Evolutionary analysis of Chironius snakes unveils cryptic diversity and provides clues to diversification in the Neotropics

Recent hypotheses to explain tropical diversity involves the Neogene and Quaternary geoclimatic dynamics, but the absence of unambiguous data permitting the choice between alternative hypotheses makes a general theory for the origin of tropical biodiversity far to be achieved. The occurrence of Chironius snakes in well-defined biogeographical regions led us to adopt Chironius as a model to unveil patterns of vertebrate diversification in the Neotropics. Here, we used molecular markers and records on geographic distribution to investigate Chironius evolution and, subsequently, providing hints on diversification in the Neotropics. To avoid analyzing nominal species that do not constitute exclusive evolutionary lineages, we firstly conducted a species delimitation study prior to carrying out the species distribution modeling analysis. We generated 161 sequences of 12S, 16S, c-mos and rag2 for 15 species and 50 specimens, and included additional data from GenBank yielding a matrix of 137 terminals, and performed the following evolutionary analyses: inference of a concatenated gene tree, estimation of gene divergence times, inference of the coalescent-based phylogeny of Chironius, estimation of effective population sizes and modeling potential distribution of species across the last millennia. We tested for species boundaries within Chironius by implementing a coalescent-based Bayesian species delimitation approach. Our analyses supported the monophyly of Chironius, although our findings underscored cryptic candidate species in C. flavolineatus and C. exoletus. The inferred timetree suggested that Chironius snakes have evolved in the early Miocene (ca. 20.2 Mya) and began to diversify from the late Miocene to the early Pliocene, values that are much older than previously reported. Following genetic divergence of virtually all extant Chironius species investigated, the effective sizes of the populations have expanded when compared to their MRCAs. The evolutionary and SDM data from C. brazili and C. diamantina provided additional evidence of the origin of species in the Neotropics. We argue that temperature, instead of altitude, has been the major driving factor in the evolution of both species, and thus we present a case for the consequences of global warming.