Eric Pante

Pierre-Yves Pascal

Vanessa Becquet

Amélia Viricel

Benoit Simon-Bouhet

Pascale Garcia

Evaluating the genetic effects of the invasive Ocenebra inornata on the native oyster drill Ocenebra erinacea

Keywords: Ocenebra erinaceus, Ocinebrellus inornatus, haplotype diversity, evolutionary 13 response, evolutionary ecology, invasive species, glacial refugium, cryptic species

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intertidal gastropod Ocenebra erinacea (Linnaeus, 1758) sampled in the presence and absence 20 of the invasive Ocenebra inornata (Recluz, 1851). Between 1999 and 2004 a total of 352 21 individuals of O. erinacea were collected from 15 sites, five of which in the presence of the 22 invasive, and genotyped at the mitochondrial locus cox1. No statistical difference was 23 observed between polymorphism levels recorded within native populations exposed to the 24 invasive, compared to populations sampled in the absence of O. inornata. No sign of native 25 population decline was detected in response to the invader. While significant shifts in native 26 O. erinacea population sizes were previously reported in the literature, genetic effects may 27 take longer to accumulate, or may be undetectable without a larger panel of genetic markers. 28

In contrast, large genetic distances and significant population differentiation were recorded 29 between Atlantic and Mediterranean O. erinacea samples, suggesting that these populations 30 have distinct evolutionary histories. Comparison of genetic divergence within the closely-31 related genus Nucella suggests that the Atlantic populations and the Mediterranean O. 32 erinacea populations from Thau Lagoon may belong to different species or subspecies. 33 34 36 Natural movements of species ranges on the surface of the world (expansions, 37 regressions, displacements) play a considerable role in the evolution of species. Most of the 38 time, these phenomena are progressive and marked by the tempo of geological processes (e.g. 39 Hewitt 1996). However, during the last few decades a growing number of species has 40 undergone changes in their natural range due to both changes at a global scale (e.g. Parmesan A total of 352 adult specimens of Ocenebra erinacea were collected between 1999 and 113 2004 at 15 sites on the French coast, along line transects (<200 m in length). At each site, 114 specimens from different rocks were collected to reduce sampling bias in favor of a particular 115 lineage. The sites were located both within oyster farming zones and unexploited areas (Table 116 1). In order to show a possible impact of the presence of Ocenebra inornata on genetic 117 diversity of O. erinacea, five locations where the two species live in sympatry and 10 sites 118 free of O. inornata have been sampled. The presence of O. inornata was assessed by direct 119 observation. After collection, specimens were stored in 95% ethanol before DNA extraction. 120 121 DNA extraction, amplification and sequencing 122

Total DNA was extracted from <15 mg of foot muscle using Dneasy TM Tissue Kit 123 according to the manufacturer's protocol (Qiagen, Germany). Part of the mitochondrial cox1 124 gene was PCR-amplified with the HCO2198/LCO1490 primers [START_REF] Folmer | DNA primers for amplification of mitochondrial 435 cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF], which 125 have proved useful for neogastropod studies (e.g. [START_REF] Zou | Comparing the Usefulness of Distance, 567 Monophyly and Character-Based DNA Barcoding Methods in Species Identification: A 568 Case Study of Neogastropoda[END_REF]Zou et al , 2012)). Polymorphism at 126 cox1 is high in Ocenebra inornata, a phylogenetically close species (Martel et al. 2004a). 127 PCRs were done in 50 µL, with 1X PCR buffer, 1.85 mM MgCl 2 , 125 µM dNTPs, 128 0.25 µM of each primer, 1.6 U of Red Hot DNA Polymerase (ABgene) and about 10 ng of 129 DNA template. The following cycling profile was performed using a MJResearch PTC 100 130 Thermal Cycler: initial 5-min denaturation step at 94°C followed by 40 cycles of 30 sec at 131 F o r P e e r R e v i e w 7 94°C, 30 sec at 50°C and 1 min at 72°C, and by a final 5-min extension period at 72°C. PCR 132 products were purified using MultiScreen-PCR MANU03010 plates (Millipore). 133

Sequencing was done by GenoScreen (Lille -France) using an ABI PRISM® 3730 XL 134 Automated DNA Sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA). Sequences 135 were aligned using ClustalX (Thompson et al. 1994). 136

137 Data analyses. 138 Genetic analyses aimed at (i) quantifying and comparing genetic diversity among 139 populations, (ii) analyzing the spatial distribution of polymorphism and genetic exchanges 140 among populations, and (iii) studying the evolutionary relationships among populations. 141

Haplotype number H, number of polymorphic sites S, haplotype diversity H e and average per 142 site nucleotide diversity π (Nei 1987) were calculated for each population using the software 143 DnaSP 5.10.1 [START_REF] Librado | DnaSP v5: A software for comprehensive analysis of DNA 492 polymorphism data[END_REF]. 144

We tested the null hypothesis of the standard neutral model in Arlequin v.3.5 (Excoffier 145 & Lischer 2010), by calculating the D and Fs statistics, as defined by Tajima (1989) and Fu 146 (1997). When these statistics are significantly different from zero, populations may have 147 undergone purifying selection, a selective sweep and/or expansion (<0), or balancing selection 148 and/or a population decline (>0). Statistical significance was tested by generating 10,000 149 random samples under the hypothesis of selective neutrality and population equilibrium. 150

These tests were performed for each sampling site separately, and also for pooled sites in 151 presence or absence of the invasive. 152

The differentiation index Φ ST [START_REF] Excoffier | Analysis of Molecular Variance Inferred From 432 Metric Distances Among DNA Haplotypes: Application to Human Mitochondrial DNA 433 Restriction Data[END_REF], an estimator of F ST [START_REF] Wright | The genetical structure of populations[END_REF]) 153 calculated from frequency values and distances between haplotypes, was computed with 154 Arlequin v.3.5. The Kimura 2 Parameter (K2P) model of nucleotide substitution was used to 155 under the null hypothesis of no difference between populations [START_REF] Excoffier | Analysis of Molecular Variance Inferred From 432 Metric Distances Among DNA Haplotypes: Application to Human Mitochondrial DNA 433 Restriction Data[END_REF]. 157 Finally, a haplotype network was built using the median-joining algorithm 158 implemented in Network 4.6.1.1 (fluxus-engineering.com, Bandelt 1999). This method is one 159 of the most accurate for inferring intra-specific networks in the absence of recombination 160 (Woolley et al. 2008). 161

