Concordant species delimitation from multiple independent evidence: A case study with the Pachytriton brevipes complex (Caudata: Salamandridae).

Mitochondrial DNA (mtDNA) sequence data are widely used to delimit species. However, owing to its strict maternal inheritance in most species, mtDNA tracks female dispersion and dispersal only. The accuracy of mtDNA-derived species delimitation is often not explicitly tested using other independent evidence, such as nuclear DNA (nDNA) data, morphological data, or ecological data. Because species are independent evolutionary lineages that can form testable hypotheses, we present a multi-evidence case study on species delimitation that combines statistical approaches with spatially explicit ecological analysis. Montane salamanders of the Pachytriton brevipes complex (Salamandridae) from southeastern China exhibit conservative morphology and variable color patterning that impede species diagnosis. Recent studies proposed splitting P. brevipes into four species based on deep mtDNA divergence but also found discordance between mtDNA and nDNA trees. In this study, we test evolutionary independence of these hypothesized species lineages using two coalescent-based Bayesian methods (Bayes factor and BP&P). Despite significant conflict between mtDNA gene tree and the species phylogeny, the results reinforce the inference of at least four species in the complex as opposed to the one species recognized for over 130 years. Correlative ecological niche modeling and statistical analysis of environmental data indicate that suitable habitats for each species are isolated by incompatible intervening lowland regions, so the likelihood of gene flow among species is very low, which means species lineages should maintain their evolutionary independence. We demonstrate that concordance among independent evidence confirms species status, which forms the basis for accurate assessment of regional biodiversity.