Stéphane Sartoretto
email: stephane.sartoretto@ifremer.fr

Thomas Schohn
email: thomas.sch@neuf.fr

Carlo Nike Bianchi
email: nbianchi@dipteris.unige.it

Carla Morri
email: morric@dipteris.unige.it

Joaquim Garrabou
email: garrabou@icm.csic.es

Enric Ballesteros

Sandrine Ruitton
email: sandrine.ruitton@mio.osupytheas.fr

Marc Verlaque
email: marc.verlaque@mio.osupytheas.fr

Boris Daniel
email: boris.daniel@afbiodiversite.fr

Eric Charbonnel
email: charbonnel.eric@parcmarincotebleue.fr

Sylvain Blouet
email: sylvain.blouet@ville-agde.fr

Romain David
email: romain.david@imbe.fr

Jean-Pierre Féral
email: jean-pierre.feral@imbe.fr

Giulia Gatti
email: gatti.giulia25@gmail.com

An integrated method to evaluate and monitor the conservation state of coralligenous habitats: The INDEX-COR approach

Keywords:

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The endemic biogenic habitat known as "coralligenous" represents an array of species diversity in the Mediterranean Sea [START_REF] Boudouresque | Marine biodiversity in the Mediterranean: status of species, populations and communities[END_REF]. Despite the lack of knowledge of the diversity of certain taxonomic groups, the first preliminary global estimation gave a total number of 1666 species [START_REF] Ballesteros | Mediterranean coralligenous assemblages: a synthesis of present knowledge[END_REF]. Such extraordinary biodiversity is mainly due to the considerable structural complexity of the coralligenous bioconstruction, the most important in the Mediterranean Sea [START_REF] Bianchi | Bioconstruction in marine ecosystems and Italian marine biology[END_REF], and the high environmental heterogeneity at small scales, which allows for the coexistence of different habitats [START_REF] Kipson | Rapid biodiversity assessment and monitoring method for highly diverse benthic communities: a case study of Mediterranean coralligenous outcrops[END_REF]. The presence of a canopy of large sessile invertebrates (e.g., gorgonians and erect sponges) further increases this complexity. Coralligenous habitats harbour several species of interest for artisanal fishing, such as crustaceans, fishes and the emblematic red coral (Corallium rubrum). Finally, they constitute a remarkable and typical Mediterranean underwater seascape, highly attractive for recreational activities (e.g., scuba-diving), but also greatly affected by anthropogenic pressures locally [START_REF] Ballesteros | Mediterranean coralligenous assemblages: a synthesis of present knowledge[END_REF].

Human activities may cause physical impacts and inputs of organic matter, sediment and pollutants. Physical impacts (anchoring, fishing and diving) damage both the large sessile invertebrates of the canopy and the associated fragile calcareous organisms, such as erect bryozoans [START_REF] Garrabou | The impact of diving on rocky sublittoral communities: a case study of a bryozoan population[END_REF][START_REF] Linares | Assessing the impact of diving in coralligenous communities: the usefulness of demographic studies of red gorgonian populations[END_REF]. Organic matter inputs may lead to an increase of bioeroders such as clionaid sponges [START_REF] Ballesteros | Mediterranean coralligenous assemblages: a synthesis of present knowledge[END_REF], while sediment deposition can modify the composition of assemblages and associated biodiversity [START_REF] Balata | Variability of Mediterranean coralligenous assemblages subject to local variation in sediment deposition[END_REF][START_REF] Balata | Increase of sedimentation in a subtidal system: effects on the structure and diversity of macroalgal assemblages[END_REF]. Finally, the presence of pollutants in the water column has been shown to cause a decrease in biodiversity [START_REF] Hong | Impact of the pollution on the benthic community[END_REF][START_REF] Ballesteros | Mediterranean coralligenous assemblages: a synthesis of present knowledge[END_REF]. Among the environmental factors affecting coralligenous habitats, extremely high temperature events have became more frequent during the last decades [START_REF] Garrabou | Mass mortality in northwestern Mediterranean rocky benthic communities: effects of the 2003 heat wave[END_REF], and have led to mass mortalities of long-living and structuring species [START_REF] Cerrano | A catastrophic mass-mortality episode of gorgonians and other organisms in the Ligurian Sea (North-western Mediterranean), summer 1999[END_REF][START_REF] Pérez | Mortalité massive d'invertébrés marins: un événement sans précédent en Méditerranée nord-occidentale[END_REF][START_REF] Garrabou | Mass mortality in northwestern Mediterranean rocky benthic communities: effects of the 2003 heat wave[END_REF][START_REF] Lejeusne | Climate change effects on a miniature ocean: the highly diverse, highly impacted Mediterranean Sea[END_REF]. The acidification of seawater caused by the increased concentration of atmospheric CO 2 has been shown to reduce the growth and calcification rates of calcareous algae involved in the bioconstruction [START_REF] Martin | Response of Mediterranean coralline algae to ocean acidification and elevated temperature[END_REF][START_REF] Lacoue-Labarthe | Impacts of ocean acidification in a warming Mediterranean Sea: an overview[END_REF], and can finally lead to the total disappearance of coralligenous reefs [START_REF] Linares | Persistent natural acidification drives major distribution shifts in marine benthic ecosystems[END_REF].

Paradoxically, despite their ecological and economical importance, European legislation never refers directly to coralligenous habitats. Annex I of the Habitat Directive (HD, 92/43/EEC) takes into account a generic "Reefs" category in the list of habitats of community interest. Only recently, coralligenous reefs have been included by the European Union in a Red List of marine habitats [START_REF] Gubbay | European Red List of Habitats. Part 1. Marine Habitats[END_REF]. The Water Framework Directive (WFD, 2000/60/EC) mentions the assessment of the composition and abundance of benthic flora and fauna only as a means to evaluate the quality of water bodies. A list of Biological Quality Elements (BQE) is defined by the WFD (phytoplankton, benthos, etc.), but only the best known benthic habitats have been considered to date: soft substrates [START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF][START_REF] Simboura | Benthic indicators to use in ecological quality classification of Mediterranean soft bottom marine ecosystems, including a new biotic index[END_REF][START_REF] Rosenberg | Marine quality assessment by use of benthic species-abundance distributions: a proposed new protocol within the European Union Water Framework Directive[END_REF][START_REF] Muxika | Using historical data, expert judgment and multivariate analysis in assessing reference conditions and benthic ecological status, according to the European Water Framework Directive[END_REF], upper infralittoral rocky reefs [START_REF] Orfanidis | Ecological evaluation of transitional and coastal waters: a marine benthic macrophytes-based model[END_REF][START_REF] Ballesteros | A new methodology based on littoral community cartography dominated by macroalgae for the implementation of the European Water Framework Directive[END_REF] and Posidonia oceanica seagrass meadows [START_REF] Romero | A multivariate index based on the seagrass Posidonia oceanica (POMI) to assess ecological status of coastal waters under the water framework directive (WFD)[END_REF][START_REF] Gobert | Assessment of the ecological status of Mediterranean French coastal waters as required by the water framework directive using Posidonia oceanica Rapid Easy Index: PREI[END_REF][START_REF] Lopez Y Royo | Application of an angiosperm-based classification system (BiPo) to Mediterranean coastal waters: using spatial analysis and data on metal contamination of plants in identifying sources of pressure[END_REF]. Finally, the Marine Strategy Framework Directive (MSFD, 2008/56/EC) introduces the concept of "seafloor integrity" as an indicator of the Ecological Status (ES) of marine environments, and studies addressing all benthic habitats, with especial attention given to biogenic substrates, are strongly encouraged. This has allowed pinpointing gaps in knowledge on the geographical and bathymetrical distribution, taxonomy, functioning and dynamics of coralligenous habitats (UNEP-MAP-RAC/SPA, 2008), thus underlining the difficulties in developing methods and indices to measure their ES.

To assess ES of a habitat, [START_REF] Borja | Assessing the environmental quality status in estuarine and coastal systems: comparing methodologies and indices[END_REF] distinguished three steps: (i) the definition of the reference conditions or of the environmental targets to define good status, (ii) the selection of the metrics and the definition of an index formula including these metrics, and (iii) the classification of the ecological status of the habitat through the application of the index. Concerning coralligenous habitats, the paucity of knowledge [START_REF] Gubbay | European Red List of Habitats. Part 1. Marine Habitats[END_REF], coupled with their high heterogeneity both at small (e.g. 1-100 m) and large (0.1-100 km) scales and with the diffused problem of shifting baselines [START_REF] Hobday | Sliding baselines and shuffling species: implications of climate change for marine conservation[END_REF][START_REF] Al-Abdulrazzak | Gaining perspective on what we've lost: the reliability of encoded anecdotes in historical ecology[END_REF]Gatti et al., 2015b), hampers defining general reference conditions and setting targets. In this context, a possible solution is to provide a large-scale current baseline as a reference for future evaluations of habitat state [START_REF] Sala | The structure of Mediterranean rocky reef ecosystems across environmental and human gradients, and conservation implications[END_REF] for infralittoral rocky reefs.

In recent years, different methods and indices focused on coralligenous habitats have been proposed [START_REF] Bavestrello | New perspectives in the study of Mediterranean coralligenous assemblages[END_REF]. The Ecological Status of Coralligenous Assemblages index (ESCA index) [START_REF] Cecchi | A new method for the assessment of the ecological status of coralligenous assemblages[END_REF][START_REF] Cecchi | Development of a new biotic index for ecological status assessment of Italian coastal waters based on coralligenous macroalgal assemblages[END_REF] and the Coralligenous Assemblages Index (CAI) [START_REF] Deter | A preliminary study toward an index based on coralligenous assemblages for the ecological status assessment of Mediterranean French coastal waters[END_REF] both adopt the state of coralligenous communities as an ES indicator of coastal waters according to the WFD. They are based on data collected by photographic sampling and subsequent image analysis using free software (CpCe-Coralligenous Assemblages Version, Image J). The Rapid Visual Assessment (RVA method) and the associated COARSE index [START_REF] Gatti | Seafloor integrity down the harbor waterfront: the coralligenous shoals off Vado Ligure (NW Mediterranean)[END_REF](Gatti et al., , 2015a) ) aim at assessing the quality of coralligenous reefs as an indicator of seafloor integrity according to the MSFD; this method is based on a seascape approach through direct SCUBA diving surveys. [START_REF] Ruitton | An ecosystem-based approach to assess the status of the Mediterranean coralligenous habitat[END_REF] developed an ecosystem approach, based on the identification of the major components at different trophic levels, whereas the OCI index [START_REF] Paoli | Capturing ecological complexity: OCI, a novel combination of ecological indices as applied to benthic marine habitats[END_REF] combines structural and functional measures to evaluate the overall ecological complexity of coralligenous ecosystems.

In this paper, a non-destructive approach for the evaluation of the ecological status of coralligenous habitats is proposed, with special consideration given to the needs of stakeholders (e.g., MPAs, Marine Protected Areas). The approach is based on the integration of three metrics: (i) the sensitivity of species to fine sedimentation and organic matter input; (ii) the taxonomic richness; and (iii) the structural complexity of the assemblages. The first objective of this paper is to describe and illustrate the method. The second is to propose a new index to assess the ecological status of coralligenous habitats, the INDEX-COR. Finally, the third objective is to test its efficiency according to different levels of anthropogenic pressure affecting the marine environment.