To help interpret the large genetic divergence observed between Atlantic and 162 Mediterranean specimens of Ocenebra erinacea, we looked for mitochondrial cox1 data from 163 the BOLD database (Ratnasingham & Hebert 2007). However, besides three other BOLD 164 cox1 sequences from Spanish specimens of O. erinacea, we produced the only available 165 mitochondrial sequences for the genus Ocenebra. We therefore used cox1 sequences from six 166 species of the closely-related genus Nucella Röding, 1798 (e.g. Pascal 2004) to measure intra-167 specific and inter-specific genetic distances. Nucella and Ocenebra are both characterized by 168 a non-planktonic larval development and lay egg capsules on hard substrates (Martel et al. 169 2004c; review by Krug 2011). We used the K2P model of nucleotide substitution (Kimura 170 1980), widely used in DNA barcoding (Hebert et al. 2003;Barrett & Hebert 2005) A single Mediterranean population (Thau) was sampled. The haplotype diversity (H e = 192 0.342) and the nucleotide diversity (π x10 -3 = 2.52) are respectively two-and five-fold higher 193 than on the whole of Atlantic populations (Table 2). Moreover, 22 polymorphic sites were 194 found among 37 individuals sampled in Thau, a value considerably higher than the 24 195 polymorphic sites observed among 315 Atlantic individuals. 196 Last, haplotype and nucleotide diversities of Ocenebra erinacea populations co-197 occurring with Ocenebra inornata were lower (H e = 0.247 ± 0.0535 and π x10 -3 = 0.57 ± 0.65) 198 than for populations located in zones where O. inornata was not detected (H e = 0.399 ± 199 0.3976, π x10 -3 = 10.04 ± 5.52) (Table 2). However, this pattern is entirely due to the higher 200 diversity encountered at Thau; when removing this site from the group of populations that 201 for π: t = -0.16, df = 12.81, p = 0.88. In both cases, results were also non-significant when 205 removing the population of Thau). 206 Except for one case, the D and Fs statistics were never positive (Table 2). Furthermore, 207 the only slightly positive D value (deviation from zero non significant) was observed when 208 sites where the invasive was absent were pooled, including Thau, and this result was therefore 209 likely influenced by the underlying population structure (see "Genetic differentiation among 210 populations" Results section). There is therefore no supporting evidence that Ocenebra 211 erinacea populations exposed to O. inornata suffered from a population decline. Some sites 212 exhibited significant negative values of D and Fs, which can be interpreted as signs of 213 purifying selection, selective sweep and/or population expansion. Particularly, the pooled 214 Atlantic sites showed significantly negative values for both tests, regardless of whether the 215 invasive was present or not. These molecular signatures must, however, be interpreted with 216 care, as they might reflect older demographic events. 217 218

Genealogical relationships and spatial distribution of haplotypes 219

Two haplogroups, separated by 18 mutational steps, were observed using the median-220 joining network (Fig. 1). The first haplogroup ( 23 values ranged between 0.94 and 0.96, corresponding to a substantial genetic differentiation 239 between the Mediterranean and Atlantic populations (Table 3). Inside the Atlantic group, no 240 significant differentiation was observed after sequential Bonferroni correction (lowest 241 corrected alpha level for Atlantic populations: 0.00055). 242 243 Levels of intra-specific divergence in Ocenebra compared to Nucella 244 Given the differences in haplotype composition and divergence between 245

Mediterranean and Atlantic sites, we investigated whether the genetic distances correspond to 246 intra-or inter-specific divergence by comparing Ocenebra to its close relative Nucella. The 247 pairwise K2P distance between Ocenebra erinacea haplotypes ranged between 0.18 and 4.54% 248 (maximum observed between haplotypes 20 from Loire and 25 from Thau Lagoon). Within 249 Nucella, K2P was calculated for 532 sequences and 117 haplotypes distributed among six 250 species, along a 434 bp stretch of cox1. Intra-specific distances ranged from 0 to 3.32%, while 251 inter-specific distance ranged from 4.81 to 12.2%, for specimens distributed over 1000 to 252 While a decrease in genetic diversity in response to invaders was reported in the past 261 (Kim et al. 2003), we did not detect such a pattern among Ocenebra erinacea exposed to the 262 invasive species Ocenebra inornata. The relatively low polymorphism levels recorded may 263 have hampered our ability to detect genetic effects of the invasive on the native species, and 264 the use of additional molecular markers such as nuclear microsatellites or SNPs might further 265 help detect possible demographic events associated with the presence O. inornata. However, 266 this remains to be tested, as even genome-wide scans can fail at detecting recent demographic 267 events and selective pressures. Riquet et al. (2013), for instance, used AFLPs to compare 268 native and invasive populations of the marine mollusk Crepidula fornicata. They reported 269 little genetic differentiation among these populations, and detected no F ST outliers out of 344 270 tested loci. An alternative hypothesis explaining the apparent absence of genetic effects of the 271 invasive on the native is that the competitive and selective pressures inflicted on O. erinacea 272 are too low to have genetic effects (e.g. Wittmann et al. 2013) species to be detectable. About ten years later, the distributional landscape of O. inornata on 280 Atlantic coasts have significantly changed, and the invasive is now found as far north as the 281 entrance of the Baltic Sea (Lützen et al. 2012). A new survey of the genetic diversity of O. 282 erinacea may today unfold the genetic consequences of the invasion by O. inornata, and this 283 study therefore provides a snapshot in time that may help better understand the temporal 284 dynamics of loss of genetic diversity. In addition to sampling in the field, we searched for 285

F
Ocenebra specimens in the collections of the Museum national d'Histoire naturelle in Paris 286 (France), in order to look for genetic diversity in O. erinacea specimens collected prior to, or 287 soon after the invasion by O. inornata (MNHN voucher numbers IM-2008-7101, IM-2008-288 7102, IM-2008-7103). Unfortunately, we were not able to amplify the cox1 marker from these 289 2). Comparatively, H e = 0.684 and π x10 -3 = 2.25 for Ocenebra 296 inornata in its natural range (data from Martel et al. 2004a), H e = 0.734 and π x10 -3 = 14.78 in 297 the gastropod Cyclope neritea (Simon-Bouhet et al. 2006), and H e = 0.720 and π x10 -3 = 89.84 298 in the bivalve Macoma balthica (Becquet et al. 2012). 299

The low genetic diversity observed at cox1 was consistent with the low diversity 300 observed using allozymes: Martel (2003) reported that the number of alleles N all and the 301 = 2.3 ± 305 1.0, H o = 0.25; Johnson &Cumming 1995), andLittorina striata (N all = 4.2 ± 1.0, H o = 0.18 ± 306 0 .17;[START_REF] De Wolf | Large scale population structure and gene flow in the 424 planktonic developing periwinkle Littorina striata (Mollusca: Gastropoda), in 425 Macronésia[END_REF]. Congruent patterns across mitochondrial and allozyme markers 307 thus suggest a low genetic diversity in these populations rather than an absence of variability 308 at cox1. Still, our sampling remains restricted compared to the native range of the species, and 309 additional monitoring may reveal new patterns of genetic diversity. 310 311

Genetic diversity and connectivity among populations 312

The genetic diversity of the Mediterranean population was among the highest (Table 313 2). The oyster farmers of the Thau lagoon, one of the main shell farming areas of the French 314 Mediterranean coasts, carry out commercial exchanges with distant production sites, and 315

Ocenebra erinacea is likely to be transferred during these exchanges. In fact, the 316 morphological survey done by Berrou et al. (2004) evidenced exchanges from Oléron Island 317 to Thau Lagoon (this is congruent with the observation of the Atlantic haplotype H26 being 318 observed at Thau; Figs. 12). In our study, the high genetic diversity observed in Thau Lagoon 319 could be the result of the introduction of Mediterranean specimens imported from other 320 production sites such as Oléron. As no Mediterranean site was sampled other than Thau 321 Lagoon, the artificial mixing induced by shellfish exchanges cannot be further evaluated here. 322

