The desiccation of the Mediterranean Sea during the Messinian Salinity Crisis 6.0 -5.3 million years ago (Ma), caused a major extinction of the marine ichthyofauna of the Mediterranean. This was followed by an abrupt replenishment of the Mediterranean from the Atlantic after the opening of the Strait of Gibraltar. In this study, we combined demographic and phylogeographic approaches using mitochondrial and nuclear DNA markers to test the alternative hypotheses of where (Atlantic or Mediterranean) and when (before or after the Messinian Salinity Crisis) speciation occurred in the Mediterranean damselfish, Chromis chromis . The closely related geminate transisthmian pair Chromis multilineata and Chromis atrilobata was used as a way of obtaining an internally calibrated molecular clock. We estimated C . chromis speciation timing both by determining the time of divergence between C . chromis and its Atlantic sister species Chromis limbata (0.93 -3.26 Ma depending on the molecular marker used, e.g. 1.23 -1.39 Ma for the control region), and by determining the time of coalescence for C . chromis based on mitochondrial control region sequences (0.14 -0.21 Ma). The time of speciation of C . chromis was always posterior to the replenishment of the Mediterranean basin, after the Messinian Salinity Crisis. Within the Mediterranean, C . chromis population structure and demographic characteristics revealed a genetic break at the Peloponnese, Greece, with directional and eastbound gene flow between western and eastern groups. The eastern group was found to be more recent and with a faster growing population (coalescent time = 0.09 -0.13 Ma, growth = 485.3) than the western group (coalescent time = 0.13 -0.20 Ma, growth = 325.6). Our data thus suggested a western origin of C . chromis , most likely within the Mediterranean. Low sea water levels during the glacial periods, the hydrographic regime of the Mediterranean and dispersal restriction during the short pelagic larval phase of C . chromis (18 -19 days) have probably played an important role in C . chromis historical colonization.

Recent studies have focused on the relationship between the marine fauna of the eastern Atlantic and the Mediterranean Sea, but within the Atlantic, little is known about genetic relationships between populations of the Macaronesian islands. In this study, we tested whether the paleo-climatology and paleo-oceanography of the region could predict the genetic relationships among three eastern Atlantic populations (Azores, Madeira, and Canaries) of a damselWsh, Chromis limbata, and compared our results with its Mediterranean and adjacent Atlantic sister species, Chromis chromis. We combined phylogeographic and coalescent approaches using the fast evolving mitochondrial control region gene. No population structure was found for the three archipelagos. The coalescence time estimated for C. limbata (0.857-1.17 Mya) was much greater than that estimated for C. chromis. We propose that this diVerence reXects diVerences in glaciating extents in the Northeastern Atlantic and the Mediterranean. Diversity indexes (Hd and genetic distances) together with historical demographic parameters of C. limbata ( and g) revealed a more stable population history when compared to C. chromis. Our results suggest that the Macaronesian populations of C. limbata have probably been less aVected by the last glaciation than the Mediterranean populations of C. chromis. Migration across the three archipelagos was estimated and a prevailing northwest trend was detected. This result supports the idea of a colonization of the Azores by warm water Wsh from Madeira or the westernmost Canary islands which acted as major glacial refugia for the tropical and subtropical marine fauna during the glaciations.