Karin Gérard

Nicolas Bierne

Philippe Borsa

Anne Chenuil

Jean-Pierre Féral

Pleistocene separation of mitochondrial lineages of Mytilus spp. mussels from Northern and Southern Hemispheres and strong genetic differentiation among southern populations

Smooth-shelled mussels, Mytilus spp., have an antitropical distribution. In the Northern Hemisphere, the M. edulis complex of species is composed of three genetically well delineated taxa: M. edulis, M. galloprovincialis and M. trossulus. In the Southern Hemisphere, morphological characters, allozymes and intron length polymorphisms suggest that Mytilus spp. populations from South America and Kerguelen Islands are related to M. edulis and those from Australasia to M. galloprovincialis. On the other hand, a phylogeny of the 16S rDNA mitochondrial locus demonstrates a clear distinctiveness of southern mussels and suggests that they are related to Mediterranean M. galloprovincialis. Here, we analysed the faster-evolving cytochrome oxidase subunit I locus. The divergence between haplotypes of populations from the two hemispheres was confirmed and was found to predate the divergence between haplotypes of northern M. edulis and M. galloprovincialis. In addition, strong genetic structure was detected among the southern samples, revealing three genetic entities that correspond to (1) South America and Kerguelen Island, (2) Tasmania, (3) New Zealand. Using the trans-Arctic interchange as a molecular clock calibration, we estimated the time since divergence of populations from the two hemispheres to be between 0.5 million years (MY) and 1.3 MY (average 0.84 MY). The contrasting patterns observed for the nuclear and the organelle genomes suggested two alternative, complex scenarios: two trans-equatorial migrations and the existence of differential barriers to mitochondrial and nuclear gene flow, or a single trans-equatorial migration and a view of the composition of the nuclear genome biased by taxonomic preconception.

Introduction

Smooth-shelled mussels (Mytilus edulis L., M. galloprovincialis Lmk., M. trossulus Gould) are among those emblematical marine invertebrates that have an antitropical distribution-i.e. present in the cold and temperate waters of both hemispheres but absent from the tropics [START_REF] Briggs | Antitropical distribution and evolution in the Indo-West Pacific Ocean[END_REF][START_REF] Vermeij | Trans-equatorial connections between biotas in the temperate eastern Atlantic[END_REF]. As for other antitropically distributed species, the analysis of the genetic relationships between populations of the two hemispheres may allow to infer the sense and timing of trans-equatorial migration [START_REF] Bowen | Phylogeography of the sardines (Sardinops spp.): assessing biogeographical models and populations histories in temperate upwelling zones[END_REF]. Although introgressive hybridization occurs in each pair of smooth-shell Mytilus species [START_REF] Skibinski | Aspects of the population genetics of Mytilus (Mytilidae; mollusca) in the British Isles[END_REF][START_REF] Rawson | Asymmetric introgression of mitochondrial DNA among European populations of blue mussels (Mytilus spp.)[END_REF][START_REF] Bierne | Introgression patterns in the mosaic hybrid zone between Mytilus edulis and M. Galloprovincialis[END_REF], the genetic composition of the three taxa is now globally well defined in the Northern Hemisphere [START_REF] Koehn | The genetics and taxonomy of species in the genus Mytilus[END_REF]Quesada et al., 1998a;[START_REF] Daguin | Phylogéographie des moules du complexe d'espèces Mytilus edulis[END_REF].

Using allozyme and morphometric characters, [START_REF] Mcdonald | Allozymes and morphometric characters of three species of Mytilus in the Northern and Southern Hemispheres[END_REF] first assigned populations from South America and Kerguelen Islands to M. edulis and those from Australasia to M. galloprovincialis. Further analysis, which added nuclear DNA markers to the available allozyme loci, globally supported this assignment but highlighted the endemic particularities of southern Mytilus spp. [START_REF] Daguin | Genetic relationships of Mytilus galloprovincialis Lmk. Populations worldwide: evidence from nuclear-DNA markers[END_REF][START_REF] Borsa | Genomic reticulation indicates mixed ancestry in Southern Hemisphere Mytilus spp. mussels[END_REF]. For instance, Kerguelen mussels present a genome of mixed M. edulis and M. galloprovincialis ancestry while Tasmanian mussels possess a predominantly M. galloprovincialis genomic background introgressed by M. edulis alleles fixed at some loci, pointing to past introgression [START_REF] Borsa | Genomic reticulation indicates mixed ancestry in Southern Hemisphere Mytilus spp. mussels[END_REF]. To date the divergence between southern and northern Mytilus spp., [START_REF] Hilbish | Origin of the antitropical distribution pattern in the marine mussels (Mytilus spp.): routes and timing of transequatorial migration[END_REF] have inferred their phylogeny at the 16S rDNA mitochondrial locus (16S). Most haplotypes from the Southern Hemisphere populations cluster into a single clade closely related to the clade D typical of Mediterranean M. galloprovincialis, and the divergence between the two clades supports a southward migration in the Pleistocene; South American populations are not differentiated from Australasian populations. In addition, some haplotypes typical of northern M. edulis were found in the Kerguelen population, suggesting a recent introduction [START_REF] Hilbish | Origin of the antitropical distribution pattern in the marine mussels (Mytilus spp.): routes and timing of transequatorial migration[END_REF]. Levels of diversity and divergence observed at the 16S locus in Mytilus spp. are low and the resolution of the phylogenetic relationships among haplotypes is poor. The cytochrome oxidase subunit I (COI) gene evolves faster than the 16S rDNA [START_REF] Mueller | Evolutionary rates, divergence dates, and the performance of mitochondrial genes in bayesian phylogenetic analysis[END_REF] and this holds true in Mytilus spp. [START_REF] Wares | Phylogeography and historical ecology of the North Atlantic interdidal[END_REF][START_REF] Riginos | Differential patterns of male and female mtDNA exchange across the Atlantic Ocean in the blue mussel, Mytilus edulis[END_REF].

The aim of the present study was to pursue the work of [START_REF] Hilbish | Origin of the antitropical distribution pattern in the marine mussels (Mytilus spp.): routes and timing of transequatorial migration[END_REF] by (i) increasing the sampling effort at the 16S locus, especially in the Kerguelen population, where the nuclear genome is supposed to be the result of reticulated evolution, (ii) analysing the faster-evolving COI locus and (iii) re-evaluating relative divergence times between southern and northern Mytilus spp. mussels, as well as attempting absolute dating with this new dataset.

Material and methods

Sampling and mtDNA sequencing

Mussels were collected between 1997 and 2004 at 13 sampling sites in Italy (CHG, equal to sample CHI of [START_REF] Daguin | The zone of sympatry and hybridization of Mytilus edulis and M. galloprovincialis, as described by intron length polymorphism at locus mac-1[END_REF]), Chile (CHL, MAU, PAT), South Africa (AFR), Kerguelen Islands (KER), Australia (AUS), Tasmania (CBL, SIM, HOB) and New Zealand (DUN, WHB, GRS). Details on sampling locations are in Table 1 and in legend to Fig. 1. Pieces of gill and mantle were preserved in 95% ethanol. DNA was extracted using Chelex 100 Ó chelating resin [START_REF] Walsh | Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material[END_REF]. The 417 bp fragment of the 16S locus was amplified by polymerase chain reaction (PCR) using the two primers 16S-RA (5 0 CGCCTGTTTATCAAAAACAT 3 0 ) and 16S-RB (5 0 CCGGTCTGAACTCAGATCACGT 3') [START_REF] Palumbi | The Simple Fool's Guide to PCR[END_REF]. The PCR program consisted of 5 min at 95 °C, then 35 cycles of 95 °C for 1 min, 54 °C for 45 s, 72 °C for 1 min and a final extension of 7 min at 72 °C. A 645 bp fragment of the COI gene was amplified using the forward primer LCO1490 (5 0 GGTCAACAAATCATAAAGA TATTGG 3 0 ) [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF], the reverse primer AM-HCO (5 0 TAAACYTCAGGGTGMCCAAAAAAYCA 3 0 ) and the same PCR program as above with an annealing temperature of 59 °C. PCR products were sequenced directly. Only female mitochondrial sequences were considered in the present study. Chromatograms were inspected visually and the sequences were aligned using CLU-STALW [START_REF] Thompson | ClustalW: improving the sensitivity of progressive multiple alignment through sequence weighting, positions-specific gap penalties and weight matrix choice[END_REF] implemented in BIOEDIT [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows95/98/NT[END_REF]. 16S and COI gene sequences were submitted to EMBL (http://www.embl-heidelberg.de/) under accession numbers AM904568-AM904606 and AM905146-AM905225, respectively (Table 1).

Population genetics analysis

Haplotype diversity and levels of nucleotide diversity estimated from the number of polymorphic sites (h W ; [START_REF] Watterson | On the number of segregating sites in genetic models without recombination[END_REF] and from pairwise differences (h p ; [START_REF] Nei | Molecular Evolutionary Genetics[END_REF] were computed using DNAsp [START_REF] Rozas | DnaSP, DNA polymorphism analysis by the coalescent and other methods[END_REF]. Genetic differentiation between populations was estimated with the U-analogues [START_REF] Excoffier | Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data[END_REF]) of [START_REF] Wright | The interpretation of population structure by F-statistics with special regard to systems of mating[END_REF] F-statistics, by using the software ARLEQUIN 3.0 [START_REF] Excoffier | Arlequin ver. 3.0: An integrated software package for population genetics data analysis[END_REF]. The null distribution of pairwise U ST values under the hypothesis of no difference among populations was obtained by permutating haplotypes between populations. The p-value given by the test is the proportion of U ST pseudo-values generated by random permutations that are larger than or equal to the observed value. In order to visualize the genetic similarities among samples, non-metric multidimensional scaling was used on the matrix of U ST values (MDS; Kruskal and Wish, 1978).

Phylogenetic analysis

To our sequence set, we added the northern and southern Mytilus haplotypes of the 16S locus and northern haplotypes of the COI gene available from the literature and from the EMBL database. The sources of the complete sequence set are detailed in Table 1. These sequences were respectively 405 bp and 384 bp long.

Phylogenetic reconstructions were performed using distance and maximum-likelihood approaches. The most appropriate model of evolution as determined by MrAIC [START_REF] Nylander | MrAIC.pl. Program distributed by the author[END_REF] was the Tamura-Nei nucleotide substitution model (TN93; [START_REF] Tamura | Estimation of the number of nucleotide substitution in the control region of mitochondrial DNA in Humans and Chimpanzees[END_REF]. Distance trees were constructed with the neighbourjoining (NJ) algorithm using TN93 distances with MEGA version 3.1 [START_REF] Kumar | MEGA 3: integrated software for molecular evolutionary genetics analysis and sequence alignment[END_REF]. Maximum-likelihood (ML) trees were built using PHYML [START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF]. Levels of support of tree nodes were determined using 1000 and 100 bootstrap replications of the original data for NJ and ML methods, respectively.