Alternatively, the difference in genetic diversity observed between the Atlantic and the 323 Mediterranean population of Thau lagoon could be explained by historical and 324 biogeographical factors, and/or selection. Ocenebra erinacea is not well adapted to cold water 325 and Belgium currently constitutes the northern limit of its natural range (Graham 1988). 326 Consequently, the species may have found, as other marine species (e.g. Nikula & Väinölä 327 2003, Ladhar-Chaabouni et al. 2010), a refugium on the Iberian coast or in the Mediterranean 328 basin during past glaciations. O. erinacea may have disappeared from the French Atlantic 329 coasts during the Würm Glacial period (115,000 to 10,000 years BP) but survived on the 330 coasts of the Iberian peninsula, which is known as one of the major Pleistocene refugia 331 (Gómez & Lunz 2007). At the end of this climatic crisis, a reduced number of individuals 332 from southern refuges may have reached northern coasts. Maggs et al. (2008), reviewing 333 molecular signatures of glacial refugia on marine species, made predictions of low genetic and 334 haplotype diversity in northern regions previously covered by ice sheets, and comparatively 335 high diversity in refugial southern regions (and see Hewitt 1996). These predictions are 336 generally met for O. erinacea, but additional sampling from the Iberian peninsula and the 337 Mediterranean Sea would be necessary to further characterize the historical biogeography of 338 this species. Given O. erinacea's maladaptation to cold-water, another possibility is that 339 selection (either purifying selection, or selective sweeps) linked to differences in water 340 temperature between Thau and the Atlantic sites produced the observed patterns of genetic 341 diversity. The negative Tajima's D and Fu's Fs observed for the Atlantic population (in 342 absence and presence of the invasive) would support this scenario. 343

One potential consequence of biogeographic divergence between Atlantic and 344

Mediterranean populations is the emergence of new species (Hewitt 1996(Hewitt , 2004)). Recently, 345 Salicini et al. (2013) Natural movements of species ranges on the surface of the world (expansions, 39 regressions, displacements) play a considerable role in the evolution of species. Most of the 40 time, these phenomena are progressive and marked by the tempo of geological processes (e.g. 41 Hewitt 1996). However, for a during the last few decades a growing number of species has 42 undergone changes in their natural range due to both global changes at the planeta global scale 43 (e.g. Parmesan & Yohe 2003) and artificial transfers due to human activities (e.g. Carlton 44 1989;Seebens et al. 2013). 45

The number of biological invasions has strongly increased during the last decades (e.g. 46 Mack et al. 2000;Mooney & Cleland, 2001;Ruiz et al. 2000). In the marine environment, 47 these phenomena are mainly due to aquaculture and especially to shellfish farming, which 48 represents a major cause of introduction, intentional or not, of exogenous species [START_REF] Elton | The ecology of invasions by animals and plants[END_REF]49 Carlton 1992). 50 Apart from potentially important economic consequences, the arrival of these 51 introduced species can also cause serious ecological impacts on local fauna. Introduced 52 species are likely to decrease the abundance of indigenous species, excluding them from part 53 of their distributional area or even causing their extinction by modifying invaded habitats, 54 hybridizing with native species, exchanging pathogens, preying on them, or competing with 55 them (e.g. Lockwood et al. 2007 and references therein). Moreover, when they exert strong 56 selective pressures, introduced species can also reduce the genetic diversity of native 57 populations (Kim et al. 2003;Wittmann et al. 2013). 58 Such impacts, although poorly known (e.g. Strauss et al. 2006) Frankham 1995; Lande 1995; Strauss et al. 2006) and may leadight contribute to extinction 62 (Wittmann et al. 2013). Thus, various authors showed a link between the fitness of a species 63 and its genetic diversity, particularly in mollusks [START_REF] Mitton | Associations among protein heterozygosity, growth rate and 516 developmental homeostasis[END_REF][START_REF] Garton | Heterozygosity, shell length and metabolism in the 443 European mussel, Dreissena polymorpha, from a recently established population in lake 444 Erie[END_REF]64 Zouros 1993;Launey & Hedgecock 2001;Hedgecock et al. 2007). ConverselyIn addition, the 65 reduced genetic variability of an indigenous population could promote the expansion process 66 of other species that are phylogenetically close. However, very few studies address changes in 67 the evolution of genetic diversity of an indigenous species under the competitive selection 68 pressure generated by a biological invader. 69

The oyster drills Ocenebra erinacea (Linnaeus, 1758) and Ocinebrellus 70 States (Martel et al. 2004a). The introduction of O. inornatusO. inornata may haves important 78 economical consequences, as it is a predator of cultivated mollusks (e.g. oysters, blue mussels, 79 Goulletquer et al. 2002). It coexists on at several French sites with an indigenous muricid, 80 O.cenebra erinacea (Linnaeus, 1758), which ranges from the straits of Gibraltar to the 81 Netherlands, and inhabits all British and Mediterranean coasts (Graham 1988). Although O. 82 erinacea and O. inornatusO. inornata differ in some life history traits (Martel et al. 2004c), 83 they these muricid gastropods fill similar ecological niches, and may compete for habitat 84 (both species live on hard substrates and drill the shell of bivalves to feed on them; e. been investigated in previous studies (Martel et al. 2004a;Martel et al. 2004b), its ecological 90 impacts and the influence ofon the local native O. erinacea species on its capacity of 91 integration are poorly known. 92

In previous studies (Martel 2003), seven allozyme loci were analyzed on populations 93 of OcenebraO. erinacea and O. inornatusOcenebra inornata collected in 7 sites of the French 94 Atlantic coast where the two species live in sympatry. These markers revealed genetic 95 diversity indices systematically weaker within the native O. erinacea than within the invasive 96 O. inornatusO. inornata. This result was counterintuitive, as (i) the founder effect linked to an 97 introduction event should lead to a low genetic diversity within the populations of the 98 introduced species, (ii) this phenomenon should be all the more marked as the invasion is 99 recent (review of Sakai et al. 2001) Total DNA was extracted from <15 mg of foot muscle using Dneasy TM Tissue Kit 128 according to the manufacturer's protocol (Qiagen, Germany). Part of the mitochondrial cox1 129 gene was PCR-amplified with the HCO2198/LCO1490 primers [START_REF] Folmer | DNA primers for amplification of mitochondrial 435 cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF], which 130 are have particularly proved useful for neogastropod studies (e.g. Harasewych et al. 1997Zou 131 et al 2011[START_REF] Inornatuso | of O. erinacea may today unfold the genetic consequences 296 of the invasion by O. inornatusO. inornata, and this study therefore provides a snapshot in 297 time that may help better understand the temporal dynamics of loss of genetic diversity[END_REF]. Polymorphism at cox1 is high in O. inornatusOcenebra inornata, a 132 phylogenetically close species (Martel et al. 2004a) Thermal Cycler: initial 5-min denaturation step at 94°C followed by 40 cycles of 30 sec at 137 94°C, 30 sec at 50°C and 1 min at 72°C, and by a final 5-min extension period at 72°C. PCR 138 products were purified using MultiScreen-PCR MANU03010 plates (Millipore). 139

Sequencing was done by GenoScreen (Lille -France) using an ABI PRISM® 3730 XL 140 Automated DNA Sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA). Sequences 141 were aligned using ClustalX (Thompson et al. 1994). 142

143 Data analyses. 144 Genetic analyses aimed at (i) quantifying and comparing genetic diversity among 145 populations, (ii) analyzing the spatial distribution of polymorphism and genetic exchanges 146 among populations, and (iii) studying the evolutionary relationships among populations. 147

Haplotype number H, number of polymorphic sites S, haplotype diversity H e He and average 148 per site nucleotide diversity π (Nei 1987) were calculated for each population using the 149 software DnaSP 5.10.1 [START_REF] Librado | DnaSP v5: A software for comprehensive analysis of DNA 492 polymorphism data[END_REF]. 150

