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.

The gut microbiota is now known as a key factor in mammalian physiology and health. Our understanding of the gut microbial communities and their effects on ecology and evolution of their hosts is extremely limited in bats which represent the second largest mammalian order. In the current study, gut microbiota of three sampling sources (small intestine, large intestine, and feces) were characterized in two sympatric and insectivorous bats (Rhinolophus sinicus and Myotis altarium) by high-throughput sequencing of the V3-V4 region of the 16S rRNA gene. Combining with published studies, this work reveals that Gammaproteobacteria may be a dominant class in the whole Chiroptera and Fusobacteria is less observed in bats although it has been proven to be dominant in other mammals. Our results reveal that the sampling source influences alpha diversity of the microbial community in both studied species although no significant variations of beta diversity were observed, which support that fecal samples cannot be used as a proxy of the microbiota in other gut regions in wild animals.

Introgression of mitochondrial DNA (mtDNA) between closely related taxa can be promoted by either neutral processes or natural selection. Since mitochondrial gene-encoded proteins play critical roles in oxidative metabolism, mtDNA genes are commonly considered to experience strong selective constraint. However, metabolic requirements vary across climatic and ecological gradients, thus modifying potential selective pressures acting on mtDNA genes. Here we conducted tests to detect adaptive evolution occurring in two mtDNA genes (Cytb and ND5) in individuals of Japanese pipistrelle bat (Pipistrellus abramus) across the mainland of China and Hainan Island. Nuclear DNA markers identified two clades in both the mainland and Hainan Island populations, whereas each of these regions had a specific mtDNA clade. This cyto-nuclear discordance is most likely caused by introgression of the mtDNA by ruling out two other alternative scenarios (incomplete lineage sorting and sex-biased gene flow). Although population-based analyses revealed purifying selection acting on Cytb and neutrality in ND5, multiple nonsynonymous substitutions in both Cytb and ND5 were suggested to have been caused by positive selection by a divergence-based analysis. Our study supports the view that molecular adaptation can occur at genes under strong purifying selection if nonsynonymous substitutions cause radical changes in the physicochemical properties of amino acids.

Echolocating bats are fascinating for their ability to ‘see’ the world in the darkness. Ultrahigh frequency hearing is essential for echolocation. In this study we collected cochlear tissues from constant-frequency (CF) bats (two subspecies of Rhinolophus affinis, Rhinolophidae) and frequency-modulated (FM) bats (Myotis ricketti, Vespertilionidae) and applied PacBio single-molecule real-time isoform sequencing (Iso-seq) technology to generate the full-length (FL) transcriptomes for the three taxa. In total of 10103, 9676 and 10504 non-redundant FL transcripts for R. a. hainanus, R. a. himalayanus and Myotis ricketti were obtained respectively. These data present a comprehensive list of transcripts involved in ultrahigh frequency hearing of echolocating bats including 26342 FL transcripts, 24833 of which are annotated by public databases. No further comparative analyses were performed on the current data in this study. This data can be reused to quantify gene or transcript expression, assess the level of alternative splicing, identify novel transcripts and improve genome annotation of bat species.

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