Genetic sex determination in three closely related hydrothermal vent gastropods, including one species with intersex individuals.

Molluscs have undergone many transitions between separate sexes and hermaphroditism, which is of interest in studying the evolution of sex determination and differentiation. Here, we combined multi-locus genotypes obtained from restriction site-associated DNA (RAD) sequencing with anatomical observations of the gonads of three deep-sea hydrothermal vent gastropods of the genus Alviniconcha living in the southwest Pacific. We found that all three species (Alviniconcha boucheti, Alviniconcha strummeri, and Alviniconcha kojimai) share the same male-heterogametic XY sex-determination system but that the gonads of XX A. kojimai individuals are invaded by a variable proportion of male reproductive tissue. The identification of Y-specific RAD loci (found only in A. boucheti) and the phylogenetic analysis of three sex-linked loci shared by all species suggested that X-Y recombination has evolved differently within each species. This situation of three species showing variation in gonadal development around a common sex-determination system provides new insights into the reproductive mode of poorly known deep-sea species and opens up an opportunity to study the evolution of recombination suppression on sex chromosomes and its association with mixed or transitory sexual systems.

Contrasted phylogeographic patterns of hydrothermal vent gastropods along South West Pacific: Woodlark Basin, a possible contact zone and/or stepping-stone.

Understanding drivers of biodiversity patterns is essential to evaluate the potential impact of deep-sea mining on ecosystems resilience. While the South West Pacific forms an independent biogeographic province for hydrothermal vent fauna, different degrees of connectivity among basins were previously reported for a variety of species depending on their ability to disperse. In this study, we compared phylogeographic patterns of several vent gastropods across South West Pacific back-arc basins and the newly-discovered La Scala site on the Woodlark Ridge by analysing their genetic divergence using a barcoding approach. We focused on six genera of vent gastropods widely distributed in the region: Lepetodrilus, Symmetromphalus, Lamellomphalus, Shinkailepas, Desbruyeresia and Provanna. A wide-range sampling was conducted at different vent fields across the Futuna Volcanic Arc, the Manus, Woodlark, North Fiji, and Lau Basins, during the CHUBACARC cruise in 2019. The Cox1-based genetic structure of geographic populations was examined for each taxon to delineate putative cryptic species and assess potential barriers or contact zones between basins. Results showed contrasted phylogeographic patterns among species, even between closely related species. While some species are widely distributed across basins (i.e. Shinkailepas tollmanni, Desbruyeresia melanioides and Lamellomphalus) without evidence of strong barriers to gene flow, others are restricted to one (i.e. Shinkailepas tufari complex of cryptic species, Desbruyeresia cancellata and D. costata). Other species showed intermediate patterns of isolation with different lineages separating the Manus Basin from the Lau/North Fiji Basins (i.e. Lepetodrilus schrolli, Provanna and Symmetromphalus spp.). Individuals from the Woodlark Basin were either endemic to this area (though possibly representing intermediate OTUs between the Manus Basin and the other eastern basins populations) or, coming into contact from these basins, highlighting the stepping-stone role of the Woodlark Basin in the dispersal of the South West Pacific vent fauna. Results are discussed according to the dispersal ability of species and the geological history of the South West Pacific.

Inter-Specific Genetic Exchange Despite Strong Divergence in Deep-Sea Hydrothermal Vent Gastropods of the Genus Alviniconcha.

Deep hydrothermal vents are highly fragmented and unstable habitats at all temporal and spatial scales. Such environmental dynamics likely play a non-negligible role in speciation. Little is, however, known about the evolutionary processes that drive population-level differentiation and vent species isolation and, more specifically, how geography and habitat specialisation interplay in the species history of divergence. In this study, the species range and divergence of Alviniconcha snails that occupy active Western Pacific vent fields was assessed by using sequence variation data of the mitochondrial Cox1 gene, RNAseq, and ddRAD-seq. Combining morphological description and sequence datasets of the three species across five basins, we confirmed that A. kojimai, A. boucheti, and A. strummeri, while partially overlapping over their range, display high levels of divergence in the three genomic compartments analysed that usually encompass values retrieved for reproductively isolated species with divergences rang from 9% to 12.5% (mtDNA) and from 2% to 3.1% (nuDNA). Moreover, the three species can be distinguished on the basis of their external morphology by observing the distribution of bristles and the shape of the columella. According to this sampling, A. boucheti and A. kojimai form an east-to-west species abundance gradient, whereas A. strummeri is restricted to the Futuna Arc/Lau and North Fiji Basins. Surprisingly, population models with both gene flow and population size heterogeneities among genomes indicated that these three species are still able to exchange genes due to secondary contacts at some localities after a long period of isolation.

