Genome-based phylogenetics and species delimitation for the narrowly distributed Pachyhynobius salamander (Caudata: Hynobiidae) reveal cryptic biodiversity.

Species delimitation is a challenging issue in taxonomy, especially in the absence of clear diagnostic morphological characters, a particular problem for cryptic species. In recent years, the rapid development of high-throughput sequencing technologies, when combined with genomic species delimitation methods such as the multispecies coalescent, has significantly improved the ability to resolve cryptic species. However, traditional methods like Bayesian phylogenetics and phylogeography often result in over-splitting due to their failure to fully consider factors such as gene flow. This study focuses on the genus Pachyhynobius, endemic to China, utilizing specific-locus amplified fragment sequencing technology to construct a high-quality nuclear genome SNP dataset. Systematic genomics combined with various species delimitation methods that incorporate the genealogical differentiation index and the isolation by distance model for geographic information was used to systematically evaluate the species boundaries of this genus. The results show that Pachyhynobius consists of four genetically independent, geographically disjunct lineages with deep genetic differentiation occurring over millions of years. Although these lineages have relatively close geographic distributions, long-term ecological niche conservatism and the sky island effect may be key driving forces responsible for their continued isolation and independent evolutionary paths. Multi-method species delimitation analysis further provides a comprehensive evaluation of the potentially distinct genetic lineages within the group, identifying them as potential cryptic species. Although new species have not yet been formally named, the evidence provided offers a new example for understanding the identification and evolutionary mechanisms of cryptic species in geographically restricted habitats.