Materials and methods

Study area

The study area is located along the French continental Mediterranean coast. The western zone, from the Gulf of Fos to Marseilles, is characterized by high turbidity and considerable sediment inputs due to the estuary of the Rhône River: thus coralligenous reefs can be found starting from relatively shallow depths (12-15 m). On the contrary, the eastern zone, from Toulon to Saint-Raphaël, is generally characterized by clear waters which allow coralligenous habitats to develop down to 100-110 m depth.

From the geological viewpoint, the study area is mainly characterized by rocky coasts composed of limestone in the Marseille area, conglomerates at La Ciotat, and phyllites from Toulon to Saint-Raphaël. The coastal waters (between the surface and the continental shelf) are dominated by the northern current [START_REF] Millot | Circulation in the Mediterranean Sea[END_REF], flowing westwards; however, coastal circulation is also constrained by two dominant winds, the north-western (upwelling favourable) and the south-eastern (downwelling favourable) [START_REF] Pairaud | Hydrology and circulation in a coastal area off Marseille: validation of a nested 3D model with observations[END_REF]. Locally (e.g., around Marseille), upwelling can lower surface water temperature by > 5 °C [START_REF] Millot | The Gulf of Lions' hydrodynamics[END_REF].

The area is characterized by the presence of two densely populated cities, Marseille (1.6 million inhabitants) and Toulon (560,000 inhabitants). Major sources of human pressure along the coast are the sewage outfalls associated with urban zones. In all, six large waste water treatment plants discharge their effluents in the study area. Outfalls are on the surface, such as at Calanque of Cortiou (Marseilles), Cap Sicié (Toulon W), Figuerolle (La Ciotat), while others are located between 35 m and 45 m depth: Sainte Marguerite (Toulon east), Cavalaire and Bonne-Terrasse (Saint-Tropez south). These sewage outfalls directly affect benthic communities of both soft and hard substrates. Human activities, such as SCUBA diving and yachting, may also be a source of disturbance, especially during summer. Other sources of impacts are professional and recreational fisheries, industry and the anchoring of tankers (mainly restricted to the Gulf of Fos).

Besides these pressures, marine protected areas are present in the study area (e.g., the Côte Bleue Marine Park, the National Park of Calanques and the National Park of Port-Cros). Human activities in these areas are forbidden (only in marine reserves) and/or regulated.

Field work

In all, 53 stations undergoing different degrees of human pressure were sampled from December to March (2012March ( -2013March ( and 2013March ( -2014)), in order to reduce seasonal variability (Fig. 1). In each station, two transects (15 m long × 1 m large) indicated by graduated rubber bands were randomly placed at the same depth, orientation and type of dominant facies (e.g., gorgonians and macroalgae). Data were collected by two SCUBA divers: a photographer and an observer. The first diver shot at least 30 photographs (hereafter called "photoquadrats"), 15 along each transect, using a 60 cm × 40 cm frame. The number and size of photoquadrats were selected according to the recommendations made by previous studies [START_REF] Cecchi | A new method for the assessment of the ecological status of coralligenous assemblages[END_REF][START_REF] Kipson | Rapid biodiversity assessment and monitoring method for highly diverse benthic communities: a case study of Mediterranean coralligenous outcrops[END_REF][START_REF] Deter | A preliminary study toward an index based on coralligenous assemblages for the ecological status assessment of Mediterranean French coastal waters[END_REF][START_REF] Cecchi | Development of a new biotic index for ecological status assessment of Italian coastal waters based on coralligenous macroalgal assemblages[END_REF]. A digital camera (Sony Alpha Nex-5 with a 16-3.5 mm zoom-lens) equipped with housing and dome was fixed over the quadrat frame using a metal structure; 4 SOLA lamps were installed at the top of each vertical side of the structure. The second diver focused on taking notes along each transect on: (i) general parameters, i.e. depth, orientation, slope (vertical, inclined and subhorizontal), presence of marine litter, dominant facies; (ii) conspicuous benthic sessile and mobile (echinoderms, crustacean decapods and nudibranchs) species recognizable visually without sampling; (iii) the percent cover of large erect species (gorgonians and sponges) (cf. Section 2.3.3). Percent cover was estimated considering five classes: 0 (absent); 1 (isolated colonies); 2 (10% < cover ≤ 25%); 3 (25% < cover ≤ 50%); 4 (50% < cover ≤ 75%); 5 (cover > 75%). The percentage of necrotic gorgonian colonies was also considered when > 10% of their surface was affected. According to the number of necrotic colonies, the values of cover classes were adjusted. The observation of > 50% of affected colonies along a transect thus implied a downgrade (e.g., from 5 to 4).

Data management

Characterization of stations and assessment of pressure level

A comprehensive definition of each sampling station, based on geomorphological, abiotic and bionomic features, is summarized in Table 1. Facies and associations characterizing the stations were identified according to the conspicuous species dominating the assemblages.

In the absence of specific criteria for quantifying the impact of human pressures affecting coralligenous habitats, a Level of Pressure (LP) was estimated for each station according to an 'expert judgment' based on distance from potential sources and field observations. Four main pressures were taken into account: (i) organic matter and sediment inputs, (ii) diving/anchoring, (iii) fishing, and (iv) litter [START_REF] Ballesteros | Mediterranean coralligenous assemblages: a synthesis of present knowledge[END_REF]. The intensity of the pressures was assessed considering 4 levels: 0 (no pressure), 1 (low), 2 (moderate) and 3 (high). Then, the LP was calculated for each station as the sum of the values obtained for the four pressures. The LP theoretically varies from 0 ('pristine') to 12 (highly impacted).

Image analysis

Photoquadrats were analyzed with the free software photoQuad [START_REF] Trygonis | photoQuad: a dedicated seabed image processing software, and a comparative error analysis of four photoquadrat methods[END_REF], using the uniform point count technique. The number of points necessary to obtain a good estimation of taxonomic richness was tested beforehand. Rarefaction curves based on the asymptotic model of Michaelis-Menten [START_REF] Keating | Estimating species richness: the Michaelis-Menten model revisited[END_REF] were used to assess the variation of taxonomic richness according to the number of points. Finally, 100 uniform points were chosen for the analyses of the photoquadrats. A library of species (or higher taxa) (Appendix A) and abiotic elements (e.g., sediment, bare rock, and black shadow) was created and uploaded in the software, then each point was directly labelled with the name of the element beneath it. Therefore the percent relative abundance of each taxon/abiotic element was obtained for each image.

Selection and definition of metrics

To assess the conservation state of coralligenous assemblages, it is necessary to consider the combined effects of human activities and environmental changes, as already suggested for Paramuricea clavata populations [START_REF] Linares | Assessing the impact of diving in coralligenous communities: the usefulness of demographic studies of red gorgonian populations[END_REF].

In this study, three metrics were selected to define a synthetic index:

(i) Taxa Sensitivity (TS), considering only sensitivity to organic matter and sediment input; (ii) Observable Taxonomic Richness (OTR), reflecting the biodiversity of coralligenous assemblages; (iii) Structural Complexity (SC), considering the stratified structure of coralligenous habitats.

TS is inspired by the existing indexes for characterizing soft substrate benthic communities, particularly the AMBI and related indexes [START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF][START_REF] Pinedo | A critical analysis on the response of macroinvertebrate communities along disturbance gradients: description of MEDOCC (MEDiterranean OCCidental) index[END_REF]. The AMBI index considers the succession of 5 groups of species showing different levels of sensitivity to the input of organic matter [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF][START_REF] Glémarec | Perturbations apportées à la macrofaune benthique de la baie de Concarneau par les effluents urbains et portuaires[END_REF]. Compared to soft substrate, knowledge on coralligenous assemblages is too scarce to allow identifying a similar succession [START_REF] Montefalcone | The two facets of species sensitivity: stress and disturbance on coralligenous assemblages in space and time[END_REF]. To overcome this problem, we selected a subset of 10 sampling stations where the pressure due to organic matter and sediment input was known, 3.

Table 1 Characterization of geomorphology, mesology and level of pressure for each station (identified by a numeric code, as in Fig. 1). Morphotype: O -outcrop, LIP -low inclination platform, SC -steep cliff. Level of pressure: O/S -organic matter/sediment inputs, D/A -diving/anchoring, F -fishing activities, ML -marine litter, LP -level of pressure; 0: no impact 1: low impact, 2: moderate impact, 3: high impact. measured by sediment traps, or assessed by models [START_REF] Sartoretto | Vitesse de croissance et bioérosion des concrétionnements "coralligènes" de Méditerranée nord-occidentale[END_REF][START_REF] Arfi | Impact du grand émissaire de Marseille et de l'Huveaune détournée sur l'environnement marin de Cortiou -Etude bibliographique raisonnée 1960-2000[END_REF]. Then, a Delta-Gamma model [START_REF] Stefansson | Analysis of groundfish survey abundance data: combining the GLM and delta approaches[END_REF] was applied to the relative abundance of each taxon (obtained from image analyses) according to the pressure of the sampling stations. This statistical approach allowed distinguishing 56 taxa distributed in four groups of sensitivity (Table 2) [START_REF] Pinedo | A critical analysis on the response of macroinvertebrate communities along disturbance gradients: description of MEDOCC (MEDiterranean OCCidental) index[END_REF]:

Code

• Group I (GI): taxa indifferent to organic matter and sediment input;

• Group II (GII): opportunistic taxa;

• Group III (GIII): tolerant taxa; • Group IV (GIV): sensitive taxa.

The computation of TS is based on a formula inspired from the AMBI index [START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF]: TS = (0.25 × % Group I + 0.5 × % Group II + 1 × % Group III + 2 × % Group IV)where % Group is obtained, for each station, by adding the percent relative cover of the species belonging to each group.

The identification of certain benthic species from images is often difficult or even impossible, especially for those taxonomic groups for which microscopic morphological studies are required. Consequently, the second metrics -OTRrefers to the total number of those conspicuous taxa that were recognizable visually on photoquadrats and in situ. Sessile and mobile macrobenthic organisms (echinoderms, nudibranchs, crustaceans) having high patrimonial value or a particular ecological role were also considered.

Finally, the third metrics -SCis based on the assumption that the impact of human activities may reduce the structural complexity of coralligenous habitats, with the progressive reduction of the long-living erect (e.g., gorgonians, sponges) and fragile (e.g., bryozoans, foliaceous Corallinaceae) species [START_REF] Zabala | Recreation in Mediterranean marine parks: limits and perspectives[END_REF][START_REF] Gatti | Seafloor integrity down the harbor waterfront: the coralligenous shoals off Vado Ligure (NW Mediterranean)[END_REF]. This assumption was also tested on our dataset (see Section 3.1). Following the approach proposed by [START_REF] Gatti | Seafloor integrity down the harbor waterfront: the coralligenous shoals off Vado Ligure (NW Mediterranean)[END_REF], three layers were considered to characterize the structure of coralligenous assemblages: (i) a basal layer, composed of encrusting organisms and those with limited vertical growth, (< 5 cm); (ii) an intermediate layer, characterized by sessile organisms with moderate vertical growth (5 cm to 15 cm); and (iii) an upper layer, constituted by sessile macrobenthic species with considerable vertical growth (> 15 cm). The scores for basal and intermediate layers were defined as the total percent abundance of the taxa belonging to them, estimated from the photoquadrats. The score of the upper layer was defined as the percent cover of gorgonians and large sponges (e.g., Axinella polypoides) observed in situ, along the transects (see Section 2.2).