We tested the null hypothesis of the standard neutral model in Arlequin v.3.5 (Excoffier 151 & Lischer 2010), by calculating the D and Fs statistics, as defined by Tajima (1989) and Fu 152 (1997). In each location, we checked the demographic equilibrium of the populations in 153 Arlequin v.3.5, by calculating the D and Fs statistics, as defined by Tajima (1989) and Fu 154 (1996). When these statistics are significantly different from zero, populations may have 155 The differentiation index Φ ST [START_REF] Excoffier | Analysis of Molecular Variance Inferred From 432 Metric Distances Among DNA Haplotypes: Application to Human Mitochondrial DNA 433 Restriction Data[END_REF], an estimator of F ST [START_REF] Wright | The genetical structure of populations[END_REF]) 161 calculated from frequency values and distances between haplotypes, was computed with 162 Arlequin v.3.5 [START_REF] Excoffier | Arlequin suite ver 3.5: A new series of programs to 429 perform population genetics analyses under Linux and Windows[END_REF]. The Kimura 2 Parameter (K2P) model of 163 nucleotide substitution was used to estimate genetic distances, and 10,000 permutations were 164 used to test statistical significance under the null hypothesis of no difference between 165 populations [START_REF] Excoffier | Analysis of Molecular Variance Inferred From 432 Metric Distances Among DNA Haplotypes: Application to Human Mitochondrial DNA 433 Restriction Data[END_REF]. 166 Finally, a haplotype network was built using the median-joining algorithm 167 implemented in Network 4.6.1.1 (fluxus-engineering.com, Bandelt 1999). This method is one 168 of the most accurate for inferring intra-specific networks in the absence of recombination 169 (Woolley et al. 2008). 170

To help interpret the large genetic divergence observed between Atlantic and 171 Mediterranean specimens of O.cenebra erinacea, we looked for mitochondrial cox1 data from 172 the BOLD database (Ratnasingham & Hebert 2007). However, besides three other BOLD 173 cox1 sequences from Spanish specimens of O. erinacea, we produced the only available 174 mitochondrial sequences for the genus Ocenebra. We therefore used cox1 sequences from 175 seven six species of the closely-related genus Nucella Röding, 1798 (e.g. Pascal 2004) to 176 measure intra-specific and inter-specific genetic distances. Nucella and Ocenebra are both 177 characterized by a non-planktonic larval development and lay egg capsules on hard substrates 178 (Martel et al. 2004c; review by Krug 2011). We used the K2P model of nucleotide 179 substitution (Kimura 1980), a widely used metric in DNA barcoding (Hebert et al. 2003;180 Barrett & Hebert 2005), to measure genetic distances among cox1 haplotypes. 181 more than five haplotypes (sample sizes provided in Table 1). Consequently,haplotype 196 diversity H e is low, except for Oléron (H e = 0.564; Table 2). In Fouras, where O. 197 inornatusOcenebra inornata and O. erinacea are sympatric, the haplotype and nucleotide 198 diversities are respectively four-and six-fold higher in the invasive drill (H e = 0.348 and π 199 x10 -3 = 0.8378; data from Martel 2003) than in the local one (H e = 0.074 and π x10 -3 = 0.14). 200

A single Mediterranean population (Thau) was sampled. The haplotype diversity (H e = 201 0.342) and the nucleotide diversity (π x10 -3 = 2.52) are respectively two-and five-fold higher 202 than on the whole of Atlantic populations (Table 2). Moreover, 22 polymorphic sites were 203 found among 37 individuals sampled in Thau, a value considerably higher than the 24 204 polymorphic sites observed among 315 Atlantic individuals. 205 2). Comparatively, H e = 0.684 and π x10 -3 = 2.25 for O. 309 inornatusOcenebra inornata in its natural range (data from Martel et al. 2004a), H e = 0.734 310 and π x10 -3 = 14.78 in the gastropod Cyclope neritea (Simon-Bouhet et al. 2006), and H e = 311 0.720 and π x10 -3 = 89.84 in the bivalve Macoma balthica (Becquet et al. 2012). 312

The low genetic diversity observed at cox1 was consistent with the low diversity 313 observed using allozymes: Martel (2003) reported that the number of alleles N all and the 314 The genetic diversity of the Mediterranean population was among the highestr than 326 that of each Atlantic population (except Oléron, Table 2). The oyster farmers of the Thau 327 commercial exchanges with distant production sites, and O.cenebra erinacea is likely to be 329 transferred during these exchanges. In fact, the morphological survey done by Berrou et al. 330 (2004) evidenced exchanges between from Oléron Island and to Thau Lagoon (this is 331 congruent with the observation of the Atlantic haplotype H26 being observed at Thau; Figs. 1-332 2). In our study, the high genetic diversity observed in Thau Lagoon could be the result of the 333 introduction of Mediterranean specimens imported from other production sites such as Oléron. 334 As no Mediterranean site was sampled other than Thau Lagoon, the artificial mixing induced 335 by shellfish exchanges cannot be further evaluated here. 336

Alternatively, the difference in genetic diversity observed between the Atlantic and the 337 Mediterranean population of Thau lagoon could be explained by historical and 338 biogeographical factors, and/or selection. Ocenebra. erinacea is not well -adapted to cold 339 water and Belgium currently constitutes the northern limit of its natural range (Graham 1988). 340 Consequently, the species may have found, as other marine species (e.g. Nikula & Väinölä 341 2003, Ladhar-Chaabouni et al. 2010), a refugium on the Iberian coast or in the Mediterranean 342 basin during past glaciations. O. erinacea may have disappeared from the French Atlantic 343 coasts during the Würm Glacial period (115,000 to 10,000 years BP) but survived on the 344 coasts of the Iberian peninsula, which is known as one of the major Pleistocene refugia 345 (Gómez & Lunz 2007). At the end of this climatic crisis, a reduced number of individuals 346 from southern refuges may have reached northern coasts. Maggs et al. (2008), reviewing 347 molecular signatures of glacial refugia on marine species, made predictions of low genetic and 348 haplotype diversity in northern regions previously covered by ice sheets, and comparatively 349 high diversity in refugial southern regions (and see Hewitt 1996). These predictions are 350 generally met for O. erinacea: a mitochondrial genealogy matching geography,, but additional 351 sampling from the Iberian peninsula and the Mediterranean Sea would be necessary to further 352 Mediterranean populations is the emergence of new species (Hewitt 1996(Hewitt , 2004)). Recently, 361 Salicini et al. (2013) have shown that in the bat Myotis naterreri, a complex of four cryptic 362 species exist in the Western Palearctic region (central and southern Europe, northwestern 363 Maghreb), each species coinciding with a glacial refugium. In Ocenebra. erinacea, inter-clade 364 divergence overlaps with the inter-specific distances observed in the closely-related genus 365 Nucella (even though the geographical distances separating Nucella specimens were greater 366 than the distances separating O. erinacea specimens; see Bergsten et al. 2012)