Estimating the time of the trans-equatorial migration

Mutation rate constancy at the COI locus in smooth-shelled mussels Mytilus spp. was checked using [START_REF] Tajima | Simple methods for testing molecular clock hypothesis[END_REF] test of relative rate implemented in MEGA 3.1. To perform this test, we used M. californianus (Accession No. U68776) as outgroup, as it represents the closest known taxon to the smooth-shelled mussels. To estimate the net nucleotide divergence, d, we assumed that the ancestral coalescence time was similar to the present one and estimated h p from the available samples. Then, h p /2 was subtracted from the average divergence d xy [START_REF] Nei | Mathematical model for studying genetic variation in terms of restriction endonucleases[END_REF]. Among the available samples, we used the five largest samples (all with size greater than 15 individuals), including two samples from the Southern Hemisphere (PAT and KER, AM905187-AM905211), two samples of M. edulis from Sweden (AY723897-AY723917) and Nova Scotia (AF241951-AF24195157, AY130016-AY130023, AY723881-AY723887, AY723889, AY723890) and a sample of M. trossulus (AY130061-AY130067, AF242027-AF242029, AF242031-AF242035). We used the opening of the Bering Strait, 3.5 million years (MY) ago as a calibration point to date the divergence between M. trossulus and North Atlantic M. edulis populations, following [START_REF] Wares | Phylogeography and historical ecology of the North Atlantic interdidal[END_REF] and [START_REF] Riginos | Differential patterns of male and female mtDNA exchange across the Atlantic Ocean in the blue mussel, Mytilus edulis[END_REF]. Although the validity of this hypothesis can be discussed, it provides at least a basis for comparison with other studies.

Results

Population genetics of southern Mytilus spp. mussels

Among the 224 sequences at the 16S locus, 20 haplotypes with 19 polymorphic sites were identified; whereas among the 171 sequences of the COI gene, we scored 59 haplotypes, with 104 polymorphic sites. Consequently, the level of polymorphism expressed by nucleotide and haplotype diversities, was much higher for the COI locus than for the 16S locus (Table 2). Nucleotidic variation at the 16S locus was low and mainly composed of singleton mutations, while the diversity of the COI locus was higher. Genetic differentiation between populations was observed at both loci but was stronger at the COI locus (global F ST = 0.8, p < 0.0001) than at the 16S locus. Fig. 2 depicts the genetic differences between southern samples for the COI locus.

Four groups of samples sharing low U ST values appeared, three of which correspond to geographic regions: South America, Tasmania and New Zealand. The fourth group was composed of geographically separated samples (CHL, AFR, AUS). Non-significant U ST values were observed among samples from New Zealand, between two samples from Tasmania (CBL and SIM), and between CHL and AFR (Fig. 2).

Phylogenetic relationships

The phylogenetic relationships among Mytilus spp. haplotypes are presented in Fig. 3A (16S locus) and Fig. 3B (COI locus). The topology of the ML tree was identical to that of the NJ tree as far as nodes supported by bootstrap scores over 50% are concerned. Only the NJ trees were presented, with the bootstrap scores of each NJ and ML phylogenies indicated on the branches. Tree nodes generally were supported by higher bootstrap values in the COI tree than in the 16S tree. The M. trossulus sample was not polluted by introgression and we were able to use it as a robust closely related outgroup. In agreement with previous results [START_REF] Hilbish | Origin of the antitropical distribution pattern in the marine mussels (Mytilus spp.): routes and timing of transequatorial migration[END_REF], the majority of haplotypes sampled in the Southern Hemisphere clustered in a single clade. We called this clade 'S' (for 'Southern'). Clade S was distinct from the haplogroup made of all the haplotypes sampled in the Northern Hemisphere and forming a clade here called N ('Northern'). This result was supported in the COI tree (Fig. 3B) with higher bootstrap values than in the 16S tree (Fig. 3A).

Clade-N haplotypes were present in four populations of the Southern Hemisphere: Chile (CHL), South Africa (AFR), Western Australia (AUS) and New Zealand (WHB). Samples CHL, AFR and AUS were exclusively composed of clade-N haplotypes (Table 2) and sample WHB was polymorphic for clade-S and clade-N haplotypes, the clade-N haplotypes being sampled in lower frequency (Table 2). Finally, the analysis of the COI locus revealed that the clade S can be subdivided into three sub-clades, two of which were well supported, corresponding to three geographic regions: S1 (South America and Kerguelen), S2 (New Zealand) and S3 (Tasmania).

Dating the trans-equatorial migration

Tajima's relative rate tests were not significant (p > 0.05). Therefore, the existence of different rates of evolution between lineages was not demonstrated and we consequently assumed a clock-like rate of evolution. The Table 3 details the different steps that led to the estimation of the net nucleotide divergence between Fig. 1. Sampling locations of Southern Hemisphere Mytilus spp. mussels. CHL, Dichato, Chile (36°32 0 S; 72°56 0 W); MAU, Maullin, Chile (41°37 0 S; 73°37 0 W); PAT, Canal de las Montañas, Patagonia, Chile (51°51 0 S; 73°20 0 W); AFR, Pater Noster Bay, South Africa (32°48 0 S; 17°54 0 E), KER, Kerguelen islands (49°20 0 S; 70°10 0 E); AUS, Nedlands, Australia (34°S; 115°E); CBL, Cloudy Bay Lagoon, Tasmania (43°26'S; 147°13 0 E); SIM, Simpson's Bay, Tasmania, (43°17 0 S; 147°18 0 E); HOB, Hobart, Tasmania (42°50 0 S; 147°20 0 E; WHB, Wellington Harbour, New Zealand (41°20 0 S; 174°48 0 E); DUN, Dunedin, New Zealand (45°54 0 S; 170°30 0 E); GRS, George Sound, New Zealand (44°52 0 S; 167°20 0 E).

Table 2

Haplogroup frequencies in Southern Hemisphere Mytilus spp. populations and parameters of nucleotide sequence variability at mitochondrial loci COI and 16S

Locus Haplogroup

Sample the M. trossulus sample and the other samples, d (1) , and the one between the M. edulis samples (from Sweden and/or Nova Scotia) and PAT and KER,d (2) . Since the ratio of the net divergences was 4.17 and assuming that the divergence time between M. trossulus and other populations was 3.5 MY, we estimated the trans-equatorial migration event to be 0.84 MY (0.54-1.31 MY) old.

CHL MAU PAT AFR KER AUS SIM CBL HOB WHB DUN GRS 16S N 1 0 0 1 0 1 0 0 0 0 . 2 0 0 S 0 1 1 0 1 0 1 1 1 0 .
COI N 1 0 0 1 0 1 0 0 0 0.14 0 0 S 1 0 1 1 0 1 0 0 0 0 0 0 0 S2 0 0 0 0 0 0 0 0 0 0.86 1 1 S 3 0 0 0 0 0 0 1 1 1 0 0 0 H d 0.

Discussion

As previously emphasized in the Northern Hemisphere, the COI locus appeared to be a more informative marker than the 16S locus for phylogeographical studies of Mytilus in the Southern Hemisphere. The divergence between Southern-and Northern-Hemisphere haplotypes predates the divergence of haplogroups typical of modern M. edulis and M. galloprovincialis. However, gene trees are not species trees [START_REF] Nichols | Gene trees and species trees are not the same[END_REF], particularly in a species complex with hybridization and introgression as observed in smoothshelled Mytilus. The results obtained with the single mitochondrial genome do not allow us to infer a general history of population divergence. The present results can nonetheless be discussed in the light of previously published results from allozymes and nuclear-DNA length polymorphisms.

Strong genetic structure among Mytilus spp. populations of the Southern Hemisphere

Three groups of samples can be recognized in the Southern Hemisphere: (i) samples exclusively composed of clade-N haplotypes, (ii) samples exclusively composed of clade-S haplotypes and (iii) samples that are polymorphic for both. We observed the first kind of samples in Chile, South Africa and Western Australia. These populations likely originate from recent introductions. The recent introduction of M. galloprovincialis mussels in South Africa has already been reported [START_REF] Grant | Mytilus galloprovincialis Lmk. In Southern Africa[END_REF][START_REF] Mcdonald | Allozymes and morphometric characters of three species of Mytilus in the Northern and Southern Hemispheres[END_REF][START_REF] Daguin | Genetic relationships of Mytilus galloprovincialis Lmk. Populations worldwide: evidence from nuclear-DNA markers[END_REF]. Recently introduced M. galloprovincialis populations have also been reported in Eastern Asia [START_REF] Wilkins | The Mediterranean mussel Mytilus galloprovincialis Lmk[END_REF][START_REF] Lee | The introduction of the Mediterranean mussel Mytilus galloprovincialis into Hong Kong[END_REF][START_REF] Daguin | Genetic relationships of Mytilus galloprovincialis Lmk. Populations worldwide: evidence from nuclear-DNA markers[END_REF], California [START_REF] Mc Donald | The mussels Mytilus galloprovincialis and M. trossulus on the Pacific coast of North America[END_REF][START_REF] Daguin | Genetic relationships of Mytilus galloprovincialis Lmk. Populations worldwide: evidence from nuclear-DNA markers[END_REF] and Chile [START_REF] Daguin | Genetic relationships of Mytilus galloprovincialis Lmk. Populations worldwide: evidence from nuclear-DNA markers[END_REF]. Here, we report the probable introduction of mussels from the Northern Hemisphere to Western Australia. The phylogenetic position of haplotypes from sample AUS indicates that these mussels are once again M. galloprovincialis. However, the same sample appeared to be rather intermediate between Southern-(Tasmania and New Zealand) and Northern Hemisphere M. galloprovincialis at the nuclear locus mac-1 [START_REF] Daguin | Genetic relationships of Mytilus galloprovincialis Lmk. Populations worldwide: evidence from nuclear-DNA markers[END_REF]. Further analyses will be needed to clarify the genetic composition of mussels from Western Australia. Samples of the second kind, exclusively composed of clade-S haplotypes, can be considered to belong to southern endemic populations. These samples comprised three genetic entities that reached reciprocal monophyly and corresponded to distinct regions: South America (MAU and PAT) and Kerguelen with haplotypes of the clade S1, New Zealand (DUN and GRS) with haplotypes of the clade S2 and Tasmania (HOB, SIM and CBL) with haplotypes of the clade S3 (Fig. 3B). In contrast to [START_REF] Hilbish | Origin of the antitropical distribution pattern in the marine mussels (Mytilus spp.): routes and timing of transequatorial migration[END_REF] who sampled exclusively clade-N haplotypes in their small sample (N = 5) from the Kerguelen Islands, we did not sample any clade-N haplotypes in our Kerguelen sample (N = 99). Population structure was also observed to a lesser extent among the three clade-S1 samples (Fig. 2), as well as between HOB and the two other samples from Tasmania (Fig. 2). The level of population differentiation was not well correlated with geographic distance. For instance, genetic differences between Tasmania and New Zealand populations were stronger than between South America and Kerguelen, in spite of being geographically closer. One explanation relies on the geographic fragmentation of landmasses, together with present and past routes of major oceanic currents that go through the mid-latitudes of the Southern Hemisphere. For instance, although the Kerguelen archipelago is isolated by the polar front and the Antarctic circumpolar current, latitudinal displacements of the front may have connected the Kerguelen Islands to South America in the recent past [START_REF] Hollyday | Surface oceanic fronts between Africa and Antarctica[END_REF]. Conversely, the eastern seaboard of Tasmania is affected by the East Australian Current, whose southern extremity pinches off warm-core anticlockwise eddies drifting south near the margin of the continental shelf through the colder waters of the Southern Ocean [START_REF] Nilsson | The formation and evolution of Eastern Australian Current eddies[END_REF]. Southeastern Tasmania may therefore be isolated by local oceanic circulation. Alternatively, barriers to mtDNA exchange may be genetic instead of geographic as it has previously been suggested for populations of the Northern Hemisphere [START_REF] Quesada | Differential introgression of mitochondrial DNA across species boundaries within the marine mussel genus Mytilus[END_REF][START_REF] Riginos | Differential patterns of male and female mtDNA exchange across the Atlantic Ocean in the blue mussel, Mytilus edulis[END_REF]. Finally, we observed a sample from New Zealand (WHB) in which clade-N haplotypes occur at low frequency within a population otherwise composed of clade-S haplotypes. Unfortunately, it is not possible with mtDNA haploid data to distinguish admixture (whereby northern migrants do not reproduce in panmixia with native Southern-Hemisphere mussels) from introgression (whereby northern alleles have invaded the genetic background of southern natives).

