Historical factors (colonization scenarios, demographic oscillations) and contemporary processes (population connectivity, current population size) largely contribute to shaping species' presentday genetic diversity and structure. In this study, we use a combination of mitochondrial and nuclear DNA markers to understand the role of Quaternary climatic oscillations and present-day gene flow dynamics in determining the genetic diversity and structure of the newt Calotriton asper (Al. Dugès, 1852), endemic to the Pyrenees. Mitochondrial DNA did not show a clear phylogeographic pattern and presented low levels of variation. In contrast, microsatellites revealed five major genetic lineages with admixture patterns at their boundaries. Approximate Bayesian computation analyses and linear models indicated that the five lineages likely underwent separate evolutionary histories and can be tracked back to distinct glacial refugia. Lineage differentiation started around the Last Glacial Maximum at three focal areas (western, central and eastern Pyrenees) and extended through the end of the Last Glacial Period in the central Pyrenees, where it led to the formation of two more lineages. Our data revealed no evidence of recent dispersal between lineages, whereas borders likely represent zones of secondary contact following expansion from multiple refugia. Finally, we did not find genetic evidence of sex-biased dispersal. This work highlights the importance of integrating past evolutionary processes and present-day gene flow and dispersal dynamics, together with multilocus approaches, to gain insights into what shaped the current genetic attributes of amphibians living in montane habitats.

Changes in chromosomal structure involving chromosomal rearrangements or copy number variation of specific sequences can play an important role in speciation. Here, we explored the chromosomal structure of two hybridizing passerine species; the common nightingale (Luscinia megarhynchos) and the thrush nightingale (Luscinia luscinia), using conventional cytogenetic approaches, immunostaining of meiotic chromosomes, fluorescence in situ hybridization as well as comparative genomic hybridization (CGH). We found that the two nightingale species show conserved karyotypes with the same diploid chromosome number of 2n = 84. In addition to standard chromosomes, both species possessed a small germline restricted chromosome of similar size as a microchromosome. Just a few subtle changes in chromosome morphology were observed between the species, suggesting that only a limited number of chromosomal rearrangements occurred after the species divergence. The interspecific CGH experiment suggested that the two nightingale species might have diverged in centromeric repetitive sequences in most macro- and microchromosomes. In addition, some chromosomes showed changes in copy number of centromeric repeats between the species. The observation of very similar karyotypes in the two nightingale species is consistent with a generally slow rate of karyotype evolution in birds. The divergence of centromeric sequences between the two species could theoretically cause meiotic drive or reduced fertility in interspecific hybrids. Nevertheless, further studies are needed to evaluate the potential role of chromosomal structural variations in nightingale speciation.