The accurate delimitation of species boundaries in non-bilaterian marine taxa is notoriously difficult, with consequences for many studies in ecology and evolution. Anthozoans are a diverse group of key structural organisms worldwide, but the lack of reliable morphological characters and informative genetic markers hampers our ability to understand species diversification. We investigated population differentiation and species limits in Atlantic (Iberian Peninsula) and Mediterranean lineages of the octocoral genus Paramuricea previously identified as P. clavata. We used a diverse set of molecular markers (microsatellites, RNA-seq derived single-copy orthologues [SCO] and mt-mutS [mitochondria]) at 49 locations. Clear segregation of Atlantic and Mediterranean lineages was found with all markers. Species-tree estimations based on SCO strongly supported these two clades as distinct, recently diverged sister species with incomplete lineage sorting, P. cf. grayi and P. clavata, respectively. Furthermore, a second putative (or ongoing) speciation event was detected in the Atlantic between two P. cf. grayi colour morphotypes (yellow and purple) using SCO and supported by microsatellites. While segregating P. cf. grayi lineages showed considerable geographic structure, dominating circalittoral communities in southern (yellow) and western (purple) Portugal, their occurrence in sympatry at some localities suggests a degree of reproductive isolation. Overall, our results show that previous molecular and morphological studies have underestimated species diversity in Paramuricea occurring in the Iberian Peninsula, which has important implications for conservation planning. Finally, our findings validate the usefulness of phylotranscriptomics for resolving evolutionary relationships in octocorals.

With the advent of genomics, sequencing thousands of loci from hundreds of individuals now appears feasible at reasonable costs, allowing complex phylogenies to be resolved. This is particularly relevant for cnidarians, for which insufficient data due to the small number of currently available markers, coupled with difficulties in inferring gene trees and morphological incongruences, encrypts species boundaries, thereby blurring the study and conservation of these organisms. Yet, can genomics alone be used to delimit species in an integrative taxonomic context? Here, focusing on the coral genus Pocillopora, which plays key roles in Indo-Pacific reef ecosystems but has challenged taxonomists for decades, we explored and discussed the usefulness of multiple criteria (genetics, morphology, biogeography and symbiosis ecology) to delimit species of this genus. Phylogenetic inferences, clustering approaches and species delimitation methods based on genome-wide single-nucleotide polymorphisms (SNPs) were first used to resolve Pocillopora phylogeny and propose genomic species hypotheses from 356 colonies sampled across the Indo-Pacific (western Indian Ocean, tropical southwestern Pacific and south-east Polynesia). These species hypotheses were then compared to previous genetic evidences, as well as to evidences based on morphology, biogeography and symbiosis. Genomics allowed to delimit 21 species hypotheses where only seven are currently recognised based on current taxonomy. Moreover, 13 species were strongly supported by all approaches, either confirming their currently recognised species status, or supporting the presence of new species that need to be formally described. Some of the other genomic species hypotheses were supported by biogeographic or symbiosis evidences, but additional investigations are needed to state on their species status. Altogether, our results support (1) the obsolescence of macromorphology (i.e., overall colony and branches shape) but the relevance of micromorphology (i.e., corallite structures) to refine Pocillopora species limits, (2) the need to identify molecularly species prior to their study, as morphology can blur species identification on the field, (3) the relevance of the mtORF (coupled with other markers in some cases) as a diagnostic marker of most species, and (4) the need for a taxonomical revision in the Pocillopora genus. These results give new insights into the usefulness of multiple criteria for resolving Pocillopora species limits and will ultimately provide helpful insights for the conservation of the species from this scleractinian genus.

The software program STRUCTURE is one of the most cited tools for determining population structure. To infer the optimal number of clusters from STRUCTURE output, the ΔK method is often applied. However, a recent study relying on simulated microsatellite data suggested that this method has a downward bias in its estimation of K and is sensitive to uneven sampling. If this finding holds for empirical datasets, conclusions about the scale of gene flow may have to be revised for a large number of studies. To determine the impact of method choice, we applied recently described estimators of K to re-estimate genetic structure in 41 empirical microsatellite datasets; 15 from a broad range of taxa and 26 focused on a diverse phylogenetic group, coral. We compared alternative estimates of K (Puechmaille statistics) with traditional (ΔK and posterior probability) estimates and found widespread disagreement of estimators across datasets. Thus, one estimator alone is insufficient for determining the optimal number of clusters regardless of study organism or evenness of sampling scheme. Subsequent analysis of molecular variance (AMOVA) between clustering solutions did not necessarily clarify which solution was best. To better infer population structure, we suggest a combination of visual inspection of STRUCTURE plots and calculation of the alternative estimators at various thresholds in addition to ΔK. Differences between estimators could reveal patterns with important biological implications, such as the potential for more population structure than previously estimated, as was the case for many studies reanalyzed here.

