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Evolutionary processes can be influenced by several factors, such as geographic isolation, environmental selection, and sensory variation. For most nocturnal bats, echolocation is the primary sensory system used to prey and communicate, and plays important roles in chiropteran diversification and evolution. Understanding the relative contribution of geography, the environment, and this sensory system to population genetic divergence can elucidate the processes involved in bat incipient speciation and evolution. In this study, we collected spatial and environmental information, echolocation calls, as well as the previously published genetic data (six microsatellite loci and the mitochondrial cytochrome b gene) of widely distributed Rhinolophus episcopus populations to test three hypotheses for nuclear and mitochondrial divergence (isolation by distance, isolation by environment, and isolation by sensory variation) and unveil the factors that drive intraspecific genetic differentiation. The moderate level of nuclear differentiation was correlated with geographic/spatial distance and acoustic variation, whereas the relatively high level of mitochondrial differentiation was mainly associated with acoustic divergence. No significant correlation was observed between genetic divergence and environmental variables. Among the three factors, acoustic divergence explained the highest percentage of both nuclear and mitochondrial divergence. Thus, our results indicate that sensory variation may have played important roles in driving population isolation early in bat speciation, which is consistent with the hypothesis of isolation by sensory variation. Our study emphasizes the need to consider more factors, especially sensory traits, and combine multiple statistical methods in landscape genetic studies to test their potential contributions to driving population divergence. [ABSTRACT FROM AUTHOR]

• Multiple evidences were used to delimit species of Rhinolophus macrotis complex. • A new taxonomic treatment is proposed for the R. osgoodi. • Evolutionary processes were reconstructed following species delimitation. • Hybridization and introgression have contributed to the reticulate evolution. Species delimitation and evolutionary reconstruction remain challenging for non-model species that have experienced reticulate evolution and exhibit conflicting patterns of differentiation among multiple lines of evidence, such as mitochondrial and nuclear data and phenotypes. Here, we applied an integrative taxonomic approach to a case study of Rhinolophus macrotis complex, whose taxonomic status remains controversial, to provide insight into the systematics and evolutionary history of these species. By integrating traditional genetic markers with different modes of inheritance, genome-wide SNPs as well as phenotypic characteristics, we clarified the presence of three closely related species, R. episcopus , R. siamensis , and R. osgoodi , within this complex, and proposed a new taxonomic treatment for R. osgoodi. Our results suggested that hybridization and introgression are the main causes of low mtDNA divergence in these species. Combined with the demographic inference, we deduced that glacial–interglacial cycles drove geographic isolation and secondary contacts of these species, then promoted hybridization and lineage fusion among them, finally resulting in a reticulate evolutionary pattern. Overall, our study highlights the importance of combining multiple types of data to delimit species, especially those with conserved morphology, and to reveal the sophisticated processes of speciation. [ABSTRACT FROM AUTHOR]