  , and 10,000 permutations were used to test statistical significance 156

  were not found in contact with O. inornata, diversity values dropped significantly (H e = 0.179 202 ± 0.0368, π x10 -3 = 0. 38 ± 0. 51). Comparing molecular diversity at the site level revealed the 203 same pattern (Welch Two Sample t-test, including Thau, for H e : t = -0.83, df = 6.22, p = 0.44; 204

  haplotypes) was mainly composed of 221 Atlantic specimens, and included one specimen from Thau, characterized by haplotype H26 222 (separated from other Mediterranean haplotypes by 20 to 22 mutational steps). Haplotypes 223 from this group diverged by ≤ 2 mutations. Haplotype H1 was common (represented in 89% 224 of Atlantic individuals) and central to the Atlantic haplogroup, while the other 22 haplotypes 225 were rare (7 individuals for H3, 3 for H13, 2 for H6, H9, H15, H19 and a single individual for 226 the others) and peripheral to H1. The second haplogroup was strictly composed of 227 Mediterranean individuals. Of six haplotypes from this group, one was common (H21, 228 of Thau individuals), and four were rare (3 individuals for H22 and a 229 single individual for H23-25). Haplotypes from this group diverged by one mutation. The 230 Atlantic samples share no haplotype with the Mediterranean sample (Fig. 2). Except for H1 231 (present at all sites except Thau), H3 (shared between Loix and Aytré) and H13 (shared 232 between Loix, Aytré and Trébeurden), all haplotypes are private (observed only within one pairs of populations was measured using Φ ST . The 237 population from Thau was significantly differentiated from all other populations. Pairwise Φ ST 238

  study were collected between 1999 and 2004. The introduction of O. inornata 278 may have been too recent at the time of sampling for genetic consequences on the native 279

  diversity of Ocenebra erinacea populations 292 Mitochondrial genetic diversity, as measured using part of cox1, was low relative to 293 what was observed in other marine mollusks. Overall, Ocenebra erinacea haplotype and 294 nucleotide diversities were (disregarding the sample from Thau, see below) H e = 0.18-0.25 295 and π x10 -3 = 0.38-0.57 (Table

  H o characterizing the Atlantic populations of Ocenebra erinacea are 302 respectively two to four times, and twenty to thirty times lower (N all = 1.1 ± 0.1; H o = 0.01 ± 303 0.01; mean ± SD) than in other marine gastropods sampled in their native range, such as 304 Bedeva hanleyi (N all = 2.2 ± 0.1, H o = 0.30 ± 0.02; Hoskin 2000), Drupella sp. (N all

  have shown that in the bat Myotis naterreri, a complex of four cryptic 346 species exist in the Western Palearctic region (central and southern Europe, northwestern 347 Maghreb), each species coinciding with a glacial refugium. In Ocenebra erinacea, inter-clade 348 divergence overlaps with the inter-specific distances observed in the closely-related genus 349 Nucella (even though the geographical distances separating Nucella specimens were greater 350 separating O. erinacea specimens; seeBergsten et al. 2012). In addition, O. 351 erinacea specimens from the Atlantic and Mediterranean can readily be distinguished using 352 morphology, and the morphological distance between Atlantic and Mediterranean O. erinacea 353 is comparable to what is observed between O. erinacea and Ocenebra brevirobusta Houart 354 2000 (Berrou et al. 2004). It is therefore possible that the Atlantic and Mediterranean clades 355 sampled for this study belong to groups in incipient stages of speciation, or even undescribed 356 species. 357 358 Acknowledgements 359 We thank Stéphane Robert (IFREMER, La Tremblade, France), Yvonne Pascal, Michel 360 Pascal (INRA Rennes, France), François Siorat (LPO), Armel Deniau (LPO), Marie-Gabrielle 361 Vitre, Paul Wendling, Francoise Dreau, Christian Bussière, Isabelle Kuhne, Jean-Paul 362 Egreteau, José Simone Guézou, Sandrine Sourisseau, Xavier de Montaudouin (University of 363 Bordeaux, France), Pierre-Guy Sauriau (IFREMER, L'Houmeau, France) for their help with 364 field sampling, Jacques Pigeot for his insights and help with identifications, Stéphane Robert 365 (IFREMER, La Tremblade, France), Philippe Goulletquer (IFREMER, La Tremblade, France) 366 and Yves Pichot (IFREMER, Sète, France) for information on shellfish transfers in France, 367 Pierre Lozouet at the Muséum national d'Histoire naturelle (Paris, France) for granting access 368 to the Ocenebra collection, and Alice Saunier for her help in the lab. This work was funded 369 by the Conseil Général de Charente-Maritime and the Ministère de l'Ecologie et du 370 Développement Durable (Program INVABIO no. D4E/SRP/01115). Salaries for EP & AV 371 were covered by a grant to the Poitou-Charentes region (Contrat de Projet État-Région 2007-372 2013), by a grant from the Fond Européen de Développement Régional (EP), and by Actions 373 Internationales et Rayonnement of the University of La Rochelle (AV). We thank the three 374 the editor, whose comments significantly improved the manuscript. 375 This work is dedicated to the memory of Michel Pascal. .T. (1992) Introduced marine and estuarine mollusks of North America: an end-of-400 the-20th-century perspective. Journal of Shellfish Research, 11, 489-505. 401 Choe, B.L., J.K. Park (1997) Description of Muricid species (Gastropoda: Neogastropoda) 402 collected from the coastal areas of south Korea. Journal of Biological Science, 1, 281-403 296. 404 De Montaudouin, X., P.-G. Sauriau (2000) Contribution to a synopsis of marine species 405 richness in the Pertuis Charentais Sea with new insights in soft-bottom macrofauna of 406 the Marennes-Oléron Bay. Cahiers de Biologie Marine, 41, 188-222. 407 De Wolf, H., R. Verhagen, et al. (2000) Large scale population structure and gene flow in the 408 planktonic developing periwinkle Littorina striata (Mollusca: Gastropoda), in 409 Macronésia. Journal of Experimental Marine Biology and Ecology, 246, 69-83. 410 Elton, C. (1958). The ecology of invasions by animals and plants. University of Chicago 411 Press. 412 Excoffier, L., H.E.L. Lischer (2010) Arlequin suite ver 3.5: A new series of programs to 413 perform population genetics analyses under Linux and Windows. Molecular Ecology 414 Resources, 10, 564-567. 415 Excoffier, L., P.E. Smouse, et al. (1992) Analysis of Molecular Variance Inferred From 416 Metric Distances Among DNA Haplotypes: Application to Human Mitochondrial DNA 417 Restriction Data. Genetics, 131, 479-491. 418 Folmer, O., M. Black, et al. (1994) DNA primers for amplification of mitochondrial 419 cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine 420 Biology and Biotechnology, 3, 294-299. 421 Frankham, R. (1995) Conservation genetics. Annual Review of Genetics, 29, 305-327. 422 Fu, Y-X. (1997) Statistical tests of neutrality of mutations against population growth, 423 hitchhiking and background selection. Genetics, 147, 915-925. 424 .G. (2000) Effects of the east Australian current on the genetic structure of a direct .B., M.

Fig. 1 .

