Quentin Jossart

Marc Kochzius

Bruno Danis

Thomas Saucède

Camille V E Moreau

Diversity of the Pterasteridae (Asteroidea) in the Southern Ocean: a molecular and morphological approach

An integrative approach is crucial in discrimination of species, especially for taxa that are difficult to identify on a morphological basis. In this study, we combined genetics and morphology to assess the diversity of the Pterasteridae, a sea star family diversified in deep-sea and polar environments. Because of their derived anatomy and the frequent loss of characters during preservation, the Pterasteridae are a case in point for an integrative study. The molecular identification of 191 specimens (mostly from the Southern Ocean) suggested 26 to 33 species from 3 genera (Hymenaster, Diplopteraster, Pteraster), which matched the morphological identification in 54 to 62 % of cases. The mismatches were either different molecular units that are morphologically indistinguishable (e.g. Pteraster stellifer units 2&4) or, conversely, nominal species being genetically identical (e.g. Hymenaster coccinatus/densus/praecoquis). Several species were shared between Northern and Southern Hemispheres (e.g. Pteraster jordani/affinis). In conclusion, the taxonomic status of some groups was confirmed but we found, for others, the need to reevaluate the taxonomy at both the genus and species levels. This work significantly increases the DNA barcode library of the Southern Ocean species and merged taxonomic information into an identification key that could be a baseline for future studies (pterasteridae-so.identificationkey.org).

INTRODUCTION

Taxonomy has a pivotal role in biology [START_REF] Costello | More taxonomists describing significantly fewer species per unit effort may indicate that most species have been discovered[END_REF]. Inaccurate identifications and naming lead to misunderstandings and spurious interpretations of biological processes in various domains of life sciences [START_REF] Dayrat | Towards integrative taxonomy[END_REF][START_REF] Pante | From integrative taxonomy to species description: one step beyond[END_REF]. Fifteen years ago, integrative taxonomy was introduced as a promising approach to complement the traditional, morphology-based taxonomy, using new data and methods [START_REF] Dayrat | Towards integrative taxonomy[END_REF]. Among these, molecular markers were in the spotlight, considering the simultaneous leaps achieved by new genetic methodologies such as DNA barcoding [START_REF] Stoeckle | Taxonomy, DNA, and the bar code of life[END_REF][START_REF] Hebert | Biological identifications through DNA barcodes[END_REF][START_REF] Hebert | The Promise of DNA Barcoding for Taxonomy[END_REF][START_REF] Ratnasingham | BOLD: The Barcode of Life Data System (http://www. barcodinglife.org)[END_REF][START_REF] Fujita | Coalescent-based species delimitation in an integrative taxonomy[END_REF]. Nowadays, the number of barcoded species is still low compared to the total number of recognized species, less than a quarter of nominal species being barcoded in most phyla [START_REF] Gong | Advances in DNA Barcoding of Toxic Marine Organisms[END_REF]). Nevertheless, a plethora of studies have shown the importance of using genetics and morphology alongside for discrimination at all taxonomic ranks (e.g. [START_REF] Richter | Collapse of a new living species of giant clam in the Red Sea[END_REF][START_REF] Laakmann | Evolution in the deep sea: biological traits, ecology and phylogenetics of pelagic copepods[END_REF][START_REF] Pante | From integrative taxonomy to species description: one step beyond[END_REF][START_REF] Christiansen | Diversity of Mesopelagic Fishes in the Southern Ocean-A Phylogeographic Perspective Using DNA Barcoding[END_REF][START_REF] Peck | Morphological variation in taxonomic characters of the Antarctic starfish Odontaster validus[END_REF][START_REF] Jossart | Dwarf brooder versus giant broadcaster: combining genetic and reproductive data to unravel cryptic diversity in an Antarctic brittle star[END_REF]. Based on the data from the World Register of Marine Species (WoRMS), [START_REF] Appeltans | The magnitude of global marine species diversity[END_REF] showed that molecular methods are significantly increasing our knowledge of marine biodiversity by helping in the detection of cryptic species and the establishment of synonymies. This is of particular importance for under-investigated taxa, such as those including numerous species that remain unrevised since their original descriptions.

