Evolutionary History of the Rhinolophus pusillus Species Complex Reveals Phylogeographic Diversification Across the East Asian Mainland–Island System

Widespread species complexes often contain hidden evolutionary diversity shaped by dispersal, isolation and climatic change. Here, we aimed to characterize geographic genetic structure within the Rhinolophus pusillus species complex, assess the concordance among genetic, morphological and acoustic patterns and the evolutionary distinctiveness of the Taiwan-endemic R. monoceros, and determine whether Quaternary changes in habitat suitability were geographically consistent with the inferred phylogeographic and demographic history. To address these objectives, we analyzed 378 individuals from 70 localities and integrated mitochondrial sequences, nuclear loci, microsatellite genotypes, external phenotypic traits comprising forearm length, body mass, forearm mass index and echolocation resting frequency, demographic inference, ancestral-area reconstruction and ecological niche modelling. Mitochondrial analyses identified five geographically structured lineages within R. pusillus, corresponding broadly to South China, East China, Central China, Hainan Island and Southwest China, while R. monoceros formed a closely related but distinctive Taiwan lineage. South China showed the highest mitochondrial diversity, occupied a central position in haplotype networks and was repeatedly inferred as an important ancestral and refugial region. Divergence-time estimates placed the early diversification of the complex in the Middle Pleistocene, followed by demographic expansion in several lineages during the Late Pleistocene. In contrast, nuclear loci and microsatellites revealed extensive allele sharing and weaker geographic subdivision, suggesting incomplete lineage sorting and historical connectivity. Our results indicate that the R. pusillus complex represents an ongoing diversification system shaped by Quaternary climatic fluctuations, sea-level changes and regional isolation, highlighting the value of integrative phylogeography for conserving hidden evolutionary diversity in widespread bats.