Reference transcriptome and Alignments of candidate gene

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.