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 (DEGs), 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.

We performed de novo transcriptome assembly based on all trimmed reads using TRINITY with default parameters. Redundant transcripts were removed using CD-HIT-EST based on a 95% sequence similarity threshold. Transcripts with the longest open reading frames (ORFs) were kept using TRANSDECODER (http://transdecoder.github.io). Functional annotation was conducted using the TRINOTATE r20190821 pipeline (http://trinotate.github.io/) by searching against UniProtKB/Swiss-Prot and Pfam database (accessed 2th February 2019) using BLASTp and BLASTx [42] (E-value < 10^-5). Gene Ontology (GO) terms were retrieved for transcripts with a positive BLAST hit. Only transcripts functionally annotated were included in the final reference transcriptome.

We screened the candidate genes (FBXL15, GATA2, CLDN11, CKB, CCDC88C, and SLC1A3) for amino acid differences, either among the three R. affinis subspecies, or the three R. ferrumequinum geographical populations.