Molecular Mechanism Of Geographic Variation In Echolocation Calls Of The Greater Horseshoe Bat

The sensory driving hypothesis assumes that animal communication signals and signal receiving systems will change with local climate conditions to adapt to the local environment.Different environmental conditions may lead to the diversity of phenotypes,genotypes,and gene expressions among different populations,and eventually lead to reproductive isolation,which is the basis for the formation of ecological species.As one of the unique biological groups in the world,bats not only play an important role in the ecosystem but also inspired the development of technology.In recent years,more and more researches have begun to pay attention to the geographic variation of sonic echolocation frequency of bats and have verified the sensory driving of echolocation signals in bats,deepening the understanding of the mechanism of species formation.However,most of these studies focus on the ecological level,and we know very little about the molecular mechanisms behind the geographic changes in bat sound signals with the development of sequencing technology,a new interdisciplinary course emerges-ecological genomics.Ecological genomics aims to combine multiple disciplines and research methods to explain the relationship between phenotypes,genes and the environment.Greater horseshoe bats use constant frequency(CF)sound waves,has a highly developed sonar system,and has a variety of vocal behaviors.It has always been an ideal species for bat echolocation sound waves,ecology,behavior,and neurophysiology research.It is widely distributed in China and can be divided into three lineages at the genetic level: northeast(NE),middle east(ME),and southwest(SW).The echolocation sound waves of these three lineages have obvious geographical differences,and this variation is considered to be related to environmental conditions.Therefore,the greater horseshoe bat is a good model to study the molecular mechanism of the geographic variation of echolocation sound waves.In this study,we used genetics,phenotype,environment data to explore the mechanism of geographic variation of echolocation calls of greater horseshoe bats in China.Considering the important role of the cochlea in the echolocation process,we obtained high-quality cochlear transcriptome data from three representative populations of the three lineages of greater horseshoe bats,and we obtained information on gene expression,sequence variation,and orthologous genes.Through differential expression analysis of different populations,weighted co-expression network analysis(WGCNA),selection of stress analysis,outlier detection,and other methods to clarify the impact of gene expression and sequence variaty on the phenotype.Also,we obtained genotyping-by-sequencing(GBS)data of 87 individuals in 11 populations.Combining environmental data and a variety of outlier testing methods,we obtained candidate genes and studied their effects on phenotype.First,we performed RNA–seq on the cochlear tissues of 14 individuals from three representative populations of greater horseshoe bats in China and compared the gene expression differences between the populations.The gene expression of RNA–seq was verified by RT–qPCR.A total of 8,190 differentially expressed genes(DEGs)were obtained.Through WGCNA analysis,we found that some DEGs modules were significantly related to resting frequency(RF)or forearm length(FL).The Unigenes in these modules were significantly enriched in a variety of pathways related to neurons and muscle functions,such as synaptic function,neuron function,ion channel function,response to sound,learning behavior,muscle and actin functions,etc.These results indicate that neural activity,vocal learning process,and muscle metabolism are related to the characteristics of echolocation calls of greater horseshoe bats.Although specific genes cannot be identified,our results still suggest that these genes are potentially important.Comparative transcriptome analysis performed using RNA–seq data showed that the three populations were under different selection pressures.Genes that are positively selected on three population branches are involved in multiple functions,such as cilia formation,oxidoreductase activity,and immune processes.But these positively selected genes are not directly related to hearing.The SNP loci in RNA-seq data provide another way to understand the geographic variation of sound signals.Combining outlier detection methods and genetic environment association analysis(GEA)methods,we found 349 candidate loci,involving multiple A variety of biological functions,including immune processes,transcription processes,and synaptic processes,18 of which are related to auditory processes or the development of auditory organs.Combining gene expression data,we found that five of these genes(ERBB4,OTOGL,IL6 R,CKMT2,and LGR6)were significantly differentially expressed among the three populations,while ERBB4 was in the DEGs module significantly associated with RF.It suggests the importance of these genes in the geographic variation of echolocation calls.SNPs were predicted and filtered from 87 individuals from 11 populations of greater horseshoe bats.The results of population structure analysis showed that the SW lineage was internally differentiated,and some individuals mingling,which suggested the existence of gene flow.We found that the geographical variation of RF OF echolocation calls is related to environmental factors(especially temperature).Using environmental data and combining four outlier detection methods,we obtained 210 candidate loci predicted by at least two methods.Among the 26 candidate loci with valid annotations,GNAI,TMPRSS,SPIN1,Wnt9 b,and UNKL are directly related to hearing or involves functions of hearing such as cochlear development and hair cell development.Among them,TMPRSS,Wnt9 b,and UNKL participate in the Wnt signaling pathway.Their functions are related to activation,regulation,or regulation of the Wnt downstream pathway,which involves plane polarization of cochlear hair cells,causing different positions of the cochlea to respond to different frequencies.It suggests that the Wnt pathway may play an important role in RF mutation.Our research has identified a large number of candidate genes and pathways.Although changes in gene expression and the function of specific amino acid substitutions in these genes are unclear,the analysis results indicate the importance of these genes in the geographic variation of echolocation acoustic waves.In this study,even if variations in RF of echolocation calls can partially explain changes in gene expression and sequence variation.So future functional experiments are essential.Also,our results suggest the importance of the Wnt pathway and other cochlear developmentrelated genes,so studying whether the cochlear structure changes between pedigrees will also help us understand the geographic variation of echolocation sound waves.In summary,our research integrates multiple methods,combining genetic,phenotypic,and environmental data to clarify differentially expressed genes or sequence mutations that may be closely related to the geographic variation of echolocation sound waves of the horsetail bat.The genetic basis of the geographic variation of bat sound signals provides further evidence for understanding the sensorydriven molecular mechanisms of sound signals,the formation of ecological species,and conservation biology.