Water eDNA metabarcoding is effective in detecting non-native species in marinas, but detection errors still hinder its use for passive monitoring.

Marinas are high-priority targets for marine non-indigenous species (NIS), where they compose a large portion of the biofouling communities. The practicality of water samples collection makes environmental DNA (eDNA) metabarcoding an interesting tool for routine NIS surveys. Here the effectiveness of water-eDNA-metabarcoding to identify biofouling NIS, in 10 marinas from western France, was examined. Morphological identification of specimens collected in quadrats brought out 18 sessile benthic NIS beneath floating pontoons. Water-eDNA-metabarcoding detected two thirds of them, failing to detect important NIS. However, sampling and bioinformatics filtering steps can be optimized to identify more species. In addition, this method allowed the detection of additional NIS from neighboring micro-habitats. Caution should, however, be taken when reporting putative novel NIS, because of errors in species assignment. This work highlights that water-eDNA-metabarcoding is effective for active (targeted) NIS surveys and could be significantly improved for its further use in marine NIS passive surveys.

Searching for genetic evidence of demographic decline in an arctic seabird: beware of overlapping generations.

Genetic data are useful for detecting sudden population declines in species that are difficult to study in the field. Yet this indirect approach has its own drawbacks, including population structure, mutation patterns, and generation overlap. The ivory gull (Pagophila eburnea), a long-lived Arctic seabird, is currently suffering from rapid alteration of its primary habitat (i.e., sea ice), and dramatic climatic events affecting reproduction and recruitment. However, ivory gulls live in remote areas, and it is difficult to assess the population trend of the species across its distribution. Here we present complementary microsatellite- and SNP-based genetic analyses to test a recent bottleneck genetic signal in ivory gulls over a large portion of their distribution. With attention to the potential effects of population structure, mutation patterns, and sample size, we found no significant signatures of population decline worldwide. At a finer scale, we found a significant bottleneck signal at one location in Canada. These results were compared with predictions from simulations showing how generation time and generation overlap can delay and reduce the bottleneck microsatellite heterozygosity excess signal. The consistency of the results obtained with independent methods strongly indicates that the species shows no genetic evidence of an overall decline in population size. However, drawing conclusions related to the species' population trends will require a better understanding of the effect of age structure in long-lived species. In addition, estimates of the effective global population size of ivory gulls were surprisingly low (~1000 ind.), suggesting that the evolutionary potential of the species is not assured.

Subtle limits to connectivity revealed by outlier loci within two divergent metapopulations of the deep-sea hydrothermal gastropod Ifremeria nautilei.

Hydrothermal vents form archipelagos of ephemeral deep-sea habitats that raise interesting questions about the evolution and dynamics of the associated endemic fauna, constantly subject to extinction-recolonization processes. These metal-rich environments are coveted for the mineral resources they harbour, thus raising recent conservation concerns. The evolutionary fate and demographic resilience of hydrothermal species strongly depend on the degree of connectivity among and within their fragmented metapopulations. In the deep sea, however, assessing connectivity is difficult and usually requires indirect genetic approaches. Improved detection of fine-scale genetic connectivity is now possible based on genome-wide screening for genetic differentiation. Here, we explored population connectivity in the hydrothermal vent snail Ifremeria nautilei across its species range encompassing five distinct back-arc basins in the Southwest Pacific. The global analysis, based on 10,570 single nucleotide polymorphism (SNP) markers derived from double digest restriction-site associated DNA sequencing (ddRAD-seq), depicted two semi-isolated and homogeneous genetic clusters. Demogenetic modeling suggests that these two groups began to diverge about 70,000 generations ago, but continue to exhibit weak and slightly asymmetrical gene flow. Furthermore, a careful analysis of outlier loci showed subtle limitations to connectivity between neighbouring basins within both groups. This finding indicates that migration is not strong enough to totally counterbalance drift or local selection, hence questioning the potential for demographic resilience at this latter geographical scale. These results illustrate the potential of large genomic data sets to understand fine-scale connectivity patterns in hydrothermal vents and the deep sea.

An introgression breakthrough left by an anthropogenic contact between two ascidians.

