Background: The flat periwinkles, Littorina fabalis and L. obtusata, are two sister species widely distributed throughout the Northern Atlantic shores with high potential to inform us about the process of ecological speciation in the intertidal. However, whether gene flow has occurred during their divergence is still a matter of debate. A comprehensive assessment of the genetic diversity of these species is also lacking and their main glacial refugia and dispersal barriers remain largely unknown. In order to fill these gaps, we sequenced two mitochondrial genes and two nuclear fragments to perform a phylogeographic analysis of flat periwinkles across their distribution range. Results: We identified two main clades largely composed by species-specific haplotypes corresponding to L. obtusata and L. fabalis, with moderate to strong support, respectively. Importantly, a model of divergence with gene flow between the two species (from L. obtusata to L. fabalis) was better supported, both in Iberia and in northern-central Europe. Three mitochondrial clades were detected within L. fabalis and two within L. obtusata, with strong divergence between Iberia and the remaining populations. The largest component of the genetic variance within each species was explained by differences between geographic regions associated with these clades. Our data suggests that overall intraspecific genetic diversity is similar between the two flat periwinkle species and that populations from Iberia tend to be less diverse than populations from northern-central Europe.Conclusions: The phylogeographic analysis of this sister-species pair supports divergence with gene flow. This system thus provides us with the opportunity to study the contribution of gene flow and natural selection during diversification. The distribution of the different clades suggests the existence of glacial refugia in Iberia and northern-central Europe for both species, with a main phylogeographic break between these regions. Although the genetic diversity results are not fully conclusive, the lower diversity observed in Iberia could reflect marginal conditions at the southern limit of their distribution range during the current interglacial period.

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Characterizing the patterns of hybridization between closely related species is crucial to understand the role of gene flow in speciation. In particular, systems comprising multiple contacts between sister species offer an outstanding opportunity to investigate how reproductive isolation varies with environmental conditions, demography and geographic contexts of divergence. The flat periwinkles, Littorina obtusata and L. fabalis (Gastropoda), are two intertidal sister species with marked ecological differences compatible with late stages of speciation. Although hybridization between the two was previously suggested, its extent across the Atlantic shores of Europe remained largely unknown. Here, we combined genetic (microsatellites and mtDNA) and morphological data (shell and male genital morphology) from multiple populations of flat periwinkles in north‐western Iberia to assess the extent of current and past hybridization between L. obtusata and L. fabalis under two contrasting geographic settings of divergence (sympatry and allopatry). Hybridization signatures based on both mtDNA and microsatellites were stronger in sympatric sites, although evidence for recent extensive admixture was found in a single location. Misidentification of individuals into species based on shell morphology was higher in sympatric than in allopatric sites. However, despite hybridization, species distinctiveness based on this phenotypic trait together with male genital morphology remained relatively high. The observed variation in the extent of hybridization among locations provides a rare opportunity for future studies on the consequences of different levels of gene flow for reinforcement, thus informing about the mechanisms underlying the completion of speciation.

Periwinkles of the family Littorinidae (Children, 1834) are common members of seashore littoral communities worldwide. Although the family is composed of more than 200 species belonging to 18 genera, chromosome numbers have been described in only eleven of them. A molecular cytogenetic analysis of nine periwinkle species, the rough periwinkles Littorina arcana, L. saxatilis, and L. compressa, the flat periwinkles L. obtusata and L. fabalis, the common periwinkle L. littorea, the mangrove periwinkle Littoraria angulifera, the beaded periwinkle Cenchritis muricatus, and the small periwinkle Melarhaphe neritoides was performed. All species showed diploid chromosome numbers of 2n = 34, and karyotypes were mostly composed of metacentric and submetacentric chromosome pairs. None of the periwinkle species showed chromosomal differences between male and female specimens. The chromosomal mapping of major and minor rDNA and H3 histone gene clusters by fluorescent in situ hybridization demonstrated that the patterns of distribution of these DNA sequences were conserved among closely related species and differed among less related ones. All signals occupied separated loci on different chromosome pairs without any evidence of co-localization in any of the species.