The Asteroidea (i.e. sea stars or starfish) is the second most diversified class of echinoderms, with around 1,900 described species assigned to 38 families [START_REF] Mah | Global Diversity and Phylogeny of the Asteroidea (Echinodermata)[END_REF][START_REF] Mah | Description of a new family, new genus, and two new species of deep-sea Forcipulatacea (Asteroidea), including the first known sea star from hydrothermal vent habitats[END_REF]. They show various ecological traits and are present in a broad variety of ecosystems [START_REF] Mah | Global Diversity and Phylogeny of the Asteroidea (Echinodermata)[END_REF][START_REF] Lawrence | Starfish -Biology and Ecology of the Asteroidea[END_REF][START_REF] Moreau | Reproductive strategy as a piece of the biogeographic puzzle: a case study using Antarctic sea stars (Echinodermata, Asteroidea)[END_REF]. For the last twenty years, considerable efforts have been made to reevaluate sea star phylogeny using molecular data [START_REF] Knott | Controversy and consensus in asteroid systematics: new insights to ordinal and familial relationships[END_REF][START_REF] Janies | Echinoderm Phylogeny Including Xyloplax, a Progenetic Asteroid[END_REF]Mah & Foltz 2011a, 2011b;[START_REF] Jr | The phylogeny of extant starfish (Asteroidea: Echinodermata) including Xyloplax, based on comparative transcriptomics[END_REF][START_REF] Moreau | Is reproductive strategy a key factor in understanding the evolutionary history of Southern Ocean Asteroidea (Echinodermata)?[END_REF]. However, there is a sharp contrast in our knowledge of sea star diversity, namely amongst families from different biogeographic regions [START_REF] Feuda | Phylogenetic signal dissection identifies the root of starfishes[END_REF]. This is the case of the Pterasteridae Perrier, 1875, the most diverse family in the Order Velatida, which includes around 130 nominal species and eight genera: Amembranaster Golotsvan, 1998, Benthaster Sladen, 1882, Calyptraster Sladen, 1882, Diplopteraster Verrill, 1880, Euretaster Fisher, 1940, Hymenaster Wyville Thomson, 1873, Hymenasterides Fisher, 1911, Pteraster Müller & Troschel, 1842[START_REF] Mah | World Asteroidea Database[END_REF]. One unique feature of the Pterasteridae is the presence of an additional (supra)dorsal membrane producing abundant quantities of mucus [START_REF] Mah | Global Diversity and Phylogeny of the Asteroidea (Echinodermata)[END_REF][START_REF] Gale | Origin and phylogeny of velatid asteroids (Echinodermata, Neoasteroidea)-new evidence from the Jurassic[END_REF]. Between the dorsal and the supradorsal membranes is a nidamental cavity where incubation of juveniles takes place in some species [START_REF] Janies | Reconstructing the evolution of morphogenesis and dispersal among velatid Asteroids[END_REF]. The taxonomy of the group is complicated for three reasons: (1) morphologies are highly derived and only few diagnostic characters are recognized. Most characters commonly used for species identification in other sea stars are not applicable to the family [START_REF] Gale | Origin and phylogeny of velatid asteroids (Echinodermata, Neoasteroidea)-new evidence from the Jurassic[END_REF]; (2) the few available characters are often indiscernible as specimens are particularly fragile and get damaged by sampling and preservation protocols [START_REF] Villier | A preliminary phylogeny of the Pterasteridae (Echinodermata, Asteroidea) and the first fossil record: Late Cretaceous of Germany and Belgium[END_REF]); (3) several species are only known from their original description based on few (poorly preserved) specimens [START_REF] Villier | A preliminary phylogeny of the Pterasteridae (Echinodermata, Asteroidea) and the first fossil record: Late Cretaceous of Germany and Belgium[END_REF]). Consequently, the Pterasteridae family is a case in point of a group that could benefit from an integrative taxonomic approach.

The family mainly occurs in cold waters from the deep sea to the Arctic and the Southern oceans [START_REF] Mah | Global Diversity and Phylogeny of the Asteroidea (Echinodermata)[END_REF]. Unfortunately, although genetic sequences were available for specimens from the Northern Hemisphere, no public data was published for Southern Ocean species (source: boldsystems.org). International initiatives of the Census of Antarctic Marine Life (CAML) and of the International Polar Year (IPY) have promoted sampling efforts in the Southern Ocean [START_REF] Schiaparelli | The Census of Antarctic Marine Life: the first available baseline for Antarctic marine biodiversity[END_REF]) and this momentum was at the origin of many biologic campaigns until today. This significantly enhanced the taxonomic and spatial coverage of the Southern Ocean biodiversity inventory, including the collection of deep-sea representatives of the Pterasteridae.

