular analyses indicate that taxonomically divergent radiolarians can contain the same symbiotic dinoflagellate. Since these analyses were conducted directly on symbionts extracted from the hosts (i.e., not cultured), the morphology of the motile stage of the symbiotic algae assigned to S. nutricula was not investigated, and has still never been reported. Gast 1996) also sequenced the SSU rDNA of the symbiont of V. velella from the Sargasso Sea and found that the sequence was very similar to those of the radiolarian symbionts (4 differences out of 1802 base pairs). They therefore also assigned this V. velella symbiont to S. nutricula.

Here we examined the morphology and molecular phylogenetic position of clonal cultures of the free-living stage of dinoflagellates isolated from several different polycystine radiolarian hosts, including Collozoum, the taxon from which Zooxanthella nutricula was originally described. In addition, we sequenced symbiont genes directly from several polycystine-symbiont holobiont specimens (including collodarian, spumellarian and nassellarian hosts) from different oceanic regions.

Accurate morpho-molecular characterization and taxonomic designation of symbionts from the genus Symbiodinium has been key for studies of the ecology and functioning of coral reef systems and is it is likewise likely to prove important for future studies on the widespread pelagic symbiosis involving polycystine radiolarian hosts.

Material and Methods

Samples and culture isolation

The radiolarian specimens from which the holobiont sequences or cultures originated were isolated from samples collected in 2010-2012 by net tows (20 to 150 micron mesh size) in the bay of Villefranche-sur-Mer (France), off Sesoko Island, Okinawa (Japan) and in the South Pacific Ocean during the Tara Oceans expedition (Table 1, Supplementary Figs 1 and2). The polycystines were first sorted from fresh net samples under a binocular microscope, cleaned by successive transfers in sterile seawater in Petri dishes, then left in an illuminated and temperature-regulated incubator for several hours to self-clean. Individual clean specimens were then 8 identified based on their morphology and imaged under an inverted microscope. Some specimens were then transferred to guanidinium isothiocyanate (GITC) buffer for direct DNA extraction from holobionts. The dinoflagellate cultures were obtained by micropipette isolation of single symbiont cells released from live radiolarian specimens that were microdissected under an inverted microscope. The resulting monoclonal cultures were maintained in filter-sterilized seawater with K/2(-Tris, -Si) medium supplements [START_REF] Keller | Media for the culture of oceanic ultraphytoplankton[END_REF]) at 22°C with an irradiance of 70-80 µmol photons m -2 s -1 in a 12:12 light:dark regime. The cultures have been deposited in the Roscoff Culture Collection (http://www.sb-roscoff.fr/Phyto/RCC). LM images of radiolarian holobionts from which sequences / cultures were obtained are shown in Supplementary Figures 1 and2. Detailed information related to each of the samples used in this study can be found in the RENKAN database at http://abims.sbroscoff.fr/renkan/.

Microscopy preparations and observations

Light micrographs of living cells were taken using a Zeiss Axiophot light microscope equipped with a Zeiss AxioCam digital camera system (Carl Zeiss, Oberkochen,

Germany). For scanning electron microscopy (SEM), dinoflagellate cells were fixed in 1% (v:v) formol for 2 hours at room temperature. Samples were then gently filtered onto 3µm pore-size Nucleopore polycarbonate filters (Pleasanton, CA, USA), washed with distilled water, dehydrated in an ethanol series (25%, 50%, 75%, 95%, 100%), and critical point dried. The filters were mounted on stubs, sputter coated with gold, and examined with a FEI Quanta™ 200 SEM (FEI, Hillsboro, Oregon, USA) Genomic DNA was extracted from exponentially growing cultures of the strains using a NucleoSpin Plant II DNA extraction kit (Macherey-Nagel), or from holobionts using the method described in De [START_REF] Vargas | A molecular approach to biodiversity and biogeography in the planktonic foraminifer Globigerinella siphonifera (d'Orbigny)[END_REF].

Partial nuclear LSU and SSU rDNA genes were PCR amplified using Phusion highfidelity DNA polymerase (Finnzymes) in a 25 µl reaction volume and the following thermocycler steps : an initial denaturation step at 98°C for 30 sec, followed by 35 cycles at 98°C for 10 sec, 30 sec at the temperature of semi-hybridization chosen for each set of primers, and 30 sec at 72°C, with a final elongation step of 10 min at 72°C. The eukaryote primer set 63F (ACGCTT GTCTCAAAGATT) / 1818R (ACGGAAACCTTGTTACGA) (Tm 50°C) [START_REF] Lepere | Whole Genome Amplification (WGA) of marine photosynthetic eukaryote populations[END_REF]) was used to amplify the SSU rDNA of the dinoflagellate cultures, whereas the dinoflagellate specific primer set DIN464F (TAACAATACAGGGCATCCAT) / S69 (CCGTCADTTCCTTTRAGDTT) (Tm 53°C) was used to target the dinoflagellates in the holobiont samples. The D1-D2 fragment of the LSU rDNA was amplified using the dinoflagellate specific primers Ldino6 (MCC CGCTGAATTTAAGCATA) / Ldino1 (AACGATTTGCAGGTCAGTACCGC) (Tm 55°C) from both cultures and holobionts. PCR products were then sequenced at the GENOSCOPE (CEA, Evry, France).

