The genus Myotis is one of the most diverse and widely distributed mammals, providing a good model for studies of speciation and diversification across large geographic scales. However, the classification within this genus has long been chaotic. Taxonomic revisions based on multiple data sources are essential and urgent. In this study, morphometrics and genetic markers with different modes of inheritance were used to clarify the taxonomy of Myotis distributed in China. Based on 173 mitochondrial Cytb sequences and five morphological characteristics, 114 specimens collected nationwide over the past 20 years were assigned to 11 Myotis species. All Chinese samples classified into M. davidii and M. longipes were revised to M. alticraniatus and M. laniger. Then, two nuclear fragments (Rag2 and Chd1) and Cytb sequences from representative individuals of Chinese Myotis were used for multilocus phylogeny reconstruction and genetic divergence evaluation. The phylogenetic relationships were clearly demonstrated in the species tree: M. alticraniatus and M. laniger; M. fimbriatus, M. pilosus, M. macrodactylus, and M. petax; and M. pequinius, M. chinensis, and M. blythii formed three strongly supported monophyletic clades. Mitochondrial divergence was almost 10 times that of nuclear divergence, with interspecific K2P distances ranging from 8% to 20% for Cytb and 0.3% to 2.3% for concatenated nuclear genes. Low levels of genetic divergence were observed between M. alticraniatus and M. laniger, as well as M. fimbriatus and M. pilosus. These results provide new insights into the taxonomy and phylogeny of Myotis bats in China and are important for the future research and conservation of Chinese Myotis.

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.

Behavioural development is an important aspect of research on animal behaviour. In bats, many studies have been conducted on the development of flight behaviour, but the postnatal behavioural development of bats remains largely unexplored. We studied the behaviours and postnatal development of infant bats by conducting controlled video recorded experiments. Our results showed that before weaning, Asian parti-coloured bats (Vespertilio sinensis) were able to exhibit four types of behaviours, namely, crawling, head moving, wing flapping, and wing spreading, and these behaviours are different from those observed in experiments with adult bats. The number of occurrences of these behaviours was correlated with age and scaled mass index. Furthermore, the number of occurrences of these behaviours in young bats could also reflect their physical developmental status. In young bats, wing flapping and spreading might be a type of play behaviour. These behaviours were negatively correlated with the time of the first flight, indicating that they might help to promote individual physical development. Our results provide fundamental data for revealing the ontogenetic and neurophysiological mechanisms of behavioural development in bats.

Vocal signals are the primary medium of intraspecific communication in bats, yet the encoding features and recognition mechanisms of distress calls remain largely unclear. This study aimed to examine sex, age, and individual signatures in noise burst to downward-frequency modulation (NB-DFM) distress calls of the Rhinolophus nippon and verify conspecific recognition ability. We recorded NB-DFM calls from 20 adult and 9 subadult bats in Jilin Province, extracted 18 acoustic parameters, built classification models with support vector machines (SVMs), evaluated feature importance using random forest, and performed habituation–dishabituation playback experiments. SVM yielded classification accuracies of 67%, 89%, and 88% for sex, age, and individual identity, respectively, all significantly above chance levels. Call duration, central minimum frequency, and root mean square (RMS) were the most diagnostic parameters, and key acoustic variables differed significantly among classification levels. Playback tests elicited distinct behavioral responses to calls of different sexes, ages, and individuals, confirming discrimination ability. These findings reveal individual, age, and sex specific acoustic markers in bat distress calls, support the utility of machine learning for bioacoustic classification, and provide empirical insight into stress-related acoustic communication in nocturnal mammals.

Acoustic divergence is widely recognized as a key driver of speciation and niche differentiation in vocal animals. In echolocating horseshoe bats (Rhinolophus), the larynx is specialized for producing high-duty-cycle signals used in foraging, navigation, and species recognition. While the ecological role of echolocation is established, the molecular mechanisms regulating laryngeal frequency remain unclear. We compared the laryngeal transcriptomes of three closely related, sympatric Rhinolophus species with distinct resting frequencies (RFs): R. episcopus (~46 kHz), R. siamensis (~66 kHz), and R. osgoodi (~85 kHz). This comparison identified 511 differentially expressed genes. High-frequency species upregulated genes involved in cytoskeletal dynamics and muscle contraction, such as cell adhesion molecules and motor proteins, while low-frequency species upregulated genes related to cellular homeostasis and metabolic maintenance. Weighted gene co-expression network analysis revealed two RF-correlated modules: a high-frequency module enriched in aerobic respiration and carbon metabolism and a low-frequency module enriched in lipid metabolism. Protein–protein interaction analysis identified ACTC1, vital for muscle contraction, as a hub gene. Evolutionary analysis showed that ACTC1 is highly conserved, with no significant positive selection, indicating that transcriptional regulation, rather than coding-sequence divergence, is the primary driver of the observed functional differences. These findings suggest that RF variation likely results from transcriptional remodeling in laryngeal superfast muscles. This study provides the first transcriptomic evidence linking laryngeal gene expression with acoustic divergence and offers new insights into the genetic basis of bat echolocation.

The evolutionary divergence of animal vocalizations is a complex process shaped by various factors, including morphology, ecology, social pressure, and phylogenetic relationships. This applies even to distress calls, which may appear to be simple expressions of emotion. However, the relative importance of these factors in shaping interspecific divergence of distress call structure remains largely unexplored. In addition, previous studies concerning the factors driving distress call divergence have been mainly conducted in birds, anurans, and lizards, whereas less is known for mammals. Here, we recorded distress calls across 32 bat species belonging to 7 families, and compiled data on body size, foraging habitat types, climatic variables, colony size, and phylogenetic components, aiming to identify the key determinants predicting the cross-species divergence in the incidence and acoustic parameter of distress calls within a comparative framework. Our results supported that bats divergence in distress vocalizations is not an adaptive evolution in response to ecological and social pressure, but constrained by phylogenetic relationship and body size. Phylogenetic components explained the most interspecific variation in the incidence and temporal parameters of distress calls, whereas body size accounts for spectral parameters. This study has revealed that distress vocalizations not only convey the caller’s emotional state, but also suffer severe phylogenetic and morphological constraint.