To summarize the layer scores in a structural complexity (SC) value, a Principal Component Analysis (PCA) was performed with the species abundances of the layers of all the stations; axes 1 and 2 explained 47.4% and 33.6% of the total variance, respectively. The formulas for calculating the coordinates of each layer along the two axes were defined by simplifying the results of the PCA (see appendix B for details) (Table 3). Then, the coordinates of each station (C1 station and C2 station ) were calculated as the sum of the coordinates of the three layers along the axes:

C(1, 2) = C(1, 2) + C(1, 2) + C(1, 2) station basal intermediate upper
The coordinates of a hypothetical reference station (RS) with scores equal to zero for each layer were calculated according to the formulas shown in Table 3 (C1 RS = 2.108 and C2 RS = 1.980). The SC value was Table 2 List of the species and higher taxa ordered alphabetically within their sensitivity group. Group I (GI): taxa indifferent to organic matter and sediment input; Group II (GII): opportunistic taxa; Group III (GIII): tolerant taxa; Group IV (GIV): sensitive taxa.

Sensitivity group

Taxon Sensitivity group Taxon

Definition of a synthetic index of conservation state

After verifying the non-redundancy of the three abovementioned metrics (TS, OTR and SC) (Pearson's r < 0.48 with a multiple correlation matrix), the INDEX-COR (IC) formula was defined using a linear model based on these metrics. The results (multiple r 2 = 0.56, p < 0.0001) allowed proposing the following formula: IC = 0.62 × TS + 0.6 × TR + 1.7 × SC Finally, the correlation between IC and LP was tested using Kendall's tau coefficient, which is more robust for non-normal data, outliers and tie observations than Pearson's r and Spearman's rho coefficients.

All statistical analyses were performed using R software (R Development Core Team, 2011).

Results

Characterization of assemblages and pressure levels

The coralligenous communities sampled in this study were mainly characterized by (i) facies with gorgonians (Paramuricea clavata and Eunicella cavolini); (ii) facies with sciaphilic to hemisciaphilic green algae, dominated by Codium spp., Flabellia petiolata and Halimeda tuna; (iii) facies with sciaphilic algae dominated by Phaeophyceae and Rhodophyceae; and (iv) "facies of impoverishment", showing highly degraded assemblages. The facies with gorgonians were mainly found on steep cliffs, which represented the most diffused geomorphological type among the sampling stations. The other facies were mainly associated with gently inclined platforms and coralligenous outcrops. The sampling stations were distributed between 15 m and 51 m depth, because of the extreme variability of the environmental conditions characterizing the study area (see Section 2.1).

In all, 23 stations were characterized as slightly impacted (LP = 2 to 5). They were mainly located in the National Park of Calanques (i.e. stations 18, 19 and 23) and within or near the National Park of 48 and 50). 24 stations were characterized by a moderate level of pressure (LP = 6 to 8), whereas the highest values of LP (9 to 12) mainly corresponded to stations located in the Gulf of Fos (e.g., stations 1 and 2) or directly exposed to sewage outfalls (e.g., stations 14 and 36) (Table 4).

Metrics and INDEX-COR assessment, correlation with LP

TS varied from 7.7 (station 1) to 76.1 (station 9). The lowest values (TS < 30) corresponded to stations particularly exposed to sediment inputs and to the Cortiou sewage outfall. On the contrary, the highest values (TS > 60) were linked to the stations located far from the coastline and to high steep cliffs (Table 4).

In total, 152 different taxa were identified for the full dataset. At each site, OTR varied from 17 (station 2) to 76 taxa (stations 12 and 46) (Table In a few cases, high numbers of taxa were observed in highly impacted stations (29 taxa in station 14 and 31 in station 1, with an LP = 10 and 12, respectively).

Finally, the values of SC ranged between 0.7 (station 2) to 5.6 (station 12) (Table 4). The lowest values (SC < 2) corresponded to highly impacted stations (sediment input or all pressures combined). On the contrary, the highest values (SC > 5) corresponded to slightly impacted sites or sites "protected" from sedimentation (high steep cliffs or reefs far from the coast). The results obtained for the three metrics are detailed in The INDEX-COR (IC) varied from 26.4 (station 1) to 97.1 (station 12) (Table 4). The lowest IC values corresponded to the most impacted stations, the highest to the least impacted ones. Five classes of conservation state were proposed (maximum IC values: 100) to respond to stakeholders' needs: IC < 20: bad status; 20 ≤ IC < 40: poor status; 40 ≤ IC < 60: moderate status; 60 ≤ IC < 80: good status; IC ≥ 80: high status.

Based on this classification, the majority of the sampled stations (38 sites) obtained a good or high conservation state (Fig. 3).

The poorest conservation state (IC < 40) mainly corresponded to the stations located in or close to the Gulf of Fos (West of Marseilles), characterized by its proximity to the Rhone river (input of sediment) and by the dense concentration of human activities (industries, fishing, anchorage of large tankers). On the contrary, the healthiest coralligenous habitats (IC > 80) were observed in 11 stations located within the boundaries of marine protected areas or offshore, mainly characterized by high steep cliffs.

The significant negative correlation between SC and LP (τ = -0.504, p < 0.0001) confirms the starting assumption that the structural complexity of coralligenous habitats decreases with the impact of the pressures on them. Also TS and OTR showed significantly correlated with LP (respectively τ = -0.513 and -0.480, p < 0.0001). Nevertheless, the best correlation resulted between IC, which integrated the three aforementioned metrics, and LP (τ = -0.642, p < 0.0001) (Fig. 2).

Discussion

Metrics and INDEX-COR assessment

The application of the INDEX-COR method proved effective in discriminating the conservation state of coralligenous habitats along the French Mediterranean coast. The accuracy of species identification can be an issue of concern, as for many groups (e.g., sponges, bryozoans, etc.) identification at species level often requires sampling and taxonomic studies in the laboratory. Nevertheless, there are plenty of examples where the monitoring of benthic assemblages has been based on photographic and video sampling (e.g., [START_REF] Edgerton | The instruments of deep-sea photography[END_REF][START_REF] Garrabou | Structure and dynamics of north-western Mediterranean benthic communities along a depth gradient[END_REF][START_REF] Mumby | Towards Reef Resilience and Sustainable Livelihoods: A Handbook for Caribbean Coral Reef Managers[END_REF]Gatti et al., 2015b). Besides, in situ observations made at the same place and time as the photographs taken may partially offset the problem. Whatever the case, when direct identification at a specific level is not possible, the use of higher taxonomic levels (genus or family) is still appropriate, as pointed out in other studies [START_REF] Bacci | Taxonomic sufficiency in two case studies: where does it work better?[END_REF][START_REF] Brind'amour | Morphospecies and taxonomic sufficiency of benthic megafauna in scientific bottom trawl surveys[END_REF]. However, taxonomic sufficiency should be applied with caution when using benthic species as 'bioindicators' [START_REF] Maurer | The dark side of taxonomic sufficiency (TS)[END_REF][START_REF] De Biasi | Analysis of macrobenthic communities at different taxonomic levels: an example from an estuarine environment in the Ligurian Sea (NW Mediterranean)[END_REF].

The use of species or higher taxa as bioindicators of organic matter input, as proposed for the TS metrics, also forms the computational basis of the AMBI and related WFD-compliance indexes [START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF][START_REF] Pinedo | A critical analysis on the response of macroinvertebrate communities along disturbance gradients: description of MEDOCC (MEDiterranean OCCidental) index[END_REF]. However, precise information about the sensitivity to sedimentation and organic matter inputs are almost nonexistent for species thriving in coralligenous assemblages [START_REF] Montefalcone | The two facets of species sensitivity: stress and disturbance on coralligenous assemblages in space and time[END_REF]. Only scant information on the sensitivity of certain species of algae, sponges, bryozoans and ascidians is available [START_REF] Muricy | Structure de peuplements de spongiaires autour de l'égout de Cortiou (Marseille, France)[END_REF][START_REF] Carballo | Taxonomic and ecological remarks on boring sponges (Clionidae) from the Straits of Gibraltar (southern Spain): tentative bioindicators?[END_REF][START_REF] Naranjo | Effects of environmental stress on ascidian populations in Algeciras Bay (southern Spain). Possible marine bioindicators?[END_REF][START_REF] Harmelin | Effects of sewage on bryozoan diversity in Mediterranean rocky bottoms[END_REF][START_REF] Balata | Variability of Mediterranean coralligenous assemblages subject to local variation in sediment deposition[END_REF][START_REF] Cecchi | Development of a new biotic index for ecological status assessment of Italian coastal waters based on coralligenous macroalgal assemblages[END_REF]. The Delta-Gamma model applied to the dataset, which allowed classifying species into four different groups of sensitivity to organic matter and sediment inputs, is based on a limited number of study sites and should be considered as a preliminary approach. Another concern is the quantification of sediment and organic matter inputs, since they may be very patchy. Expert judgment was used as the main criterion to assess these inputs as precise quantitative data were difficult to obtain in situ.

Coralligenous habitats represent a 'hotspot' of biodiversity in the Mediterranean. Spatial competition is strong and epibiosis is extremely frequent [START_REF] Ballesteros | Mediterranean coralligenous assemblages: a synthesis of present knowledge[END_REF]. These features led [START_REF] Laubier | Le coralligène des Albères: monographie biocénotique[END_REF] to consider coralligenous habitats as an ecological crossroads rather than as a single 'biocoenosis'. Consequently, it could be very difficult to perceive and measure species loss due to human activities [START_REF] Hong | Impact of the pollution on the benthic community[END_REF] and to define adequate metrics to this purpose. The second metrics of INDEX-COR (OTR), considering visually recognizable species, gives an image of the richness of coralligenous assemblages. The level of identification of the organisms adopted for OTR is in accordance with the taxonomic sufficiency recommended for the study of macrobenthic assemblages [START_REF] Bacci | Taxonomic sufficiency in two case studies: where does it work better?[END_REF]. In addition, the OTR values followed, with few exceptions, the impact due to human activities measured as LP (Level of Pressure). OTR was quite high in two highly impacted stations (1 and 14, LP = 12 and 10 respectively): these values are likely due to cryptic species present in cavities sheltered from sediment inputs and physical impacts.