The sequences generated from the studied strains and holobionts (GenBank accession Phylogenetic analyses were conducted with Maximum Likelihood (ML) and Bayesian methods. The ML analysis was carried out using MEGA v. 5.1 [START_REF] Tamura | MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods[END_REF] with the General Time Reversible (GTR) as the best model of nucleotide substitution and considering a gamma distribution with a proportion of invariable sites (I) set at 5 by default. Bootstrap supports for the tree were obtained after 1000 replicates. The

Results

Microscopy Observations

In our culture conditions, our the clonal strains of polycystine symbionts tended to contain a mixture of motile thecate cells and larger, irregularly-shaped non-motile cells devoid of the typical features of motile cells (theca, cingulum, sulcus), the latter more closely resembling the in hospite symbiotic state. The proportion of motile and non-motile cells varied between strains and through growth cycles for each strain. The Cells are 10.5 to 15 µm in length (average 13.1 µm, n=30) and 9.1 to 11.2 µm in width (average 10.4 µm, n=30). The epitheca is larger than the hypotheca. Observed under LM, cells have a slightly convex conical epitheca with a well-pronounced apical horn (Fig. 1A,1B,1D). The hypotheca is rounded (Fig. 1A,1D). The nucleus is large and occupies the center of the cells (Fig. 1B,1D). One or two golden-yellow chloroplasts are present around the cell periphery, sometimes appearing as a single plastid bordering the cell periphery (Fig. 1D). One large circular pyrenoid (sometimes two) is often visible in LM (Fig. 1A-D). No eyespot is visible in light microscopy.

Cells swim steadily in a straight line, rotating around the transapical axis. They suddenly stop, change direction at different angles from the original path, often backtracking.

In SEM, the epitheca appears conical (Fig. 2A) to rounded (Fig. 2C), and the smaller hypotheca is symmetrical and rounded in ventral (Fig. 2A) and dorsal (Fig. 2C) view.

The plate tabulation is Po, X, 4', 3a, 7'', 5C, 4S, 5''', 1'''' (Figs 2A-E 2C, 3A-B). It is very wide and shallow and is constituted by a single series of five rectangular plates, the first being much narrower than the others (Fig. 2A-C, 2E, 3A-B). The sulcus is fairly shallow and narrows towards the antapical end (Fig. 2A-B). The sulcal area comprises four plates (Fig. 2B,3A). One of these (Sd) forms a conspicuous flange extending over the median area of the sulcus, partially covering the sulcal area (Fig. 2B). There appears to be a single plate (Ss) beneath this flange (Fig. 2B). Flagella were not preserved in our SEM preparations. In the hypotheca, a series of 5 trapezoid plates of similar size borders the cingulum. A single six-sided antapical plate completes the hypothecal tabulation (Fig. 2E,3D). The cell surface is mostly smooth. We have never observed a peduncule in either LM or SEM preparations.

Phylogenetic Analyses

PCR amplifications of DNA extracts from culture strains and uncultured holobionts led to generation of 35 partial SSU rDNA (~650 bp) and 22 partial LSU rDNA (~675 bp) sequences of dinoflagellate symbionts from spumellarian, collodarian and nassellarian hosts collected in the Mediterranean Sea and in the North and South Pacific oceans (Table 1). For each gene the vast majority of these sequences were identical (see below) and hence only a subset of 15 SSU rDNA and 10 LSU rDNA sequences, representing a cross-section of host diversity, were included in datasets for phylogenetic reconstructions. Phylogenetic analyses on the SSU and LSU rDNA datasets demonstrated that all of our sequences grouped together in a distinct and highly supported clade (hereafter called clade B) within the dinoflagellate order Peridiniales (full ML and Bayesian statistical support; Figs. 4 and5). In both SSU and LSU rDNA phylogenies, this clade included two distinct sub-clades, B1 and B2, each 13 containing sequences that are 100% identical irrespective of host taxon and oceanic region. In our SSU rDNA phylogenetic tree (Fig. 4), sub-clade B1 included the majority of symbiont sequences recovered in this study (including those from five culture strains isolated from Collozoum colonies from the Mediterranean Sea and Pacific Ocean), as well as published sequences that correspond to the symbionts of five collodarians and one spumellarian collected in the Atlantic Ocean [START_REF] Gast | Molecular phylogeny of symbiotic dinoflagellates from planktonic foraminifera and radiolaria[END_REF]. Sub-clade B2 contained the sequences generated in the present study of the symbionts of two collodarian holobionts as well as one published sequence (U52357) of the symbiont of the jellyfish Velella velella [START_REF] Gast | Molecular phylogeny of symbiotic dinoflagellates from planktonic foraminifera and radiolaria[END_REF]. In both phylogenetic reconstructions, the monophyletic clade B containing the sequences of polycystine symbionts was phylogenetically distinct from the well-supported clade containing members of the genus Scrippsiella (including the holotype species S. sweeneyae Loeblich III), but overall the phylogenetic relationships between clades within the Peridiniales were not clearly resolved in our analyses. When sequences of members of the genus Bysmatrum, which have a plate tabulation pattern similar to Scrippsiella-like peridinaleans (Table 2), were included in phylogenetic analyses, they formed a distinct mono-generic clade which fell on a long branch that altered overall tree topology (Supplementary Figure 3). In the SSU rDNA phylogeny (Figure 4), note that the sequence labeled "uncultured alveolate from Nasselaria" (DQ916409) and the two sequences labeled "Dinophyceae from Collodaria" (DQ116021 and DQ116022) correspond to non-photosynthetic dinoflagellate parasites of Radiolaria (Gast 2006).

