Aim: In the Mediterranean Biodiversity Hotspot freshwater fishes are key biogeographical indicators while their habitats are vulnerable to ongoing anthropogenic pressures. The freshwater blenny is a widespread endemic taxon in this area. However, our understanding of the overall diversity of specific populations and their phylogenetic relationships is sparse. Thus, we aim to investigate the genetic diversity, infer relationships among sampled populations related to major paleoenvironmental changes and suggest insights for future research and conservation targets.

Location: Mediterranean Basin

Methods: We studied 171 Salaria specimens from 51 rivers and lakes, and from 13 countries surrounding the Mediterranean Sea. For assessing the phylogenetic relationships among different locations, we used mtDNA d-loop and Intron S7 sequences. Furthermore, we inferred absolute divergence times as well as demographic changes using secondary calibrations and investigated the diversity within major lineages using haplotype networks as well as several geographical and genetic clustering methods.

Results: We found eight well differentiated lineages, each of which being confined to a particular geographic region. The onset of the freshwater blenny radiation was dated around the Messinian salinity crisis. Further differentiation happened during the Plio- and Pleistocene with signatures of population expansion after the Last Glacial Maximum in some of the main lineages.

Main Conclusions: Due to their unusual widespread distribution, the freshwater blennies represent an excellent model for studying phylogeographic structure across the Mediterranean basin biodiversity hotspot. The extant diversity and distribution of the freshwater Salaria species mirrors