These newly and well-preserved specimens have offered the opportunity to reinvestigate the taxonomy of the family. Based on these new samples, we combined morphological and molecular approaches to verify whether their joint use could better assess the diversity within the Pterasteridae. After an initial morphological investigation, we used a mitochondrial gene (Cytochrome c Oxidase subunit I -COI) to verify how it confirms or complements the morphological identification. Subsequently, we reinvestigated (a posteriori) specimens using a morphological approach in order to identify new characters that may differentiate species. Finally, we synthesized, for the first time, the revised taxonomy of the family and made it available to all potential users in an open-access identification key that includes all the Southern Ocean species (pterasteridae-so.identificationkey.org).

MATERIAL & METHODS

Sampling

Specimens were collected during seventeen international campaigns undertaken at sea from 1998 to 2017 (ACE, ANDEEP-3, ANDEEP-SYSTCO, ARGOS, CEAMARC 2007-2008, JR144, JR179, JR262, JR275, JR15005, MD208, MUSORSTOM 10, POKER II, PS77, PS81, PS96). Available specimens cover a wide distribution within the Southern Ocean (Figure 1), including the Patagonian shelf, South Sandwich Islands, South Georgia, South Orkney, Shag Rocks, Kerguelen, Crozet and the Antarctic continental shelf (Adélie Land, Amundsen Sea, Antarctic Peninsula, Weddell Sea). A total of 174 specimens from these locations were included in the analysis. Moreover, in order to increase the taxonomic and geographical scope, as well as the phylogenetic resolution, 20 additional genetic sequences (see below) from specimens outside the Southern Ocean were also added to the dataset (i.e. Fiji, South Africa, Pacific and Atlantic coasts of North America, Norway, Russia). Metadata documenting all the 191 samples can be found in Supplementary Material 1.

Morphological identification

A total of 124 Southern Ocean individuals (preserved in ethanol or frozen) were identified morphologically by the authors using both original descriptions (e.g. [START_REF] Sladen | The Asteroidea of H.M.S. Challenger Expedition, pt. 1. Pterasteridae[END_REF][START_REF] Koehler | Astéries, Ophiures et Echinides de l'Expedition Antarctique National Ecossaise. Scottish National Antarctic Expedition[END_REF], identification books [START_REF] Clark | Asteroidea. Reports of B.A.N.Z. Antarctic Research Expedition, 1929-1931[END_REF][START_REF] Clark | Starfishes of the Atlantic[END_REF][START_REF] Mcknight | The marine fauna of New Zealand, Echinodermata: Asteroidea (Sea-stars)[END_REF] and contemporary scientific literature [START_REF] Villier | A preliminary phylogeny of the Pterasteridae (Echinodermata, Asteroidea) and the first fossil record: Late Cretaceous of Germany and Belgium[END_REF][START_REF] Gale | Origin and phylogeny of velatid asteroids (Echinodermata, Neoasteroidea)-new evidence from the Jurassic[END_REF].

Subsequently to genetic analyses (see below), an "a posteriori" morphological investigation was carried out to look for new characters to differentiate species when new species delineations and synonymies were suggested by genetic data. Finally, the taxonomy of the family was synthesized and made available online, building an interactive identification key through the Xper 3 portal (Figure 2). Xper 3 is a web portal with an easy-to-use interface allowing multiple accesses (the key can be started using any character; [START_REF] Vignes-Lebbe | Xper 3 : nouveaux outils pour le travail collaboratif, la formation et la transmission des connaissances sur les phénotypes végétaux[END_REF]. Specimens and characters were also illustrated by drawings and macro pictures (photographed using a camera with a macro lens, two flashes and accessories to diffuse or reflect the light,Figure 2, Figure 3).

Genetic data

A fragment of the mitochondrial gene "Cytochrome c Oxidase subunit I (COI)" was sequenced (601 base pairs) for the 191 individuals. These genetic sequences were obtained through laboratory works in our institutes (80 individuals, see protocol below), through our private Barcode of Life Data System project (BOLD; 97 individuals) or mined from public BOLD projects (17 individuals).