Discussion

Dinoflagellates that form symbiotic relationships with metazoan or protistan hosts are characterized by complex life cycles, with an alternation of symbiotic and free-living [START_REF] Trench | Symbiodinium microadriaticum Freudenthal[END_REF]). In the freeliving stage, cells tend to regain their original morphology [START_REF] Freudenthal | Symbiodinium gen. nov. and Symbiodinium microadriaticum sp. nov., a zooxanthella: Taxonomy, life cycle and morphology[END_REF][START_REF] Spero | Symbiosis in the planktonic foraminifer, Orbulina universa, and the isolation of its symbiotic dinoflagellate, Gymnodinium béii sp. nov[END_REF][START_REF] Siano | Pelagodinium gen. nov. and P. beii comb. nov., a dinoflagellate symbiont of planktonic foraminifera[END_REF]. Since the taxonomy of dinoflagellates is largely based on comparison of the number, shape and arrangement of the thecal plates (or amphiesmal vesicles in athecate species) that form the periplast of free-living motile cells, the establishment of clonal cultures from symbionts extracted from their hosts is critical for accurate taxonomic assignation.

The genus Zooxanthella was originally created to collectively describe the symbionts of diverse hosts from the Mediterranean Sea, including polycystines, corals and hydrozoans [START_REF] Brandt | Ueber das Zusammenleben von Thieren und Algen[END_REF]) and Z. nutricula was created to describe the symbionts of the collodarian polycystine Collozoum inerme [START_REF] Brandt | Ueber die morphologische und physiologische Bedeutung des Chlorophylls bei Thieren[END_REF]. The taxonomic history of Zooxanthella has subsequently been confusing, with Z. nutricula being alternately combined within Endodinium, Amphidinium Claperède et Lachmann (see review of the nomenclatural history of endosymbiotic dinoflagellates by [START_REF] Blank | Nomenclature of endosymbiotic dinoflagellates[END_REF] and most recently (albeit technically invalidly) within Scrippsiella [START_REF] Banaszak | Scrippsiella velellae sp. nov. (Peridiniales) and Gloeodinium viscum sp. nov (Phytodiniales), dinoflagellate symbionts of hydrozoans (Cnidaria)[END_REF].

Our observations of the plate tabulation pattern of cultured motile cells of the freeliving stage of the dinoflagellate isolated from diverse polycystine hosts clearly show that it is a member of the order Peridiniales (bilateral symmetry, cingulum only slightly displaced, presence of Po and X plates, presence of 3 intercalary plates in the epitheca) and that it should not be classified in the genus Scrippsiella, nor in the related genera Calciodinellum, Bysmatrum, Pentapharsodinium, or Ensiculifera. All of these latter genera are described as possessing 2 antapical plates, whereas the The polycystine symbionts also differ from Scrippsiella and Bysmatrum (but not from Pentapharsodinium and Ensiculifera) in possessing 5 (rather than 6) cingular plates.

The wing-like flange that covers the sulcal area has not been described in any of these related genera. This structure resembles the peduncule cover plate (PC) of heterotrophic dinoflagellates in the peridinialean family Pfiestereaceae Steidinger et Burkholder emend. Litaker. Motile forms of members of the Pfiestereaceae feed myzocytotically by means of a peduncule that emerges close to the flagella and that can attach to microalgal prey or epidermal cells of live fish (e.g. [START_REF] Steidinger | Cryptoperidiniopsis brodyi gen. et sp. nov. (Dinophyceae), a small lightly armored dinoflagellate in the Pfiesteriaceae[END_REF]). We have not observed a peduncle in the taxon described here, but should it be present, the Sd plate should rather be termed PC and the plate formula would become: Po, X, 4', 3a, 7'', 5c, , 3s, PC, 5''', 1''''.

