Amniote vertebrates possess various mechanisms of sex determination, but their variability is not equally distributed. The large evolutionary stability of sex chromosomes in viviparous mammals and birds was believed to be connected with their endothermy. However, some ectotherm lineages seem to be comparably conserved in sex determination, but previously there was a lack of molecular evidence to confirm this. Here, we document a stability of sex chromosomes in advanced snakes based on the testing of Z-specificity of genes using quantitative PCR (qPCR) across 37 snake species (our qPCR technique is suitable for molecular sexing in potentially all advanced snakes). We discovered that at least part of sex chromosomes is homologous across all families of caenophidian snakes (Acrochordidae, Xenodermatidae, Pareatidae, Viperidae, Homalopsidae, Colubridae, Elapidae and Lamprophiidae). The emergence of differentiated sex chromosomes can be dated back to about 60 Ma and preceded the extensive diversification of advanced snakes, the group with more than 3000 species. The Z-specific genes of caenophidian snakes are (pseudo)autosomal in the members of the snake families Pythonidae, Xenopeltidae, Boidae, Erycidae and Sanziniidae, as well as in outgroups with differentiated sex chromosomes such as monitor lizards, iguanas and chameleons. Along with iguanas, advanced snakes are therefore another example of ectothermic amniotes with a long-term stability of sex chromosomes comparable with endotherms.

Hybridization and incomplete lineage sorting are common confounding factors in phylogeny and speciation resulting in mitonuclear disparity. Mitochondrial introgression, a particular case of hybridization, may, in extreme cases, lead to replacement of the mitochondrial genome of one species with that of another (mitochondrial capture). We investigated mitochondrial introgression involving two species of the cyprinid genus Squalius in the western Peloponnese region of Greece using molecular and morphological data. We found evidence of complete mitochondrial introgression of Squalius keadicus into two populations recognized as Squalius peloponensis from the Miras and Pamissos River basins and a divergence of mitochondrial genomes of S. keadicus from the Evrotas basin from that of the introgressed populations dating from the Pleistocene. Secondary contact among basins is a possible factor in connection of the species and the introgression event. Morphological analyses support the hypothesis of mitochondrial introgression, as S. keadicus was different from the other three populations recognized as S. peloponensis, although significant differences were found among the four populations. Isolation by geographical barriers arose during Pleistocene in the western Peloponnese were the source of the evolution of the two reciprocally monophyletic subclades found in the S. keadicus mitochondrial clade, and the morphological differences found among the four populations. Along with the lack of structure in the nuclear genome in the three populations ascribed to S. peloponensis, this suggests an incipient speciation process occurring in these Squalius species in the western Peloponnese.