 1 Fig. 1. Median-joining haplotype network. Each circle represents a haplotype, which 579

  in genetic diversity are generally considered detrimental (e.g. 61

  inornatusOcenebra inornata(Recluz, 1851) constitute a noteworthy model to study the 71 genetic effects of indigenous-invasive interactions on indigenous populations. A native of the 72 northwestern Pacific (Choe & Park 1997; Amano & Vermeij 1998), Ocinebrellus inornatusO. 73 inornata (previously known as Ocinebrellus inornatus; see Houart and Sirenko 2003; Bouchet 74 and Houart 2014) (Recluz, 1851) has recently invaded European coasts, probably following 75 massive oyster imports (Pigeot 2000; De Montaudouin & Sauriau 2000; review of Lützen et 76 al. 2012). Genetic data suggest that French populations may come from Asia and the United 77

  2012).Pigeot et al. (2000) recorded a decrease in population density of O. 86 erinacea, in parallel to an in increase in numbers of O. inornata in Marennes-Oléron 87 (Charente-Maritime, France), between 1997 and 1999 (two years after the invasive was first 88 detected). While the introduction and expansion patterns of O. inornatusO. inornata have 89

Page

  . Consequently, a lower genetic diversity within the 100 populations of the exogenous species compared to the populations of the indigenous species 101 was expected. It is thus of importance to test whether the genetic diversity of the indigenous 102 species O. erinacea is correlated with the presence of the introduced species O. inornatusO. 103 inornata in zones of sympatry. Indeed, O. inornatusO. inornata could induce a selective 104 pressure on O. erinacea leading to decrease of polymorphism in this local species. 105 Here, we test this hypothesis by sampling O.cenebra erinacea from the Atlantic and 106Mediterranean French coasts, in the presence and absence of O. inornatusOcenebra inornata, 107 and by measuring genetic diversity of the native species using the mitochondrial marker cox1. 108 While investigating the genetic effects that the presence of O. inornatusO. inornata may have 109 on sympatric populations of O. erinacea, we came across a very strong genetic break between 110 352 adult specimens of Ocenebra erinacea were collected between 1999 and 117 2004 at 15 sites on the French coast, along line transects (<200 m in length). At each site, 118 specimens from different rocks were collected to reduce sampling bias in favor of a particular 119 lineage. The sites were located both within oyster farming zones and unexploited areas (Table120

  1). In order to show a possible impact of the presence of O. inornatusOcenebra inornata on 121 genetic diversity of O. erinacea, five locations where the two species live in sympatry and 10 122 sites free of O. inornatusO. inornata have been sampled. The presence of O. inornata was 123 assessed by direct observation. After collection, specimens were stored in 95% ethanol before 124 DNA extraction. 125 126 DNA extraction, amplification and sequencing 127

  in 50 µL, with 1X PCR buffer, 1.85 mM MgCl 2 , 125 µM dNTPs, 134 0.25 µM of each primer, 1.6 U of Red Hot DNA Polymerase (ABgene) and about 10 ng of 135 DNA template. The following cycling profile was performed using a MJResearch PTC 100 136

  undergone purifying selection, a selective sweep and/or expansion (<0), or balancing selection 156 and/or a population declineor overdominant selection and/or a bottleneck (>0)by generating 10,000 random samples under the hypothesis of 158 selective neutrality and population equilibrium. These tests were performed for each sampling 159 site separately, and also for pooled sites in presence or absence of the invasive. 160

  fragment of cox1 was sequenced for 352 individuals, and 29 haplotypes were 185 identified (GenBank accession numbers AY995771-99; Popset 63109090). Sequences include 186 37 polymorphic sites, 20 of which are parsimony informative, and one of which has three 187 character states. No indels were observed (Table 2). 188 189 Population-level genetic diversity and demographic stability 190 Genetic diversity is comparable among Atlantic populations of Ocenebra. erinacea, 191 but values for the different diversity indices are low compared to other recently-studied 192 marine mollusks. The number of polymorphic sites between two different sequences varies 193 between 0 and 6 among Atlantic populations. Two of these populations (Morbihan and St 194 Quay) are each characterized by a single haplotype, and the 12 other Atlantic sites have no 195

  and spatial distribution of haplotypes 230 Two haplogroups, separated by 18 mutational steps, were observed using the median-231 joining network (Fig. 1). The first haplogroup (23 haplotypes) was mainly composed of 232 Atlantic specimens, and included one specimen from Thau, characterized by haplotype H26 233 (separated from other Mediterranean haplotypes by 20 to 22 mutational steps). Haplotypes 234 from this group diverged by ≤ 2 mutations. Haplotype H1 was common (represented in 89% 235 of Atlantic individuals) and central to the Atlantic haplogroup, while the other 22 haplotypes 236 were rare (7 individuals for H3, 3 for H13, 2 for H6, H9, H15, H19 and a single individual for 237 the others) and peripheral to H1. The second haplogroup was strictly composed of 238 Mediterranean individuals. Of six haplotypes from this group, one was common (H21, 239 represented in 82% of Thau individuals), and four were rare (3 individuals for H22 and a 240 single individual for H23-25). Haplotypes from this group diverged by one mutation. The 241 Atlantic samples share no haplotype with the Mediterranean sample (Fig. 2). Except for H1 242 (present at all sites except Thau), H3 (shared between Loix and Aytré) and H13 (shared 243 between Loix, Aytré and Trébeurden), all haplotypes are private (observed only within one pairs of populations was measured using Φ ST . The 248 population from Thau was significantly differentiated from all other populations. Pairwise Φ ST 249values ranged between 0.94 and 0.96, corresponding to an important substantial genetic 250 differentiation between the Mediterranean and Atlantic populations (Table3). Inside the 251 Atlantic group, no significant differentiation was observed after sequential Bonferroni 252 correction (lowest corrected alpha level for Atlantic populations: 0diversity of Ocenebra. erinacea populations 305 Mitochondrial genetic diversity, as measured using part of cox1, was low relative to 306 what was observed in other marine mollusks. Overall, OcenebraO. erinacea haplotype and 307 nucleotide diversities were (disregarding the sample from Thau, see below) H e = 0.18-0.25 308 and π x10 -3 = 0.38-0.57 (Table

  observed heterozygosity H o characterizing the Atlantic populations of O.cenebra erinacea are 315 respectively two to four times, and twenty to thirty times lower (N all = 1.1 ± 0.1; H o = 0.01 ± 316 0.01; mean ± SD) than in other marine gastropods sampled in their native range, such as 317 Bedeva hanleyi (N all = 2.2 ± 0.1, H o = 0.30 ± 0.02; Hoskin 2000), Drupella sp. (N all = 2.3 ± 318 1.0, H o = 0.25; Johnson & Cumming 1995), and Littorina striata (N all = 4.2 ± 1.0, H o = 0.18 ± 319 0.17; De Wolf et al. 2000). Congruent patterns across mitochondrial and allozyme markers 320 thus suggest a low genetic diversity in these populations rather than an absence of variability 321 at cox1. Still, our sampling remains restricted compared to the native range of the species, and 322 additional monitoring may reveal new patterns of genetic diversity. 323 324 Genetic diversity and connectivity among populations 325

  of the main shell farming areas of the French Mediterranean coasts, carry out 328

  biogeography of this species. Given O. erinacea's maladaptation to 353 cold-water, another possibility is that selection (either purifying selection, or selective sweeps) 354 linked to differences in water temperature between Thau and the Atlantic sites produced the 355 observed patterns of genetic diversity. The negative Tajima's D and Fu's Fs observed for the 356 Atlantic population (in absence and presence of the invasive) would support this scenario.and 357 Hewitt's "northern purity, southern richness" genetic diversity pattern (Magg's et al. Model 358 IIa). 359 One potential consequence of biogeographic divergence between Atlantic and 360

H26

  Fig.ure 1. Median-joining haplotype network. Each circle represents a haplotype, which 601 frequency is proportional to circle diameter (legend: bottom right). 602 Distances between haplotypes are proportional to the number of mutation events (see text). 603 The Atlantic haplogroup contains one Mediterranean population, represented by haplotype 604 H26 (marked with **). 605