Comparison of morphological characters strongly supports a generic level separation of the polycystine symbiont reported here from other described Peridiniales taxa, a conclusion that is corroborated by phylogenetic analyses. In both SSU and LSU In light of both morphological and genetic differences from existing genera, this taxon should clearly be classified in a distinct genus. Although S. nutricula was previously classified within the genus Endodinium, this genus was created to describe the symbiont of Velella velella from the Mediterranean and there is sufficient doubt as to whether these organisms are actually closely related (see below) to preclude reinstatement of this combination, which in any case should be considered synonymous with Z. nutricula. Strict adherence to nomenclatural rules would hence dictate the use of the genus Zooxanthella for this species, but we agree with numerous previous authors (e.g. [START_REF] Blank | Nomenclature of endosymbiotic dinoflagellates[END_REF][START_REF] Trench | Symbiodinium microadriaticum Freudenthal[END_REF][START_REF] Banaszak | Scrippsiella velellae sp. nov. (Peridiniales) and Gloeodinium viscum sp. nov (Phytodiniales), dinoflagellate symbionts of hydrozoans (Cnidaria)[END_REF]) who have convincingly argued that Zooxanthella should be rejected as a confusing name that has been widely applied to divergent taxa. We therefore propose the erection of a new genus, which we name Brandtodinium Probert et Siano in reference to Karl Brandt who first described this species [START_REF] Brandt | Ueber die morphologische und physiologische Bedeutung des Chlorophylls bei Thieren[END_REF], and the transfer of Z. nutricula to this new genus as Brandtodinium nutriculum comb. nov.. In the absence of a holotype, not provided in the original description of the species, we designate Fig. 2, SEM illustrations of plate tabulation of the motile stage of the culture strain VFR1-1RCC3387 of this species, as the neotype for the species.

Whereas the generic level distinction of Brandtodinium from other peridinialeans is obvious, the relationship of this genus to other genera within the Peridiniales is not clear. In terms of overall morphology of the motile stage (e.g. cell size and shape, plate tabulation), Brandtodinium has several features in common with members of the Calciodinellaceae Taylor, a family that includes Scrippsiella. The Calciodinellaceae, Pfiestereaceae. An unexpectedly close genetic relationship between B. nutriculum (as Z. nutricula) and a small group of taxa in which photosynthesis takes place by a tertiary endosymbiont derived from a diatom [START_REF] Horiguchi | Ultrastructure of a new marine sand-dwelling dinoflagellate, Gymnodinium quadrilobatum sp. nov. (Dinophyceae) with special reference to its endosymbiotic alga[END_REF], the 'dinotoms' [START_REF] Imanian | The complete plastid genomes of the two 'dinotoms' Durinskia baltica and Kryptoperidinium foliaceum[END_REF], was recently reported [START_REF] Gottschling | New home for tiny symbionts: Dinophytes determined as Zooxanthella are Peridiniales and distantly related to Symbiodinium[END_REF]. These investigators employed a 'maximal taxon sample' approach by inferring relationships based on a concatenated SSU, LSU and ITS rDNA sequence alignment irrespective of whether all of these sequences were available for the taxa included (i.e.

an alignment with significant gaps). Our individual SSU and LSU phylogenies do not recover this relationship. The present study provides strong evidence from two highly conserved phylogenetic markers (SSU and LSU rDNA) to support the conclusion from our observations of the morphology of free-living cells that Brandtodinium is a taxonomically distinct genus within the Peridiniales. We chose not to employ an approach comparable to that of [START_REF] Gottschling | New home for tiny symbionts: Dinophytes determined as Zooxanthella are Peridiniales and distantly related to Symbiodinium[END_REF] because in-depth assessment of evolutionary and phylogenetic relationships between Brandtodinium and other members of the order Peridiniales goes beyond the scope of our research.

