Climatic oscillations triggered post-Messinian speciation of Western Palearctic brown frogs (Amphibia, Ranidae).

Oscillating glacial cycles over the past 2.4 million years are proposed to have had a major impact on the diversity of contemporary species communities. We used mitochondrial and nuclear DNA sequence data to infer phylogenetic relationships within Western Palearctic brown frogs and to test the influence of Pliocene and Pleistocene climatic changes on their evolution. We sequenced 1976bp of the mitochondrial genes 16S rRNA and cytochrome b and of the nuclear rhodopsin gene for all current species and subspecies. Based on an established allozyme clock for Western Palearctic water frogs and substitution rate constancy among water frogs and brown frogs, we calibrated a molecular clock for 1425bp of the 16S and rhodopsin genes. We applied this clock to date speciation events among brown frogs. Western Palearctic brown frogs underwent a basal post-Messinian radiation about 4 million years ago (mya) into five major clades: three monotypic lineages (Rana dalmatina, Rana latastei, Rana graeca), an Anatolian lineage, and a lineage comprising Rana italica, Rana arvalis, and all Iberian taxa. Polytypic lineages radiated further in concordance with the onset of climatic oscillations ca. 3.2, 2.0, and 1.0-0.6 mya, respectively. The dated fossil record corroborates our paleobiogeographic scenario. We conclude that drastic climatic changes followed by successive temperature oscillations "trapped" most brown frog species in their southern European glacial refugia with enough time to speciate. Substantial dispersal was only possible during extensive interglacial periods of a constant subtropical climate.