Finally, the third metrics (SC) is based on the structural approach proposed for the COARSE index, aimed at evaluating the state of conservation of coralligenous habitats [START_REF] Gatti | Seafloor integrity down the harbor waterfront: the coralligenous shoals off Vado Ligure (NW Mediterranean)[END_REF](Gatti et al., , 2015a)). This metrics combines the information obtained from both in situ observations (as in the RVA method) and image analysis. The SC value was obtained by combining the three layers recognizable in the coralligenous structure (basal, intermediate and upper layers) and it was shown to be correlated with the level of pressure on coralligenous habitats, as seen for the COARSE index (Gatti et al., 2015a). This correlation is particularly influenced by sensitivity to physical impacts (e.g., diving, fishing and anchoring) and the mass mortality events of sessile invertebrates composing the intermediate and upper layers, like bryozoans, ascidians and gorgonians [START_REF] Garrabou | The impact of diving on rocky sublittoral communities: a case study of a bryozoan population[END_REF][START_REF] Linares | Life history and population viability of a long-lived marine invertebrate: the octocoral Paramuricea clavata[END_REF][START_REF] Franco | Scuba diver behavior and its effects on the biota of a Mediterranean marine protected area[END_REF][START_REF] Luna-Pérez | Halocynthia papillosa (Linnaeus, 1767) as an indicator of SCUBA diving impact[END_REF][START_REF] Pairaud | Impacts of climate change on coastal benthic ecosystems: assessing the current risk of mortality outbreaks associated with thermal stress in NW Mediterranean coastal areas[END_REF]. At last, a negative influence of sedimentation can be observed on the basal and the upper layer of coralligenous assemblages due to the balance between algae and invertebrate cover [START_REF] Irving | Sedimentation and light penetration interact to maintain heterogeneity of subtidal habitats: algal versus invertebrate dominated assemblages[END_REF][START_REF] Balata | Variability of Mediterranean coralligenous assemblages subject to local variation in sediment deposition[END_REF].

The integration of different metrics in a unique index may be criticized because of the loss of information, which makes the interpretation of index values difficult [START_REF] Moss | Shallow lakes, the water framework directive and life. What should it all be about ?[END_REF]. By contrast, other authors support that only a synthetic index can detect departure from biological integrity caused by anthropogenic pressures [START_REF] Karr | Defining and measuring river health[END_REF]. This is the case of INDEX-COR, which resulted to respond to the overall level of pressure on coralligenous assemblages better than the individual metrics. This result underlines the complementarity of TS, OTR and SC metrics despite the lower weight given to the SC metrics in the linear model used to define the IC.

Reference conditions and robustness of INDEX-COR

Building a relevant index to assess the conservation state of a marine habitat requires: (i) defining reference conditions, and (ii) estimating the level of pressure affecting it. The definition of reference conditions mainly depends on the availability of 'pristine' areas, historical references, or models based on extrapolations of the biological attributes characterizing a certain zone in natural conditions [START_REF] Borja | The importance of setting targets and reference conditions in assessing marine ecosystem quality[END_REF]. If none of the previous criteria is applicable, reference conditions can be determined by 'best professional judgment' (expert judgment), or statistically from available datasets [START_REF] Romero | A multivariate index based on the seagrass Posidonia oceanica (POMI) to assess ecological status of coastal waters under the water framework directive (WFD)[END_REF][START_REF] Gobert | Assessment of the ecological status of Mediterranean French coastal waters as required by the water framework directive using Posidonia oceanica Rapid Easy Index: PREI[END_REF][START_REF] Teixeira | Assessing coastal benthic macrofauna community condition using best professional judgement-developing consensus across North America and Europe[END_REF]. Considering coralligenous habitats, it would be possible to obtain theoretical reference values INDEX-COR by combining the highest values obtained for each metric and for each ecoregion. Unfortunately, as already underlined, a larger dataset would be necessary to better define the possible range of variation of each metric. It is necessary to take into account that the reference conditions of coralligenous habitats situated close to areas with a naturally high sediment and organic matter inputs (i.e. Gulf of Fos) are different from those situated offshore or far away from river mouths (e.g. [START_REF] Curiel | Species composition, and spatial variability of macroalgal assemblages on biogenic reefs in the northern Adriatic Sea[END_REF][START_REF] Falace | Calcareous bio-concretions in the northern Adriatic Sea: habitat types, environmental factors that influence habitat distributions and predictive modeling[END_REF]. Furthermore, coralligenous assemblages are characterized by long-lived species with parsimonious population dynamics (e.g., low growth and mortality rates) [START_REF] Ballesteros | Mediterranean coralligenous assemblages: a synthesis of present knowledge[END_REF][START_REF] Teixidó | Low dynamics, high longevity and persistence of sessile structural species dwelling on Mediterranean coralligenous outcrops[END_REF]. Due to these features, assemblages can display both limited resilience in the case of impact from strong disturbances and apparent stability in the absence of disturbances, even when they are in fact immersed in declining trajectories [START_REF] Hughes | Living dangerously on borrowed time during slow, unrecognized regime shifts[END_REF][START_REF] Gatti | Observational information on a temperate reef community helps understanding the marine climate and ecosystem shift of the 1980-90s[END_REF]. Therefore, the assessment of conservation state is a basic step for the management of coralligenous habitats. INDEX-COR provided the first assessment in a specific area as a 'current baseline' that can be used to compare future evaluations of state [START_REF] Sala | The structure of Mediterranean rocky reef ecosystems across environmental and human gradients, and conservation implications[END_REF]. Stakeholders should take care to ensure management in view not to drop below it. This solution implies the definition of an acceptable range of variation of the index independently of the variation of the level of pressure. The results obtained during the present study constitute the first dataset for the INDEX-COR. The acquisition of other annual datasets in the same sampling stations with no notable variations of human impact will allow estimating the robustness of the index. Thus, future perspectives include the enlargement of the dataset, tests on local variability and bias due to operators, in order to improve the index and to make it as robust as possible.

To link the results of an index of conservation to human pressures, it is necessary to quantify the level of the impact affecting the habitat. One or several pressures can be considered. On soft bottoms, the main source of pressure is the presence of organic matter in the sediment, which is directly measured in samples and can be easily compared between sites [START_REF] Borja | A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF]. In the case of hard substrates, and especially for coralligenous reefs, benthic assemblages are affected by different human pressures (Cánovas [START_REF] Molina | Combining literature review, acoustic mapping and in situ observations: an overview of coralligenous assemblages in Liguria (NW Mediterranean Sea)[END_REF]. Unfortunately, their characterization in a complex three dimensional habitat is very difficult, because their impact can differ according to the scale considered. Furthermore, many species belonging to coralligenous communities are also affected by extreme high temperatures, which may cause extensive mortality events [START_REF] Cerrano | A catastrophic mass-mortality episode of gorgonians and other organisms in the Ligurian Sea (North-western Mediterranean), summer 1999[END_REF][START_REF] Pérez | Mortalité massive d'invertébrés marins: un événement sans précédent en Méditerranée nord-occidentale[END_REF][START_REF] Cupido | Response of a gorgonian (Paramuricea clavata) population to mortality events: recovery or loss?[END_REF][START_REF] Coma | Global warmingenhanced stratification and mass mortality events in Mediterranean[END_REF][START_REF] Garrabou | Mass mortality in northwestern Mediterranean rocky benthic communities: effects of the 2003 heat wave[END_REF]. Other species such as coralline algae, the main builders of the calcareous substrate, may also be impacted by water acidification [START_REF] Martin | Response of Mediterranean coralline algae to ocean acidification and elevated temperature[END_REF][START_REF] Noisette | Physiological responses of three temperate coralline algae from contrasting habitats to near-future ocean acidification[END_REF]. In this context, [START_REF] Elliott | Force majeure: will climate change affect our ability to attain good environmental status for marine biodiversity?[END_REF] emphasized that marine management has to accommodate 'shifting baselines' caused by changes at planetary scale, such as warming and acidification. In our opinion and in the absence of quantitative data for each pressure, the expert judgment seems to be the only solution possible at present.

Practical use

The INDEX-COR approach proposes a methodology and an index to assess the conservation state of coralligenous reefs. Its application requires SCUBA diving experience and knowledge of marine organisms. More or less 2 days are required for each station to acquire and analyze the data. Moreover, the level of efficacy of an index is directly linked to knowledge of the habitat considered (e.g., distribution, natural dynamics, functionality, response to the different pressures, etc.). Unfortunately, little knowledge is available on coralligenous habitats and their associated communities [START_REF] Ballesteros | Mediterranean coralligenous assemblages: a synthesis of present knowledge[END_REF]. Basic studies to fill these gaps in knowledge are strongly recommended.

Fig. 1 .

 1 Fig. 1. Study area with the sampling stations represented by a numeric code as in Table3.

  Figuerolle

Fig. 3 .

 3 Fig. 3. Color-coded representation of the conservation state of coralligenous habitats along the coasts of Provence. Orange: poor status; yellow: moderate status; green: good status; blue: high status. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 4

 4

(see appendix C for further details. The whole database is available here: https://www.dropbox.com/s/ a7rj0ckcarkwbbh/INDEXCOR.accdb?dl=0).

Table 4

 4 INDEX-COR (IC) values, metrics (TS = Taxa Sensitivity; OTR = Observed Taxonomic Richness; SC = structural complexity) scores, level of pressure (LP) and conservation status defined with 5 classes, for the 53 stations sampled along the French Mediterranean coast. Classes' boundaries: bad (IC < 20), poor (20 ≤ IC < 40); moderate (40 ≤ IC < 60); good (60 ≤ IC < 80); high (IC ≥ 80).

	Station	TS	OTR	SC	IC	LP	Conservation status	Station	TS	OTR	SC	IC	LP	Conservation status
	1	7.7	31	1.6	26.4	12	Poor	28	48.9	51	2.1	65.3	6	Good
	2	29.6	17	0.7	28.4	12	Poor	29	52.7	56	4.0	74.2	5	Good
	3	51.8	28	1.5	50.7	8	Moderate	30	64.4	41	2.0	67.7	7	Good
	4	48.4	33	1.6	52.4	8	Moderate	31	36.1	59	3.8	66.7	8	Good
	5	21.3	33	3.0	37.3	7	Poor	32	56.2	61	3.4	79.2	6	Good
	6	62.6	53	5.1	80.4	4	High	33	39.6	48	2.2	58.5	8	Moderate
	7	48.8	59	2.3	71.4	7	Good	34	53.6	51	3.6	70.3	5	Good
	8	36.5	50	2.1	57.8	9	Moderate	35	71.8	42	5.5	79.6	4	Good
	9	76.1	55	5.3	90.1	5	High	36	32.2	37	1.7	45.2	9	Moderate
	10	36.6	45	3.7	58.1	8	Moderate	37	37.7	61	3.3	67.8	5	Good
	11	42.7	51	2.9	63.2	6	Good	38	51.6	63	4.7	79.4	5	Good
	12	62.3	76	5.6	97.1	3	High	39	57.5	54	3.1	74.7	6	Good
	13	27.5	59	2.9

Acknowledgement

We would like to thank the French Protected Marine Areas Agency (AAMP) for its financial support to this project (convention n°3211545/ F). We also thank the Marine Protected Areas involved in the INDEX-

COR program for their technical support (boats and divers), especially the directors and the staff of the "Parc Marin de la Côte Bleue", the "Observatoire Marin du Littoral des Maures" and the "Aire Marine Protégée de la Côte Agathoise". This work was also supported by the ERA-NET (SeasEra) CIGESMED Program (contract 2014/3/3211985). Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.marpolbul.2017.05.020.