DNA extractions were performed on one tube foot (podia) per sample and were based on the salting-out protocol of [START_REF] Sunnucks | Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae)[END_REF]. Amplification step was performed using the forward primers "F-COI-PTE-28" (5'-GCTGGAATGATTGGAACTGC-3') or "LCOech1aF1" (5' TTTTTTCTACTAAACACAAGGATATTGG") and the reverse universal primer "R-HCO2198" (5′-TAAACTTCAGGGTGACCAAAAAATCA′; [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome c oxidase sub-unit I from diverse metazoan invertebrates[END_REF]. Each PCR mix (25 μl) included 12.5 μl of a VWR Mastermix (2.5 units of VWR Taq polymerase, 0.4 mM of each dNTP and 1.5mM of MgCl2), 10.5 µl of molecular water, 0.5 μl each primer (10 μM), and 1 μl of the DNA extract. PCR conditions consisted of 35 cycles for each of the three temperature steps [30 s at 95 °C (denaturation), 30 s at 48 °C (annealing), and 30 s at 72 °C (elongation)]. These cycles were preceded by 2 min at 95 °C and were followed by 10 min at 72 °C. EXOSAP purification (incubation at 37 °C for 15 minutes followed by another at 80°C for 15 minutes) was done before the sending of PCR products to the MACROGEN sequencing service. Sequence editing and alignment were done using the software Geneious [START_REF] Kearse | Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data[END_REF]. The absence of stop codon in the sequence was checked in the same software in order to exclude the presence of nuclear pseudogenes.

PartitionFinder2 (BIC criterion, [START_REF] Lanfear | PartitionFinder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses[END_REF]) was used within the CIPRES portal [START_REF] Miller | Creating the CIPRES Science Gateway for inference of large phylogenetic trees in Proceedings of the Gateway Computing Environments Workshop[END_REF] to select the most suitable substitution models (i.e. TRNEF+I+G for codon position 1, HKY+I+X for codon position 2 and TRN+I+G+X for codon position 3). A Bayesian phylogeny was subsequently produced using BEAST 1.8.4 [START_REF] Drummond | BEAST: Bayesian evolutionary analysis by sampling trees[END_REF] within the CIPRES portal. Based on a previous phylogeny using multiple genes, Remaster gourdoni Koehler, 1912 was used as the outgroup [START_REF] Jr | The phylogeny of extant starfish (Asteroidea: Echinodermata) including Xyloplax, based on comparative transcriptomics[END_REF]. Parameters of the analysis were: partitioned dataset, Yule process tree prior, Markov chain Monte Carlo (MCMC) of 100 x 10 6 generations sampled every 10,000 trees. Tracer v1.6 was used to ensure an appropriate effective sampling size (ESS all above 200). TreeAnnotator v1.8.4 was used to find the most likely tree that was visualized in FigTree v1.4.3 (tree.bio.ed.ac.uk/software/figtree). Node support was assessed through posterior probability (pp), with values lower than 0.75 not retained and collapsed into polytomies [START_REF] Huelsenbeck | Frequentist properties of Bayesian posterior probabilities of phylogenetic trees under simple and complex substitution models[END_REF]. Moreover, the software DensiTree 2.2 was used to verify the potentiality of competing topologies among the set of trees [START_REF] Bouckaert | DensiTree: making sense of sets of phylogenetic trees[END_REF]. The ABGD analysis (bioinfo.mnhn.fr/abi/public/abgd) was performed with a relative gap width of 1 and K80 as the genetic distance. The bPTP analysis (species.h-its.org/ptp) was applied using 500,000 generations of MCMC, a thinning of 100 and a burn-in of 25 %. Finally, the GMYC analysis (species.h-its.org/gmyc) was performed using the single threshold method. Haplotype diversity and nucleotide diversity were calculated within