4.2. The isolation between mtDNA lineages of populations from the two hemispheres predates the divergence between M. edulis and M. galloprovincialis haplotypes [START_REF] Hilbish | Origin of the antitropical distribution pattern in the marine mussels (Mytilus spp.): routes and timing of transequatorial migration[END_REF] previously showed with the 16S locus that haplotypes from endemic Southern-Hemisphere Mytilus spp. clustered together into a single clade. In their topology the southern clade was related to haplotypes sampled in Mediterranean M. galloprovincialis. With our new dataset for the 16S locus, we obtained a slightly different although poorly supported topology, where the clade-S branched ancestrally to the clade N. We interpret the discrepancy between the 16S tree of [START_REF] Hilbish | Origin of the antitropical distribution pattern in the marine mussels (Mytilus spp.): routes and timing of transequatorial migration[END_REF] and ours by the lack of phylogenetic signal with this locus. Much better statistical support was obtained with the COI locus. The COI tree not only reveals the distinction between endemic southern and Fig. 3. Phylogenetic relationships of (A) 16S rDNA and (B) COI haplotypes in Mytilus spp. mussels inferred using NJ algorithm. Bootstrap scores larger than 50% are indicated on branches for NJ and ML, respectively. Tree-length scales given at the bottom of each tree. Note that the scale of 16S tree is four times larger than that of the COI tree. See Table 1 for site abbreviations.

northern haplotypes but also indicates that this dichotomy predates the divergence of typical M. edulis and M. galloprovincialis haplotypes. An attempt to date the divergence between populations of the two hemispheres led to an estimate of 0.84 MY (0.54-1.31 MY). This divergence dates from the Pleistocene (0.01-1.8 MY) as already inferred by [START_REF] Hilbish | Origin of the antitropical distribution pattern in the marine mussels (Mytilus spp.): routes and timing of transequatorial migration[END_REF].

Can we reconcile mitochondrial and nuclear data into a single scenario?

From the genetic data on southern Mytilus spp. mussels provided by markers of various types, two conflicting scenarios of trans-equatorial migration can be inferred. The first scenario is supported by allozymes [START_REF] Mcdonald | Allozymes and morphometric characters of three species of Mytilus in the Northern and Southern Hemispheres[END_REF] and two introns (mac-1 and EFbis, [START_REF] Daguin | Phylogéographie des moules du complexe d'espèces Mytilus edulis[END_REF][START_REF] Borsa | Genomic reticulation indicates mixed ancestry in Southern Hemisphere Mytilus spp. mussels[END_REF]. It distinguishes South American and Kerguelen populations that share similarities with M. edulis, from Australasian populations (Australia, New Zealand, Tasmania) that share similarities with M. galloprovincialis. This pattern suggests two independent trans-equatorial migrations, one each from M. edulis-like and M. galloprovincialis-like populations that would have occurred after the separation of the two species. The second scenario comes from the most parsimonious interpretation of the mtDNA data and suggests a unique transequatorial migration, which would be older than the coalescence of all the northern haplotypes.

Although conflicting at first view, cyto-nuclear discordance may reveal interesting details about the evolution of species and may have important consequences for evolutionary inference if undetected [START_REF] Sang | Testing hybridization hypotheses based on incongruent gene trees[END_REF][START_REF] Funk | Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA[END_REF]. We here propose to consider how the scenario suggested by one genome (nuclear vs. organelle) can fit the results observed with the other one.

Under the first scenario, we need to explain why the divergence of the southern and northern haplotypes appears more ancient than the divergence between typical M. edulis and M. galloprovincialis haplotypes, and why the two trans-equatorial migrations did not result in a paraphyletic pattern of the southern and northern lineages. Pre-divergence coalescence can be invoked to explain that the southern clade branches deeply in the tree. The present mtDNA differences between M. edulis and M. galloprovincialis are not strong and one can hypothesize that they were less pronounced at the time of migrations. Post-migration lineage sorting would then have resulted in the successful retention of an old lineage in southern populations-i.e. a gene tree that does not match the population tree. However, this hypothesis of mismatched lineage sorting does not explain the reciprocal monophyly of the clades S and N under the two-migration hypothesis. This is why one may invoke the effect of semi-permeable barriers to gene flow either in the northern or in the southern populations. The barrier to gene flow between M. edulis and M. galloprovincialis is semi-permeable [START_REF] Skibinski | Aspects of the population genetics of Mytilus (Mytilidae; mollusca) in the British Isles[END_REF][START_REF] Bierne | Can habitat specialisation maintain a mosaic hybrid zone in marine bivalves?[END_REF] and strong mitochondrial introgression has been documented (Quesada et al., 1998b). The flow of mtDNA genes is likely to have continued while the flow of some nuclear genes was already impeded. Alternatively, hybridization and introgression within the Southern Hemisphere would have erased the signature of one trans-equatorial migration on the mtDNA genome. Be they localised in the North or in the South, the presence of semi-permeable barriers leading to differential organelle and nuclear gene flow greatly helps in reconciling the data obtained with the two genomes under the twomigration scenario.

The second scenario consists of a single southward trans-equatorial migration early in the Pleistocene before the presumed speciation of M. edulis and M. galloprovincialis. Under this scenario the distinction, based on nuclear markers, of M. edulis-like and M. galloprovincialis-like mussels in the Southern Hemisphere would be merely virtual, constrained by human's wish to always assign new samples to reference populations assumed to represent the genetic composition of a given taxon (e.g. Hey, 2001a,b). In addition, the few nuclear markers used to date might suffer from an ascertainment bias as they have often been chosen to discriminate M. edulis and M. galloprovincialis populations in the Northern Hemisphere. However, it is difficult to rule out that the high stochasticity of the coalescence process on a limited number of loci did not result in some populations being apparently closely related to one of the references.

Table 1

 1 Mytilus spp. nucleotide sequences of COI and 16S loci used in the present study. N, sample size

	Locus species	Sampling locations	N	Name of sequences	GenBank Accession Nos.	References
	16S
	M. trossulus	Northeastern Pacific: Alaska, Oregon	5 Tshel, Tore, Tak	AY636148, AY636150-AY636153	Rawson (2005)
	M. trossulus	Northeastern Pacific: Oregon	3 TU1, TU2, TU3	U22879-U22881	Rawson and Hilbish (1995)
	M. edulis	Northwestern Atlantic: Delaware	3 E1, E3, E4	U22864, U22866, U22867	Rawson and Hilbish (1995)
	M. edulis	Northwestern Atlantic: Delaware	6 EDe6-11	AF023542, AF023544, AF023545,	Rawson and Hilbish (1998)
					AF023547, AF023549, AF023550
	M. edulis	-	1 Ew22F	AY350784	Cao et al. (2004)
	M. edulis	-	1 E38	AJ293738	Quesada et al. (2003)
	M. galloprovincialis	Mediterranean: Sète (France)	5 GFr1-5	AF023552, AF023554, AF023556,	Rawson and Hilbish (1998)
					AF023558, AF023560
	M. galloprovincialis	Atlantic: Spain	3 GSp1-3	AF023563-AF023565	Rawson and Hilbish (1998)
	M. galloprovincialis	Northeastern Pacific: San Diego, CA	2 GM-1, GM-2	U22884-U22885	Rawson and Hilbish (1995)
	M. galloprovincialis	Northeastern Pacific: San Diego, CA	7 GAL2-4, GAL6-9	U22870-U22872, U22874-U22877	Rawson and Hilbish (1995)
	M. galloprovincialis	-	1 Mgal-mtgenome	AY497292	Mizi et al. (2005)
	M. galloprovincialis	Chioggia, Italy	9 CHG	AM904603-AM904606	Present study
	Mytilus sp.	Northeastern Atlantic: Ex River, UK	2 HEx1, HEx2	AF023589-AF023590	Rawson and Hilbish (1998)
	Mytilus sp.	Northeastern Atlantic: Whitsand Bay,	8 HWb1-8	AF023572, AF023574, AF023576,	Rawson and Hilbish (1998)
		UK			AF023577, AF023579, AF023581,
					AF023582, AF023584
	Mytilus sp.	Southern Ocean: Chile, Falklands,	20 CH, FA, K, AU, NZ	AF179443-AF179462	Hilbish et al. (2000)
		Kerguelen, Australia, New Zealand
	Mytilus sp.	Dichato, Chile	8 CHL	AM904600-AM904602	Present study
	Mytilus sp.	Maullin, Chile	10 MAU	AM904582-AM904584	Present study
	Mytilus sp.	Canal de las Montañas, Patagonia, Chile	39 PAT	AM904585-AM904588	Present study
	Mytilus sp.	Pater Noster Bay, South Africa	8 AFR	AM904595-AM904599	Present study
	Mytilus sp.	Kerguelen islands	99 KER	AM904589-AM904590	Present study
	Mytilus sp.	Nedlands, Australia	5 AUS	AM904591-AM904594	Present study
	Mytilus sp.	Cloudy Bay lagoon, Tasmania	9 CBL	AM904577, AM904578	Present study
	Mytilus sp.	Simpson's Bay, Tasmania	6 SIM	AM904579-AM904581	Present study
	Mytilus sp.	Hobart, Tasmania	9 HOB	AM904576	Present study
	Mytilus sp.	Wellington Harbour, New Zealand	10 WHB	AM904570-AM904572	Present study
	Mytilus sp.	Dunedin, New Zealand	10 DUN	AM904568, AM904569	Present study
	Mytilus sp.	George Sound, New Zealand	10 GRS	AM904573-AM904575	Present study
	COI
	M. trossulus	Mahone Bay, Nova Scotia	3 669NS, 671NS, 672NS	AY130061-AY130063	Riginos et al. (2004)
	M. trossulus	Penn Cove, WA, USA	4 764WA, 766WA, 768WA,	AY130064-AY130067
				769WA
	M. trossulus	North eastern Pacific	2 MMT	AF242031-AF242032	Wares and Cunningham
						(2001)
	M. trossulus	North America	6 JMT, PMT	AF242027-AF242029, AF242033-	Wares and Cunningham
					AF242035	(2001)
	M. edulis	Northwestern Atlantic: Newfoundland,	46 NF, NS, ME, NC, RI, MA	AF241936-AF241981	Wares and Cunningham
		Nova Scotia, Maine, North Carolina,				(2001)
		Rhode Island, Massachusetts
	M. edulis	Northwestern Atlantic: Massachusetts	4 MA	AY101417, AY101425, AY101426,	Riginos et al. (2004)
					AY101429
	M. edulis	Northwestern Atlantic: Newfoundland,	40 NF, NS, ME, MA, NC	AY130011-AY130037, AY723880-	Riginos et al. (2004)
		Nova Scotia, Maine, Massachusetts,			AY723892
		North Carolina
	M. edulis	Northeastern Atlantic: Iceland, Norway,	45 Ice, No, Fr	AF241982-AF242026	Wares and Cunningham
		France				(2001)
	M. edulis	Northeastern Atlantic: Iceland, Norway,	28 Ice, No, Sw	AY723893-AY723901, AY723903-	Riginos et al. (2004)
		Sweden			AY723917, AY101421-AY101424
	M. edulis	Northeastern Atlantic: Ireland, Finland,	21 Ire, Fi, Fr	AY723918-AY723922, AY130038-	-
		France			AY130053
	M. galloprovincialis	Greece: Samos	7 GR	AY130054-AY130060	-
	M. galloprovincialis	-	1 Mgal-mtgenome	AY497292	Mizi et al. (2005)
	M. galloprovincialis	Chioggia, Italy	10 CHG	AM905220-AM905225	Present study
	Mytilus sp.	Dichato, Chile	9 CHL	AM905174-AM905179	Present study
	Mytilus sp.	Maullin, Chile	7 MAU	AM905180-AM905186	Present study
	Mytilus sp.	Canal de las Montañas, Patagonia,	20 PAT	AM905187-AM905195	Present study
		Chile
	Mytilus sp.	Pater Noster Bay, South Africa	7 AFR	AM905216-AM905219	Present study
	Mytilus sp.	Kerguelen islands	83 KER	AM905196-AM905211	Present study
	Mytilus sp.	Nedlands, Australia	7 AUS	AM905212-AM905215	Present study
	Mytilus sp.	Cloudy Bay lagoon, Tasmania	5 CBL	AM905161-AM905164	Present study
	Mytilus sp.	Simpson's Bay, Tasmania	3 SIM	AM905165-AM905167	Present study
	Mytilus sp.	Hobart, Tasmania	8 HOB	AM905168-AM905173	Present study
	Mytilus sp.	Wellington Harbour, New Zealand	7 WHB	AM905149-AM905154	Present study
	Mytilus sp.	Dunedin, New Zealand	6 DUN	AM905146-AM905148	Present study
	Mytilus sp.	George Sound, New Zealand	9 GRS	AM905155-AM905160	Present study
	Hyphens indicate missing informations.

