Although the phylogenetic relationship of Western Palearctic brown frogs has been repeatedly studied, the taxonomic status and phylogenetic relationship of Anatolian-Hyrcanian brown frogs is still not fully resolved. Here, we assess the phylogenetic status of these species among Western Palearctic brown frogs with special emphasize on Iranian populations based on two partial mitochondrial DNA sequences (16S rRNA and cytochrome b genes) and the application of a molecular clock. Our results clearly show that Western Palearctic brown frogs underwent a basal radiation in to two main monophyletic clades, the European brown frogs plus the Asian R. asiatica and the Anatolian-Hyrcanian brown frogs, during Early Miocene ca. 20.2 mya. The Hyrcanian (R. pseudodalmatina) and the Anatolian lineage diverged approximately 16.6 mya. The further diverged into two subclades, R. tavasensis and R. macrocnemis, during the Middle Miocene, 14.5 mya. Our results suggest that diversification within these lineages may be closely linked to the formation of Neotethys and Paratethys and the subsequent uplift of the Turkish-Iranian plateau during the Early Miocene which led to restricted gene flow among brown frogs in these regions. Contrary to previous studies, we conclude that the Plio-Pleistocene epoch seems to be not associated to further significant speciation events within Anatolian-Hyrcanian brown frogs.
