Phylogenomics Resolves Deep Phylogenetic Uncertainties and Hybridization in Rapidly Radiated Horseshoe Bats (Rhinolophus)

Phylogenetic reconstruction is indispensable for inferring evolutionary trajectories of novel traits and historical biogeography of extant species. However, resolving phylogenetic relationships remains particularly challenging during episodes of rapid radiation, as exemplified by Rhinolophus—the second-largest genus of Chiroptera. This Old World bat lineage underwent rapid diversification, resulting in persistent ambiguities in its species-level phylogeny. To date, limited taxonomic sampling and insufficient molecular markers have precluded robust identification of the ancestral clade within Rhinolophus. Here, we address this knowledge gap using genome-wide nuclear datasets with comprehensive taxon sampling. Phylogenetic reconstructions integrating concatenation and coalescent-based approaches robustly recovered two strongly supported sister clades within Rhinolophus—the Afro-Palaearctic clade and the Asian clade—and resolved R. hipposideros as the ancestral lineage of the Afro-Palaearctic clade. This topology received further validation from PhyloNet analyses accounting for gene flow. Notably, mitochondrial phylogenomics exhibited significant topological discordance with nuclear DNA, suggesting widespread mito-nuclear discordance attributable to historical introgression. Genome-scale introgression analyses revealed pervasive cross-lineage nuclear gene flow, occurring not only among sister taxa but also between distantly related lineages. Crucially, highly introgressed genes (RANBP2 and SERINC3) functionally associated with antiviral defence mechanisms were previously shown to be under positive selection in bats. This pattern supports the occurrence of adaptive introgression facilitating viral resistance across the genus. Overall, our findings demonstrate the power of genome-scale data in resolving deep evolutionary relationships within rapidly radiating groups and underscore the importance of hybridization and introgression as key mechanisms in mammal diversification.