Human-driven translocations of species have diverse evolutionary consequences such as promoting hybridization between previously geographically isolated taxa. This is well illustrated by the solitary tunicate, Ciona robusta, native to the North East Pacific and introduced in the North East Atlantic. It is now co-occurring with its congener Ciona intestinalis in the English Channel, and C. roulei in the Mediterranean Sea. Despite their long allopatric divergence, first and second generation crosses showed a high hybridization success between the introduced and native taxa in the laboratory. However, previous genetic studies failed to provide evidence of recent hybridization between C. robusta and C. intestinalis in the wild. Using SNPs obtained from ddRAD-sequencing of 397 individuals from 26 populations, we further explored the genome-wide population structure of the native Ciona taxa. We first confirmed results documented in previous studies, notably (i) a chaotic genetic structure at regional scale, and (ii) a high genetic similarity between C. roulei and C. intestinalis, which is calling for further taxonomic investigation. More importantly, and unexpectedly, we also observed a genomic hotspot of long introgressed C. robusta tracts into C. intestinalis genomes at several locations of their contact zone. Both the genomic architecture of introgression, restricted to a 1.5 Mb region of chromosome 5, and its absence in allopatric populations suggest introgression is recent and occurred after the introduction of the non-native species. Overall, our study shows that anthropogenic hybridization can be effective in promoting introgression breakthroughs between species at a late stage of the speciation continuum.

High-throughput sequencing on preservative ethanol is effective at jointly examining infraspecific and taxonomic diversity, although bioinformatics pipelines do not perform equally.

High-throughput sequencing of amplicons (HTSA) has been proposed as an effective approach to evaluate taxonomic and genetic diversity at the same time. However, there are still uncertainties as to how the results produced by different bioinformatics treatments impact the conclusions drawn on biodiversity and population genetics indices.We evaluated the ability of six bioinformatics pipelines to recover taxonomic and genetic diversity from HTSA data obtained from controlled assemblages. To that end, 20 assemblages were produced using 354 colonies of Botrylloides spp., sampled in the wild in ten marinas around Brittany (France). We used DNA extracted from preservative ethanol (ebDNA) after various time of storage (3, 6, and 12 months), and from a bulk of preserved specimens (bulkDNA). DNA was amplified with primers designed for targeting this ascidian genus. Results obtained from HTSA data were compared with Sanger sequencing on individual zooids (i.e., individual barcoding).Species identification and relative abundance determined with HTSA data from either ebDNA or bulkDNA were similar to those obtained with traditional individual barcoding. However, after 12 months of storage, the correlation between HTSA and individual-based data was lower than after shorter durations. The six bioinformatics pipelines were able to depict accurately the genetic diversity using standard population genetics indices (HS and FST), despite producing false positives and missing rare haplotypes. However, they did not perform equally and dada2 was the only pipeline able to retrieve all expected haplotypes.This study showed that ebDNA is a nondestructive alternative for both species identification and haplotype recovery, providing storage does not last more than 6 months before DNA extraction. Choosing the bioinformatics pipeline is a matter of compromise, aiming to retrieve all true haplotypes while avoiding false positives. We here recommend to process HTSA data using dada2, including a chimera-removal step. Even if the possibility to use multiplexed primer sets deserves further investigation to expand the taxonomic coverage in future similar studies, we showed that primers targeting a particular genus allowed to reliably analyze this genus within a complex community.

Sexual isolation with and without ecological isolation in marine isopods Jaera albifrons and J. praehirsuta.

Sexual barriers associated with mate choice are often found to be associated with some level of ecological isolation between species. The independence and relative strength of sexual isolation are thus difficult to assess. Here, we take advantage of a pair of marine isopod species (Jaera albifrons and J. praehirsuta) that show sexual isolation and coexist in populations where they share the same microhabitat or not (i.e. without or with ecological isolation). We estimated the strength of sexual isolation between J. albifrons and J. praehirsuta using no-choice trials and a multiple-choice experimental population. We found that sexual isolation is strong in both the presence and the absence of ecological isolation, but that it is asymmetric and fails to prevent interspecific gene flow entirely. First-generation intrinsic post-zygotic barriers were low, and there was no sexual isolation within J. praehirsuta across habitats. The J. albifrons/J. praehirsuta species pair thus provides an example where the role of sexual isolation as a barrier to gene flow (a) does not depend upon current ecological isolation, (b) seems to have evolved independently of local ecological conditions, but (c) is insufficient to complete speciation entirely on its own.

