Patterns of intraspecific geographic variation of signaling systems provide insight into the microevolutionary processes driving phenotypic divergence. The acoustic calls of bats are sensitive to diverse evolutionary forces, but processes that shape call variation are largely unexplored. In China, Rhinolophus ferrumequinum displays a diverse call frequency and inhabits a heterogeneous landscape, presenting an excellent opportunity for this kind of research. We quantified geographic variation in resting frequency (RF) of echolocation calls, estimated genetic structure and phylogeny of R. ferrumequinum populations, and combined this with climatic factors to test three hypotheses to explain acoustic variation: genetic drift, cultural drift, and local adaptation. Our results demonstrated significant regional divergence in frequency and phylogeny among the bat populations in China's northeast (NE), central-east (CE) and southwest (SW) regions. The CE region had higher frequencies than the NE and SW regions. Drivers of RF divergence were estimated in the entire range and just the CE/NE region (since these two regions form a clade). In both cases, RF divergence was not correlated with mtDNA or nDNA genetic distance, but was significantly correlated with geographic distance and mean annual temperature, indicating cultural drift and ecological selection pressures are likely important in shaping RF divergence among different regions in China.

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Palaeoclimatic oscillations and different landscapes frequently result in complex population-level structure or the evolution of cryptic species. Elucidating the potential mechanisms is vital to understanding speciation events. However, such complex evolutionary patterns have rarely been reported in bats. In China, the Rhinolophus macrotis complex contains a large form and a small form, suggesting the existence of a cryptic bat species. Our field surveys found these two sibling species have a continuous and widespread distribution with partial sympatry. However, their evolutionary history has received little attention. Here, we used extensive sampling, morphological and acoustic data, as well as different genetic markers to investigate their evolutionary history. Genetic analyses revealed discordance between the mitochondrial and nuclear data. Mitochondrial data identified three reciprocally monophyletic lineages: one representing all small forms from Southwest China, and the other two containing all large forms from Central and Southeast China, respectively. The large form showed paraphyly with respect to the small form. However, clustering analyses of microsatellite and Chd1 gene sequences support two divergent clusters separating the large form and the small form. Moreover, morphological and acoustic analyses were consistent with nuclear data. This unusual pattern in the R. macrotis complex might be accounted for by palaeoclimatic oscillations, shared ancestral polymorphism and/or interspecific hybridization.

Rhinolophids (horseshoe bats) are a widespread and diverse family. Their high level of morphological convergence has made taxonomic identification and estimation of species diversity problematic. The Rhinolophus philippinensis group, which is characterized with huge ears and low echolocation frequency relative to body size, is a specific group among all the rhinolophid groups, but the phylogeny of this group is poorly understood. In this study, we performed integrated analyses of the morphology, acoustic and genetic data and constructed the first multilocus phylogeny for the philippinensis group using four mitochondrial genes, four autosomal introns and one Y‐linked intron in 95 specimens representing nine taxa. Phylogenies were constructed based on concatenated phylogenetic methods and a multilocus coalescence approach. The estimated phylogenies revealed the polyphyly of the philippinensis group. Except for R. philippinensis, all the other species in this group formed a monophyletic cluster, named herewith as “R. macrotis” species group. Phylogenies, genetic and phenotypic divergence, and species delimitation analyses supported the revised status of R. paradoxolophus as a subspecies of R. rex, R. cf. macrotis as a subspecies of R. macrotis, and R. huananus and R. cf. siamensis as junior synonyms of R. siamensis. Significant discordances were found between the mitochondrial and nuclear gene trees, suggesting incomplete lineage sorting or ancient introgression events within the philippinensis group. The macrotis group appeared to have undergone a rapid radiation approximately 2.57 million years ago during the early Pleistocene period due to palaeoclimatic oscillations. Reconstruction of the ancestral ranges suggested a wide distribution of the common ancestor for the philippinensis and macrotis groups.