Hiding in the highlands: evolution of a frog species complex of the genus Ptychadena in the Ethiopian highlands.

The Ethiopian highlands are a biodiversity hotspot characterized by a high level of endemism, particularly in amphibians. Frogs of the genus Ptychadena have experienced an evolutionary radiation in these highlands. Thus, this group provides an excellent opportunity to study the process of speciation in this important biogeographic area. We sequenced two mitochondrial (16S and COI) and four nuclear (Rag-1, CXCR4, NCX1 and Tyr) genes in a sample of 236 frogs from 49 Ethiopian localities. Phylogenetic analyses of the mitochondrial genes revealed the presence of eight divergent mitochondrial lineages. We uncovered a near perfect concordance between these lineages and genetic clusters based on nuclear sequences. A Bayesian species delimitation analysis confirmed that these eight lineages correspond to eight genetically isolated populations which may represent eight species. Some of these species have already been recognized due to their distinct morphology (P. cooperi, P. nana and P. erlangeri) but we determined that the species P. neumanni is a complex of 5 cryptic species, thus increasing substantially the number of species in this genus and for this country. We resolved the phylogeny of Ethiopian highland Ptychadena using a species tree approach and determined that Ptychadena species group on the phylogeny according to their habitat preference. We propose that the diversity of Ethiopian Ptychadena results from an early phase of specialization to distinct elevations followed by a phase of ecological diversification within each elevational range. We estimated that the early phase of diversification of Ethiopian Ptychadena probably occurred in the late Miocene and the most recent speciation events in the late-Pliocene or Pleistocene.