  Abbreviations for samples as in legend to Fig.1. It should be noted that the haplogroup frequencies of the sample AUS strongly differs between the two loci due to different subsets of individuals successfully sequenced.

		889	1	0.75	0.81	0.85	0.81	1	0.90	0.893	0.95	0.60	0.83
	h p	0.01559	0.00753	0.00453	0.01521	0.00341	0.01801	0.00413	0.00279	0.00604	0.02110	0.00270	0.00172
	h w	0.01711	0.00949	0.00524	0.01392	0.00590	0.01455	0.00413	0.00298	0.00777	0.00285	0.00272	0.02974
	(N)	(9)	(7)	(20)	(7)	(83)	(8)	(3)	(5)	(8)	(7)	(6)	(9)
	Clades-N and -S (S1, S2, S3) were defined in the results section. H

d , haplotype diversity; h p , nucleotide diversity; h w , Watterson's estimate of polymorphism (1975); N, sample size.

  Bidimensional representations of multidimensional scaling (MDS) based onUst values calculated between all pairs of southern samples at COI locus (stress 0.17). Circles group samples significantly differentiated from excluded sample and between which Ust values are non-significant (P > 0.05). See Fig.1for sample abbreviations.

					0.6	KER
					0.4	PAT	South
		Tasmania			MAU	America
		CBL	HOB		0.2
	2
	Dimension	-0.6	SIM -0.4	-0.2		0.2	WHB 0.4	0.6
					-0.2		DUN GRS
			AUS	-0.4		New Zealand
					AFR
				CHL
					-0.6
					Dimension 1
	Fig. 2.

Table 3

 3 Estimation of relative divergence between Mytilus spp. samples from Northern and Southern Hemispheres /2 was calculated for each of the five samples (M. trossulus, M. edulis from Sweden and from Nova Scotia, PAT and KER). d xy : average divergence; d: net nucleotide divergence. For each parameter, the average was calculated from all possible values, among which are the indicated extreme values (min., max.). Standard error is indicated in brackets.

	Comparison, Parameter	Average	Min.	Max.
	h p /2	0.0046	0.0018 (±0.0005)	0.0076 (±0.0015)
	M. trossulus vs. any other sample
	d xy	0.146	0.144 (±0.012)	0.149 (±0.013)
	d (1)	0.141	0.123	0.161
	M. edulis * vs. any other sample
	d xy	0.040	0.036 (±0.002)	0.043 (±0.004)
	d (2)	0.035	0.025	0.046
	d (1) /d (2)	4.17	2.67	6.44
	h p

* M. edulis from Sweden or Nova Scotia.

Acknowledgments

We are very grateful to C. Gilbert, J. Beesley, J.P.A. Gardner, C. Perrin, C. J. Morales and S. Faugeron, and C. Riquelme and D. Moraga for providing mussels from, respectively, Paternoster Bay (South Africa), Australia, Wellington Harbour, Dunedin and George Sound (NZ), Maullin and Dichato (Chile); to J. Lallemand, R.D. Ward and H. Weimerskich for helpful assistance to P.B. in Tasmania; to C. Daguin for providing a number of DNA extracts and to C. Rocher for lab work aid. We also thank the French Polar Institute (IPEV) for fieldwork logistics in Kerguelen (program 195) and Gilles Santantonio for fieldwork logistic in Patagonia. We also thank two anonymous reviewers for helpful comments on the manuscript.


Thomas Saucède
email: thomas.saucede@u-bourgogne.fr

Angie Díaz

Benjamin Pierrat

Javier Sellanes

Bruno David

Jean-Pierre Féral

Elie Poulin

The phylogenetic position and taxonomic status of Sterechinus bernasconiae Larrain, 1975 (Echinodermata, Echinoidea), an enigmatic Chilean sea urchin

Keywords: Sterechinus bernasconiae, Gracilechinus multidentatus, Echinoidea, Antarctic, Phylogeny, Biogeography

Sterechinus is a very common echinoid genus in benthic communities of the Southern Ocean. It is widely distributed across the Antarctic and South Atlantic Oceans and has been the most frequently collected and intensively studied Antarctic echinoid. Despite the abundant literature devoted to Sterechinus, few studies have questioned the systematics of the genus. Sterechinus bernasconiae is the only species of Sterechinus reported from the Pacific Ocean and is only known from the few specimens of the original material. Based on new material collected during the oceanographic cruise INSPIRE on board the R/V Melville, the taxonomy and phylogenetic position of the species are revised. Molecular and morphological analyses show that S. bernasconiae is a subjective junior synonym of Gracilechinus multidentatus (Clark). Results also show the existence of two genetically distinct subclades within the so-called Sterechinus clade: a Sterechinus neumayeri subclade and a subclade composed of other Sterechinus species. The three nominal species Sterechinus antarcticus, Sterechinus diadema, and Sterechinus agassizi cluster together and cannot be distinguished. The species Sterechinus dentifer is weakly differentiated from these three nominal species. The elucidation of phylogenetic relationships between G. multidentatus and species of Sterechinus also allows for clarification of respective biogeographic distributions and emphasizes the putative role played by biotic exclusion in the spatial distribution of species.

Introduction

For more than a century, species of the genus Sterechinus have been among the most frequently collected and commonly studied Antarctic echinoids [START_REF] Agassiz | Preliminary reports on the Echini and star-fishes dredged in deep water between Cuba and the Florida reef by L.F. de Pourtales[END_REF][START_REF] Agassiz | Report on the Echinoidea dredged by H.M.S. Challenger[END_REF][START_REF] Studer | U ¨ber Echinodermen aus Antarkischen Meere und zwei neue seeigel von Papoua-Inseln, gesammelt auf der Reise S.M.S. Gazelle um die Erde[END_REF][START_REF] Studer | Gazelle-Echinoidea[END_REF][START_REF] Meissner | Echinoideen[END_REF][START_REF] Koehler | Re ´sultats du voyage de S.Y. Belgica en 1897-1898-1899[END_REF], 1906[START_REF] Koehler | (ed) Australasian Antarctic expedition 1911-1914[END_REF][START_REF] Do ¨derlein | Die Echinoiden der deutschen Tiefsee-Expedition[END_REF][START_REF] Mortensen | The Echinoidea of the Swedish South Polar Expedition[END_REF]. The genus includes six nominal species that were treated in many ecological and biogeographic studies devoted to Antarctic marine life [START_REF] Brey | The genus Sterechinus (Echinodermata, Echinoidea) on the Weddell Sea shelf and slope (Antarctica)distribution, abundance and biomass[END_REF][START_REF] Arnaud | Semi-quantitative study of macrobenthic fauna in the region of the South Shetland Islands and the Antarctic Peninsula[END_REF]; [START_REF] Barnes | Zoobenthic biodiversity, biomass and abundance at Adelaide Island, Antarctica[END_REF][START_REF] Lee | Molecular phylogeny and divergence time of the Antarctic sea urchin (Sterechinus neumayeri) in relation to the South American sea urchins[END_REF][START_REF] David | Antarctic Echinoidea. Synopses of the Antarctic Benthos. Koeltz Scientific Books, Ko ¨nigstein Del Rio CJ[END_REF][START_REF] Brandt | First insights into the biodiversity and biogeography of the Southern Ocean deep sea[END_REF][START_REF] Linse | Biodiversity of echinoids and their epibionts around the Scotia Arc, Antarctica[END_REF][START_REF] Dı ´az | Evolutionary pathways among shallow and deep-sea echinoids of the genus Sterechinus in the Southern Ocean[END_REF]Gonza ´lez-Wevar et al. 2012a;Pierrat et al. 2012a, b). The genus Sterechinus is widely distributed across the Southern Ocean and extends northward along the coasts of Argentina until 35°S latitude [START_REF] David | Antarctic Echinoidea. Synopses of the Antarctic Benthos. Koeltz Scientific Books, Ko ¨nigstein Del Rio CJ[END_REF]Pierrat et al. 2012b;[START_REF] Sauce `de | Biogeographic atlas of the Southern Ocean[END_REF]). In the Antarctic, Sterechinus neumayeri [START_REF] Meissner | Echinoideen[END_REF], Sterechinus antarcticus [START_REF] Koehler | Re ´sultats du voyage de S.Y. Belgica en 1897-1898-1899[END_REF], and Sterechinus dentifer [START_REF] Koehler | (ed) Australasian Antarctic expedition 1911-1914[END_REF] inhabit the inner (0-800 m depth), outer (200-1500 m depth), and deep shelf and slope (500-2400 m depth) of the Antarctic continent, respectively. Sterechinus diadema [START_REF] Studer | U ¨ber Echinodermen aus Antarkischen Meere und zwei neue seeigel von Papoua-Inseln, gesammelt auf der Reise S.M.S. Gazelle um die Erde[END_REF] is restricted to sub-Antarctic islands (Marion and Prince Edward Islands, Crozet and the Kerguelen Plateau), and the distribution range of Sterechinus agassizi [START_REF] Mortensen | The Echinoidea of the Swedish South Polar Expedition[END_REF] extends from sub-Antarctic islands (Falkland Islands, South Georgia, Shag Rocks, Bouvet Island, and Marion and Prince Edward Islands) to the coasts of Argentina as far northward as the mouth of Rio de la Plata at ca. 35°S latitude [START_REF] David | Antarctic Echinoidea. Synopses of the Antarctic Benthos. Koeltz Scientific Books, Ko ¨nigstein Del Rio CJ[END_REF]Pierrat et al. 2012b). Finally, Sterechinus bernasconiae [START_REF] Baillie `re | Los Equinoı ´deos regulares fo ´siles y recientes de Chile[END_REF] was only described from few localities off the coasts of southern Chile. It is the only species of Sterechinus recorded from the Pacific Ocean [START_REF] Baillie `re | Los Equinoı ´deos regulares fo ´siles y recientes de Chile[END_REF].