Claudio González-Wevar

Nicolás Segovia

Sebastián Rosenfeld

Jaime Ojeda

Mathias Hüne

Javier Naretto

Thomas Saucède

Paul Brickle

Simon Morley

Jean-Pierre Feral

Claudio A Gonz Alez-Wevar

| Nicol

I Segovia

| Sebasti An Rosenfeld

Mathias H€ Une

| Thomas

| Paul Brickle

| Jean-Pierre

Hamish G Spencer

Elie Poulin

Jonathan Waters

Unexpected absence of island endemics: Long-distance dispersal in higher latitude sub-Antarctic Siphonaria

Keywords: Antarctic circumpolar current, direct developers, long-distance dispersal, oceanic biogeography, pulmonate, rafting, Siphonaria, sub-Antarctic

come   L'archive ouverte pluridisciplinaire

| INTRODUCTION

Biogeographical mechanisms explaining the distribution of southern temperate taxa have been debated for more than a century [START_REF] Crame | An evolutionary perspective on marine faunal connection between southernmost South America and Antarctica[END_REF][START_REF] Darwin | Journal of researches into the natural history and geology of the countries visited during the voyage of H.M.S. Beagle round the world[END_REF][START_REF] Dell | Antarctic benthos[END_REF][START_REF] Knox | Littoral ecology and biogeography of the Southern Ocean[END_REF][START_REF] Strugnell | The thermohaline expressway: The Southern Ocean as a centre of origin for deep-sea octopuses[END_REF]. Recently, the literature has been characterized by disagreement over the relative importance of vicariance versus dispersal in the biogeography of the Southern Ocean biota but consensus has emerged about the combined significance of these processes [START_REF] Clarke | How isolated is Antarctica?[END_REF][START_REF] Fraser | Kelp genes reveal effects of subantarctic sea during the last glacial maximum[END_REF][START_REF] Gonz Alez-Wevar | Molecular phylogeny and historical biogeography of Nacella (Patellogastropoda: Nacellidae) in the Southern Ocean[END_REF][START_REF] Gonzalez-Wevar | Following the Antarctic circumpolar current: patterns and processes in the biogeography of the limpet Nacella (Mollusca: Patellogastropoda) across the Southern Ocean[END_REF][START_REF] Moon | Reconsidering connectivity in the sub-Antarctic[END_REF][START_REF] Nikula | Circumpolar dispersal by rafting in two subantarctic kelp-dwelling crustaceans[END_REF][START_REF] Nikula | Passive rafting is a powerful driver of transoceanic gene flow[END_REF][START_REF] Poulin | Divergence between Antarctic and South American marine invertebrates: What molecular biology tells us about Scotia Arc geodynamics and the intensification of the Antarctic Circumpolar Current[END_REF][START_REF] Sauc Ede | Biogeographic processes in the Southern Ocean[END_REF][START_REF] Waters | Driven by the west wind drift? A synthesis of southern temperate marine biogeography, with new directions for dispersalism[END_REF].

In the marine realm, dispersal plays a central role in the evolution of species with spatially structured populations, resulting in genetic cohesion, global persistence despite local extinction and the tracking of favourable environments in a changing world [START_REF] Ayre | Does life history predict past and current connectivity for rocky intertidal invertebrates across a marine biogeographic barrier?[END_REF][START_REF] Cowen | Larval dispersal and marine population connectivity[END_REF][START_REF] Puritz | Life-history predicts past and present population connectivity in two sympatric sea stars[END_REF][START_REF] Ronce | How does it feel to be like a Rolling Stone? Ten questions about dispersal evolution[END_REF]. The presence of pelagic dispersive larval stages in species with low autonomous vagility is often considered a valuable proxy for dispersal potential [START_REF] Haye | Genetic signatures of rafting dispersal in algal-dwelling brooders Limnoria spp. (Isopoda) along the SE Pacific (Chile)[END_REF][START_REF] Puritz | Life-history predicts past and present population connectivity in two sympatric sea stars[END_REF][START_REF] Thorson | Reproductive and larval ecology of marine bottom invertebrates[END_REF]. Species with free-living planktonic stages (broadcast-spawners) are expected to be more effective dispersers than those lacking larval stages (direct developers) and to exhibit less population differentiation [START_REF] Haye | Genetic signatures of rafting dispersal in algal-dwelling brooders Limnoria spp. (Isopoda) along the SE Pacific (Chile)[END_REF][START_REF] Marko | What's larvae got to do with it?' Disparate patterns of post-glacial population structure in two benthic marine gastropods with identical dispersal potential[END_REF]. Nevertheless, meta-analyses and comparative studies, particularly in the Southern Ocean, have challenged such assumptions [START_REF] Cumming | Transoceanic genetic similarities of kelp-associated sea slug populations: Long-distance dispersal via rafting?[END_REF][START_REF] Helmuth | Long-distance dispersal of a subantarctic brooding bivalve (Gaimardia trapesina) by kelp-rafting[END_REF][START_REF] Nikula | Circumpolar dispersal by rafting in two subantarctic kelp-dwelling crustaceans[END_REF][START_REF] Weersing | Population genetics, larval dispersal, and connectivity in marine systems[END_REF].

The role of long-distance dispersal by rafting has been especially controversial [START_REF] Gillespie | Long-distance dispersal: A framework for hypothesis testing[END_REF][START_REF] Thiel | The ecology of rafting in the marine environment. III. Biogeographical and evolutionary consequences[END_REF], with its rarity and unpredictability often hindering the development of testable hypotheses [START_REF] Cowie | Dispersal is fundamental to biogeography and the evolution of biodiversity on oceanic islands[END_REF][START_REF] Crisp | Hypothesis testing in biogeography[END_REF].

In the sub-Antarctic, rafting events are mainly associated with buoyant kelps, which facilitate dispersal of different groups from invertebrates [START_REF] Cumming | Transoceanic genetic similarities of kelp-associated sea slug populations: Long-distance dispersal via rafting?[END_REF][START_REF] Gonzalez-Wevar | Following the Antarctic circumpolar current: patterns and processes in the biogeography of the limpet Nacella (Mollusca: Patellogastropoda) across the Southern Ocean[END_REF][START_REF] Helmuth | Long-distance dispersal of a subantarctic brooding bivalve (Gaimardia trapesina) by kelp-rafting[END_REF][START_REF] Nikula | Circumpolar dispersal by rafting in two subantarctic kelp-dwelling crustaceans[END_REF] to non-buoyant kelps [START_REF] Fraser | Genetic affinities between Trans-Oceanic populations of non-buoyant macroalgae in the high latitudes of the Southern Hemisphere[END_REF].

Members of the pulmonate limpet genus Siphonaria occur on rocky intertidal shores worldwide, except in the Arctic. In a recent revision, [START_REF] Dayrat | Diversity of Indo-West Pacific Siphonaria (Mollusca: Gastropoda: Euthyneura)[END_REF] evaluated species richness through molecular and morphological analyses, recording a total of 41 genetic units (species), although many of them were morphologically cryptic and could not be reliably identified by shell characters. Together with this, they showed that no Siphonaria species was broadly distributed across the Indo-West Pacific. Three clades were recorded in southern South America: S. lessonii and two sister cryptic species which had previously been considered as S. lateralis [START_REF] Dayrat | Diversity of Indo-West Pacific Siphonaria (Mollusca: Gastropoda: Euthyneura)[END_REF], also known as Kerguelenella lateralis [START_REF] White | Checklist of genus-and species-group names of the false limpets Siphonaria (Mollusca: Gastropoda: Euthyneura)[END_REF]. These cryptic species showed 6.9% mtDNA divergence [START_REF] Dayrat | Diversity of Indo-West Pacific Siphonaria (Mollusca: Gastropoda: Euthyneura)[END_REF], and were recognized by [START_REF] G€ Uller | How many Siphonaria species (Gastropoda: Euthyneura) live in southern South America?[END_REF] as S. lateralis and the newly described S. fuegiensis.

Based on morphology, S. lateralis is broadly distributed across the sub-Antarctic from southern South America to Macquarie Island where it is dominant in intertidal rocky environments [START_REF] Simpson | Egg masses of three gastropods, Kerguelenella lateralis (Siphonariidae), Laevilitorina caliginosa and Macquariella hamiltoni (Littorinidae), from Macquarie Island (sub-Antarctic)[END_REF][START_REF] Griffiths | The first comprehensive description of the biodiversity and biogeography of Antarctic and Sub-Antarctic intertidal communities[END_REF]; Figure 1a). Published mtDNA sequences for S. lateralis include individuals from southern South America (n = 10) and South Georgia (n = 2) [START_REF] Dayrat | Diversity of Indo-West Pacific Siphonaria (Mollusca: Gastropoda: Euthyneura)[END_REF][START_REF] G€ Uller | How many Siphonaria species (Gastropoda: Euthyneura) live in southern South America?[END_REF]. Genetically identified individuals of S. fuegiensis (n = 6) have only been recorded from the southern tip of South America [START_REF] Dayrat | Diversity of Indo-West Pacific Siphonaria (Mollusca: Gastropoda: Euthyneura)[END_REF][START_REF] G€ Uller | How many Siphonaria species (Gastropoda: Euthyneura) live in southern South America?[END_REF]. However, on the basis of subtle differences in shell shape, other specimens have been assigned to S. fuegiensis from the Strait of Magellan, the Falkland/Malvinas Islands and Isla de los Estados in southern Argentina (G€ uller et al., 2015), where the two species are sympatric (Figure 1b).

Like many marine invertebrates in the sub-Antarctic, S. lateralis and probably S. fuegiensis are direct developers, with benthic development and low dispersal capacity [START_REF] Chambers | A review of larval development in the intertidal limpet genus Siphonaria (Gastropoda: Pulmonata)[END_REF][START_REF] Simpson | Egg masses of three gastropods, Kerguelenella lateralis (Siphonariidae), Laevilitorina caliginosa and Macquariella hamiltoni (Littorinidae), from Macquarie Island (sub-Antarctic)[END_REF]. Direct development is considered a key evolutionary trait in Southern Ocean marine invertebrates enhancing potential for speciation [START_REF] Pearse | Brooding and species diversity in the Southern Ocean: Selection for brooders or speciation within brooding clades?[END_REF][START_REF] Thatje | Effects of capability for dispersal on the evolution of diversity in Antarctic benthos[END_REF]. Consequently, and as demonstrated in several marine invertebrate groups in this region [START_REF] Allcock | Cryptic speciation and the circumpolarity debate: A case study on endemic Southern Ocean octopuses using the COI barcode of life. Deep-Sea Research Part II[END_REF][START_REF] Janosik | Evolutionary history of Southern Ocean Odontaster sea star species (Odontasteridae; Asteroidea)[END_REF][START_REF] Leese | Cryptic speciation in a benthic isopod from Patagonian and Falkland Island waters and the impact of glaciations on its population structure[END_REF][START_REF] Wilson | Ocean barriers and glaciation: Evidence for explosive radiation of mitochondrial lineages in the Antarctic sea slug Doris kerguelenensis (Mollusca, Nudibranchia)[END_REF], molecular comparisons of widely separated populations might be expected to reveal additional unrecognized cryptic species. Nevertheless, both species are commonly associated with buoyant and broadly dispersing kelps, often being encased in the holdfasts of Durvillaea antarctica [START_REF] Cumming | Transoceanic genetic similarities of kelp-associated sea slug populations: Long-distance dispersal via rafting?[END_REF]Figure 1c-e), and might have a high potential for rafting. As recently demonstrated in crustaceans and gastropods, rafting can be a major biogeographical process shaping patterns of genetic diversity and structure in direct developers across distant areas of the Southern Ocean [START_REF] Cumming | Transoceanic genetic similarities of kelp-associated sea slug populations: Long-distance dispersal via rafting?[END_REF][START_REF] Nikula | Circumpolar dispersal by rafting in two subantarctic kelp-dwelling crustaceans[END_REF][START_REF] Nikula | Passive rafting is a powerful driver of transoceanic gene flow[END_REF]. Such long-distance dispersal could contribute to the broad geographical distribution of Siphonaria at high latitudes.