RESULTS

Initial morphological identification

Among the 124 morphologically investigated individuals, 91 were identified to species level. Thirty-two individuals were identified to genus level and one to family level due to the small size of specimens (juveniles) or poor preservation not permitting observation of diagnostic characters. Thirteen different species and three genera were identified (Figure 3). Four species of Pteraster were found: Pteraster affinis Smith, 1876, Pteraster gibber [START_REF] Sladen | The Asteroidea of H.M.S. Challenger Expedition, pt. 1. Pterasteridae[END_REF], Pteraster rugatus Sladen, 1882, and Pteraster stellifer Sladen, 1882. The number and type of marginal oral spines were important diagnostic characters to discriminate the different Pteraster species and these characters are usually well preserved. Specimens of Diplopteraster were only identified up to the genus level due to the absence of observable characters (e.g. between Diplopteraster verrucosus [START_REF] Sladen | The Asteroidea of H.M.S. Challenger Expedition, pt. 1. Pterasteridae[END_REF] and Diplopteraster semireticulatus [START_REF] Sladen | The Asteroidea of H.M.S. Challenger Expedition, pt. 1. Pterasteridae[END_REF]; see discussion). Nine species of Hymenaster were identified: Hymenaster campanulatus Koehler, 1907, Hymenaster coccinatus Sladen, 1882, Hymenaster densus [START_REF] Koehler | Astéries, Ophiures et Echinides de l'Expedition Antarctique National Ecossaise. Scottish National Antarctic Expedition[END_REF], Hymenaster edax Koehler, 1907, Hymenaster formosus Sladen, 1882, Hymenaster latebrosus Sladen, 1882, Hymenaster perspicuus Ludwig, 1903, Hymenaster praecoquis Sladen, 1882 and Hymenaster sacculatus Sladen, 1882. Some of these Hymenaster species were discriminated based on tenuous morphological differences, such as H. densus and H. praecoquis only differentiated based on slight variations in the morphology of the segmental papillae and number of marginal spines [START_REF] Clark | Asteroidea. Reports of B.A.N.Z. Antarctic Research Expedition, 1929-1931[END_REF].

Species delimitation (COI)

Species delimitation methods applied to molecular data gave 26 species hypotheses for ABGD and 33 for bPTP and GMYC (Figure 4). For each species hypothesis, the relationship between haplotype diversity and nucleotide diversity fitted the expected variation for a single species, except for Pteraster stellifer unit 1 (Goodall-Copestake et al. 2012). Morphological species identification only matched with molecular species delimitation in 54 % and 62 % of species for ABGD and GMYC/bPTP, respectively.

Within the genus Pteraster, species complexes were suggested within the nominal species P. stellifer (3 to 5 units) and P. affinis (2 units). Within the genus Hymenaster, three morphological species appeared to be undifferentiated genetically, suggesting that the diversity of the genus was over-estimated (H. coccinatus, H. densus and H. praecoquis). Moreover, four species are present in both the Southern and Northern Hemispheres: Pteraster jordani/affinis, P. militaris/affinis, Diplopteraster sp1 and P. gibber. In every case, Northern and Southern Hemisphere specimens were closely related within these species (p-distance of 0.3 % for P. jordani/affinis and P. militaris/affinis, 0.7 % for Diplopteraster sp1 and 1.2 % for P. gibber).

COI phylogeny

Three main groups were identified: one Hymenaster group (A) and two Diplopteraster/Pteraster groups (B and C; Figure 4). The relationship of group B with the two other groups was unclear, as illustrated by the low posterior probability and the competing topologies from the DensiTree output (Figure 4; Supplementary material 2).

Within group A (Hymenaster), H. campanulatus and unidentified specimens was the sister group of all other Hymenaster (pp = 1). Among these, H. sacculatus formed a subclade with H. formosus, H. perspicuus and H. pellucidus, while H. coccinatus/densus/praecoquis formed another subclade with H. edax and H. latebrosus (pp = 0.85). Group B (Diplopteraster/Pteraster) included Pteraster rugatus as well as the Diplopteraster/Pteraster stellifer complex (pp = 1). Within group C (Diplopteraster/Pteraster), Pteraster gibber was close to P. obscurus, P. tesselatus units 1&2 and Diplopteraster sp1 (pp = 1); while the other subclade included the Pteraster affinis and P. militaris complexes (pp = 1).

A posteriori morphological re-investigation

Subsequently to species delimitation and phylogenetic analysis, new morphological investigations were performed to clarify the mismatch between morphological and molecular data and the status of ambiguous taxa. For unrecognized molecular units, new discriminant morphological characters were potentially identified. First, we found a different number of marginal oral spines (5 versus 3) in two genetic entities initially identified under the same species name P. affinis (i.e. P. jordani/affinis and P. militaris/affinis). Secondly, the presence of a large, clavate suboral spine was found in several specimens of the Diplopteraster/P. stellifer complex, all belonging to P. stellifer unit 1 and P. stellifer unit 5. Thirdly, the morphological re-examination of two closely related molecular units (Pteraster gibber and Diplopteraster sp1) showed that a character state was shared by all specimens of these units. In fact, these specimens had a single web (for two oral plates) among marginal oral spines while the other Pteraster/Diplopteraster specimens harbored free marginal oral spines or a separate web for each plate.

