Speciation in a biodiversity hotspot: phylogenetic relationships, species delimitation, and divergence times of the Patagonian ground frogs of Eupsophus roseus group (Alsodidae)

The alsodid ground frogs genus Eupsophus is divided into the roseus (2n=30) and vertebralis (2n=28) groups, distributed throughout the temperate Nothofagus forests of South America. Currently, the roseus group is composed by four species, while the vertebralis group consists of two. Phylogenetic relationships and species delimitation within each group are controversial. In fact, previous analyses considered that roseus group was composed between four to nine species. In this work, we evaluated phylogenetic relationships, diversification times, and species delimitation within roseus group using a multi-locus dataset. For this purpose, mitochondrial (D-loop, Cyt b, and COI) and nuclear (POMC and CRYBA1) partial sequences, were amplified from 164 individuals, representing all species. Maximum Likelihood (ML) and Bayesian approaches were used to reconstruct phylogenetic relationships. Species tree was estimated using BEAST and singular value decomposition scores for species quartets (SVDquartets). Species limits were evaluated with six coalescent approaches. Diversification times were estimated using mitochondrial and nuclear rates with LogNormal relaxed clock in BEAST. Nine well-supported monophyletic lineages were recovered in Bayesian, ML, and SVDquartets, including eight named species and a lineage composed by specimens from Villarrica population (Bootstrap: >90, PP:> 0.9). Single-locus species delimitation analyses overestimated the species number in E. migueli, E. calcaratus and E. roseus lineages, while multi-locus analyses recovered as species the nine lineages observed in phylogenetic analyses (>0.95). It is hypothesized that Eupsophus diversification occurred during Mid-Pleistocene (0.42-0.14 Mya), with most species originated after of the Last Southern Patagonian Glaciation (0.18 Mya). Our results revitalize the hypothesis that E. roseus group is composed by eight species and support to Villarrica lineage as a new putative species.