We nevertheless provide evidence that Brandtodinium is distinct from the dinotom velellae as pp (=Po, X), 4', 3a, 7'', 5c, 3s, 5''', 2'''', which corresponds neither to that of Scrippsiella nor to that of Brandtodinium (Table 2). The spine-like protuberance on the first cingular plate illustrated in Figure 11 (page 520) of [START_REF] Banaszak | Scrippsiella velellae sp. nov. (Peridiniales) and Gloeodinium viscum sp. nov (Phytodiniales), dinoflagellate symbionts of hydrozoans (Cnidaria)[END_REF] is a characteristic feature of the genus Ensiculifera, to which we believe this species should have been assigned. However, the SEM images illustrated in [START_REF] Banaszak | Scrippsiella velellae sp. nov. (Peridiniales) and Gloeodinium viscum sp. nov (Phytodiniales), dinoflagellate symbionts of hydrozoans (Cnidaria)[END_REF] do not permit verification of whether this organism really has 3 sulcal plates (as stated in the description), rather than 5, as diagnostic for members of the genus Ensiculifera. It could also be inferred that S. chattonii, the symbiont of Mediterranean V. velella, might also be transferred to Ensiculifera, but unfortunately no morphological data has ever been provided for the free-living stage of this taxon. It is noteworthy that the only existing sequence (SSU rDNA) of a symbiont of V. velella (from the Sargasso Sea, Atlantic Ocean) produced by [START_REF] Gast | Molecular phylogeny of symbiotic dinoflagellates from planktonic foraminifera and radiolaria[END_REF] falls within our Brandtodinium clade, in the sub-clade B2 composed of three identical sequences, two of which we generated from Pacific polycystine holobionts. This sub-clade is distinct from the sub-clade B1 formed by the group of identical sequences from all of our Pacific (South and North) and Mediterranean culture strains of B. nutriculum isolated from polycystines, from several Pacific polycystine holobionts that we sequenced, and from the Sargasso Sea polycystine symbionts sequenced by [START_REF] Gast | Molecular phylogeny of symbiotic dinoflagellates from planktonic foraminifera and radiolaria[END_REF]. [START_REF] Gast | Molecular phylogeny of symbiotic dinoflagellates from planktonic foraminifera and radiolaria[END_REF] did not observe the morphology of the dinoflagellate symbionts of Sargasso Sea V. velella that they sequenced, but we predict that they would have plate tabulation [START_REF] Siano | Pelagodinium gen. nov. and P. beii comb. nov., a dinoflagellate symbiont of planktonic foraminifera[END_REF]Decelle et al. 2012b). A comparison of genetic sequences from morphologically characterized cultured V. velella symbionts from the Pacific Ocean, Sargasso Sea and Mediterranean Sea could be helpful in establishing the validity of historical descriptions of these symbionts and their relationship to B.

nutriculum.

Brandtodinium has been found (in this and previous studies) in association with diverse polycystine radiolarian hosts from the North and South Pacific Ocean, Sargasso Sea, and Mediterranean Sea. In light of the abundance of symbiotic polycystines in the world ocean, Brandtodinium likely plays a key ecological role in primary and secondary production at a global scale. Putting aside associations with parasitic alveolates (Gast 2006;[START_REF] Bråte | Radiolaria associated with large diversity of marine alveolates[END_REF]) that can be considered as a form of symbiosis, all Collodaria investigated so far harbor only Brandtodinium species as symbionts. At present, Brandtodinium is the only symbiont identified for Nassellaria, but information for this radiolarian group remains extremely scarce. Brandtodinium has now been found in association with numerous spumellarian hosts, but unlike the other polycystine lineages, other types of (non-dinoflagellate) microalgal and cyanobacterial symbionts have also been reported for this group [START_REF] Anderson | Radiolaria[END_REF][START_REF] Gast | Photosymbiotic associations in planktonic foraminifera and radiolaria[END_REF]Yuasa et al. 2005). With Brandtodinium also probably found in symbiosis with jellyfish, it is clear that Brandtodinium, like the suessialean dinoflagellates Pelagodinium and Symbiodinium, is a generalist symbiont. In this context it is interesting to note that the known genetic diversity (in terms of SSU and LSU rDNA sequences) of Brandtodinium and Pelagodinium, both of which form symbiotic relationships with planktonic hosts, is relatively low (2 clades described within each of these genera) compared to that of Symbiodinium (9 divergent clades and multiple sub-clades, [START_REF] Stat | Symbiont acquisition strategy drives host-symbiont associations in the southern Great Barrier Reef[END_REF][START_REF] Pochon | A new Symbiodinium clade (Dinophyceae) from soritid foraminifera in Hawai'i[END_REF]) that is predominately found in association with benthic host organisms. This apparent trend might be explained by the relatively low number of studies on symbiosis in the pelagic realm, but might also be real and reflect inherent differences between life and symbiotic processes in planktonic and benthic ecosystems [START_REF] Decelle | New perspectives on the functioning and evolution of photosymbiosis in plankton: Mutualism or parasitism?[END_REF]. Diagnosis: Plate tabulation: Po, X, 4', 3a, 7'', 5c, 4s, 5''', 1''''. Epitheca larger than hypotheca. Epitheca convex conical with well-pronounced apical horn. Hypotheca rounded. Wide and shallow cingulum located in the median portion of the cell, displaced by a small fraction of its own width. Sulcal area with 4 plates, one of which forms a wing-like flange over the median part of the sulcus. Single antapical plate.

Taxonomic appendix

Brandtodinium

Cells on average 13.1µm in length by 10.4µm in width. Symbiont of polycystine radiolarians.

Type locality: Bay of Villefranche sur Mer (France), Western Mediterranean Sea Authentic culture strain: RCC3387 in the Roscoff Culture Collection. 1 and2).

