We investigated the phylogeographical patterns of Lissotriton italcus, a newt endemic to the Italian peninsula, aiming to determine why hotspots of intraspecific diversity so 'hot'. We found two main mitochindrial DNA lineages (net sequence divergence of 6.8% at two fragments of total length of 1897 bp): one restricted to part of the Calabrian peninsula (i.e the southernmost portion of the species range) and the other widespread throughout the rest of the species range. Both lineages, which had a parapatric distribution, showed evidence of further subdivisions, with an overall number of eight terminal haplogroups, most of whose times to the most recent common ancestors were estimated at the Late Pleistocene. Analysis of molecular variance suggested that partitioning populations according to the geographical distribution of these haplogroups can explain 97% of the observed genetic variation. These results suggest that L. italicus underwent repeated cycles of allopatric fragmentation throughout the Pleistocene, and that it likely survived the Late Pleistocene paleoenvironmental changes within eight separate refugia. Thus, the current hotspot of intraspecific diversity of L. italicus (within the Calabrian peninsula) has not been moulded by long-term stability of large populations but rather by multiple events of allopatric fragmentation and divergence. When compared with the patterns recently identified in other species, these results suggest that the occurrence of phases of allopatric divergence (eventually followed by secondary admixture) could be a common, albeit probably underrated feature in the history of formation of hotspots of intraspecific diversity.

Hotspots of genetic diversity are regions of utmost importance for species survival and conservation, and their intimate link with the geographic location of glacial refugia has been well established. Nonetheless, the microevolutionary processes underlying the generation of hotspots in such regions have only recently become a fervent field of research. We investigated the phylogeographic and population genetic structure of the agile frog, Rana dalmatina, within its putative refugium in peninsular Italy. We found this region to harbour far more diversity, phylogeographic structure, and lineages of ancient origin than that by the rest of the species' range in Europe. This pattern appeared to be well explained by climate-driven microevolutionary processes that occurred during both glacial and interglacial epochs. Therefore, the inferred evolutionary history of R. dalmatina in Italy supports a view of glacial refugia as ‘factories' rather than as repositories of genetic diversity, with significant implications for conservation strategies for hotspots.