Comparative Transcriptomics Reveals Molecular Basis Of Hearing Frequency Variation Within Subspecies Of The Intermediate Horseshoe Bat

Many natural organisms have phenotypic variation.Genetic variation of the phenotype is the basis of biodiversity.Studying the molecular basis of adaptive phenotypic variation is the hotspot and major challenge in evolutionary biology.In echolocating bats,call frequency is crucial in predation,adaptation to the environment and communication with each other.Although call frequency varies greatly among species or subspecies in bats,the molecular basis of this phenotypic variation is still poorly known.Rhinolophus belongs to constant frequency-frequency modulation(CF-FM)bat.Due to the Doppler effect,echolocation call frequency of bats in this genus is consistent with the hearing frequency.In this study,by focusing on three recently diverged subspecies of the intermediate horseshoe bat(Rhinolophus affinis)that displayed divergent echolocation call frequencies,we aimed to explore the molecular basis of call frequency variation using comparative transcriptomics of cochlea.Four adult males were sampled for each of the three subspecies and we used two methods(edgeR and DESeq2)to identify different expressed genes(DEGs).First,by pairwise comparison of gene expression profiles from the three subspecies,we found that call frequency difference between subspecies was related to differential expression patterns of genes.In comparisons of taxa with relatively greater difference of call frequency(R.a.himalayanus vs.R.a.hainanus,16.3 kHz call frequency difference;R.a.himalayanus vs.R.a.macruruss,13.4 kHz call frequency difference)we found relatively more number of DEGs(R.a.himalayanus vs.R.a.hainanus,799 DEGs;R.a.himalayanus vs.R.a.macruruss,750 DEGs),whereas in comparison of taxa with relatively smaller difference of call frequency less number of DEGs were identified(R.a.macrurus vs.R.a.hainanus,2.8 kHz call frequency difference,39 DEGs).Second,we identified 252 DEGs occurring in both comparisons of taxa with divergent call frequencies(R.a.himalayanus vs.R.a.macrurus and R.a.himalayanus vs.R.a.hainanus),which were considered as candidate loci associated with hearing frequency variation with 157 upregulated and 95 downregulated in R.a.himalayanus relative to the other two subspecies(R.a.macrurus and R.a.hainanus).Gene Ontology enrichment analysis found a total of 6 significant GO terms with FDR < 0.05,but they were not related to inner ear development or hearing.By comparing with the list of hearing or deafness genes identified in human/mouse,six DEGs(FBXL15,GATA2,CLDN11,CKB,CCDC88 C and SLC1A3)were found.Among them,FBXL15 may be the best candidate gene associated with hearing frequency variation.This gene has been shown to be associated with hearing at relatively high frequencies(above 24 kHz)in the ABR test(IMPC).We performed quantitative real-time PCR on this gene in 5 taxa with different call frequencies.Results revealed that the expression level of FBXL15 might be associated with call frequency variation in four taxa.However,this conclusion needs to be tested in more species in the future.Finally,67 DEGs were also found to be differentially expressed in pairwise comparisons of three Rhinolophus ferrumequinum geographic populations with significant differences of call frequencies using comparative transcriptomics of cochlear,including 4 DEGs(GATA2,CKB,SLC1A3 and CCDC88C)discussed in details in this study.The 6 DEGs identified here represent candidate loci associated with hearing frequency variation in bats.Further studying the function and evolutionary patterns of those candidate genes is helpful to understand the mechanism of complex phenotypic variation which is common in nature.In addition,gene editing and functional verification of these candidate genes in model organisms will provide valuable insights into the research of high frequency hearing recovery in human deafness.