S

5

  Polycystine radiolarians (including the orders Spumellaria, Collodaria and

  further characterized the in hospite stage of E. chattonii and the latter authors proposed the reclassification of the polycystine symbionts (Z. nutricula) as E. nutricola (Brandt) Hollande et Carré (E. nutricula under ICBN Art. 73), despite the fact that Hovasse (1924) had in fact previously recombined E. chattonii as Z. chattonii (Hovasse) Hovasse. Banaszak et al. (1993) isolated a culture of the symbiont of V. velella from the Pacific, which they considered slightly different from E. chattonii (larger cell size and presence of trichocysts in hospite and in culture). Based on SEM observations of the morphology and arrangement of thecal plates in the motile stage, Banaszak et al. (1993) classified their organism in the genus Scrippsiella as a new species, S. velellae Banaszak, Iglesias-Prieto et Trench (a name later validated by Trench 2000). These authors also transferred E. chattonii and E. nutricula to Scrippsiella as S. chattonii (Hovasse) Banaszak, Iglesias-Prieto et Trench and S.

  numbers: XXXX KF557491 to KF557545 to XXXX) were aligned with other LSU and SSU rDNA sequences from GenBank (release 194.0, February 2013) attributed to Scrippsiella and related Peridiniales genera, as well as representatives of the Suessiales as an outgroup. Alignments were generated using MUSCLE implemented in Seaview v.4.0 (Gouy et al. 2010) with subsequent manual verification. The LSU contained 48 sequences (675 unambiguously aligned positions) and the SSU rDNA data set contained 57 sequences (652 unambiguously aligned positions).

  tree was visualized and edited in Fig Tree v 1.3.1 (Rambaut 2010). Bayesian analyses were conducted using Mr Bayes v.3.2.1 (Huelsenbeck and Ronquist 2001) using the same model of evolution. For each gene marker, two Markov Chain Monte Carlo (MCMC) chains were run for 1 million generations, sampling every 500 generations (diagnostic frequency = 5000). The standard deviation of split frequencies between the 2 runs was <0.01 in both LSU and SSU rDNA analyses. TFor both ML and Bayesian analyses, the trees wasere visualized and edited in Fig Tree v 1.3.1 (Rambaut 2010). The In the trees presented herein the posterior probabilities (PP) associated to each node in the Bayesian topologies were are reported on the ML topologies.

  thecal plate pattern of motile cells was identical for several different strains observed. The following descriptions and illustrations are based on observations of strain VFR1-1RCC3387.

  , 3A-D). The pore plate (Po) is circular and surrounded by a high collar and is connected to the first apical plate by a long well-defined rectangular canal plate (X)(Figs 2A, 3A, 3C).Three intercalary plates are interposed on the dorsal side of the cell between the apical series and the second epithecal (precingular) series (Figs2C-D, 3B-C). The first intercalary plate (1a) is five-sided and borders only one of the apical plates (2'), whereas the second and third intercalary plates (2a and 3a) are six-sided and both border two apical plates (Figs2C-D, 3C). The cingulum is located in the median portion of the cell and descends slightly, displaced by approximately one third of its

  morphological and physiological differentiation between them. Within the host cells, the symbionts are typically coccoid without flagella, and the cingulum and sulcus are no longer apparent

  here possesses a single antapical plate(Table 2, Figs 2E and 3D). The presence of a single antapical plate is rare in the order Peridiniales, occurring notably in a group of heterotrophic genera (Podolampas Stein, Blepharocysta Ehrenberg, and Lissodinium Matzenauer) characterized by the absence of both a cingulum and a depressed sulcus[START_REF] Gómez | Molecular phylogeny of the dinoflagellates Podolampas and Blepharocysta (Peridiniales, Dinophyceae)[END_REF]) and a group of heterotrophic taxa (Diplopsalis Bergh, Preperidinium Mangin, Boreadinium Dodge et Hermes) characterized by having large lenticular-shaped cells. The radiolarian symbionts are clearly morphologically and ecologically distinct from these other peridinialeans with that have a single antapical plate.

  phylogenies(Figs 4 and 5), the analyzed polycystine symbionts (including several cultures isolated from Collozoum colonies) formed a well-supported clade within the distinct from Scrippsiella and related genera and distant from other dinoflagellate taxa known to form symbiotic relationships such as the suessialeans Symbiodinium and Pelagodinium.

  characterized by the production of calcified resting cysts, a feature that we have not observed in Brandtodinium. As discussed above, Brandtodinium also has certain morphological similarities with members of other groups such as the

  genera (Durinskia Carty et Cox, Galeidinium Tamura et Horiguchi, Kryptoperidinium Lindemann, and some species currently assigned to Peridiniopsis Lemmermann or Peridinium Ehrenberg) on the basis of morphological criteria, notably because dinotom genera all have 2 antapical plates whereas B. nutriculum possesses a single antapical plate, but also because the characteristic highly visible eyespot of dinotoms is absent in B. nutriculum.

