At a time when biodiversity is threatened, we are still discovering new species, and particularly in the marine realm. Delimiting species boundaries is the first step to get a precise idea of diversity. For sympatric species which are morphologically undistinguishable, using a combination of independent molecular markers is a necessary step to define separate species. Amphipholis squamata, a cosmopolitan brittle star, includes several very divergent mitochondrial lineages. These lineages appear totally intermixed in the field and studies on morphology and colour polymorphism failed to find any diagnostic character. Therefore, these mitochondrial lineages may be totally interbreeding presently. To test this hypothesis, we characterized the genetic structure of the complex in the French Mediterranean coast using sequences of mitochondrial DNA (16S) and for the first time, several nuclear DNA markers (introns and microsatellites). The data revealed six phylogenetic lineages corresponding to at least four biological species. These sibling species seem to live in syntopy. However, they seem to display contrasted levels of genetic diversity, suggesting they have distinct demographic histories and/or life-history traits. Genetic differentiation and isolation-by-distance within the French Mediterranean coasts are revealed in three lineages, as expected for a species without a free larval phase. Finally, although recombinant nuclear genotypes are common within mitochondrial lineages, the data set displays a total lack of heterozygotes, suggesting a very high selfing rate, a feature likely to have favoured the formation of the species complex.
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