Here we present phylogenetic and population-based analyses of high-latitude Siphonaria populations from South America, the Falkland/Malvinas Islands, South Georgia, Kerguelen and Macquarie Islands. We provide a clear picture of (1) the evolutionary relationships among the sub-Antarctic Siphonaria species and (2) the geographical distribution of each taxon, to depict biogeographical patterns of the genus at high latitudes. As a consequence of direct development in these higher latitude pulmonates, the main biogeographical patterns in Siphonaria across the Indo-West Pacific [START_REF] Dayrat | Diversity of Indo-West Pacific Siphonaria (Mollusca: Gastropoda: Euthyneura)[END_REF], and patterns recorded in Southern Ocean near-shore marine molluscs including Nacella [START_REF] Gonzalez-Wevar | Following the Antarctic circumpolar current: patterns and processes in the biogeography of the limpet Nacella (Mollusca: Patellogastropoda) across the Southern Ocean[END_REF] and Doris [START_REF] Wilson | Ocean barriers and glaciation: Evidence for explosive radiation of mitochondrial lineages in the Antarctic sea slug Doris kerguelenensis (Mollusca, Nudibranchia)[END_REF], we expected to find closely related cryptic species, endemic to each island group.

Alternatively, long-distance dispersal mediated by rafting could play a key role in the biogeography of direct developers like Siphonaria permitting a broad distribution of its representatives across the sub-Antarctic. S1 in Appendix S2). All individuals were identified following [START_REF] Dayrat | Diversity of Indo-West Pacific Siphonaria (Mollusca: Gastropoda: Euthyneura)[END_REF][START_REF] G€ Uller | How many Siphonaria species (Gastropoda: Euthyneura) live in southern South America?[END_REF]. Whole specimens were fixed in ethanol (95%) and DNA was extracted from the mantle using standard salting-out methodologies [START_REF] R E F E R E N C E S Aljanabi | Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques[END_REF].

| MATERIAL AND METHODS

Universal primers were used to amplify partial fragments of the mitochondrial gene cytochrome c oxidase subunit I (COI) [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF], the nuclear 28S rRNA (Littlewood, [START_REF] Littlewood | The interrelationships of Proseriata (Platyhelminthes: Seriata) tested with molecules and morphology[END_REF] and the internal transcribed spacer region (ITS1 & ITS2) [START_REF] Oliverio | Contrasting morphological and molecular variation in Coralliophila meyendorffii (Muricidae, Coralliophilinae)[END_REF]. Sequences for each of the analysed fragments were assembled independently and edited using GENEIOUS 5.1.7 [START_REF] Kearse | Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data[END_REF]. Similarly, alignments and base composition of nucleotide sequences were analysed for each marker separately in MEGA 6.0 [START_REF] Tamura | MEGA6: Molecular evolutionary genetics analysis version 6.0[END_REF] using MUSCLE [START_REF] Edgar | MUSCLE: A multiple sequence alignment method with reduced time and space complexity[END_REF] with standard settings. Mitochondrial codon usage was determined using the Effective Number of Codons value (ENC; [START_REF] Wright | The "effective number of codons" used in a gene[END_REF] using DNASP v5 [START_REF] Librado | DnaSP v5: A software for comprehensive analysis of DNA polymorphism data[END_REF]. New high latitude Siphonaria sequences are available at Genbank under the following Accession Numbers: COI (MF651995-MF652021), ITSs (MF652022-MF652042) and 28S rRNA (MF652043-MF652059). [START_REF] Dayrat | Diversity of Indo-West Pacific Siphonaria (Mollusca: Gastropoda: Euthyneura)[END_REF] Siphonaria haplotypes from the sub-Antarctic. For comparative purposes, we also performed multi-locus phylogenetic reconstructions using mtDNA (COI) and nucDNA (ITS1 & ITS2) sequences. For this we amplified each marker from at least eight individuals of S. lateralis, S. fuegiensis and S. lessonii, and we analysed them together with available sequences from S. japonica [START_REF] Wang | Causations of phylogeographic barrier of some rocky shore species along the Chinese coastline[END_REF]. Maximum parsimony (MP), maximum likelihood (ML) and Bayesian analyses (BA) were used to estimate phylogenetic relationships. MP analyses were performed using PAUP * [START_REF] Swofford | PAUP*: Phylogenetic analysis using parsimony (* and other methods)[END_REF] following [START_REF] Gonz Alez-Wevar | Molecular phylogeny and historical biogeography of Nacella (Patellogastropoda: Nacellidae) in the Southern Ocean[END_REF]. The program JMODELTEST 2 [START_REF] Darriba | jModelTest 2: More models, new heuristics and parallel computing[END_REF] was used to select the GTR+I+G (COI) and GTR+G (ITS1 & ITS2) substitution models for the ML and BA. ML and BA reconstructions based on mtDNA (COI) and multi-locus data sets were carried out using PHYML [START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF][START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF]MRBAYES v.3.1.2 (Huelsenbeck &[START_REF] Huelsenbeck | MRBAYES: Bayesian inference of phylogenetic trees[END_REF] respectively. Nodal support for MP and ML analyses was inferred from non-parametric bootstrap (BS) using 1,000 pseudo-replicates [START_REF] Felsenstein | Evolutionary trees from DNA sequences: A maximum likelihood approach[END_REF]. Bayesian-inference posterior probabilities were estimated using the Metropolis coupled Markov chain Monte Carlo algorithm (MCMC) running four chains for 100 9 10 6 generations, with trees sampled every 1,000 generations. Stationarity was inferred when the average standard deviation of split frequencies was less than 0.01 [START_REF] Huelsenbeck | MRBAYES: Bayesian inference of phylogenetic trees[END_REF]. The initial 10% of the trees were discarded (burn-in) and posterior probabilities were estimated as the fraction of trees showing a particular node. Posterior probability density was summarized as a maximum clade credibility tree using TREEANNOTATOR v.1.6.1 (http:// beast.bio.ed.ac.uk/TreeAnnotator) and visualized with FIGTREE v.1.4 (http://tree.bio.ed.ac.uk/software/figtree).

The best fit model for the mtDNA COI data set, a relaxed molecular-clock analysis with an uncorrelated lognormal (ucln) model of evolutionary rate heterogeneity, was implemented using the GTR+I+G substitution model in BEAST v.1.6.2 [START_REF] Drummond | Bayesian phylogenetics with BEAUti and the BEAST 1.7[END_REF]. A birth-death speciation prior was used for branching rates in the phylogeny, four chains were run twice for 100 9 10 6 generations, and trees were sampled every 1,000 generations. Because of the absence of a clear fossil record in Siphonaria we used a conservative mutation rate (1%), following previous studies in siphonariids [START_REF] Teske | Climate-driven genetic divergence of limpets with different life histories across a southeast African marine biogeographic disjunction: Different processes, same outcome[END_REF][START_REF] Wang | Causations of phylogeographic barrier of some rocky shore species along the Chinese coastline[END_REF]. Convergence of model parameters was estimated by plotting the marginal posterior probabilities versus the generations in TRACER v.1.5 (http://beast.bio.e d.ac.uk/Tracer). Effective sample-size values were estimated for each parameter to ensure adequate mixing of the MCMC (ESSs > 1,000).

| Estimating levels of genetic diversity and structuring

Levels of mtDNA genetic polymorphism were determined using standard diversity indices including number of haplotypes (k), number of segregating sites (S), haplotype diversity (H), average number of pairwise differences (Π) and nucleotide diversity (p) for each locality and species with DNASP. Genealogical relationships in sub-Antarctic Siphonaria populations were reconstructed using maximum parsimony networks in HAPVIEW (http://www.cibiv.at/~greg/haploviewer).

For comparative purposes, we also reconstructed haplotype networks based on 28S rRNA (n = 60) and ITS1 & ITS2 (n = 40) sequences, independently. We estimated the levels of mtDNA differentiation following [START_REF] Pons | Measuring and testing genetic differentiation with ordered Versus unordered alleles[END_REF] through mean pairwise differences (N ST ) and haplotype frequencies (G ST ) with ARLEQUIN v. 3.5 [START_REF] Excoffier | Arlequin (version 3.0): An integrated software package for population genetics data analysis[END_REF]. The statistical significance of these analyses was assessed using permutation tests (20,000 iterations). We estimated the levels of mtDNA phylogeographical differentiation between subpopulations using the nearest-neighbor statistic (S nn ) [START_REF] Hudson | A new statistic for detecting genetic differentiation[END_REF] and its statistical significance was estimated through a permutation test (10,000 iterations). We inferred the spatial pattern of COI genetic structure in each species by estimating the number and the composition of groups that were the most differentiated based on sequence data with SAMOVA (Spatial Analysis of Molecular Variance) following [START_REF] Dupanloup | A simulated annealing approach to define the genetic structure of population[END_REF].

| RESULTS

Mitochondrial DNA alignment within the clearly resolved Siphonaria

| Population-based analyses

The complete COI data set for S. lateralis and S. fuegiensis Haplotype networks for S. lateralis and S. fuegiensis showed similar topologies with short genealogies and the presence of specific dominant haplotypes and several endemic ones (Figure 3).

S. lateralis S. fuegiensis

In the case of S. lateralis, PP, AP, KI and MI shared a highly dominant haplotype (H1). Individuals from SG showed a single private haplotype (H2) separated by a single substitution from H1. Finally, in the FI, we recorded two closely related private haplotypes (H3 and H4) that are separated by seven substitutional steps from H1 (Figure 3). Similarly, in the case of S.

fuegiensis, PP, KI and MI shared a dominant COI haplotype (HI; Figure 3a). A less common haplotype (HV) was recorded in both PP and KI. In addition, PP and KI exhibited private medium-frequency haplotypes (HII and HVII respectively). In contrast to S.

lateralis, the dominant haplotype was not found in AP localities.

In fact, AP and FI shared two medium-frequency haplotypes (HVIII and HIX) separated by one and two substitutions from HI respectively (Figure 3a).

