Hidden species diversity in Pachyhynobius: A multiple approaches species delimitation with mitogenomes

The lack of distinct morphological features of cryptic species is a hard problem for taxonomy, especially when the taxa are closely related with considerable amounts of ancestral polymorphism. Lately, intensive coalescent-based analyses involving multiple loci have become the preferred method to assess the extent of genetic distinctiveness in otherwise phenotypically similar populations. Previously, phylogenetic studies on Pachyhynobius shangchengensis uncovered five extremely deeply divergent clades, which suggested that this species may be a cryptic species complex. In this study, we used the complete mitochondrial genome data and samples from the entire range of stout salamander (Pachyhynobius), as well as publicly available mitochondrial genomes to assess species boundaries within this genus using a suite of diverse methodologies (e.g. general mixed Yule coalescent model, Automatic Barcode Gap Discovery). The phylogenetic relationships recovered two major groups within P. shangchengensis, with one group formed by four of the six extant populations and corresponding to the central and eastern range of the Dabie mountains, while the other group encompassed two other lineages in the north west of the Dabie mountain range. The species delimitation comparison within Pachyhynobius supported the presence of recognized species within the genus, and consensus was observed across methods for the existence of up to five cryptic species within what has been traditionally considered to be P. shangchengensis. While this implies the existence of four taxa in addition to the described P. shangchengensis species, morphological data and life history information are further required to contribute to the species definition. The observed pattern of genetic variation is likely the outcome of a discontinuous habitat combined with niche conservatism, which produced the sky-island effect observed in Pachyhynobius, and which led to formation of a hidden species diversity in this genus.