  Genetic diversity is comparable among Atlantic populations ofOcenebra erinacea, but 182 values for the different diversity indices are low compared to other recently-studied marine 183 mollusks. The number of polymorphic sites between two different sequences varies between 0 184 and 6 among Atlantic populations. Two of these populations (Morbihan and St Quay) are each 185 characterized by a single haplotype, and the 12 other Atlantic sites have no more than five 186 haplotypes (sample sizes provided in Table1). Consequently, haplotype diversity H e is low, 187 except for Oléron (H e = 0.564; Table2). In Fouras, where Ocenebra inornata and O. erinacea 188 are sympatric, the haplotype and nucleotide diversities are respectively four-and six-fold 189 higher in the invasive drill (H e = 0.348 and π x10 -3 = 0.83; data from Martel 2003) than in the 190 local one (H e = 0.074 and π x10 -3 = 0.14). 191

	181	Population-level genetic diversity and demographic stability
	171 172 173 174 175	, to A 550 bp fragment of cox1 was sequenced for 352 individuals, and 29 haplotypes were F measure genetic distances among cox1 haplotypes. Results o r P e e r R e v i e w
		9

Table 1 .

 1 C. Grant (1984) Associations among protein heterozygosity, growth rate and 496 developmental homeostasis. Annual review of Ecology and Systematics, 15, 479-499. Ocenebra erinacea collection sites (listed from North to South, following the 556 coastline), site name abbreviations (Ab., as used in Figure2), number of O. erinacea 557 specimens collected and sequenced (n), geographical coordinates, year of collection, and 558 characteristics of the locations: presence (+) or absence (-) of oyster farms and of O. inornata.

	F o
	r
	P
	e e r
	R e v i e w

Table 2 .

 2 Molecular diversity of populations and results of the neutrality tests. The number of 563 segregating sites (S), the number of haplotypes (H), the haplotype diversity (H e ± one standard 564 deviation), and the nucleotide diversity (π ± one standard deviation) are given for each 565 sampling site, site groups in presence and in absence of the invasive (with and without the 566 Mediterranean population of Thau), and for the entire data set. Sites where Ocenebra inornata 567 was present are indicated by an asterisk. For neutrality tests ofTajima and Fu, statistical 568 significance after sequential Bonferroni correction is indicated with an asterisk.

	569
	570
	571
	Sampling sites	S	H	He ± SD	π ± SD (x10 -3 ) Tajima's D Fu's Fs
	Blainville	1	2 0.065 ± 0.059	0.12 ± 0.28	-1.14	-1.24*
	Chaussey	3	4 0.230 ± 0.110	0.44 ± 0.58	-1.73	-3.08*
	Saint Malo	1	2 0.091 ± 0.081	0.17 ± 0.34	-1.16	-0.96
	Saint Quay	0	1	0	0	0	0
	Trébeurden	5	5 0.377 ± 0.122	0.90 ± 0.89	-1.83	-2.80*
	Crozon	2	3 0.316 ± 0.118	0.60 ± 0.70	-0.86	-0.87
	Le Croisic	4	4 0.333 ± 0.124	0.81 ± 0.84	-1.67	-1.74
	Morbihan	0	1	0	0	0	0
	Bourgneuf *	2	2 0.083 ± 0.075	0.30 ± 0.47	-1.51	-0.19
	Loix *	5	5 0.270 ± 0.109	0.65 ± 0.72	-2.01*	-3.57*
	Aytré *	4	4 0.363 ± 0.131	0.89 ± 0.89	-1.64	-1.61
	Fouras *	1	2 0.074 ± 0.067	0.14 ± 0.30	-1.15	-1.12
	Oléron *	2	3 0.564 ± 0.112	1.12 ± 1.06	-0.13	-0.17
	Arcachon	1	2 0.095 ± 0.084	0.17 ± 0.35	-1.16	-0.92
	Thau	22	6 0.342 ± 0.098	2.52 ± 1.76	-2.50*	-0.42

Sites in presence of invasive 11 9 0.247 ± 0.0535 0.57 ± 0.65 -2.10* -9.42*

	Sites in absence	31 22 0.399 ± 0.3976 10.04 ± 5.52	0.22	-0.06
	of invasive
	Sites in absence	16 16 0.179 ± 0.0368 0. 38 ± 0. 51	-2.38*	< -10*
	of invasive
	(without Thau)
	All populations 37 29 0.356 ± 0.0325 7.63 ± 4.20	-0.81	-4.97
	572
	573

Table 3 .

 3 Pairwise Φ ST values calculated using the Kimura 2 Parameter model of nucleotide substitution. Only pairwise comparisons involving the 574 population of Thau are statistically significant after sequential Bonferroni correction.

	Page 27 of 61							Marine Ecology
	575
	576
		1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
	1. Blainville	0
	2. Chausey	0.0048	0
			-
	3. Saint Malo	0.00242	0.00244	0
		-	-
	4 . Saint Quay	0.01001	0.00356	0.00207	0
	5. Trebeurden	0.02194	0.01045	0.00921	0.01207	0
	6. Crozon	0.05037	0.02772	0.03452	0.04529	0.02682	0
	7. Le Croisic	0.03001	0.01442	0.01577	0.02118	0.01654	0.01135	0
		-	-	-		-		-
	8. Morbihan	0.03708	0.03371	0.03067	0	0.02156	0.00476	0.01543	0
			-	-	-				-
	9. Bourgneuf	0.00458	0.00021	0.00102	0.00181	0.01083	0.0316	0.01598	0.03274	0
				-	-					-
	10. Loix	0.00242	-0.0008	0.00559	0.00747	0.00258	0.02217	0.012	-0.0358	0.01337	0
									-	-	-
	11. Aytré	0.03811	0.01838	0.02156	0.0293	0.00956	0.03281	0.02045	0.01049	0.00555	0.01096	0
	12. Oleron	0.28743	0.18725	0.23275	0.28709	0.1342	0.16138	0.13706	0.18898	0.15611	0.12163	0.04004	0
					-				-		-
	13. Fouras	0.0004	0.00162	0.00096	0.00611	0.01656	0.04351	0.02394	0.03502	0.00196	0.00096	0.03104	0.26425	0
			-						-	-	-
	14. Arcachon	0.0032	0.00323	0.00014	0.00443	0.00763	0.03266	0.01404	0.02941	0.00153	0.00658	0.01955	0.22606	0.00145	0
	15. Thau	0.96208	0.95559	0.95617	0.95856	0.94988	0.95236	0.95001	0.94928	0.95614	0.9547	0.9484	0.94167	0.95959	0.95541	0
	577
	578
								Marine Ecology

Table 1 .

 1 . In addition, O. 367 erinacea specimens from the Atlantic and Mediterranean can readily be distinguished using 368 morphology, and the morphological distance between Atlantic and Mediterranean O. erinacea 369 is comparable to what is observed between O. erinacea and Ocenebra. brevirobusta Houart 370 2000 (Berrou et al. 2004). It is therefore possible that the Atlantic and Mediterranean clades 371 sampled for this study belong to groups in incipient stages of speciation, or even undescribed 372 Sampling sites.Ocenebra erinacea Collection collection sites (listed from North to 576

	373	species.

Table 2 .

