Molecular Evolution Of High-Frequency Hearing In Echolocating Bats

Bats mainly occupy the night sky niches and have evolved special echolocating ability for navigating,detection and hunting when the visual and other sensory information are limited,which makes the echolocation to be the typical case of adaptive evolution.Most bats are capable of well-developed echolocation except for a small group of bats from Pteropodidae.Generally,echolocating bats can be divided into three broad categories according to the dominant frequency ranges and the structures of their echolocation calls: frequency modulated(FM)bats,constant-frequency modulation(CF-FM)bats(also named CF bats),and tongue-click bats(also named click bats).Correspondingly,echolocating bats have evolved remarkable high-frequency hearing ability to perceive their ultrasonic signals.Cochlea acts as a crucial component of the peripheral auditory system,playing important roles in sound perception,signal processing and transmission.Previous studies have demonstrated that special morphological structures and physiological activities are occurred in the cochleae of echolocating bats for the reception of ultrasonic signals,however,our knowledge about the internal genetic bases and molecular mechanism underlying the phenotypic and physiological adaptations are still limited.In this study,we took use of the comparative cochlear transcriptomics to assess the variations and patterns of expressed genes in the cochleae of three types of echolocating bats,meanwhile,to uncover the changes of important hearing related physiological processes dominated by differentially expressed genes.Subsequently,we combined the cochlear transcriptome data of the three types of echolocating bats with the genome data of 16 other mammals with or without echolocating ability.Based on the variations of gene sequence to investigate the adaptive evolution of hearing related genes expressed in the cochleae.The origin and development of echolocation in mammals involved with multiple physiological processes and genes.Therefore,besides those hearing related genes obtained by cochlear transcriptome sequencing,two other important candidate hearing related genes—Shh and SK2 were further studied for their molecular evolution in echolocating bats and whales.Firstly,numerous differentially expressed genes(DEGs)were identified between every two types of echolocating bats.Further analyses of DEGs indicated that the largest molecular difference was detected between CF and click bats,followed by the difference between CF and FM bats and the smallest difference was found between FM and click bats;a large number of upregulated genes in the cochleae of CF bats were found to be related with neuron production,nervous system activity and various ion transport activities,implying unique nervous system activities and abundant ion transport activities in the cochleae of CF bats.Subsequently,intersection and cluster analyses of all DEGs identified two groups of genes,including 426 and 1,504 DEGs,respectively,showing consistent upregulation in the cochleae of two CF bats,indicating those genes were essential molecular bases underlying hearing physiological functions in CF bats' cochleae.Downstream GO and KEGG enrichment analyses indicated that both of the 426 and 1,504 gene sets were responsible for the differences of nervous activities in the cochleae of CF bats.In addition,another gene set of 1,764 DEGs was also identified,showing upregulation in laryngeal echolocating bats.Functional enrichment analyses indicated that those 1,764 DEGs playing crucial hearing related physiological functions in laryngeal echolocating bats,implying those genes were important molecular bases for the high-frequency hearing perception in laryngeal echolocating bats.Secondly,a total of 144 genes were identified to be under positively selected in different types of echolocating bats and whales,offering important candidate genes for following studies in future.Large number of parallel/convergent sites were detected among every two comparative echolocating bats and whales,however,most parallel/convergent sites were not under positively selected,indicating those parallel/convergent signals were caused by neutral evolution rather than natural selection.According to the relevant studies,34 out of the 144 positively selected genes were further identified to be closely related with hearing behavior or auditory processes,at the same time,17 out of the 34 hearing related genes were also identified to have parallel sites.Combined with the results of various analyses,enrichment analyses,site location and protein network interaction analyses,multiple hearing related physiological processes were found to be adaptively evolved in echolocating bats and toothed whales,including cochlear bony development,antioxidant activity,ion balance and homeostatic process and signaling transduction.In addition,we also found an important hearing related gene named TECPR2,two parallel evolved sites of this gene were identified between the CF and FM bats lineages,more importantly,these two sites were under positively selected,indicating these two sites could be functionally important for the origin and evolution of ultrasonic hearing in laryngeal echolocating bats.Finally,we identified and analyzed two other important hearing related genes: Shh and SK2.The topologies of phylogenetic trees conducted by these two genes were different.The results of molecular evolution analyses showed that these two genes suffered stronger selective pressures and showing faster evolutionary rates in echolocating bats and toothed whales compared with non-echolocating mammals.At the same time,several positively selected sites were detected in the non-coding functional domain of SK2 gene,similar result was also concluded by sliding window analyses,indicating different strengths of selective pressures were acted on the different parts of this gene: functional domains of SK2 were relatively conserved through evolution,however,non-coding functional domain showing faster evolutionary rates.The discovery and research of these two genes could be an important complement for previous studies.Taken together,in this study,we mainly focus on the echolocating bats,combining analyses with other mammal species with or without echolocating ability and paying close attention to the variations of gene expression and gene sequence,our study totally uncovered the molecular bases and evolutionary mechanisms underlie the adaptive evolution of high-frequency hearing in echolocating mammals.Our study offered important candidate genes and research ideas for related studies in future.Furthermore,all methods used in this study for the research of adaptive evolution at molecular level could be the guidance for following study with other sense organs and phenotypes.