A genome-wide investigation of the worldwide invader Sargassum muticum shows high success albeit (almost) no genetic diversity.

Twenty years of genetic studies of marine invaders have shown that successful invaders are often characterized by native and introduced populations displaying similar levels of genetic diversity. This pattern is presumably due to high propagule pressure and repeated introductions. The opposite pattern is reported in this study of the brown seaweed, Sargassum muticum, an emblematic species for circumglobal invasions. Albeit demonstrating polymorphism in the native range, microsatellites failed to detect any genetic variation over 1,269 individuals sampled from 46 locations over the Pacific-Atlantic introduction range. Single-nucleotide polymorphisms (SNPs) obtained from ddRAD sequencing revealed some genetic variation, but confirmed severe founder events in both the Pacific and Atlantic introduction ranges. Our study thus exemplifies the need for extreme caution in interpreting neutral genetic diversity as a proxy for invasive potential. Our results confirm a previously hypothesized transoceanic secondary introduction from NE Pacific to Europe. However, the SNP panel unexpectedly revealed two additional distinct genetic origins of introductions. Also, conversely to scenarios based on historical records, southern rather than northern NE Pacific populations could have seeded most of the European populations. Finally, the most recently introduced populations showed the lowest selfing rates, suggesting higher levels of recombination might be beneficial at the early stage of the introduction process (i.e., facilitating evolutionary novelties), whereas uniparental reproduction might be favored later in sustainably established populations (i.e., sustaining local adaptation).

Population genomics of the introduced and cultivated Pacific kelp Undaria pinnatifida: Marinas-not farms-drive regional connectivity and establishment in natural rocky reefs.

Ports and farms are well-known primary introduction hot spots for marine non-indigenous species (NIS). The extent to which these anthropogenic habitats are sustainable sources of propagules and influence the evolution of NIS in natural habitats was examined in the edible seaweed Undaria pinnatifida, native to Asia and introduced to Europe in the 1970s. Following its deliberate introduction 40 years ago along the French coast of the English Channel, this kelp is now found in three contrasting habitat types: farms, marinas and natural rocky reefs. In the light of the continuous spread of this NIS, it is imperative to better understand the processes behind its sustainable establishment in the wild. In addition, developing effective management plans to curtail the spread of U. pinnatifida requires determining how the three types of populations interact with one another. In addition to an analysis using microsatellite markers, we developed, for the first time in a kelp, a ddRAD-sequencing technique to genotype 738 individuals sampled in 11 rocky reefs, 12 marinas, and two farms located along ca. 1,000 km of coastline. As expected, the RAD-seq panel showed more power than the microsatellite panel for identifying fine-grained patterns. However, both panels demonstrated habitat-specific properties of the study populations. In particular, farms displayed very low genetic diversity and no inbreeding conversely to populations in marinas and natural rocky reefs. In addition, strong, but chaotic regional genetic structure, was revealed, consistent with human-mediated dispersal (e.g., leisure boating). We also uncovered a tight relationship between populations in rocky reefs and those in nearby marinas, but not with nearby farms, suggesting spillover from marinas into the wild. At last, a temporal survey spanning 20 generations showed that wild populations are now self-sustaining, albeit there was no evidence for local adaptation to any of the three habitats. These findings highlight that limiting the spread of U. pinnatifida requires efficient management policies that also target marinas.

Geographically distinct patterns of reproductive isolation and hybridization in two sympatric species of the Jaera albifrons complex (marine isopods).

Sympatric species that in some populations hybridize and in other populations remain reproductively isolated open interesting research possibilities for the study of hybridization and speciation. Here, we test for such a situation in two littoral isopods (Jaera albifrons and J. praehirsuta) that occur in mixed populations and where past morphological descriptions suggested that the two species are generally reproductively isolated except in rare populations where hybridization may be happening. Using field surveys and microsatellite genetic structure analyses in two regions from France (Normandy and Brittany), we confirmed that introgressive hybridization occurs in a subset of mixed J. albifrons/J. praehirsuta populations (region Normandy) where the two species are found in the same habitat (pebbles on the shore). Moreover, we found that introgression in these populations is differential, 21 of 23 microsatellite markers showing little genetic divergence between species (hierarchical analysis of molecular variance FCT = 0.017) while the remaining two loci were strongly differentiated (FCT = 0.428). By contrast, J. albifrons and J. praehirsuta in mixed populations from region Brittany occupied distinct habitats (pebbles and seaweeds, respectively) with little overlap and showed stronger genetic divergence (FCT = 0.132). In hybridizing populations, the majority of individuals show morphological traits that are characteristic of one or the other species. This raises the question of the forces that act to maintain this polymorphism, noting that hybridizing populations seem to be geographically isolated from potential source parental populations and show no detectable habitat divergence between species.