Despite the abundant literature devoted to Sterechinus, recent molecular and morphological results have shown that the three nominal species S. diadema, S. agassizi, and S. antarcticus cannot be distinguished [START_REF] David | Antarctic Echinoidea. Synopses of the Antarctic Benthos. Koeltz Scientific Books, Ko ¨nigstein Del Rio CJ[END_REF][START_REF] Dı ´az | Evolutionary pathways among shallow and deep-sea echinoids of the genus Sterechinus in the Southern Ocean[END_REF]). In the past, the distinction between species often relied on the geographic origin of specimens as well as on subtle differences in morphology. However, most of the morphological characters show a wide range of within-species variation that tend to overlap among species and make the distinctions between them unclear (Mortensen 1943; [START_REF] David | Antarctic Echinoidea. Synopses of the Antarctic Benthos. Koeltz Scientific Books, Ko ¨nigstein Del Rio CJ[END_REF].

In this context, S. bernasconiae has attracted our attention as it is the only species of Sterechinus reported from the Pacific Ocean. It is recorded from a narrow area off the coast of southern Chile. In contrast, S. agassizi is much more widely distributed on the Atlantic side of southern South America; it extends from sub-Antarctic islands to the coasts and continental shelf of Argentina. Considering the few specimens of S. bernasconiae ever collected as compared with the numerous samples available for other Sterechinus species [START_REF] Baillie `re | Los Equinoı ´deos regulares fo ´siles y recientes de Chile[END_REF], the taxonomic status and distribution range of the species were not revised in studies devoted to the systematics and biogeography of Austral echinoids [START_REF] David | Antarctic Echinoidea. Synopses of the Antarctic Benthos. Koeltz Scientific Books, Ko ¨nigstein Del Rio CJ[END_REF][START_REF] Dı ´az | Evolutionary pathways among shallow and deep-sea echinoids of the genus Sterechinus in the Southern Ocean[END_REF]Gonza ´lez-Wevar et al. 2012a;Pierrat et al. 2012b[START_REF] Pierrat | Comparative Biogeography of echinoids, bivalves, and gastropods from the Southern Ocean[END_REF]. In light of the taxonomic issues raised in a recent study [START_REF] Dı ´az | Evolutionary pathways among shallow and deep-sea echinoids of the genus Sterechinus in the Southern Ocean[END_REF], the taxonomic status of S. bernasconiae and its phylogenetic relationships with S. agassizi and other species of Sterechinus had to be clarified.

The oceanographic cruise INSPIRE was led off the coasts of Chile (February-March 2010, R/V Melville) to explore chemosynthetic systems (i.e. hydrothermal vents and methane seeps) and study the associated marine life [START_REF] Zapata-Herna ´ndez | New insights on the trophic ecology of bathyal communities from the methane seep area off Concepcio ´n, Chile (*36°S)[END_REF]). The sampling effort was led between 400 and 3300 m depth and from * 33°S to * 46°S and allowed for obtaining specimens of rare species, among which specimens of S. bernasconiae were collected. Based on these newly collected specimens along with original samples including the type material [START_REF] Baillie `re | Los Equinoı ´deos regulares fo ´siles y recientes de Chile[END_REF], the present work aims to revise the systematics of S. bernasconiae, clarify phylogenetic relationships with other species of Sterechinus, and re-examine the biogeographic distribution of the genus Sterechinus in the Southern Ocean.

Materials and methods

Material studied

New specimens of S. bernasconiae were collected off the coasts of Chile during the oceanographic cruise INSPIRE aboard the R/V Melville along the Chilean continental margin (Fig. 1; Table 1). Eight specimens were caught by trawling at six stations, between about 600 and 1100 m depth (Tables 1,2). They are housed at the Biological Collection of Universidad Cato ´lica del Norte (CBUCN, Coquimbo, Chile). The analysis of specimens was complemented by the examination of specimens of S. bernasconiae formerly collected in Patagonia during the Hero 72, 4b cruise [START_REF] Baillie `re | Los Equinoı ´deos regulares fo ´siles y recientes de Chile[END_REF]. They are housed at the Museum of Zoology, Universidad de Concepcio ´n, Chile (Table 1). To perform the phylogenetic analysis, specimens of S. bernasconiae were sequenced along with specimens belonging to the five other nominal species of Sterechinus that were collected in six areas of the Southern Ocean (Ade ´lie Land, South Shetland Islands, Antarctic Peninsula, Weddell/Bellingshausen seas, and around the Kerguelen Islands) and off the coasts of Argentina (Table 2). This material is housed at the Molecular Ecology Laboratory, Faculty of Ecology Sciences at Universidad de Chile, Santiago, Chile.

Morphological study

New material was fixed in 96 % ethanol, the old specimens housed at the University of Concepcio ´n being preserved in formaldehyde. Morphological observations were performed with a binocular microscope, test measurements were taken with a digital caliper to the nearest 0.1 mm. Statistical tests were performed with PAST v. 1.93 [START_REF] Hammer | PAST: paleontological Statistics software package for education and data analysis[END_REF]. Echinoid test plating was drawn using a binocular microscope equipped with a camera lucida. Secondary spines and pedicellariae were removed from tests of the newly collected specimens and placed into 96 % ethanol. They were bleached with a 10 % solution of sodium hypochlorite to remove soft tissue and to separate individual valves of pedicellariae. Then, they were washed in water, dried for 24 h, and mounted on SEM stubs. The entire process was performed under a binocular microscope. Details of pedicellariae and spines were examined with a tabletop scanning electron microscope, and digital images were recorded.

Extraction, amplification, and sequencing

Total DNA was extracted from gonadal tissue or spines of 30 specimens of Sterechinus including S. bernasconiae, i.e. 5 specimens per species, using the salt method (Aljanabi and Martı ´nez 1997). PCR was used to amplify a fragment of the mitochondrial cytochrome oxidase I gene, using the primers described by [START_REF] Lee | Molecular phylogeny and divergence time of the Antarctic sea urchin (Sterechinus neumayeri) in relation to the South American sea urchins[END_REF], LCOI1490 (5 0 -TCTA CAA ACC ACA ARG AYA TTG G-3 0 ) and HCOIN (5 0 -CCC ATT GAA AGA ACG TAG TGA AAG TG-3 0 ). For some samples that had degraded DNA, we used the intermediate primers designed by [START_REF] Dı ´az | Evolutionary pathways among shallow and deep-sea echinoids of the genus Sterechinus in the Southern Ocean[END_REF], ERZin-F (5 0 -GAC CGA CTG CCC TTA TTT-3 0 ) [START_REF] Filatov | Processing and population genetic analysis of multigenic datasets with ProSeq3 software[END_REF] and ClustalX 1.8 [START_REF] Thompson | The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools[END_REF]).

Phylogenetic analysis

The relatively best-fit DNA substitution model was selected by the Akaike Information Criterion deployed in jModelTest v. 0.1.1 [START_REF] Posada | JModelTest: phylogenetic model averaging[END_REF], and the model (GTR I ? C) was used for subsequent Bayesian inference (BI) and maximum likelihood (ML) phylogeny inference. To root the phylogram, sequences of outgroups were chosen within the order Echinoidea following [START_REF] Lee | Molecular phylogeny and divergence time of the Antarctic sea urchin (Sterechinus neumayeri) in relation to the South American sea urchins[END_REF]. Three species were chosen within the family Echinidae: Loxechinus albus (GenBank access AY275550.1), Paracentrotus lividus (GenBank access NC_001572.1), and Gracilechinus multidentatus (GenBank access EU8699-27.1, 28.1, 29.1 and 31.1), along with two species within the family Strongylocentrotidae: Strongylocentrotus purpuratus (Gen-Bank access X12631.1) and Hemicentrotus pulcherrimus (GenBank access JQ742947.1). Finally, the species Pseudechinus magellanicus of the order Temnopleuroida (Gen-Bank access AY275549.1) was selected as outgroup for the order Echinoidea. Maximum likelihood analysis was carried out using the software RAxML v7.03 [START_REF] Stamatakis | A rapid bootstrap algorithm for the RAxML web-servers[END_REF], and branch support values were estimated using 1000 bootstrap replicates [START_REF] Felsenstein | Related articles, links evolutionary trees from DNA sequences: a maximum likelihood approach[END_REF]. Bayesian phylogenetic analyses were performed using the program MrBayes v.3.1.2 [START_REF] Huelsenbeck | MRBAYES: Bayesian inference of phylogenetic trees[END_REF]. This method estimates the posterior probability that each recovered taxon forms a monophyletic unit. All Markov Chain Monte Carlo analyses were performed twice using independent runs of random trees for a total of 8 9 10 6 generations, and trees were sampled every 500 generations after discarding the first 25 % as a burn-in. Average standard deviation of split frequencies converged below 0.01. All remaining trees were used to construct a majority rule consensus phylogram.

To test for the reliability of the clades obtained in phylogenetic inferences we used the Automatic Barcode Gap Discovery (ABGD) species delineation tool (Puillandre et al. 2012a). Pairwise comparisons of COI sequences were performed within and among species, the shortest genetic distances being expected within species. Within the range of computed values, a barcode gap was expected between intra-and interspecific distance values. It provides a set of a priori threshold values for delineating genetically distinct species (Puillandre et al. 2012a, b). In addition to within and among species comparisons, genetic distances were calculated within and among clades and subclades. They were computed among the clades ''GRA'' (including sequences of G. multidentatus and S. bernasconiae), ''LOX'' (representing the sequence of L. albus), and ''STER'' (including sequences of the five remaining species of Sterechinus), within the clade ''GRA'' and ''STER'', and within the two subclades ''NEU'' (sequences of S. neumayeri) and ''Stere'' (including the four remaining species of Sterechinus). The software MEGA v6.06 [START_REF] Tamura | MEGA6: molecular evolutionary genetics analysis version 6.0[END_REF]) with Kimura's two-parameter (K2P) model of base substitution [START_REF] Kimura | A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[END_REF]) was used.

Results

Phylogenetic relationships

The length of the COI section obtained for the species of Sterechinus was 774 bp. Maximum likelihood analysis (ML) and the Bayesian inference (BI) resulted in a tree with similar topology in terms of branching and robustness of the nodes (Fig. 2). As expected, all the species of Echinidae included in the analysis form one single monophyletic group with respect to the representatives of the Strongylocentrotidae and Temnopleuroida. The Echinidae clade comprises the species P. lividus as sister group and two clades: a first one groups sequences of G. multidentatus and S. bernasconiae together (the ''GRA'' clade), and the second one is composed of the remaining Sterechinus sequences (the ''STER'' clade that includes S. neumayeri, S. antarcticus, S. diadema, S. agassizi, and S. dentifer). This second clade is composed of two well-differentiated and supported subclades. The first one includes all sequences of S. neumayeri, while the second one group together the sequences of other Sterechinus species. In the latter, sequences of S. dentifer form a supplementary grouping that it is not supported by posterior probability values (Fig. 2). L. albus forms a third group (''LOX''), but the clade is not well supported (Fig. 2).