In spite of the low number of individuals analysed, ITS sequences in S. lateralis showed the presence of a dominant shared haplotype across PP, AP, KI and MI. Again, individuals from FI showed a different haplotype (n = 3) separated by four substitutional steps from the dominant one. Similarly, in the case of S.

fuegiensis ITS sequences recorded a dominant haplotype broadly distributed in PP, AP, KI and MI, whereas FI individuals (n = 2) carried a single haplotype separated by two mutational steps from the dominant one (Figure 3b). [START_REF] Gonzalez-Wevar | Following the Antarctic circumpolar current: patterns and processes in the biogeography of the limpet Nacella (Mollusca: Patellogastropoda) across the Southern Ocean[END_REF][START_REF] Janosik | Evolutionary history of Southern Ocean Odontaster sea star species (Odontasteridae; Asteroidea)[END_REF][START_REF] Leese | Cryptic speciation in a benthic isopod from Patagonian and Falkland Island waters and the impact of glaciations on its population structure[END_REF][START_REF] Wilson | Ocean barriers and glaciation: Evidence for explosive radiation of mitochondrial lineages in the Antarctic sea slug Doris kerguelenensis (Mollusca, Nudibranchia)[END_REF], macroalgae [START_REF] Billard | Deep genetic divergence between austral populations of the red alga Gigartina skottsbergii reveals a cryptic species endemic to the Antarctic continent[END_REF][START_REF] Fraser | Genetic affinities between Trans-Oceanic populations of non-buoyant macroalgae in the high latitudes of the Southern Hemisphere[END_REF] and fishes [START_REF] Dornburg | Cryptic species diversity in Sub-Antarctic islands: A case study of Lepidonotothen[END_REF]. In these latter cases, there appears to be a limited connectivity among benthic marine populations, regardless of dispersal capacity [START_REF] Moon | Reconsidering connectivity in the sub-Antarctic[END_REF]. For instance South American and South Georgia populations of the broadcastspawning nemertean Parborlasia corrugatus are genetically distinct, despite the high dispersal capacity (evidenced by population panmixia within the sub-Antarctic and Antarctic regions) [START_REF] Thornhill | Open-ocean barriers to dispersal: A test case with the Antarctic Polar Front and the ribbon worm Parborlasia corrugatus (Nemertea: Lineidae)[END_REF]. Similarly, populations of the sea spider Pallenopsis from the Falkland/Malvinas Islands, Antarctica and South America are genetically differentiated [START_REF] Harder | Diversity and distribution within the sea spider genus Pallenopsis (Chelicerata: Pycnogonida) in the Western Antarctic as revealed by mitochondrial DNA[END_REF][START_REF] Weis | Pallenopsis patagonica (Hoek, 1881)a species complex revealed by morphology and DNA barcoding, with description of a new species of Pallenopsis Wilson, 1881[END_REF]. the observed distribution of direct developers [START_REF] Ayre | Does life history predict past and current connectivity for rocky intertidal invertebrates across a marine biogeographic barrier?[END_REF]. In fact, several direct developing species in the sub-Antarctic including molluscs [START_REF] Cumming | Transoceanic genetic similarities of kelp-associated sea slug populations: Long-distance dispersal via rafting?[END_REF] and crustaceans [START_REF] Leese | Long-distance island hopping without dispersal stages: Transportation across major zoogeographic barriers in a Southern Ocean isopod[END_REF][START_REF] Nikula | Circumpolar dispersal by rafting in two subantarctic kelp-dwelling crustaceans[END_REF] exhibit a marked genetic homogeneity across geographically distant areas.

|

Sub-Antarctic Siphonaria often live closely associated with, and even encased in bull-kelp (Durvillaea antarctica) holdfasts [START_REF] Morton | The New Zealand sea shore[END_REF][START_REF] Simpson | Physical and biotic factors limiting the distribution and abundance of littoral mollusks on Macquarie Island (Sub-Antarctic)[END_REF]; E. Poulin & T. Sauc ede observations at Kerguelen Islands). Such observations strongly suggest that bull-kelp provides the mechanism for long-distance dispersal as has been inferred for other sub-Antarctic taxa [START_REF] Cumming | Transoceanic genetic similarities of kelp-associated sea slug populations: Long-distance dispersal via rafting?[END_REF][START_REF] Fraser | Oceanic rafting by a coastal community[END_REF][START_REF] Moon | Reconsidering connectivity in the sub-Antarctic[END_REF][START_REF] Nikula | Circumpolar dispersal by rafting in two subantarctic kelp-dwelling crustaceans[END_REF]. As also noted by [START_REF] Griffiths | The first comprehensive description of the biodiversity and biogeography of Antarctic and Sub-Antarctic intertidal communities[END_REF], the distribution of several sub-Antarctic molluscs (Laevilitorina caliginosa, Nacella and Mytilus)

matches the distribution of the bull-kelp. Alternative mechanisms (e.g., rafting on other substrates and anthropogenic introductions) might explain our data, but they seem unlikely [START_REF] Cumming | Transoceanic genetic similarities of kelp-associated sea slug populations: Long-distance dispersal via rafting?[END_REF]. We conclude that for species with no dispersive larval stage, long-distance dispersal mediated by rafting is a key biogeographical mechanism explaining the low, or even the absence, of phylogeographical structure across geographically distant areas in the sub-Antarctic connected by the flow of the Antarctic circumpolar current (ACC).

Divergence time estimates suggest that the separation of S. lateralis and S. fuegiensis took place during the Pliocene 4 Ma (3-8 Ma) and after that they followed different evolutionary trajectories in sympatry-and even in syntopy-across almost their whole range.

At SG only a single species, S. lateralis, was recorded; the apparent absence of S. fuegiensis needs to be confirmed. If genuine, this distributional discrepancy could be due to different thermal tolerances between them. At the sampling locations where both species occurred, we noted that S. fuegiensis was more prevalent at lower latitudes on the Atlantic coast and in the FI than S. lateralis, which was more common at higher latitudes of PP and KI. Thus, the absence of S. fuegiensis in SG, the locality of our sampling area with the lowest winter sea-surface temperatures and that was heavily glaciated during the Last Glacial Maximum (LGM) [START_REF] Hodgson | Terrestrial and submarine evidence for the extent and timing of the Last Glacial Maximum and the onset of deglaciation on the maritime-Antarctic and sub-Antarctic islands[END_REF], could be due to a restricted lower thermal tolerance. This hypothesis will be tested by future physiological experiments.

Quaternary glacial processes in the Southern Ocean severely affected the demography of shallow benthic populations as has been demonstrated in several population-based studies (e.g. [START_REF] Allcock | Southern Ocean diversity: New paradigms from molecular ecology[END_REF][START_REF] Fraser | Kelp genes reveal effects of subantarctic sea during the last glacial maximum[END_REF][START_REF] Gonz Alez-Wevar | Extinction and recolonization of maritime Antarctica in the limpet Nacella concinna (Strebel, 1908) during the last glacial cycle: Toward a model of Quaternary biogeography in shallow Antarctic invertebrates[END_REF][START_REF] Gonz Alez-Wevar | Towards a model of postglacial biogeography in shallow marine species along the Patagonian Province: Lessons from the limpet Nacella magellanica (Gmelin, 1791)[END_REF], 2016;[START_REF] Nikula | Circumpolar dispersal by rafting in two subantarctic kelp-dwelling crustaceans[END_REF][START_REF] Thornhill | Open-ocean barriers to dispersal: A test case with the Antarctic Polar Front and the ribbon worm Parborlasia corrugatus (Nemertea: Lineidae)[END_REF]. Repeated ice advances and retreats would have generated massive reductions in population sizes (or even local extinctions) and consequent severe declines in genetic diversity. A key signature of populations formerly confined to ancient glacial refugia should be higher and older levels of genetic diversity compared to populations from recently recolonized areas [START_REF] Allcock | Southern Ocean diversity: New paradigms from molecular ecology[END_REF][START_REF] Gonz Alez-Wevar | Extinction and recolonization of maritime Antarctica in the limpet Nacella concinna (Strebel, 1908) during the last glacial cycle: Toward a model of Quaternary biogeography in shallow Antarctic invertebrates[END_REF][START_REF] Provan | Phylogeographic insights into cryptic glacial refugia[END_REF]. Moreover, populations confined to geographically separate refugia are expected to show genetic differentiation due to genetic drift [START_REF] Provan | Phylogeographic insights into cryptic glacial refugia[END_REF]. Accordingly, we hypothesize that both species of Siphonaria became extinct at KI and MI during the LGM, before they recolonized by rafting during the glacial-interglacial transition from geographically distant refugia, such as southern South America, where glacial impact is known to have been lower, particularly along the Atlantic coast [START_REF] Rabassa | Chronology of the Late Cenozoic Patagonian glaciations and their correlation with biostratigraphic units of the Pampean region (Argentina)[END_REF]. Such a process could have occurred several times, during the Plio-Pleistocene, and generated a 'demographic reset' during glacial maxima on the sub-Antarctic islands followed by interglacial recolonization.

Unexpectedly, our results showed that haplotypes of S. lateralis from FI are clearly differentiated (≥8 steps away) from those of other localities, including adjacent areas such as Patagonia. Evidence of genetic differentiation between PP and FI populations has been recorded in crustaceans like Parawaldeckia kidderi [START_REF] Nikula | Circumpolar dispersal by rafting in two subantarctic kelp-dwelling crustaceans[END_REF], and Serolis paradoxa [START_REF] Leese | Cryptic speciation in a benthic isopod from Patagonian and Falkland Island waters and the impact of glaciations on its population structure[END_REF] and limpets of the genus Nacella [START_REF] Gonz Alez-Wevar | Towards a model of postglacial biogeography in shallow marine species along the Patagonian Province: Lessons from the limpet Nacella magellanica (Gmelin, 1791)[END_REF], 2016). Due to their geographical position, the FI were less affected by Quaternary glaciations than other areas of the Southern Ocean [START_REF] Hodgson | Terrestrial and submarine evidence for the extent and timing of the Last Glacial Maximum and the onset of deglaciation on the maritime-Antarctic and sub-Antarctic islands[END_REF]. Hence, Siphonaria populations could have persisted and differentiated there during the LGM. In S. fuegiensis, populations from the FI and those of AP share two unique haplotypes, suggesting a closer connectivity between the two areas. There is, therefore, a contrast between the two Siphonaria species and their dispersal patterns between these areas. This is an unexpected finding considering the overall morphological and phylogeographical similarities recorded between the two taxa. In S. lateralis, the presence of a fixed SG haplotype indicates that this island may be currently isolated from other areas analysed with the Antarctic Polar Front acting as an oceanographic barrier between the Antarctic and the sub-Antarctic [START_REF] Poulin | Divergence between Antarctic and South American marine invertebrates: What molecular biology tells us about Scotia Arc geodynamics and the intensification of the Antarctic Circumpolar Current[END_REF]. Such a barrier could also explain the absence of S.

fuegiensis in SG. Alternatively, the stochasticity of rafting could also be used to explain the differences in phylogeographical structure recorded among Patagonia, FI and SG. Just by chance, S. fuegiensis may have rafted between FI and AP, but S. lateralis not. Similarly, by chance, just a single S. lateralis haplotype may have reached SG since the last deglaciation but no S. fuegiensis individuals successfully colonized this sub-Antarctic island during that period.

| CONCLUSION S

New biogeographical studies in Southern Ocean near-shore marine benthic invertebrates help us understand the evolution of biota at higher latitudes [START_REF] Allcock | Southern Ocean diversity: New paradigms from molecular ecology[END_REF][START_REF] Allcock | Cryptic speciation and the circumpolarity debate: A case study on endemic Southern Ocean octopuses using the COI barcode of life. Deep-Sea Research Part II[END_REF][START_REF] Fraser | Kelp genes reveal effects of subantarctic sea during the last glacial maximum[END_REF][START_REF] Fraser | Oceanic rafting by a coastal community[END_REF][START_REF] Gonzalez-Wevar | Following the Antarctic circumpolar current: patterns and processes in the biogeography of the limpet Nacella (Mollusca: Patellogastropoda) across the Southern Ocean[END_REF][START_REF] Moon | Reconsidering connectivity in the sub-Antarctic[END_REF][START_REF] Poulin | Divergence between Antarctic and South American marine invertebrates: What molecular biology tells us about Scotia Arc geodynamics and the intensification of the Antarctic Circumpolar Current[END_REF][START_REF] Sauc Ede | Biogeographic processes in the Southern Ocean[END_REF][START_REF] Waters | Driven by the west wind drift? A synthesis of southern temperate marine biogeography, with new directions for dispersalism[END_REF].

