Geographic and genetic factors shape acoustic divergence and dialect-like variation in communication calls in the greater horseshoe bats

Acoustic divergence is a fundamental component of signal-based communication in animals, including humans, and has major implications for individual recognition, mate choice, and speciation. Bats have recently become an important model system for investigating acoustic communication due to their structurally complex vocal repertoires. This study explored communication calls of greater horseshoe bats (Rhinolophus nippon) to determine whether geographically structured dialects occur and to identify factors associated with dialect formation. Communication calls from nine populations distributed across three regions were analyzed, and 12 syllable types shared among populations were identified. The associations of dialect differentiation with acoustic, climatic, morphological, and genetic distances, together with the influence of geographic barriers, were investigated. Results demonstrated significant acoustic variation among populations and regions for each syllable type, and this divergence was driven by diverse factors. As bats typically emit syllables in combination, all shared syllable types were subsequently analyzed as an integrated dataset. At this broader level, acoustic distance showed significant correlations with both geographic and genetic distance. Causal modeling further revealed that geographic distance and geographic barriers exerted direct effects on dialect differentiation. The sampled populations were classified into three dialect regions: Northeast, Central-East, and Southwest. The Qinling Mountains formed the boundary between the Central-East and Southwest regions. These findings provide clear evidence for the existence of dialect structure in communication calls of greater horseshoe bats and identify geographic isolation as a major force in the formation and preservation of vocal divergence. This study advances current understanding of animal acoustic evolution and offers insight into mechanisms linking vocal diversification with the generation of biological diversity.