Of the 12 species analysed, eight genetically distinct units (OTUs) were identified using the ABGD method. Threshold values for delineating the distinct species are comprised between 0.01 and 0.03 % (Kimura distance K80) (Table 3). The 8 OTUs comprise (1) P. magellanicus, (2) H. pulcherrimus, (3) S. purpuratus, (4) P. lividus, (5) L. albus, (6) the nominal species G. multidentatus and S. bernasconiae, (7) S. neumayeri, and (8) the cluster S. agassizi, S. antarcticus, S. diadema, and S. dentifer. These results are in line with the phylogenetic analyses (Fig. 2). The single OTU composed of the sequences of G. multidentatus and S. bernasconiae corresponds to the ''GRA'' clade identified in the phylogeny; OTUs 7 and 8 composed of other Sterechinus species correspond to the two subclades ''NEU'' and ''Stere'' of the ''STER'' clade (Fig. 2). Pairwise comparisons among the ''GRA'', ''LOX'' and ''STER'' clades show similar values for the distances ''LOX''-''STER'' and ''GRA''-''STER'' (0.2 %) and a lower value for the distance ''LOX''-''GRA'' (0.1 %). Within-clade comparisons give a genetic distance of 0.004 % (SE 0.001) for the ''GRA'' clade and 0.027 % (SE 0.004) for the ''STER'' clade. Genetic distances are 0.003 % (SE 0.001) within the ''NEU'' subclade and 0.004 % (SE 0.001) within the ''Stere'' subclade (Table 3).

Systematics

Measurement and examination of morphological characters of Larrain's original material (Universidad de Concepcio ´n) as well as of newly collected specimens of S. bernasconiae (Tables 1,4) show that they all fall into the diagnosis of both S. bernasconiae Larrain, 1975 and G. multidentatus [START_REF] Clark | A catalogue of the recent sea-urchins (Echinoidea) in the collection of the British Museum (Natural History)[END_REF]. Hence, molecular and morphological results agree with each other and support the synonymy of the two species. The species is here assigned to the genus Gracilechinus multidentatus [START_REF] Clark | A catalogue of the recent sea-urchins (Echinoidea) in the collection of the British Museum (Natural History)[END_REF] Echinus acutus [START_REF] Agassiz | Report on the Echinoidea dredged by H.M.S. Challenger[END_REF]p. 114 Holotype Specimen housed at the Natural History Museum, collected during the cruise of the H.M.S. Challenger near the Kermadec Islands (northeast of New Zealand), station 170, 1135 m depth, on a rocky bottom.

Material See Tables 1,2 for examined material.

Diagnosis One primary tubercle on each ambulacral plate, primary tubercles forming two distinct vertical rows in each ambulacrum. Primary tubercles irregular in size and arrangement; they can be replaced by secondary tubercles adorally and adapically on every alternate or third plate. Valves of globiferous pedicellariae with two to five lateral teeth near the tip. Large tridentate pedicellariae with long (up to 2.5 mm), narrow, and slightly curved valves, blade edges finely serrate at the tip. Shaft of secondary spines smooth. Buccal plates without spines.

Description Test measurements are shown in Table 4. Test circular at the ambitus. In lateral view, test hemispherical in shape, flattened orally (Fig. 3). Mean test height 57 % the diameter of the test but relative test height highly variable, test profile varying from high subconical to flattened aborally. There is an allometric relationship between test diameter and relative test height (H/D), relative test height increasing with size, as shown in Fig. 6 where a natural logarithm regression model is fit to morphological data. The regression model was tested significant (10,000 replicate permutation test; p \ 0.005), but the value of the coefficient of determination is low (R 2 = 0.26) due to strong among-individual variability. This is in agreement with very first studies [START_REF] Clark | A catalogue of the recent sea-urchins (Echinoidea) in the collection of the British Museum (Natural History)[END_REF]. Mean peristome diameter 32 % the diameter of the test, relative peristome size (P/D) decreasing with size (Fig. 6). The allometric relationship between relative peristome size and test diameter can be described by a natural logarithm regression model (R 2 = 0.83, p \ 0.005, 10,000 replicate permutation test). Mean apical disc diameter 0.28 % the diameter of the test, relative apical size (A/D) decreasing with size (Fig. 6) following an allometric relationship (R 2 = 0.7, p \ 0.005, 10,000 replicate permutation test). The peristome diameter significantly exceeds the size of the apical disc (paired t test, p \ 0.01). Peristome with five separated pairs of buccal plates carrying no spines, but with ophicephalous and some tridentate pedicellariae. Beside the buccal plates, pedicellariae also scattered on plates of the peristomial membrane. There is no significant difference in test measurements between the specimens originally attributed to S. bernasconiae by Larrain and the other specimens of G. multidentatus (NPMANOVA not significant, p [ 0.1), both those recently collected off the coasts of Chile and those described in the literature [START_REF] Mcknight | Additions to the echinoid fauna of New Zealand[END_REF].

All studied specimens along with specimens described in the literature [START_REF] Clark | A catalogue of the recent sea-urchins (Echinoidea) in the collection of the British Museum (Natural History)[END_REF][START_REF] Mcknight | Additions to the echinoid fauna of New Zealand[END_REF]) have dicyclic apical systems, that is, the five ocular plates are exsert (out of the genital plate ring) and do not contact the periproct (Fig. 4a; Table 1). Some ocular plates covered with one small tubercle, genital plates covered with one to five small tubercles. Surface of genital plate 2 almost entirely covered with hydropores. Periproctal membrane without spines, anus excentric towards genital plate 5, and surrounded by small periproctal plates.

About two ambulacral plates to each interambulacral plate ambitally, ambulacra one-third the width of interambulacra. Ambulacral plates trigeminate, pore pairs arranged in arcs of three, with one tubercle on each compound plate (Fig. 4b), primary tubercles forming two distinct vertical rows in each ambulacrum. Size of primary tubercles decreases from the ambitus to the peristome adorally, and to the apex adapically. However, primary tubercles are irregular in size and arrangement, and can be replaced by secondary tubercles adorally and adapically on every alternate or third plate. Secondary tubercles and granules scattered over the plates above the ambitus, except in the bare perradial area, which is slightly sunken. Secondary tubercles numerous adorally where they partially encircle primary tubercles. Aborally, one primary tubercle on each interambulacral plate, located near the adoral and adradial plate sutures (Fig. 4c). Primary tubercles form two distinct vertical rows in each interambulacrum. Secondary tubercles and granules scattered over the plates above the ambitus, except in the bare interradial area. Adorally and ambitally, several series of primary tubercles form up to three distinct vertical rows in Ophicephalous and globiferous pedicellariae abundant over the test. Valves of ophicephalous pedicellariae low, wide, scarcely constricted in the middle, blade edges indented and finely serrate (Fig. 5a-b). Valves of globiferous pedicellariae with blade sharply differentiated from base, forming a more or less tubular rostrum (Fig. 5e-f). Crossbeams linking the sides of the rostrum. Tip of the blade forming a robust hook, presence of two to five lateral teeth on each side of the rostrum near the tip. Basal part with an irregular, thorny apophysis, and rounded outer corners. Two forms of tridentate pedicellariae (Fig. 5c-d). Small rostrate-like form with slender, curved valves (Fig. 5c). Large forms with long (up to 2.5 mm-Clark 1925), narrow valves, slightly curved, and finely serrate at the tip (Fig. 5d). Edges of the blade thick in the lower part, with small teeth arranged in transverse series. Triphyllous pedicellariae present, of the classical type of the family. Primary spines straight, ovoid to flatten distally, length of primary spines up to 60 % (3/5) the test diameter, the longest ones near the ambitus in interambulacra. Secondary spines few, short, with smooth shaft (Fig. 5g-h), although small forms may have a finely serrate shaft and tip (Fig. 5i). Below the ambitus, primary and secondary spines may have flattened and slightly expanded distal parts (Fig. 5g-h), while others have pointed tips (Fig. 5i).

Living specimens pale cream. Specimens preserved in ethanol whitish, pale pink or light brown. Dried specimens straw-coloured to light brown, denuded test and spines white.

Distribution G. multidentatus is known between 35.05°S and 50.94°S latitude, between 73.38°W and 117.21°E longitude, and between 300 and 1800 m depth (see Fig. 7 and Pierrat et al. 2012b for occurrence records).

Remarks

The species now included in the genus Gracilechinus were formerly assigned to the genus Echinus Linnaeus, 1758. The genus Gracilechinus was erected by [START_REF] Fell | Echinacea. In: Moore RC (ed) A treatise on invertebrate paleontology. Part U Echinodermata 3. Asterozoa-Echinozoa[END_REF][START_REF] Fell | Echinacea. In: Moore RC (ed) A treatise on invertebrate paleontology. Part U Echinodermata 3. Asterozoa-Echinozoa[END_REF]Pawson 1966, p. 431, Fig. 322-1) based on the type species Echinus gracilis A. [START_REF] Agassiz | Preliminary reports on the Echini and star-fishes dredged in deep water between Cuba and the Florida reef by L.F. de Pourtales[END_REF] Diagnosis is as follows: (1) presence of primary tubercles on each ambulacral plate, (2) a peristome larger than the apical disc, (3) secondary spines with smooth shafts, and (4) valves of globiferous pedicellariae with blade sharply differentiated from base, forming a tubular rostrum. [START_REF] Mcknight | Additions to the echinoid fauna of New Zealand[END_REF] noticed that the original description of the holotype of G. multidentatus [START_REF] Clark | A catalogue of the recent sea-urchins (Echinoidea) in the collection of the British Museum (Natural History)[END_REF] is not in line with the diagnosis of the genus Gracilechinus. [START_REF] Clark | A catalogue of the recent sea-urchins (Echinoidea) in the collection of the British Museum (Natural History)[END_REF] states that primary tubercles are present on every second or third ambulacral plate, which partly corresponds to the amended diagnosis of the genus Echinus (Fell and Pawson 1966, p. 431, Fig. 322-2). However, in his re-examination and re-description of G. multidentatus, [START_REF] Mcknight | Additions to the echinoid fauna of New Zealand[END_REF] indicates that primary tubercles are irregular in size and arrangement aborally, but are present on every ambulacral plate, a situation typical of the genus Gracilechinus. Species of the genus Gracilechinus are characterized by the presence of primary tubercles on every ambulacral plate, whereas primary tubercles are on every alternate, or every third ambulacral plate in the genus Echinus Linnaeus, 1758. Gracilechinus also differs from Sterechinus Koehler, 1901, in which primary and secondary spines are well-differentiated, secondary spines are thorny, numerous, and densely packed. Moreover, in Sterechinus the apical system is monocyclic to hemicyclic, with oculars I and V insert, and test and spines are bright red in life. Species of Dermechinus Mortensen, 1942 have primary tubercles on every ambulacral plate as in Gracilechinus, but the test is extremely high in the former, equalling or exceeding the diameter of the test. Moreover, secondary spines are thorny, numerous, densely distributed (such as Sterechinus), and the peristome is smaller than the apical disc. Gracilechinus is different from Parechinus Mortensen, 1903 only in the shape of globiferous pedicellariae, which have triangular valves, with blade not sharply differentiated from base in Parechinus. Specimens of both genera that lack pedicellariae (e.g. denuded tests and fossil material) cannot be distinguished from each other [START_REF] Fell | Echinacea. In: Moore RC (ed) A treatise on invertebrate paleontology. Part U Echinodermata 3. Asterozoa-Echinozoa[END_REF]. In this work, morphological and molecular analyses are congruent and provide evidence for synonymizing S. bernasconiae and G. multidentatus. Molecular results show an inconsistency within Sterechinus, sequences of S. bernasconiae and G. multidentatus clustering together (only 0.004 % of intraspecific genetic distance), while other Sterechinus species do form a distinct, robust clade (0.2 % of genetic distance to G. multidentatus) composed of two subclades. The first one corresponds exclusively to the Antarctic species S. neumayeri, which is separated from other species of Sterechinus (0.1 % of genetic distance), both Antarctic and sub-Antarctic. The second one groups together S. agassizi from the continental shelf of Argentina, S. diadema from the Kerguelen Plateau, S. antarcticus from the deep Antarctic shelf, and the Antarctic deep-sea S. dentifer (only 0.004 % of genetic distance within the subclade). This challenges their different taxonomic status as the four nominal species could be considered a single group or subclade (i.e. S. diadema). Within this subclade, all sequences of S. dentifer cluster together. Interestingly, Dı ´az et al. (2011) showed that specimens of S. dentifer could be distinguished from other Sterechinus based on the morphology of globiferous pedicellariae. However, the cluster is not well-supported and the short genetic distances within the S. diadema subclade suggest that S. dentifer is not much differentiated and does not form a distinct species.

