Whole-genome sequencing uncovers genomic divergence and gene flow in closely related virus-harbouring horseshoe bats ( Rhinolophus )

Abstract Phylogenetic reconstruction is fundamental for evolutionary inference, yet it remains challenging to establish robust relationships among closely related taxa, often due to incomplete lineage sorting and post-divergence introgression. Here, we employed genomic approaches, including target resequencing and whole-genome resequencing, to simultaneously infer phylogenetic relationships and to quantify historical gene-flow among six recently diverged horseshoe bats in the genus Rhinolophus. Our updated phylogeny revealed two distinct clades: one comprising East R. sinicus, Hainan R. s. sinicus, and Central R. s. sinicus, and the other consisting of R. s. septentrionalis, R. thomasi, and R. s. ssp.. This topology contrasts with previous analyses that identified R. s. ssp. as the ancestral lineage. Comparative analysis of nuclear and mitochondrial phylogenies revealed widespread mito-nuclear discordance, consistent with both ancient and recent mitochondrial introgression events. Genome-wide introgression tests further uncovered pervasive nuclear introgression across the six taxa. Notably, multiple lines of evidence suggest a hybrid origin for Central R. s. sinicus, probably resulting from past hybridization between East R. s. sinicus and R. s. septentrionalis. These results underscore the prevalence of hybridization and introgression throughout the evolutionary history of these taxa—a dynamic that may have facilitated cross-species’ transmission of viruses hosted by these bats.