| Species differentiation and adaptation are important for species diversity.Many studies have focused on the role of neutral drift and natural selection in the process of evolution,which could elucidate the molecular mechanisms of species differentiation and adaptation.The studies in this field extensively focus on the adaptive evolution at DNA sequence level,and rarely on the evolutionary forces of expression variation.Furthermore,those studies are mostly limited to a single scale of macroevolution(at the species level)or microevolution(at the subspecies or population level).Therefore,it is necessary to investigate the molecular mechanisms of species differentiation and adaptation at both expression and sequence levels,and on both macroevolutionary and microevolutionary scales.Chiropterans,commonly known as bats,are the only mammals with the true ability of flight.Most bat species have echolocation ability.Many closely related species have formed due to the chiropteran adaptive radiation.Those closely related species are usually at early stage of species differentiation,and are as the ideal models for exploring the species differentiation and adaptation due to their similar genotypes but different phenotypes.In this study,we selected the typical closely related species of Rhinolophus as objects,including Rhinolophus episcopus,Rhinolophus siamensis,and Rhinolophus osgoodi.Based on comparative transcriptomes,we combined the phenotypic and acoustic data,and analyzed the molecular basis of the differentiation and adaptation for three closely related horseshoe bats from the perspectives of gene differential expression and its evolutionary forces,and DNA sequence variation and adaptation on both species and subspecies scales using the analysis methods at the omics and single gene levels.The results would be helpful for the deeply understanding of chiropteran adaptive evolution and formation of species diversity.To explore the expression variation and clarify its functional patterns,we selected sympatric taxa for minimizing the influence of environment on gene expression at the species level,and the taxa inhabiting heterogeneous environment at the subspecies level.We sequenced fifty-seven transcriptome data of brain,cochlea,and liver samples from R.siamensis and R.osgoodi,R.e.episcopus and R.episcopus spp.using reference sequence.Two inter-specific comparisons(H,R.siamensis vs R.e.episcopus;Y,R.osgoodi vs R.episcopus spp.)and the inter-subspecific comparison(Re,R.episcopus spp.vs R.e.episcopus)were conducted for the analysis of pairwise gene expression variation in this study.The results indicated a stronger organ specificity than species specificity of gene expression.Little divergence of the expression variation was detected between the inter-specific and inter-subspecific comparisons for brain and cochlear samples.While,for liver samples,the gene expression variation between subspecies was greater than that between species,indicating the organ-specific variation in gene expression.The results of functional enrichment using differentially expressed genes(DEGs)showed the similar functional patterns of expression variation between brain and cochlea samples.The expression variation between taxa were associated with many functions,such as disease and immune processes,ion activities and signal channels,nervous system,and multi-sensory system regulations,including auditory sense,vision,optesthesia,and gustation.In addition,we found genes associated with senses were uniquely expressed for R.siamensis and R.episcopus,which implied the importance of sensory-related differentiation at the expression level among species.Furthermore,a certain amount of DEGs related to body size or acoustic signal were screened combined with the data of forearm length and dominant frequency of echolocation calls in each comparisons for each organ.To further clarify the evolutionary forces of expression variation,we screened the DEGs with expression variation governed by directional selection,stabilizing selection,and balancing selection based on the variation between taxa and among individuals within taxon using ANOVA and a rank-based method.The relative influence of natural selection on expression variation was evaluated.The results indicated that the expression variation was extensively governed by natural selection(> 63.128%)between taxa.The proportion of expression variation governed by directional selection was the highest,followed by stabilizing selection,and balancing selection was the lowest.The percentages of expression variation forced by natural selection were similar between two taxonomic levels.Furthermore,using the DEGs with expression variation forced by natural selection in three comparisons,the GO categories and KEGG pathways related to disease and immune regulations were significant enriched in three comparisons,suggesting that the adaptive expression variation related to immune process might play an important