The present molecular phylogeny totally agrees with results of Dı ´az et al. ( 2011) who showed that distinct Sterechinus subclades could be distinguished also using morphological criteria, and more precisely using the morphology of globiferous pedicellariae. In contrast, the three nominal species S. antarcticus, S. diadema, and S. agassizi cannot be distinguished from each other based on either genetics or morphology. [START_REF] David | Antarctic Echinoidea. Synopses of the Antarctic Benthos. Koeltz Scientific Books, Ko ¨nigstein Del Rio CJ[END_REF] had already noticed that morphological differences between Sterechinus species were unclear and that patterns of geographic distribution provide us with more significant features to distinguish between certain species (e.g. between S. diadema and S. antarcticus). They suggested that only details of the morphology of globiferous pedicellariae could be used to distinguish among the three species S. neumayeri, S. dentifer, and S. antarcticus, while S. antarcticus, S. diadema, and S. agassizi only differ in their respective geographic distributions.

Within the genus Sterechinus, the taxonomy established by previous authors [START_REF] Koehler | Re ´sultats du voyage de S.Y. Belgica en 1897-1898-1899[END_REF][START_REF] Koehler | (ed) Australasian Antarctic expedition 1911-1914[END_REF][START_REF] Do ¨derlein | Die Echinoiden der deutschen Tiefsee-Expedition[END_REF][START_REF] Mortensen | Maximum entropy modeling of species geographic distributions[END_REF]) relied on the examination of a small number of specimens [START_REF] David | Antarctic Echinoidea. Synopses of the Antarctic Benthos. Koeltz Scientific Books, Ko ¨nigstein Del Rio CJ[END_REF]. Subsequently, the morphological characters they regarded as diagnostic (mostly the structure of the apical system) proved to be highly variable, with wide and overlapping ranges of variation [START_REF] David | Antarctic Echinoidea. Synopses of the Antarctic Benthos. Koeltz Scientific Books, Ko ¨nigstein Del Rio CJ[END_REF]. Hence, there is no real discrepancy between molecular data and morphology in Sterechinus; the inconsistency between the current taxonomic status of species and recent molecular results (Dı ´az et al. 2011, this work) is due to an insufficient appraisal of morphological variations.

Comparative biogeography of G. multidentatus and Sterechinus

Previously, G. multidentatus was known exclusively in deepsea areas off the coasts of New Zealand, south of Australia, and Tasmania (McKnight 1968;Pierrat et al. 2012b-Fig. 7). In light of the present taxonomic revision, it turns out that the distribution area of G. multidentatus is much broader than previously thought. It extends across the South Pacific Ocean from the southwest of Australia to the coasts of Chile (Fig. 7). Interestingly, Chilean specimens from Patagonia were collected in relatively shallow waters considering the average depth (ca. 900 m) and range (300-1800 m) of the species. Larrain's specimens were collected in Patagonia at 300 m and recent specimens from Patagonia (INSPIRE AGT-1 and AGT-2) were collected at 628 and 579 m depth, respectively (Table 1). The huge expanse of water that separates the continental shelves of New Zealand and Chile suggests that G. multidentatus can disperse with currents or across the ocean floor. Considering its known bathymetric range, the species can be expected on seamounts of the South Pacific Ocean as well. New occurrence data are needed to address this topic and better characterize the apparent, fragmented pattern of species distribution. Future oceanographic programmes in this part of the Ocean are expected to provide such data. They also could The occurrence of G. multidentatus off the coasts of both Australia and New Zealand is a common biogeographic pattern in echinoids. The similarity of echinoid faunas between Australia and New Zealand has been explained by the existence of recurring trans-Tasman faunal exchanges between southeastern Australia and New Zealand throughout the Cenozoic [START_REF] Foster | Tertiary holasteroid echinoids from Asutralia and new Zealand[END_REF][START_REF] Pierrat | Comparative Biogeography of echinoids, bivalves, and gastropods from the Southern Ocean[END_REF]. The existence of a faunal connectivity between New Zealand and southern South America is more atypical, though it was demonstrated by previous authors [START_REF] Beu | Opening of Drake Passage gateway and Late Miocene to Pleistocene cooling reflected in Southern Ocean molluscan dispersal: evidence from New Zealand and Argentina[END_REF]Del Rio 2002;[START_REF] Pierrat | Comparative Biogeography of echinoids, bivalves, and gastropods from the Southern Ocean[END_REF] to have occurred in some benthic invertebrates since the Oligocene owing to dispersal through the Antarctic circumpolar current at its early stages of development [START_REF] Lawver | Evolution of Cenozoic seaways in the circum Antarctic region[END_REF]. In echinoids, the two genera Pseudechinus and Austrocidaris are present off the coasts of New Zealand and southern Chile, but they both diversified into distinct species on each side of the South Pacific Ocean (Pierrat et al. 2012b). It is postulated that the two genera originated in Australasia then dispersed to the west after the Early Miocene [START_REF] Pierrat | Comparative Biogeography of echinoids, bivalves, and gastropods from the Southern Ocean[END_REF]. Dermechinus horridus is the only echinoid species to have a circum-polar, sub-Antarctic distribution. It is known from off southern Chile, off southern Africa, on the Kerguelen Plateau, and between Tasmania and Victoria [START_REF] David | Antarctic Echinoidea. Synopses of the Antarctic Benthos. Koeltz Scientific Books, Ko ¨nigstein Del Rio CJ[END_REF]Pierrat et al. 2012b). Hence, like G. multidentatus, it occurs on both the eastern and western sides of the South Pacific Ocean, and interestingly, both species have similar depth ranges (between 150 and 1800 m depth and mean depth of ca. 800 m for D. horridus). Most deep-sea species of pourtalesiid and plexechinid echinoids are known to occur below 2500 m all around Antarctica. Therefore, some of them could also extend across the South Pacific Ocean, but occurrence records are too sparse to provide a clear representation of distribution patterns [START_REF] David | Antarctic Echinoidea. Synopses of the Antarctic Benthos. Koeltz Scientific Books, Ko ¨nigstein Del Rio CJ[END_REF].

To date, no species of Sterechinus has ever been recorded along the coast of southern Chile, the specimens previously described as S. bernasconiae being reassigned to G. multidentatus here (Fig. 7). The absence of Sterechinus in Pacific Patagonia contrasts with the extended distribution of the genus along the southwestern Atlantic coast. On the Atlantic side, the genus is represented by S. agassizi (S. diadema subclade) that is known from off Atlantic Patagonia and over the Argentinian continental shelf as far northward as 35°S latitude (Pierrat et al. 2012b). Based on presence data and on a set of 10 oceanographic variables, Pierrat et al. (2012a) modelled the distribution of two Sterechinus species, S. antarcticus and S. neumayeri, using the ''maximum entropy'' modelling procedure [START_REF] Mortensen | Maximum entropy modeling of species geographic distributions[END_REF], which is a correlative approach for assessing species distribution probabilities [START_REF] Gutt | Correlative and dynamic species distribution modelling for ecological predictions in the Antarctic: a cross-disciplinary concept[END_REF]. The distribution of S. antarcticus was modelled all around Antarctica, on the Campbell Plateau (South New Zealand), and on both the Pacific and Atlantic sides of southern South America (Pierrat et al. 2012a). According to the model, areas suitable to Sterechinus occur on both the Atlantic and Pacific sides of southern South America. The discrepancy observed between the modelled distribution and true absence data can be explained alternatively by the accuracy of the model, by the impact of biogeographic processes and/or by the results of biotic interactions.

Along the shorelines of Patagonia, interactions among winds, tides, freshwater discharges, local geomorphology, and oceanographic currents determine coastal marine fronts that have a key role in local ecology (e.g. areas of large primary production, retention areas for larvae of benthic species) and structure the spatial distribution of marine species [START_REF] Acha | Marine fronts at the continental shelves of austral South America[END_REF][START_REF] Miloslavich | Marine biodiversity in the Atlantic and Pacific Coasts of South America: knowledge and gaps[END_REF]Gonza ´lez-Wevar et al. 2012b). In the biogeographic Magellanic Province, interactions between the Cape Horn Current and the Patagonian cold estuarine front can promote dispersal of organisms from the Pacific to the Atlantic Ocean [START_REF] Acha | Marine fronts at the continental shelves of austral South America[END_REF]Gonza ´lez-Wevar et al. 2012b), while the Atlantic Patagonian cold estuarine front constitutes an oceanographic barrier to dispersal between Pacific and Atlantic Patagonia [START_REF] Miloslavich | Marine biodiversity in the Atlantic and Pacific Coasts of South America: knowledge and gaps[END_REF]Gonza ´lez-Wevar et al. 2012b). In contrast to Sterechinus, many invertebrates are known to occur along both Pacific and Atlantic Patagonia among which the echinoids Arbacia dufresni and P. magellanicus, the gastropod Nacella magellanica, the octopus Enteroctopus megalocyathus, and the bivalve Aequipecten tehuelchus [START_REF] Real | Genetic variability of Tehuelche scallop, Aequipecten tehuelchus, populations from the Patagonian coasts (Argentina)[END_REF]Gonza ´lez-Wevar et al. 2012b;Pierrat et al. 2012b). Therefore, the role played by oceanographic currents cannot be considered as decisive in explaining the absence of Sterechinus along the Chilean coast. Pierrat et al. (2012a) proposed a putative exclusion pattern between G. multidentatus and S. antarcticus to explain the absence of S. antarcticus on the Campbell Plateau (Figs. 1,7) although it is a suitable area according to models. Such an exclusion pattern between G. multidentatus and S. agassizi (S. diadema subclade) can be also invoked to explain the absence of Sterechinus in Pacific Patagonia. The clear-cut distinct distribution areas of G. multidentatus and Sterechinus subclades also suggest the existence of an exclusion pattern between the two clades at ocean scale (Fig. 7). Sterechinus subclades occur in the Southern Ocean and in sub-Antarctic areas of the Atlantic and Indian Oceans, while G. multidentatus is present exclusively in the South Pacific Ocean. The two distinct distribution areas are adjacent, especially in the Magellanic region, and do not seem to overlap.

