Recently diverged taxa with contrasting phenotypes offer opportunities for unravelling the genetic basis of phenotypic variation in nature. Horseshoe bats are a speciose group that exhibit a derived form of high-duty cycle echolocation in which the inner ear is finely tuned to echoes of the narrowband call frequency. Here, by focusing on three recently diverged subspecies of the intermediate horseshoe bat ( Rhinolophus affinis ) that display divergent echolocation call frequencies, we aim to identify candidate loci putatively involved in hearing frequency variation. We used de novo transcriptome sequencing of two mainland taxa ( himalayanus and macrurus ) and one island taxon ( hainanus ) to compare expression profiles of thousands of genes. By comparing taxa with divergent call frequencies (around 15 kHz difference), we identified 252 differentially expressed genes, of which six have been shown to be involved in hearing or deafness in human/mouse. To obtain further validation of these results, we applied quantitative reverse transcription–PCR to the candidate gene FBXL15 and found a broad association between the level of expression and call frequency across taxa. The genes identified here represent strong candidate loci associated with hearing frequency variation in bats.

Seasonality can cause changes in many environmental factors which potentially affects gene expression. Here, we used a bat species ( Rhinolophus sinicus ) from eastern China as a model to explore the molecular mechanisms of seasonal effects, in particular during phenological shifts in the spring and autumn. Based on the analysis of 45 RNA‐seq samples, we found strong seasonal effects on gene expression, with a large number of genes identified as either specific or biased to each season. Weighted gene co‐expression network analysis also identified multiple modules significantly associated with each season. These seasonal genes were further enriched into different functional categories. Consistent with effects of phenological shifts on bats, we found that genes related to promoting food intake were highly expressed in both autumn and spring. In addition, immunity genes were also highly expressed in both seasons although this seasonal immune response had tissue specificity in different seasons. In female bats, genes related to the delay of ovulation (e.g., NPPC , natriuretic peptide precursor type C) were highly expressed in October, while genes associated with the promotion of reproduction (e.g., DIO2 , iodothyronine deiodinase 2) were biasedly expressed in April. Lastly, we found multiple known core clock genes in both October‐biased and April‐biased expressed genes, which may be involved in regulating the start and end of hibernation, respectively. Overall, together with studies in other land and aquatic animals, our work supports that seasonal gene expression variations may be a general evolutionary response to environmental changes in wild animals.

Mitochondrial function needs strong interactions of mitochondrial and nuclear (mitonuclear) genomes, which can be disrupted by mitonuclear mismatch due to mitochondrial DNA (mtDNA) introgression between two formerly isolated populations or taxa. This mitonuclear disruption may cause severe cellular stress in mismatched individuals. Gene expression changes and alternative splicing (AS) are two important transcriptional regulations to respond to environmental or cellular stresses. We previously identified a naturally introgressed population in the intermediate horseshoe bat (Rhinolophus affinis). Individuals from this population belong to R. a. himalayanus and share almost identical nuclear genetic background; however, some of them had mtDNA from another subspecies (R. a. macrurus). With this unique natural system, we examined gene expression changes in six tissues between five mitonuclear mismatched and five matched individuals. A small number of differentially expressed genes (DEGs) were identified, and functional enrichment analysis revealed that most DEGs were related to immune response although some may be involved in response to oxidative stress. In contrast, we identified extensive AS events and alternatively spliced genes (ASGs) between mismatched and matched individuals. Functional enrichment analysis revealed that multiple ASGs were directly or indirectly associated with energy production in mitochondria which is vital for survival of organism. To our knowledge, this is the first study to examine the role of AS in responding to cellular stress caused by mitonuclear mismatch in natural populations. Our results suggest that AS may play a more important role than gene expression regulation in responding to severe environmental or cellular stresses.

MicroRNAs (miRNAs) are important post‐transcriptional regulators of gene expression and play key roles in many biological processes, such as development and response to multiple stresses. However, little is known about their roles in generating novel phenotypes and phenotypic variation during the course of animal evolution. Here, we, for the first time, characterized the miRNAs of the cochlea in an echolocating bat ( Rhinolophus affinis ). We sampled eight individuals from two R. affinis subspecies with significant echolocation call frequency differences. We identified 365 miRNAs and 121 of them were novel. By searching sequences of these miRNAs precursors in multiple high‐quality mammal genomes, we found one specific miRNA shared by all echolocating bats but not present in all other nonecholocating mammals. The targeted genes of this miRNA included several known hearing genes (e.g., KCNQ4 and GJB6 ). Together with the matched mRNA‐seq data, we identified 1766 differentially expressed genes (DEGs) between the two subspecies and 555 of them were negatively regulated by differentially expressed miRNAs (DEMs). We found that almost half of known hearing genes in the list of all DEGs were regulated negatively by DEMs, suggesting an important role of miRNAs in call frequency variation of the two subspecies. These targeted DEGs included several important hearing genes (e.g., Piezo1 , Piezo2, and CDH23 ) that have been shown to be important in ultrasonic hearing of echolocating mammals.