role in the process of species differentiation and adaptation.The vision-related terms and pathways were also enriched in the inter-specific comparison.In the inter-subspecific comparison,the terms and pathways associated with ion activities,signal transduction,nervous system,and the process of growth and development were enriched as well.It implied that there were differentiation and adaptation related to these functions at the expression level in the process of evolution for three closely related horseshoe bats.Furthermore,the analysis results of the evolutionary forces of expression variation related to phenotypes showed that the expression variation related to acoustic signal and body size were widely forced by natural selection(> 69.091%).The adaptive expression variation related to traits were focused on directional selection,followed by stabilizing selection,and no balancing selection.The enrichment analysis suggested that the functional patterns of adaptive expression variation associated with phenotypes were different between two taxonomic levels,manifested as cellular processes at the species level and immunoregulation at the subspecies level.To elucidate the genetic basis of species and subspecies differentiation at the sequence level,we performed comparative transcriptomics study for four taxa combined with the sequences of Rhinolophus ferrumequinum and Hipposideros armiger.We obtained 647 orthologous genes among six taxa.The results of selection pressure analysis showed that sixteen,eight,twelve and twelve positively selected genes(PSGs)were found for R.siamensis,R.osgoodi,R.e.episcopus and R.episcopus spp.,respectively,and 369 taxon-specific selection sites were detected.The enrichment results using PSGs showed that the adaptive evolution related to disease and immune regulations existed in four taxa.Combined with the results at the expression level,five genes(MCEMP1,IL10 RA,BCL3,GPM6 A,and TMEM182)were with expression variation forced by natural selection and showed positively selected signals at the sequence level,implying the adaptive evolution of these genes during the differentiation and adaptation of three closely related horseshoe bats.For each of above five genes,we performed the branch-site model of positive selection analysis using more Rhinolophus species,and found that three immune genes(MCEMP1,IL10 RA,and BCL3)suffered strong selective pressure in R.e.episcopus,and GPM6 A was under positive selection in R.siamensis,however,no selective signals were detected for TMEM182.Finally,based on the samples taken at a large geographic scale,we verified that the adaptive evolution of GPM6 A,which encode neuronal glycoprotein M6 a,among taxa by performing the molecular evolution analysis of coding region sequence and secondary structure simulation of 5’ untranslated region(5’ UTR)sequence.The results of molecular evolution analysis showed that the sequence in protein-coding region was conserved in the evolutionary process of GPM6 A among three closely related horseshoe bats.The strong selective pressure in R.siamensis was detected,and A65 V on the small extracellular loop might be the key amino acid site for the adaptive evolution of GPM6 A in R.siamensis.The prediction results of the effect of amino acid mutation on protein stability manifested that the amino acid mutation,from alanine(A)to valine(V),on this site leaded to the lower stability of GPM6A(ΔΔG=-0.370),which might originate from the stronger hydrophobicity and more average buried area during the process of protein folding for valine.In addition,the secondary structure simulation results of GPM6 A 5’UTR showed that it was highly conserved for R.siamensis and R.osgoodi,however,more stem rings and extension directions were found in the secondary structure for R.episcopus.In conclusion,based on the theory and methods of comparative transcriptomes,our study clarified the variation in gene expression and its functional patterns,and identified the evolutionary forces of expression variation,and evaluated the positively selected genes and sites at both species and subspecies levels.The role of natural selection on the differentiation and adaptation of three closely related horseshoe bats was investigated at both gene expression and sequence levels.At the same time,combined with the analysis results at the omics and single gene levels,this study broadly revealed the molecular basis of differentiation and adaptation for three closely related horseshoe species,which is helpful for the deeply understanding of adaptive evolution and species diversity.Furthermore,we analyzed the impacts of natural selection on the expression variation associated with phenotypes,and obtained the adaptive functional patterns of expression variation related to phenotypes,which could be helpful to better understand the role of natural selection on the phenotypic evolution of bats and provide scientific basis for the protection of bat species. |