Conclusion

The molecular phylogeny performed in this work resulted in synonymizing the two species S. bernasconiae and G. multidentatus. Molecular results are congruent with morphological data and imply that the species name S. bernasconiae should not be used anymore and that echinoids formerly identified as S. bernasconiae belong to the species G. multidentatus.

Molecular results also show the existence of two genetically distinct subclades within the Sterechinus clade: the S. neumayeri and S. diadema subclades. This totally agrees with previous results of Dı ´az et al. ( 2011) who showed that the two subclades could be distinguished also upon morphological criteria, and more precisely upon the morphology of globiferous pedicellariae. In contrast, the three nominal species S. antarcticus, S. diadema, and S. agassizi belong to the same subclade; they cannot be genetically or morphologically distinguished from each other [START_REF] Dı ´az | Evolutionary pathways among shallow and deep-sea echinoids of the genus Sterechinus in the Southern Ocean[END_REF]. The genus Sterechinus requires more comprehensive taxonomic revision.

The elucidation of phylogenetic relationships between Gracilechinus and Sterechinus also allowed for clarification of their respective biogeographic distributions and emphasized the putative role played by biotic exclusion in the spatial structuring of marine biodiversity. The family Echinidae is represented by two more genera in the Antarctic and sub-Antarctic regions: Loxechinus and Dermechinus. If phylogenetic relationships among Sterechinus, Gracilechinus, and Loxechinus seem clear [START_REF] Lee | Molecular phylogeny and divergence time of the Antarctic sea urchin (Sterechinus neumayeri) in relation to the South American sea urchins[END_REF]; this study), the position of Dermechinus is still uncertain. Forthcoming studies should identify phylogenetic relationships among all representatives of Antarctic and sub-Antarctic Echinidae, thereby clarifying biogeographic patterns in the family.

Fig. 1

 1 Fig. 1 Distribution map of the 413 specimens of Sterechinus spp. studied for this work. The number of studied specimens is given for each nominal species

Gracilechinus

  [START_REF] Fell | Echinacea. In: Moore RC (ed) A treatise on invertebrate paleontology. Part U Echinodermata 3. Asterozoa-Echinozoa[END_REF] Pawson 1966, not Sterechinus Koehler 1901.

Fig. 2

 2 Fig. 2 Bayesian inference (BI) tree based on COI sequences. Branch support values are given for main nodes, Bayesian posterior probabilities preceding bootstrap values (analysis with 1000 replicates)

  Fig. 3 Gracilechinus multidentatus. a Apical and, b oral views of specimen Hero 72.4b GP 7969. c View of the denuded ambulacrum I of specimen Hero 72.4b GP 7973

Fig. 4

 4 Fig. 4 Gracilechinus multidentatus, details of test plating. a Apical disc of specimen Hero 72.4b GP 7969. b Aboral plates of ambulacrum I, specimen Hero 72.4b GP 7971. c Aboral plates of interambulacrum 5, specimen Hero 72.4b GP 7971

Fig. 5

 5 Fig. 5 Gracilechinus multidentatus. a, b Isolated valves of ophicephalous pedicellariae, a specimen AGT-1 426 and b AGT-1 423. c, d Valves of tridentate pedicellariae, specimen SO-210 64, c three valves of the small form in connection, d isolated valve of the large form. e, f Isolated valves of globiferous pedicellariae, e specimen

Fig. 6

 6 Fig. 6 Regression plot of test relative height (H/D), relative peristome size (P/D), and relative apical size (A/D) against test diameter (D). See Table 4 for values and sample details. Data taken from the literature about Gracilechinus multidentatus are shown as grey shaded triangles and circles. Natural logarithm models best fit the regressions

Fig. 7

 7 Fig. 7 Distribution map and depth range of Gracilechinus multidentatus along with Sterechinus subclades. The orange dotted line symbolizes the biogeographic boundary between the two adjacent, but not overlapping distribution areas of G. multidentatus and of Sterechinus subclades. Depth range shown as a box plot. Each box shows the 25-75 % quartiles with the median value (horizontal line

Table 1

 1 Origin of samples and number of specimens of S.

		Location	Cruise	Station Latitude	Longitude	Depth (m) Specimen Number
	bernasconiae studied for the morphological analysis	Chilean margin So-210 Chilean margin INSPIRE	64 AGT-1 -45°55.17 -75°35.12 -35°58.56 -73°38.43 1034 628	1 2
		Chilean margin INSPIRE	AGT-2 -45°53.83 -75°35.97	579	1
		Gulf of Penas a	Hero 72, 4b -	-47°50	-74°40	300	8 b
		Topar Island	Hero 72, 4b -	-50°08.5	-74°41	360	9
		Newly collected specimens are from cruises So-210 and INSPIRE. Larrain's specimens are from cruise
		Hero 72, 4b
		a S. bernasconiae's type locality
		b Including the holotype and the seven paratypes

Table 2

 2 Origin of specimens analysed for the molecular analysis (labelled as in the tree)

	Species	Location	Programme	Station	Latitude	Longitude	Depth (m)	Specimens
	S. neumayeri	Ade ´lie Land	BENTHADEL	Emperor Bay	-66°40.19	140°00.61	26	1
		King George Is.	INACH 13-05	Ardley Pen. Bay	-62°07.79	-58°08.37	10	2
		King George Is.	ECOS C06B02	Ardley Pen. Bay	-62°07.79	-58°08.37	10	3
		Antarctic Pen.	INACH 02-02	O'Higgins	-63°19.23	-57°53.87	20	4
		Weddell Sea	INACH B01-07	Red Island	-63°44.22	-57°52.85	10	5
	S. agassizi	Argentina	INIDEP 1608	Reclutas	-39°50.05	-56°12.24	100-200	1
		Argentina	INIDEP 1608	SAO	-40°45.22	-57°00.39	100-200	2
		Argentina	INIDEP 1608	Tango B	-43°38.12	-59°49.97	100-200	3
		Argentina	INIDEP 1608	Valde ´s	-43°21.55	-59°36.18	100-200	4
		Argentina	INIDEP 1608	SW SAO	-41°39.06	-58°05.40	100-200	5
	S. diadema	Kerguelen Islands	BENTHOS-MAC	Portes Noires	-49°29.65	69°08.97	15	1
		Kerguelen Islands	BENTHOS-MAC	Ile Longue	-49°32.19	69°53.03	20	2
		Kerguelen Islands	BENTHOS-MAC	Ile Haute	-49°23.25	69°56.49	20	3
		Kerguelen Islands	BENTHOS-MAC	Port Couvreux	-49°17.38	69°42.67	20	4
		Kerguelen Islands	BENTHOS-MAC	Port Matha	-48°56.10	69°02.29	10	5
	S. antarcticus	Weddell Sea	CHESSO	ISIS 152	-59°41.00	-28°21.00	1400	4
		Weddell Sea	BIOPEARL I	SG-EBS-35	-53°35.85	-37°54.13	502	5
		Ade ´lie Land	CEAMARC	CEAMARC-08	-66°33.64	142°19.93	372	1
		Weddell Sea	ANT XXIII/8	PS69/603-5	-70°30.99	-08°48.08	285	2
		Weddell Sea	ANT XXIII/8	PS69/603-5	-70°30.99	-08°48.08	285	3
	S. dentifer	Weddell Sea	CHESSO	ISIS 142	-60°02.00	-29°98.00	2400	4
		Weddell Sea	CHESSO	ISIS 152	-59°41.00	-28°21.00	1400	5
		Bellinghausen Sea	BENTART'06	MB 30	-69°58.00	-87°31.00	1814	1
		Bellinghausen Sea	BENTART'06	MB 30	-69°58.00	-87°31.00	1814	2
		Bellinghausen Sea	BENTART'06	MB 30	-69°58.00	-87°31.00	1814	3
	S. bernasconiae	Chilean margin	So-210	69	-36°24.95	-73°42.15	680	1
		Chilean margin	INSPIRE	AGT-4	-36°23.59	-73°42.91	700	2
		Chilean margin	INSPIRE	AGT-3	-36°22.31	-73°43.00	854	3
		Chilean margin	So-210	64	-35°58.56	-73°38.43	1034	4
		Chilean margin	INSPIRE	AGT-1	-45°55.17	-75°35.12	628

  (pro parte, St. 170, non Echinus acutus Lamarck, 1816).

	Echinus multidentatus Clark 1925; p. 115-116, Pl. VI.
	1-2.
	Gracilechinus multidentatus McKnight 1968: 94-99;
	Figs. 4-6.
	Sterechinus bernasconiae Larrain 1975: 94-105;
	Figs. 109-129; Tabs. 9-11, maps 9, 11, and 15.

Table 3

 3 Pairwise genetic distances within and between OTUs based on the Automatic Barcode Gap Discovery (ABGD)

			7									0.1 ± 0.01
			6								0.2 ± 0.01	0.2 ± 0.02
			5						0.1 ± 0.01		0.2 ± 0.02	0.2 ± 0.02
			4					0.2 ± 0.02	0.2 ± 0.02		0.2 ± 0.02	0.2 ± 0.02
			3				0.3 ± 0.02	0.2 ± 0.02	0.2 ± 0.02		0.2 ± 0.02	0.3 ± 0.02
			2			0.1 ± 0.01	0.2 ± 0.02	0.2 ± 0.02	0.2 ± 0.02		0.2 ± 0.02	0.2 ± 0.02
	% K2P sequence divergence	Within OTU Between OTUs	1	n/a	n/a 0.2 ± 0.02	n/a 0.2 ± 0.02	n/a 0.2 ± 0.02	n/a 0.2 ± 0.02	0.004 ± 0.001 0.2 ± 0.02		0.003 ± 0.001 0.2 ± 0.02	0.004 ± 0.001 0.2 ± 0.02
	Number of sequences			1	1	1	1	1	4	5	5	5	5	5
	Species			P. magellanicus	H. pulcherrimus	S. purpuratus	P. lividus	L. albus	G. multidentatus	S. bernasconiae	S. neumayeri	S. agassizii	S. antarcticus	S. diadema	S. dentifer
	OTUs			1	2	3	4	5	6		7	8

Acknowledgments Samples were collected during oceanographic campaigns through IPEV (formerly IFRTP) programmes No. 195 BENTHOS-MAC, No. 345 BENTHADEL, and No. 1044 PROTE-KER; Chilean programmes INACH 13-05, INACH 02-02, INACH B01-07, ECOS C06B02, and INIDEP 1608; and oceanographic cruises INSPIRE, Antarktis XXIII/8, CEAMARC, CHESSO, BIO-PEARL I, and Bentart'06. The authors are indebted to the staff of the Marine Research Station of Las Cruces (Chile), CBUCN (Biological Collection of Universidad Cato ´lica del Norte, Coquimbo, Chile), ZMUC (Concepcio ´n, Chile), NIWA (Wellington, New Zealand), BAS (Cambridge, UK), and AWI (Bremerhaven, Germany). This study was supported by the European Community through an ASSEMBLE Grant (Grant No. 22779). This is Contribution No. 4 to the vERSO project (www.versoproject.be), funded by the Belgian Science Policy Office (BELSPO, Contract No. BR/132/A1/vERSO). This is a contribution to team BioME of the CNRS laboratory Bioge ´osciences (UMR 6282).