Multi-locus phylogeny and evolution of a Sky Island species complex reveal Pliocene diversification and unexpected incongruence between morphological and molecular data

Abstract

Sky islands ecosystems are known to promote high speciation rates and consequently hold high numbers of endemic species. The treefrog Ololygon machadoi is a sky island dweller that occurs in the Brazilian highlands of the Espinhaço Range and the Canastra Range. We have collected adult specimens morphologically similar to O. machadoi but with tadpoles presenting conspicuous differences in color pattern, which raised doubts about the taxonomic identity of these populations. To clarify these taxonomic uncertainties, we redescribed O. machadoi based on the type series and topotype specimens using a refined character analyses and used multi-locus phylogenetic analyses of samples from all the species distribution range and five other Ololygon species to test its monophyly. The phylogenetic tree recovered O. machadoi as paraphyletic due to the placement of populations from the Canastra Range. Within the large clade formed by samples from the Espinhaço, we have found that the conspicuous differences in the tadpole color pattern correspond to a remarkable intraspecific variation since one of the distinct morphotypes is nested within the other in the phylogeny. On the other hand, the morphological analyses of adult individuals revealed important character differences between the type and topotype specimens of O. machadoi from the specimens collected at the Quadrilátero Ferrífero (QF), an iron mountain massif in the South region of the Espinhaço Range, suggesting a second undescribed species. To investigate population structure and evolutionary patterns of the sister taxa O. machadoi and Ololygon sp. QF, we performed three species delimitation methods (GMYC, BPP and STACEY) and also obtained a dated phylogeny. The most conservative method was the STACEY, which recovered 12 highly structured populations within O. machadoi and three within the Ololygon sp. QF. Most of these populations are clearly in accordance with the sky island configuration, suggesting that climatic vicariant events have played a major role in the current population structure. This hypothesis was also supported by the dated phylogeny, which suggests that the majority of the populational diversification occurred during the Pliocene, a period 2–3°C warmer than today. This warmer period probably restricted the populations to mountaintops, where the temperature remained cooler.