Presence of the tunicate Asterocarpa humilis on ship hulls and aquaculture facilities in the coast of the Biobío Region, south central Chile.

Non-native ascidians are important members of the fouling community associated with artificial substrata and man-made structures. Being efficient fouling species, they are easily spread by human-mediated transports (e.g., with aquaculture trade and maritime transports). This is exemplified by the ascidian Asterocarpa humilis which displays a wide distribution in the Southern Hemisphere and has been recently reported in the Northern Hemisphere (NW Europe). In continental Chile, its first report dates back from 2000 for the locality of Antofagasta (23°S). Although there was no evidence about the vectors of introduction and spread, nor the source, some authors suggested maritime transport by ship hulls and aquaculture devices as putative introduction pathways and vectors. In the present study, we report for the first time the presence of A. humilis on the hull of an international ship in a commercial port in Concepción bay (36°S), south central Chile. We also found one individual associated to a seashell farm, 70 km far from Concepción bay. Further individuals were subsequently identified within Concepción bay: one juvenile settled upon international harbor pilings and a dozen individuals along aquaculture seashell longlines. For the first specimens sampled, species identification was ascertained using both morphological criteria and molecular barcoding, using the mitochondrial gene cytochrome c oxidase subunit I (COI) and a nuclear gene (ribosomal RNA 18S). The nuclear 18S gene and the mitochondrial gene COI clearly assigned the specimens to A. humilis, confirming our morphological identification. Two haplotypes were obtained with COI corresponding to haplotypes previously obtained with European and Northern Chilean specimens. The present study thus reports for the first time the presence of A. humilis in the Araucanian ecoregion, documenting the apparent expansion of this non-native tunicate in Chile over 2,000 km, spanning over three ecoregions. In addition we reveal the potential implication of the international maritime transport as a vector of spread of this species along the Eastern Pacific coast, and the putative role of aquaculture facilities in promoting local establishments of non-native tunicates.

Microsatellite markers for the Jaera albifrons species complex (marine isopods).

BACKGROUND: The Jaera albifrons complex contains five species of marine isopods (J. albifrons, J. praehirsuta, J. ischiosetosa, J. forsmani, and J. posthirsuta). These species, occurring on the shores of the North-Atlantic Ocean, are partially reproductively isolated by barriers due to sexual isolation (mate choice), genetic incompatibilities, and ecological specialization. Microsatellite loci would be useful for parentage-based analyses of sexual selection and studies of genetic structure in the context of speciation. FINDINGS: Twenty-four microsatellite markers were developed for J. albifrons using pyrosequencing of enriched libraries. Patterns of polymorphisms were analyzed in 49 J. albifrons adult males sampled in two populations from Brittany (Western France). The average number of alleles per locus was 4.73 ± 2.45 and the average gene diversity was 0.55 ± 0.23. Most markers also successfully amplified in the three sibling species J. praehirsuta, J. ischiosetosa, and J. forsmani. CONCLUSIONS: These polymorphic and cross-amplifiable markers will be useful for population genetics and parentage studies in the J albifrons complex.

Contrasting patterns of genome-wide polymorphism in the native and invasive range of the marine mollusc Crepidula fornicata.

Selection processes are believed to be an important evolutionary driver behind the successful establishment of nonindigenous species, for instance through adaptation for invasiveness (e.g. dispersal mechanisms and reproductive allocation). However, evidence supporting this assumption is still scarce. Genome scans have often identified loci with atypical patterns of genetic differentiation (i.e. outliers) indicative of selection processes. Using microsatellite- and AFLP-based genome scans, we looked for evidence of selection following the introduction of the mollusc Crepidula fornicata. Native to the northwestern Atlantic, this gastropod has become an emblematic invader since its introduction during the 19th and 20th centuries in the northeastern Atlantic and northeastern Pacific. We examined 683 individuals from seven native and 15 introduced populations spanning the latitudinal introduction and native ranges of the species. Our results confirmed the previously documented high genetic diversity in native and introduced populations with little genetic structure between the two ranges, a pattern typical of marine invaders. Analysing 344 loci, no outliers were detected between the introduced and native populations or in the introduced range. The genomic sampling may have been insufficient to reveal selection especially if it acts on traits determined by a few genes. Eight outliers were, however, identified within the native range, underlining a genetic singularity congruent with a well-known biogeographical break along the Florida. Our results call into question the relevance of AFLP genome scans in detecting adaptation on the timescale of biological invasions: genome scans often reveal long-term adaptation involving numerous genes throughout the genome but seem less effective in detecting recent adaptation from pre-existing variation on polygenic traits. This study advocates other methods to detect selection effects during biological invasions-for example on phenotypic traits, although genome scans may remain useful for elucidating introduction histories.