Xper 3 identification key

The Xper3 identification key included 33 distinct species (Figures 2 and3), namely all the species currently accepted in the Register of Antarctic Marine Species (RAMS; [START_REF] Jossart | The Register of Antarctic Marine Species (RAMS): a ten-year appraisal[END_REF][START_REF] Broyer | Register of Antarctic Marine Species[END_REF]. Moreover, an asterisk (and related comment) was attached to each species name, for which there was a mismatch between genetic and morphological identification (i.e. Pteraster affinis, P. stellifer, Diplopteraster clarki, D. hurleyi, D. peregrinator, D. semireticulatus, D. verrucosus, Hymenaster coccinatus, H. densus, H. praecoquis). Fourteen characters were selected, namely the number of arms, the type and number of paxillar spines, the type and number of adambulacral spines, the number of rows of tube feet, the type and number of suboral/marginal oral spines, the morphology of the segmental papillae, the presence of granular bodies in the supradorsal membrane, the opacity of the supradorsal membrane ,the presence of muscle fibres holding the supradorsal membrane and the number of oscular spines.

Several previously used descriptors were evaluated but not retained, because they were not accurately quantifiable (e.g. osculum diameter, density of spiraculae) or undistinguishable in most specimens (e.g. body convexity). Potential new diagnostic characters that could be used in the Pteraster affinis (different number of marginal oral spines) and Diplopteraster/Pteraster stellifer (clavate suboral spine) complexes were mentioned as comments within the key. A particular attention was devoted to make this identification key as user-friendly as possible: (1) the Xper 3 platform allows the user to easily detect the remaining taxa and characters throughout the identification process (Vignes-Lebbe et al. 2016); (2) Any number of characters can be used in any order (multiple accesses key); (3) Numerous macro-pictures and drawings are available, illustrating both whole specimens, characters and character states. This identification key is accessible through the following link: pterasteridae-so.identificationkey.org.

DISCUSSION

Our integrative approach was successful to revise species identity and phylogenetic relationships. The results call for a revision of the taxonomic status of both species and genus within the family Pterasteridae. Such a revision would not be possible without a joint use of morphological investigations and molecular analyses. We identified three of the four genera of Pterasteridae documented in the Southern Ocean: Diplopteraster, Hymenaster and Pteraster, with Calyptraster the only genus not being encountered [START_REF] Mah | World Asteroidea Database[END_REF]. Thirty-three species were identified by the bPTP and GMYC molecular approaches, which was concordant with 54 to 62 % of the morphology-based identifications. Mismatches between morphological and genetic identifications were either due to different molecular units that are morphologically similar or, conversely, to morphological species being genetically identical (see below). Several cases of species shared between high latitudes of the Northern and Southern Hemispheres were found, which could correspond either to cosmopolitanism or bipolarity (species with disjunct distribution sensu [START_REF] Darling | Molecular evidence for genetic mixing of Arctic and Antarctic subpolar populations of planktonic foraminifers[END_REF]. Following the molecular analyses, the return to morphological samples allowed identification of potential new characters that can be used as diagnostic features to define molecular species that were previously undifferentiated based on morphology alone. Merging available morphological and molecular results, we synthesized the taxonomy of the Pterasteridae from the Southern Ocean and built a synthetic, polytomous and open-access identification key that is intended to serve as a precious baseline for future taxonomic and ecological studies.

Finally, this work significantly increases the DNA barcode library for Southern Ocean species. Prior to this study, no genetic sequence of Southern Ocean Pterasteridae was publicly available on GenBank or BOLD platforms. Moreover, the addition of 174 sequences extends by more than three times the public barcode library of the whole family, which also represents a 2.5 % extension for the Asteroidea class and a 0.6 % extension for the Echinodermata phylum (source: boldsystems.org).

