Research On Population Genetics And Phenotypic Differentiation Of Hipposideros Larvatus

Speciation and evolutionary processes of organisms are one of the core questions in evolutionary biology field.During the long evolutionary process of species,many factors,including palaeoclimate,geologic events and environmental changes etc.,affect the phenotypic and genetic diversities among populations.Uncovering the genetics and evolutionary history of populations and revealing the potential drivers,such as drift,selection and colonization history,which lead to genetic and phenotypic variations of populations are always the interesting topics in evolutionary biology.Those studies will be very important for revealing the formation of biodiversity and maintaining mechanisms.As the unique mammals which can really fly,Chiroptera(bats)widely distribute in the world and are the important biological indicators in the ecosystems.Previous studies mainly studied on the genetic structure and evolutionary history of some bat species.While,comparing to the rich biodiversity of Chiroptera(more than1400 species),those studies are very limited.Furthermore,the potential factors driving population differentiation still need to be explored.In this study,we targeted on Hipposideros larvatus,which widely distributed in southern China and other regions in east-south Asia.We collected the samples of this species from China Mainland and Hainan Island.Based on mitochondrial DNA,microsatellites and genotyping-by-sequencing data,we detected the mode of population genetic differentiation and deduced the evolutionary history,as well as analyzed the role of island in population differentiation.Combined the geographic and climatic factors,we revealed the drives of genetic differentiation,including isolation by distance(IBD),isolation by adaptation(IBA)and isolation by colonization(IBC).We used multiple methods to test the relative importance of geographic,environmental and colonization factors for population divergence.And we evaluated the mode of morphological and acoustic differentiation among different geographic populations,and revealed the potential factors which drive those phenotypic differentiation integrating the geographic,climatic and genetic factors,as well as illustrated the relative importance of drift and selection.Our results provide the scientific evidences for deeply understanding the formation of biodiversity and maintaining mechanisms.We collected 165 individuals of H.larvatus from 18 populations in China Mainland and Hainan Island and studied the evolutionary history and genetic structure among populations based on two mitochondrial markers(cytochrome b and control region),nuclear microsatellites(seven loci)and genomic SNPs.Our results showed the high genetic diversities of H.larvatus populations and obvious genetic structure.In this species,two evolutionary lineages were obtained.The phylogenetic trees based on mitochondrial(mt)DNA showed that most of populations from China Mainland formed a monophyletic lineage(clade A),the populations from Hainan Island and part populations from Yunnan Province formed another lineage(clade B).In clade B,the populations from Hainan Island formed a monophyletic subclade(subclade B1).The STRUCTURE analysis based on microsatellites showed the similar genetic differentiation with mt DNA.While,the phylogenetic trees based on SNPs showed some gene flow between Hainan Island and China Mainland,even if the existence of genetic differentiation between them.The population demorgraphic analyses showed that the most recent common ancestor(TMRCA)of H.larvatus could be traced back to 1.70 Ma,and the TMRCA of clade A and subclade B1 could be traced back to 0.40 Ma and 0.21 Ma,respectively.The clade A and subclade B1 occurred the population expansion in 0.146 Ma and 0.041 Ma,respectively.The results of Mantel tests showed that the nuclear genetic distances among H.larvatus populations were significantly correlated with geographic,climatic and mitochondrial genetic distances(all P_adj<0.05).When controlling with geographic,climatic or mitochondrial genetic distances,the nuclear genetic distances still have significant correlations with those distances(all P_adj<0.05),which suggested that IBD,IBA and IBC drive the genetic differentiation of H.larvatus populations.Our further RDA analysis suggested that the mitochondrial variables(R²_adj=0.543,P=0.004)and climatic variables(R²_adj=0.307,P=0.001)significantly explained the greatest fraction of the nuclear divergence;however,geographic variation explained a smaller fraction with no significant pure effect on nuclear genetic divergence(R²_adj=0.183,P=0.186).Those results indicated that the colonization history,different climatic conditions between China Mainland and Hainan Island,and geographic distances play important roles in population genetic differentiation in H.larvatus.We detected the geographic variation of morphological characters based on the 116individuals with morphological phenotypes(61 females,55 males)from 14 H.larvatus populations.Population significantly affected the geographic variation of morphological phenotypes,but no sexual dimorphism was detected among populations.We found a significant correlation between morphological phenotypes and latitude,and significant correlations between forearm length and latitude and four climatic factors,including mean diurnal range,temperature annual range,precipitation of wettest month and precipitation of wettest quarter(all P_adj<0.05).When controlling with climatic factors and latitude,forearm length was still correlated with latitude and two climatic factors(precipitation of wettest month and precipitation of wettest quarter),suggesting local adaptation drives the geographic variation of morphological characters in H.larvatus.Based on the genotype variation induced by phenotypes and environment,a pathway named glutamatergic snapse was significantly enriched,suggesting its important role in adaptation evolution in H.larvatus.For 7 populations of 36 individuals with 13 skull parameter measurements,using principal component analysis,MDS analysis and Wilcox variance analysis,we found that the skulls of two populations from Hainan Island are significantly larger than those from China Mainland,but no significant difference within Hainan Island and China Mainland.Combined with the genetic data,the bats in Hainan island and China Mainland should be different subspecies.We detected the geographic variation of echolocation calls based on 102 individuals(55females,47 males)from 14 H.larvatus populations.Population significantly affected the geographic variation of acoustic phenotypes,but no sexual dimorphism was detected among populations.Mantel tests showed that the resting frequencies of echolocation calls in H.larvatus were significantly correlated with geographic,longitude,isothermality and genetic distances(all P_adj<0.05),but not morphological distances(P_adj>0.05).When controlling with any other variables,resting frequency was always correlated with mitochondrial genetic distance(all P_adj<0.05),suggesting the maternal transmission hypothesis drives the echolocation calls variation among H.larvatus populations,but genetic drift,ecological selection and body size did not have obvious effects on acoustic variation.In conclusion,this study shows that:1)H.larvatus has obvious genetic structure,and obvious genetic differentiation exist between Hainan Island and China Mainland.The Pleistocene glaciation affected the evolutionary history and population dynamics in H.larvatus;2)significant phenotypic differentiation of H.larvatus in morphology and skull features was detected between Hainan Island and China Mainland,suggesting the existence of two different subspecies;3)genetic drift,selection and colonization history drive the genetic differentiation among H.larvatus populations;and 4)obvious geographic variation of morphological and acoustic phenotypes were detected in H.larvatus.The maternal transmission hypothesis drives the acoustic variation and local adaptation drives the morphological variation among populations in H.larvatus.