 2 Molecular diversity of populations and results of the neutrality tests. The number of 584 segregating sites (S), the number of haplotypes (H), the haplotype diversity (H e ± one standard 585 deviation), and the nucleotide diversity (π ± one standard deviation) are given for each 586 sampling site, site groups in presence and in absence of the invasive (with and without the 587 Mediterranean population of Thau), and for the entire data set. Sites where O. 588 inornatusOcenebra inornata was present are indicated by an asterisk. For neutrality tests of 589 Tajima and Fu, statistical significance after sequential Bonferroni correction is indicated with 590

	591	an asterisk.
	592
	593
		Sampling sites	S	H	H e ± ± ± ± SD	π ± ± ± ± SD (

x10 -3 ) Tajima's D Fu's Fs

	Page 57 of 61				Marine Ecology
		Saint Quay	0	1	0	0	0	0
		Trébeurden	5	5 0.377 ± 0.122	0.90 ± 0.89	-1.83	-2.80*
		Crozon	2	3 0.316 ± 0.118	0.60 ± 0.70	-0.86	-0.87
		Le Croisic	4	4 0.333 ± 0.124	0.81 ± 0.84	-1.67	-1.74
		Morbihan	0	1	0	0	0	0
		Bourgneuf *	2	2 0.083 ± 0.075	0.30 ± 0.47	-1.51	-0.19
		Loix *	5	5 0.270 ± 0.109	0.65 ± 0.72	-2.01*	-3.57*
		Aytré *	4	4 0.363 ± 0.131	0.89 ± 0.89	-1.64	-1.61
		Fouras *	1	2 0.074 ± 0.067	0.14 ± 0.30	-1.15	-1.12
		Oléron *	2	3 0.564 ± 0.112	1.12 ± 1.06	-0.13	-0.17
		Arcachon	1	2 0.095 ± 0.084	0.17 ± 0.35	-1.16	-0.92
		Thau	22	6 0.342 ± 0.098	2.52 ± 1.76	-2.50*	-0.42
		Sites in presence of invasive Sites in absence of invasive of invasive Sites in absence	11 31 22 0.399 ± 0.3976 10.04 ± 5.52 9 0.247 ± 0.0535 0.57 ± 0.65 F o r 16 16 0.179 ± 0.0368 0. 38 ± 0. 51	-2.10* 0.22 -2.38*	-9.42* -0.06 < -10*
		(without Thau)
	594	All populations 37 29 0.356 ± 0.0325 7.63 ± 4.20 P	-0.81	-4.97
	595	Blainville Chaussey Saint Malo Saint Quay	1 3 1 0	2 0.065 ± 0.059 4 0.230 ± 0.110 2 0.091 ± 0.081 1 0	0.12 ± 0.28 0.44 ± 0.58 0.17 ± 0.34 0 r e e	-1.14 -1.73 -1.16 0.00	-1.24* -3.08* -0.96 0.00
		Trébeurden Crozon Le Croisic Morbihan Bourgneuf * Loix * Aytré * Fouras * Oléron * Arcachon Thau Sites in presence of invasive	5 2 4 0 2 5 4 1 2 1 22 11	5 0.377 ± 0.122 3 0.316 ± 0.118 4 0.333 ± 0.124 1 0 2 0.083 ± 0.075 5 0.270 ± 0.109 4 0.363 ± 0.131 2 0.074 ± 0.067 3 0.564 ± 0.112 2 0.095 ± 0.084 6 0.342 ± 0.098 9 0.247 ± ± ± ± 0.0535 0.57 ± 0.90 ± 0.89 0.60 ± 0.70 0.81 ± 0.84 0 0.30 ± 0.47 0.65 ± 0.72 0.89 ± 0.89 0.14 ± 0.30 1.12 ± 1.06 0.17 ± 0.35 2.52 ± 1.76 ± ± ± 0.65 R -1.83 -0.86 -1.67 0.00 -1.51 -2.01* -1.64 -1.15 -0.13 -1.16 -2.50* -2.10* e v -2.80* -0.87 -1.74 0.00 -0.19 -3.57* -1.61 -1.12 -0.17 -0.92 -0.42 -9.42* i e w
		Sites in absence of invasive	31 22 0.399 ± ± ± ± 0.3976 10.04 ± ± ± ± 5.52	0.22	-0.06
		Sites in absence	16 16 0.179 ± ± ± ± 0.0368 0. 38 ± ± ± ± 0. 51	-2.38*	< -10*
		of invasive
		(without Thau)
		All populations 37 29 0.356 ± ± ± ± 0.0325 7.63 ± ± ± ± 4.20	-0.81	-4.97
		Sampling sites	S	H	He ± SD	π ± SD (x10 -3 ) Tajima's D Fu's Fs
		Blainville	1	2 0.065 ± 0.059	0.12 ± 0.28	-1.14	-1.24*
		Chaussey	3	4 0.230 ± 0.110	0.44 ± 0.58	-1.73	-3.08*
		Saint Malo	1	2 0.091 ± 0.081	0.17 ± 0.34	-1.16	-0.96
								29
						Marine Ecology

Page 58 of 61 Marine Ecology Marine Ecology F o r P e e r R e v i e w 30Table 3 .

 3 Pairwise Φ ST values calculated using the Kimura 2 Parameter model of nucleotide substitution. Only pairwise comparisons involving the 596 population of Thau are statistically significant after sequential Bonferroni correction (p=0).

	Page 59 of 61									Marine Ecology
	597
	598
		1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
	1. Blainville	0
	2. Chausey	0.0048	0
			-
	3. Saint Malo	0.00242	0.00244	0
		-	-
	4 . Saint Quay	0.01001	0.00356	0.00207	0
	5. Trebeurden	0.02194	0.01045	0.00921	0.01207	0
	6. Crozon	0.05037	0.02772	0.03452	0.04529	0.02682	0
	7. Le Croisic	0.03001	0.01442	0.01577	0.02118	0.01654	0.01135	0
		-	-	-		-		-
	8. Morbihan	0.03708	0.03371	0.03067	0	0.02156	0.00476	0.01543	0
			-	-	-				-
	9. Bourgneuf	0.00458	0.00021	0.00102	0.00181	0.01083	0.0316	0.01598	0.03274	0
				-	-					-
	10. Loix	0.00242	-0.0008	0.00559	0.00747	0.00258	0.02217	0.012	-0.0358	0.01337	0
									-	-	-
	11. Aytré	0.03811	0.01838	0.02156	0.0293	0.00956	0.03281	0.02045	0.01049	0.00555	0.01096	0
	12. Oleron	0.28743	0.18725	0.23275	0.28709	0.1342	0.16138	0.13706	0.18898	0.15611	0.12163	0.04004	0
					-				-		-
	13. Fouras	0.0004	0.00162	0.00096	0.00611	0.01656	0.04351	0.02394	0.03502	0.00196	0.00096	0.03104	0.26425	0
			-						-	-	-
	14. Arcachon	0.0032	0.00323	0.00014	0.00443	0.00763	0.03266	0.01404	0.02941	0.00153	0.00658	0.01955	0.22606	0.00145	0
	15. Thau	0.96208	0.95559	0.95617	0.95856	0.94988	0.95236	0.95001	0.94928	0.95614	0.9547	0.9484	0.94167	0.95959	0.95541	0
	599
	600
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developing muricid snail (Bedeva hanleyi, Angas): variability within and among local

polymorphism data. Bioinformatics, 25, 1451-1452.

Acknowledgements

Marine Ecology

Marine Ecology

Marine Ecology

Marine Ecology

F o r P e e r R e v i e w

Discussion 269