Species delimitation

The molecular identification confirmed the species status of several species previously described on a morphological basis only (e.g. Hymenaster campanulatus, H. sacculatus, Pteraster rugatus). On the other hand, discrepancies between morphological and genetic delineations were observed. These are not surprising and illustrate taxonomic uncertainties of the family already discussed in other studies (Clarke & Downey 1992;[START_REF] Mcknight | The marine fauna of New Zealand, Echinodermata: Asteroidea (Sea-stars)[END_REF]. For example, [START_REF] Clark | The echinoderms of southern Africa[END_REF] highlighted the 'ludicrous' situation in Hymenaster, to which more than 50 nominal species have been ascribed over the last 100 years. This is well illustrated in our study by the two genetic entities gathering distinct nominal species: the unit Hymenaster coccinatus/densus/praecoquis and the unit Diplopteraster peregrinator/semireticulatus/verrucosus. Such a taxonomic issue can be related to the inadequacy of morphological characters used to discriminate species, to diagnostic characters between species rather corresponding to intraspecific variations or to the lack of taxonomic investigations of these species since they were originally described [START_REF] Clark | Asteroidea. Reports of B.A.N.Z. Antarctic Research Expedition, 1929-1931[END_REF][START_REF] Clark | Starfishes of the Atlantic[END_REF]. On the other extreme, an interesting case study is provided by the species Pteraster steliffer that corresponds to a species complex (3 to 5 species). Variations within P. stellifer were already noticed by [START_REF] Clark | Asteroidea. Reports of B.A.N.Z. Antarctic Research Expedition, 1929-1931[END_REF] who had proposed to distinguish two subspecies, "Pteraster steliffer stellifer Sladen, 1882" and "Pteraster stellifer hunteri Koehler, 1920", distinct from each other by their geographic distribution and the shape of the paxillar spinelets. Our results also suggest that

Pteraster affinis should encompass several distinct species that, interestingly, are closely related to species recorded in the Northern Hemisphere (see below). Some species within these complexes correspond to unrecognized diversity as we found (a posteriori) potential diagnostic characters (e.g. clavate suboral spines in Pteraster stellifer units 1&5). Some others might represent true cases of cryptic diversity as the morphological re-investigation did not reveal any diagnostic character (e.g. among Pteraster stellifer unit 2 and 4).

Phylogenetic relationships

As expected for a single gene phylogeny, some relationships remained partially unresolved [START_REF] Gontcharov | Are combined analyses better than single gene phylogenies? A case study using SSU rDNA and rbc L sequence comparisons in the Zygnematophyceae (Streptophyta)[END_REF]Christiansen 2008;[START_REF] Sands | Assessing meiofaunal variation among individuals utilising morphological and molecular approaches: an example using the Tardigrada[END_REF]), but most of them are supported by high posterior probabilities. Considering the uncertainty associated with the placement of group B, on the one hand, the relationship between the Hymenaster genus and the two other genera remains unresolved. On the other hand, both Diplopteraster and Pteraster were retrieved in groups B and C. This was also found in previous multiple-gene phylogeny based on Northern Hemisphere species of these two genera (Mah & Foltz 2011b). This also matches our morphological observations that diagnostic characters of the genus Diplopteraster, are doubtful [START_REF] Clark | Starfishes of the Atlantic[END_REF][START_REF] Villier | A preliminary phylogeny of the Pterasteridae (Echinodermata, Asteroidea) and the first fossil record: Late Cretaceous of Germany and Belgium[END_REF]). First, the presence of four rows of tube feet per arm might be incorrect. The close examination of all specimens at hand reveals that they rather possess two rows of overlapping tubefeet. Secondly, the alternating arrangement of adambulacral plates is very difficult to observe. Finally, the presence of an enlarged central paxillar spinelet could not be observed on most specimens. Therefore, we recommend a taxonomic revision of these two genera using both genetic and morphological data. Finally, we did not find any species belonging to the genus Calyptraster in our collection. According to previous studies, it is the sister taxon of the genus Hymenaster, but only a small number of species, records and diagnostic characters are recognized [START_REF] Clark | Starfishes of the Atlantic[END_REF][START_REF] Villier | A preliminary phylogeny of the Pterasteridae (Echinodermata, Asteroidea) and the first fossil record: Late Cretaceous of Germany and Belgium[END_REF]). Moreover, there is no genetic data available (regardless of the gene) for a Calyptraster species. We thus recommend a thorough re-investigation of this genus in order to verify whether it should not be synonymized with Hymenaster.

The molecular phylogeny confirmed the relationships of species already recognized as closely related species such as Hymenaster sacculatus and H. perspicuus [START_REF] Clark | Asteroidea. Reports of B.A.N.Z. Antarctic Research Expedition, 1929-1931[END_REF].