  . (1993) described the dinoflagellate symbiont of the jellyfish Velella velella from the Pacific as Scrippsiella velellae and also (albeit invalidly) transferred Endodinium (=Zooxanthella) chattonii, the symbiont of Mediterranean V. velella, to Scrippsiella, as S. chattonii. These authors gave the thecal plate formula for S.

  description of Brandtodinium. If this were the case, it would mean that V. velella is capable of forming symbiotic associations with different dinoflagellate genera (Brandtodinium and Scrippsiella (or Ensiculifera)), possibly with a biogeographical pattern (Brandtodinium in the Atlantic and possibly Mediterranean, Scrippsiella (or Ensiculifera) in the Pacific). The capacity of hosts to form associations with different symbionts has already been observed for other pelagic organisms

  Probert et Siano gen. nov. Diagnosis: Photosynthetic dinoflagellate. Motile cells covered by 6 series of thecal plates: 3 in the epitheca, 2 in the hypotheca (including single antapical plate), and 1 in the cingulum. One transverse and one longitudinal flagellum. Large nucleus located in central part of cell. One or two peripheral chloroplasts, golden-yellow in color. One or two large circular pyrenoids. Type species: Brandtodinium nutriculum (Brandt) Probert et Siano comb. nov. Etymology: the genus name for this dinoflagellate (= dinos) derives from Karl Brandt who first described Zooxanthella in 1882. Brandtodinium nutriculum (Brandt) Probert et Siano comb. nov. Basionym: Zooxanthella nutricula Brandt in Brandt (1882Endodinium nutricula (Brandt) Hollande et Carré in Holland and Carré (1974); Scrippsiella nutricula (Brandt) Banaszak, Iglesias-Prieto et Trench in Banaszak et al. (1993). Neotype : Fig. 2 in this publication.

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. SEM micrographs of Brandtodinium nutriculum gen. nov., comb. nov.. (enumeration of plates follows the Kofoidian tabulation system). A. Ventral view of a cell (flagella lost during fixation). B. Detail of the sulcal region. C. Dorsal view. D. Apical view. E. Antapical view. Scale bars = 2µm.

Figure 3 .

 3 Figure 3. Schematic representation of plate patterns of Brandtodinium nutriculum gen. nov., comb. nov. (enumeration of plates follows the Kofoidian tabulation system). A. Ventral view (generalized). B. Dorsal view (generalized). C. Apical view (generalized). D. Antapical view (generalized).

Figure 4 .

 4 Figure 4. SSU rDNA phylogenetic tree inferred by Maximum Likelihood (ML) analysis. 650 652 unambiguously aligned positions were considered from an alignment of 57 sequences, including Brandtodinium gen. nov.. Sequences obtained in this study are indicated in bold (followed by the type of host from which the sequence was obtained and the number of holobiont specimens or culture strains in parentheses). The tree was rooted with Suessiales (Symbiodinium spp. and Pelagodinium béii) as the outgroup. Branch lengths are drawn to scale, with the scale bar indicating the number of nucleotide substitutions per site. Numbers on branches

Figure 5 .

 5 Figure 5. LSU rDNA phylogenetic tree inferred by Maximum Likelihood (ML) analysis. 675 unambiguously aligned positions were considered from an alignment of 48 sequences, including Brandtodinium gen. nov.. Sequences obtained in this study are indicated in bold (followed by the type of host from which the sequence was obtained and the number of holobiont specimens or culture strains in parentheses). The tree was rooted with Suessiales (Symbiodinium spp. and Pelagodinium beii) as the outgroup. Branch lengths are drawn to scale, with the scale bar indicating the number of nucleotide substitutions per site. Numbers on branches are statistical

  Figure 1.

  Figure 5.

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .

 12345 Figure 1. Light micrographs of Brandtodinium nutriculum gen. nov., comb. nov.. (arrow indicates large pyrenoid). A, B. Ventral view of the cell showing the large nucleus in the central portion of the cell. C. Lateral (slightly antapical) view of the cell. D. Dorsal view of the cell. Scale bars = 5µm. 44x49mm (300 x 300 DPI)

chattoni

  , who initially described the in hospite symbionts of Mediterranean V. velella as Endodinium Hovasse (E. chattonii under ICBN Art. 73).