The development of population genomic approaches in non-model species allows for renewed studies of the impact of reproductive systems and genetic drift on population diversity. Here, we investigate the genomic signatures of partial clonality in the deep water kelp Laminaria rodriguezii, known to reproduce by both sexual and asexual means. We compared these results with the species Laminaria digitata, a closely related species that differs by different traits, in particular its reproductive mode (no clonal reproduction). We analysed genome-wide variation with dd-RAD sequencing using 4077 SNPs in L. rodriguezii and 7364 SNPs in L. digitata. As predicted for partially clonal populations, we show that the distribution of FIS within populations of L. rodriguezii is shifted toward negative values, with a high number of loci showing heterozygote excess. This finding is the opposite of what we observed within sexual populations of L. digitata, characterized by a generalized deficit in heterozygotes. Furthermore, we observed distinct distributions of FIS among populations of L. rodriguezii, which is congruent with the predictions of theoretical models for different levels of clonality and genetic drift. These findings highlight that the empirical distribution of FIS is a promising feature for the genomic study of asexuality in natural populations. Our results also show that the populations of L. rodriguezii analysed here are genetically differentiated and probably isolated. Our study provides a conceptual framework to investigate partial clonality on the basis of RAD-sequencing SNPs. These results could be obtained without any reference genome, and are therefore of interest for various non-model species.

Genetic diversity is crucial for species’ maintenance and persistence, yet is often overlooked in conservation studies. Species diversity is more often reported due to practical constraints, but it is unknown if these measures of diversity are correlated. In marine invertebrates, adults are often sessile or sedentary and populations exchange genes via dispersal of gametes and larvae. Species with a larval period are expected to have more connected populations than those without larval dispersal. We assessed the relationship between measures of species and genetic diversity, and between dispersal ability and connectivity. We compiled data on genetic patterns and life history traits in nine species across five phyla. Sampling sites spanned 600 km in the northwest Mediterranean Sea and focused on a 50 km area near Marseilles, France. Comparative population genetic approaches yielded three main results. (1) Species without larvae showed higher levels of genetic structure than species with free-living larvae but the role of larval type (lecithotrophic or planktotrophic) was negligible. (2) A narrow area around Marseilles, subject to offshore advection, limited genetic connectivity in most species. (3) We identified sites with significant positive contributions to overall genetic diversity across all species, corresponding with areas near low human population densities. In contrast, high levels of human activity corresponded with a negative contribution to overall genetic diversity. Genetic diversity within species was positively and significantly linearly related with local species diversity. Our study suggests that local contribution to overall genetic diversity should be taken into account for future conservation strategies.

Sexual reproduction is widespread among eukaryotes, and the sex-determining processes vary greatly among species. While genetic sex determination (GSD) has been intensively described in bilaterian species, no example has yet been recorded among non-bilaterians. However, the quasi-ubiquitous repartition of GSD among multicellular species suggests that similar evolutionary forces can promote this system, and that these forces could occur also in non-bilaterians. Studying sex determination across the range of Metazoan diversity is indeed important to understand better the evolution of this mechanism and its lability. We tested the existence of sex-linked genes in the gonochoric red coral (Corallium rubrum, Cnidaria) using restriction site-associated DNA sequencing. We analysed 27 461 single nucleotide polymorphisms (SNPs) in 354 individuals from 12 populations including 53 that were morphologically sexed. We found a strong association between the allele frequencies of 472 SNPs and the sex of individuals, suggesting an XX/XY sex-determination system. This result was confirmed by the identification of 435 male-specific loci. An independent test confirmed that the amplification of these loci enabled us to identify males with absolute certainty. This is the first demonstration of a GSD system among non-bilaterian species and a new example of its convergence in multicellular eukaryotes.

Defining the scale of connectivity among marine populations and identifying the barriers to gene flow are tasks of fundamental importance for understanding the genetic structure of populations and for the design of marine reserves. Here we investigated the population genetic structure at three spatial scales of the red gorgonian Paramuricea clavata (Cnidaria, Octocorallia) a key species dwelling in the coralligenous assemblages of the Mediterranean Sea. Colonies of P. clavata were collected from 39 locations across the Mediterranean Sea from Morocco to Turkey and analysed using microsatellite loci. Within three regions (Medes, Marseille and North Corsica) sampling was obtained from multiple locations and at different depths. Three different approaches (measures of genetic differentiation, Bayesian clustering and spatially explicit maximum-difference algorithm) were used to determine the pattern of genetic structure. We identified genetic breaks in the spatial distribution of genetic diversity which were concordant with oceanographic conditions in the Mediterranean Sea. We revealed a high level of genetic differentiation among populations and a pattern of isolation by distance across the studied area and within the three regions, underlining short effective larval dispersal in this species. We observed genetic differentiation among populations in the same locality dwelling at different depths which may be explained by local oceanographic conditions and which may allow a process of local adaptation of the populations to their environment. We discuss the implications of our results for the conservation of the species which is exposed to various threats.