Moreover, the phylogeny highlighted species relationships that were previously unknown, such as the close relationships between Pteraster gibber and P. affinis, rather than with P. stellifer. Another unprecedented result is the affinity of the species P.

jordani/affinis, P. militaris/affinis, Diplopteraster sp1 and P. gibber, recorded both in the Northern and Southern Hemispheres. Depending on the species, this could correspond either to cosmopolitanism or bipolarity. This might indicate a recent migration between the two hemispheres, which would notably be facilitated by deep-sea dispersal routes [START_REF] Strugnell | The thermohaline expressway: the Southern Ocean as a centre of origin for deep-sea octopuses[END_REF][START_REF] Strugnell | The Southern Ocean: source and sink?[END_REF][START_REF] Laakmann | Evolution in the deep sea: biological traits, ecology and phylogenetics of pelagic copepods[END_REF]. In fact, the Pterasteridae are known to be highly diverse and abundant in the deep sea, being one of the most represented sea star families in abyssal basins worldwide [START_REF] Sibuet | Distribution and diversity of asteroids in Atlantic abyssal basins[END_REF][START_REF] Danis | Antarctic Starfish (Echinodermata, Asteroidea) from the ANDEEP3 expedition[END_REF].

CONCLUSION

Our work has confirmed the relevance of using molecular tools to complement morphology-based taxonomy. This is especially true for taxa that are complex to identify on a morphological basis such as the Pterasteridae. In every genus investigated, we found several species for which the taxonomy should be reevaluated and revised. These taxonomic issues generate either unrecognized or conversely, overestimated diversity.

Formal taxonomic revision of these species and genera would be premature at this stage. Further analyses are therefore needed to obtain a better picture of the diversity of the family and precise phylogenetic relationships. This implies an extensive investigation of numerous specimens, a thorough taxonomic revision and morphological survey of holotypes. Some characters seem promising as new diagnostic features of species (e.g. oral spines), while other characters currently used should be abandoned (e.g. opacity of the supradorsal membrane), considering their intraspecific variability and their problematic preservation in collection specimens. The investigation of arm ossicles through X-ray photography and electron microscopy (SEM) also constitutes a promising prospect. Former studies of ossicle arrangement and morphology provided useful taxonomic information in different asteroid groups [START_REF] Gale | Origin and phylogeny of velatid asteroids (Echinodermata, Neoasteroidea)-new evidence from the Jurassic[END_REF][START_REF] Fau | Comparative anatomy and phylogeny of the Forcipulatacea: insights from ossicle morphology[END_REF]). In the case of Pterasteridae, primary radials (supporting the osculum) and adambulacrals seem the most pertinent ossicles to be investigated [START_REF] Gale | Origin and phylogeny of velatid asteroids (Echinodermata, Neoasteroidea)-new evidence from the Jurassic[END_REF]. Besides morphological studies, the use of additional nuclear genes would be interesting to properly turn the primary species hypotheses proposed here into secondary species hypotheses [START_REF] Sands | Assessing meiofaunal variation among individuals utilising morphological and molecular approaches: an example using the Tardigrada[END_REF][START_REF] Abdelkrim | Delimiting species of marine gastropods (Turridae, Conoidea) using RAD sequencing in an integrative taxonomy framework[END_REF]). In addition, the use of multiple genomic markers would be key to fully resolve phylogenetic relationships within the family and analyze phylogeographic patterns within species. This is, for example, a necessary condition to test different colonization scenarios between the two hemispheres for cosmopolitan or bipolar species.

  Three different methods of species delimitation were used to propose primary species hypotheses: one distance-based (ABGD-Automatic Barcode Gap Discovery: Puillandre et al. 2012) and two tree-based (bPTP-Bayesian Poisson Tree Process: Zhang et al. 2013 and GMYC-Generalized Mixed Yule Coalescent: Fujisawa & Barraclough 2013).

FIGURE 2 -

 2 FIGURE 2 -Interface of the Xper 3 identification key (top) and two examples of integrated drawings illustrating diagnostic characters (adambulacral spines: bottom left; oscular spines: bottom right).

FIGURE 3 -FIGURE 4 -

 34 FIGURE 3 -Aboral view of Pterasteridae species illustrating their Southern Ocean diversity. A: Diplopteraster sp; B: Hymenaster campanulatus; C: Hymenaster praecoquis; D: Hymenaster edax; E: Hymenaster sacculatus; F: Pteraster gibber; G: Pteraster affinis; H: Pteraster koelheri; I: Pteraster stellifer. Scale bars : 1 cm.

each species using DnaSP 6[START_REF] Rozas | DnaSP 6: DNA sequence polymorphism analysis of large data sets[END_REF]).