	S u
	b m
	i t t
	e d
	M
	a n u
	s c r i
	p
	t
	6

25

  22 the CNRS, the Muséum National d'Histoire Naturelle, the INRA and the CEA (Centre National de Séquençage), the EU FP7 projects ASSEMBLE (grant agreement 227799) and MACUMBA, and the French Investissements d'Avenir project EMBRC-Gast, R.J. 2006. Molecular phylogeny of a potentially parasitic dinoflagellate isolated from the solitary radiolarian, Thalassicola nucleate. J. Eukaryot. Microbiol. 53: 43-29 dinoflagellate symbionts of marine invertebrates. J. Phycol. 23:469-81. Yuasa, T., Takahashi, O., Honda, D. & Mayama, S. 2005. Phylogenetic analyses of the polycystine Radiolaria based on the 18s rDNA sequences of the Spumellarida and the Nassellarida. European J. Protistology 41:287-98.

	France.
	u S u S u S u S u S u S u S u S u
	b m b m b m b m b m b m b m b m b m
	i t t i t t i t t i t t i t t i t t i t t i t t i t t
	e d e d e d e d e d e d e d e d e d
	M M M M M M M M M
	a n u a n u a n u a n u a n u a n u a n u a n u a n u
	s c r i s c r i s c r i s c r i s c r i s c r i s c r i s c r i s c r i
	p p p p p p p p p
	t t t t t t t t t
	30

Table 1 .

 1 List of specimens used to obtain symbiont sequences (images of host cells 1 are shown in Supplementary Figures

Table 2 .

 2 Kofoidian plate tabulation of Brandtodinium and related genera

		(colony)	23°42.289 S, 131°12.744 W			xxxxx	xxx
	PAC19	Collodaria	South Pacific	n.a.		KF557516xx	KF557542xxxx
		(colony)	23°42.289 S, 131°12.744 W			xxxxx	xxx
	PAC21	Collodaria	South Pacific	n.a.		KF557517xx	KF557543xxxx
		(colony)	23°42.289 S, 131°12.744 W			xxxxx	xxx
	PAC22	Collodaria	South Pacific	n.a.		KF557518xx	KF557544xxxx
	PAC24 PAC26 PAC27	(colony) Collodaria S (colony) u 23°42.289 S, 131°12.744 W South Pacific 23°42.289 S, 131°12.744 W Collodaria (colony) South Pacific 23°42.289 S, 131°12.744 W b (colony) 23°42.289 S, 131°12.744 W m Collodaria South Pacific	n.a. n.a. n.a.		xxxxx KF557519xx xxxxx KF557520xx xxxxx xxxxx KF557521xx	xxx n.a. n.a. n.a.
	SES47 SES19 SES28	Collodaria (colony) Spumellaria Nassellaria	Sesoko, Japan i t 26°37'20 N, 127°52'15 E t n.a. Sesoko, Japan 26°37'20 N, 127°52'15 E n.a. e d Sesoko, Japan n.a.	KF557502xx xxxxx KF557501xx xxxxx n.a.	KF557546xxxx xxx n.a. KF557545xxxx
			26°37'20 N, 127°52'15 E				xxx
	Vil 210	Spumellaria?	43°41'10 N, 7°18'50 E Villefranche-sur-Mer, France	n.a.	xxxxx M KF557522xx	n.a.
	Vil 217 Vil 219	Spumellaria Spumellaria	Villefranche-sur-Mer, France 43°41'10 N, 7°18'50 E 43°41'10 N, 7°18'50 E Villefranche-sur-Mer, France	n.a. n.a.		KF557523xx a xxxxx u n.a. xxxxx n KF557524xx n.a.
	Vil 231 Culture strains SES46	Spumellaria Collodaria (Collozoum	Villefranche-sur-Mer, France 43°41'10 N, 7°18'50 E Sesoko, Japan 26°37'20 N, 127°52'15 E	n.a. RCC3378	KF557525Xx xxxxx KF557500xx xxxxx	n.a. s c r i KF557526xxxx p xxx
		colony)		RCC3379	KF557499	n.a.	t
	SES46B	Collodaria	Sesoko, Japan	RCC3379	xxxxxxx	n.a.
		(Collozoum	26°37'20 N, 127°52'15 E
		colony)
	VFPO14-8	Collodaria	Villefranche-sur-Mer, France	RCC3380	KF557494xx	KF557530xxxx
		(Collozoum	43°41'10 N, 7°18'50 E			xxxxx	xxx
			31

Acknowledgements

We thank staff (in particular John Dolan and Sophie Marro) of the Laboratoire d'Océanographie de Villefranche-sur-Mer (UPMC-CNRS) and of the Sesoko Marine Station (University of Ryukyus) as well as the Tara Oceans Expedition (doi:10.1371/journal.pbio.1001177) for providing sampling facilities. We thank Nicolas Gayet of the Laboratoire Environnement Profond (PDG-REM-EEP-LEP) of Ifremer Centre de Brest for his technical support for electron microscopy analyses and Julien Quéré of the Dyneco/Pelagos laboratory (PDG-ODE-DYNECO-PELAGOS) for cultivating stains at Ifremer. This research was supported by a JST-CNRS exchange program to F.N and N.S., the "Bibliothèque du Vivant" network funded by