Impact of the Miocene orogenesis on Kaloula spp. radiation and implication of local refugia on genetic diversification

The phylogeography of the Kaloula genus in East Asia is still poorly understood. One of the difficulties is the absence of fossils to corroborate molecular dating estimates. Here, we examined the mitochondrial structure of Kaloula spp. in East Asia and focused on the impact of glaciations on the northernmost species: Kaloula borealis. We determined the phylogenetic relationships, molecular dating, and genetic connectivity assessments within the genus from 1211 bp of concatenated mitochondrial 12S and 16S. The relaxed clock analyses reveal the emergence of Kaloula spp. common ancestor in East and Southeast Asia between the Eocene and Oligocene, c. 38.47 Ma (24.69–53.65). The genetic diversification of lineages then increased on the East Asian Mainland during the Lower Miocene, c. 20.10 (8.73–30.65), most likely originating from the vicariance and radiation triggered by the orogeny of the Qinghai–Tibetan Plateau. Later, the dispersal towards the North East Asian Mainland during the Upper Miocene drove the population diversification of K. borealis c. 9.01 Ma (3.66–15.29). Finally, the central mainland population became isolated following orogenesis events and diverged into K. rugifera during the Pliocene, c. 3.06 Ma (0.02–10.90). The combination of population genetic and barrier analyses revealed a significant genetic isolation between populations of Kaloula spp. matching with the massive Qinling–Daba Mountain chain located in south-central China. Finally, we highlight a young divergence within the Eastern Mainland population of K. borealis, possibly attributed to refugia in south eastern China from which populations later expanded.