Direct developing species like the pulmonates S. lateralis and S.

fuegiensis show clear evidence of long-distance dispersal across the sub-Antarctic, whereas broadcast-spawning species like Nacella are clearly isolated and separated in the sub-Antarctic islands [START_REF] Gonzalez-Wevar | Following the Antarctic circumpolar current: patterns and processes in the biogeography of the limpet Nacella (Mollusca: Patellogastropoda) across the Southern Ocean[END_REF][START_REF] Gonz Alez-Wevar | Molecular phylogeny and historical biogeography of Nacella (Patellogastropoda: Nacellidae) in the Southern Ocean[END_REF]. We argue that the geographical distribution of genetic lineages in the Southern Ocean depends on idiosyncratic life-history traits related to the ecology of the species, such as the association with buoyant kelps. In this context, our study suggests that species combining direct development and rafting may be more effective for long-distance dispersal than those species with free-living planktotrophic larval stages. Hence, biogeographical patterns in these organisms also depend on the stochasticity of long-distance dispersal that is mediated by particular vectors. Moreover, in intertidal and shallow water species, the processes of local extinction and recolonization driven by glacial cycles may have prevented speciation across the sub-Antarctic islands.

ACKNOWLEDG EMENTS

2. 1 |

 1 Sample collection, DNA preparation and sequence editing Siphonaria individuals were sampled from around the Southern Ocean (Figure 1; Figure S1 in Appendix S1) including Pacific Patagonia (PP), Atlantic Patagonia (AP), the Falkland/Malvinas Islands (FI), South Georgia (SG), Kerguelen Islands (KI) and Macquarie Island (MI) (Table

  U R E 1 (a) Distribution of Siphonaria lateralis around the Southern Ocean showing major oceanographic currents and subdivisions where STF, subtropical front, SAF, sub-Antarctic front, PF, polar front. Stars indicate records of the species (Griffiths & Waller, 2016) and coloured areas show sampling sites in different provinces of the Southern Ocean (red, Pacific Patagonia; orange, Atlantic Patagonia; blue, South Georgia; yellow, Falkland/Malvinas Islands; green, Kerguelen archipelago; purple, Macquarie Island). (b) Syntopic individuals of S. lateralis (red) and S. fuegiensis (yellow) recorded at Kerguelen Islands. (c) Individuals of Siphonaria living closely associated with the bull-kelp Durvillaea antartica. (de) Siphonaria individuals living encased in kelp holdfasts at Kerguelen archipelago 2.2 | Phylogenetic reconstructions and divergence time estimates Mitochondrial (COI) DNA reconstructions used new and previously published

  Clade A[START_REF] Dayrat | Diversity of Indo-West Pacific Siphonaria (Mollusca: Gastropoda: Euthyneura)[END_REF] included 268 variable positions (40.79%) of which 259 (96.64%) were parsimony informative. Mitochondrial sequences were A-T rich (65.7%). In contrast, nuclear 28S rRNAs (n = 36) and ITSs sequences (n = 40) were C-G rich (55.5% and 60.7% respectively). Mitochondrial and nuclear sequences were not saturated at any position. No evidence for mtDNA codon bias was detected (ENC = 39.70).3.1 | Phylogenetic reconstructionsMitochondrial reconstructions corroborated previous molecular studies in Siphonaria and discriminated major taxonomic lineages within Clade A of Dayrat et al. (2014), with high bootstrap values and posterior probabilities (Figure 2). All the reconstruction methods (MP, ML and BA) and markers showed S. lateralis and S. fuegiensis as sister species (Figure 2 and Figure S2 in Appendix S1). Uncorrected pvalues between these species are 6.4% and 3.0% for COI and ITS (Figure 3a & b) respectively. In contrast, S. lateralis and S. fuegiensis differed by just a single base pair for 28S rRNA (Figure 3c), highlighting the low resolution of this marker in siphonariids. Even while COI and ITS registered high levels of genetic divergence (>12%), the 28S rRNA recorded only three substitutions between S. lessonii and S. fuegiensis/S. lateralis. Divergence time estimates based on mtDNA (COI) sequences, under a 95% Bayesian confidence credible interval for BCI for relative divergence times (Figure S3 in Appendix S1), suggest that the separation between S. lateralis and S. fuegiensis occurred during the Pliocene, ~4 Ma (3-8 Ma).

  Within S. lateralis, mtDNA reconstructions showed three subclades: (1) Pacific Patagonia (PP) + Atlantic Patagonia (AP) + Kerguelen Islands (KI) + Macquarie Island (MI), (2) South Georgia (SG) and (3) the Falkland/Malvinas Islands (FI) (Figure 2). Within S. fuegiensis, the COI phylogeny showed two subclades: (1) PP+KI+MI and (2) FI+AP (Figure 2), being this species absent in SG. Multi-locus reconstructions recovered similar subdivisions within S. lateralis and S. fuegiensis (Figure S2 in Appendix S1). Considering the low resolution of the 28S rRNAs, this marker failed to resolve any grouping within high latitude Siphonaria.

  included 395 and 319 individuals, respectively, and consisted of 687 nucleotide positions coding for 229 amino acids. Neither indels or stop codons, nor any amino acid change were recorded in the COI data matrix, even when S. lateralis and S. fuegiensis were separated by a total of 44 substitutions (Figure 3a). ITS sequences for siphonariids (n = 40) consisted of 1,282 nucleotide positions. Several indels were recorded in the ITS data matrix and S. lateralis and S. fuegiensis were separated by 39 substitutions. Finally, 28S rRNA sequences included a total of 60 individuals and consisted of 698 nucleotide positions.Across their respective distributions, very low levels of mtDNA genetic variation characterized the standard diversity indices (S, H, k, Π and p) in both S. lateralis and S. fuegiensis (TablesS2 and S3in Appendix S2). In fact, 10 out of 14 S. lateralis localities showed no genetic diversity (H = 0). Similarly, H indices in S. fuegiensis were relatively low and fluctuated between 0.171 (Baie du Prince de Monaco) and 0.605 (Diego Ram ırez Island).

  U R E 3 (a) mtDNA (COI) Maximum parsimony network including 714 sub-Antarctic individuals of Siphonaria (S. lateralis and S. fuegiensis) collected at different localities in the Southern Ocean. (b) & (c) nucDNA Maximum parsimony networks based on ITS and 28S rRNA sequences respectively. Each haplotype is represented by a coloured circle indicating the main area where it was collected. The size of the circle is proportional to its frequency in the whole sampling effort

  4 | DISCUSSION Our results show that two species of pulmonate limpet, Siphonaria lateralis and S. fuegiensis, occur across vast distances of the Southern Ocean, from the Pacific and Atlantic coasts of Patagonia eastward to the Falkland/Malvinas (FI), South Georgia (SG), Kerguelen (KI) and Macquarie Islands (MI). The presence of two such widespread directdeveloping species is a surprise considering that several studies have revealed locally endemic cryptic species on different island groups in the Southern Ocean across different marine invertebrate groups

  The vast distributions recorded in both S. fuegiensis and S. lateralis, together with the extensive sharing of mitochondrial haplotypes among far-flung populations (Patagonia-Kerguelen-Macquarie) and the low levels of nucleotide polymorphism, imply that both species are good long-distance dispersers. It might seem unlikely that directdeveloping pulmonates would be able to travel across several thousand kilometres of open ocean. Nevertheless, previous studies have demonstrated striking discrepancies between dispersal potential and

  This work was supported by Fondecyt Initiation Project 11140087 and Program FONDAP, Project N°15150003 to C.A.G-W; Projects P05-002 ICM, PFB 023 (Instituto de Ecolog ıa y Biodiversidad IEB, Universidad de Chile), and project GAB (ACT172065) to C.A.G-W and E.P. Fondecyt Regular Project 1151336 to E.P and C.A.G-W. The Allan Wilson Centre supported H.G.S. Field access facilities at Kerguelen Archipelago were supported by IPEV program 1044 Proteker to E.P, C.A.G-W, J-P. F. and T.S. We mostly appreciate the editorial assistance of Dr. Jon Waters and Dr. Peter Linder for their comments and suggestions. At the same time, four anonymous reviewers helped us significantly to improve the article. This article contributes to the SCAR 'State of Antarctic Ecosystem' (AntEco) programme.

  ST and G ST comparisons, S nn and SAMOVA

	S nn and SAMOVA all clearly discriminated three groups in this
	species: (1) PP/AP/KI, (2) FI and (3) SG. SAMOVA analyses of S.
	lateralis showed that among-group variation accounts for 95.01%
	of the total variance (Table S6 in Appendix S2). Similarly, the
	nearest-neighbour statistic algorithm recorded high levels of phylo-
	geographical signal (S nn = 1.000 p < .0001) among these SAMO-
	VA's recognized groups.
	Within S. fuegiensis, average G ST (0.229) and N ST (0.300) values
	for COI were also high as a consequence of the marked differentia-
	tion of FI/AP from the remaining populations (Table S5 in
	Appendix S2). Pairwise N

Genetic structure in Siphonaria species across the sub-Antarctic Mean general values of COI differentiation measured over 14 populations of S. lateralis were high, especially average G ST

(0.239)

and N ST (0.259) (Table

S4 in Appendix S2

). Nevertheless, these results must be regarded with caution because of the presence of endemic haplotypes at SG and FI that strongly separated these localities from the rest. In fact, pairwise N ST and G ST comparisons, in S. fuegiensis showed two clearly differentiated groups: (1) PP/KI and (2) FI/AP. SAMOVA analyses indicated that variation among these groups accounted for 64.41% of the total variance (Table

S7

in Appendix S2). Similarly, the S nn statistic recorded high levels of phylogeographical signal (S nn = 1.000 p > .0001) among the recognized SAMOVA groups.

Fondecyt Initiation Project, Grant/Award Number: 11140087; Instituto de Ecolog ıa y Biodiversidad, Grant/Award Number: P05-002 ICM, PFB 023; Fondecyt Regular Project, Grant/Award Number: 1151336; IPEV program 1044 Proteker

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