Genetics and taxonomy of Chilean smooth-shelled mussels, Mytilus spp. (Bivalvia: Mytilidae).

It has been previously established that native smooth-shelled mussels in southern South America possess close evolutionary affinities with Northern-Hemisphere Mytilus edulis L. 1758 (McDonald et al. (1991) [5]). This result has since been challenged by authors claiming that Chilean mussels should be considered a local subspecies of M. galloprovincialis Lmk. 1819. Moreover, morphological, physiological, ecotoxicological and molecular genetic studies on Chilean smooth-shelled mussels still frequently refer to 'M. chilensis' Hupé 1854, even though the previous discovery of alien M. galloprovincialis and considerable heterogeneity in shell morphology among samples collected along the Chilean shores raise concerns that different Mytilus spp. species might have been included under 'M. chilensis'. Here we reviewed the molecular and morphological data available on smooth-shelled mussels from Chile in an attempt to clarify both their genetic composition and their taxonomic status. Using multivariate analysis on sample×allozyme-frequency matrices, we confirmed the widespread occurrence of the Southern-Hemisphere form of M. edulis along the shores from the North Patagonia region of Chile to the southern tip of the South American continent. The populations sampled in southern central Chile showed some evidence of slight introgression from Southern-Hemisphere M. galloprovincialis. Morphological characterization of a sample from Dichato in southern central Chile was consistent with its previous genetic identification as Mediterranean M. galloprovincialis. The occurrence of Southern-Hemisphere M. galloprovincialis in Punta Arenas at the southern tip of the South American continent was also reported. Southern-Hemisphere M. edulis, including native Chilean smooth-shelled Mytilus, should be assigned subspecific rank and named M. edulis platensis d'Orbigny 1846.

GENETIC POPULATION STRUCTURE AND MATING SYSTEM IN CHONDRUS CRISPUS (RHODOPHYTA)1.

Chondrus crispus Stackh. has been intensely studied, yet no study to date has elucidated its population structure or mating system despite many populations in which there was a haploid bias and lack of male gametophytes. Therefore, 12 nuclear microsatellite loci were identified in this red alga. Microsatellite markers were developed and tested against a panel of specimens collected from two shore levels at two sites in Brittany, France: Pointe de Primel and Pointe de la Jument, Concarneau. Single locus genetic determinism was verified at eight polymorphic loci, as only one band was observed for haploid genotypes, whereas one or two bands were observed for diploids. These markers enabled the detection of unique genotypes within sampled populations, indicating that very few fronds shared the same multilocus genotype. This finding suggests that asexual reproduction was not the prevailing mode of reproduction. In addition, we explored the hierarchical population structure showing that gene flow is restricted at small spatial scales (<50 m) between upper and lower Chondrus-range populations within a shore. Sexual reproduction predominated in the populations of C. crispus studied, but probably due to fine-scale spatial substructuring, inbreeding was also significant. In conclusion, this study reveals that fine-scale genetic variation is of major importance in C. crispus, suggesting that differences between microhabitats should be essential in understanding evolutionary processes in this species.

Isolation of 11 microsatellite markers in Crepidula convexa (Gastropoda, Calyptraeideae) for parentage analyses.

Crepidula convexa, a calyptreid gastropod with direct embryonic development, changes sex from male to female in the course of its lifetime (protandry). Under sex-allocation theory, male reproductive success should be independent from age and size (a proxy used for age). However, this may be counterbalanced by female cryptic choice or gregarious behaviour. Eleven polymorphic microsatellite loci were thus developed to examine paternity of embryos and larvae. This set of loci appears suitable to carry out paternity analyses due to the high exclusion probability of unrelated males given the maternal genotype.