| The genetic mechanisms and evolutionary processes associated of speciation have always been a central goal of evolutionary biology.In recent years,global warming has a great effects on many aspects of biodiversity.Understanding the effects of natural selection,drift and other factors on speciation and the mechanisms by which organisms respond to climate change becomes more important.Tree shrew（Tupaia belangeri）belongs to the the genus of Tupaia of the family of Tupaiidae of the order of Scandentia,and is a unique species of Scandentia in China.It is widely distributed throughout Yunnan,Guizhou,Sichuan,Guangxi and Hainan island of China.Molecular techniques have shown that it has more closely related to primates than rodents,and has shorter life span than Macaca,special than the Macaca used as experimental animals,and because of its unique characteristics,such as small body size,high brain-to-body mass ratio,short reproductive cycle,tree shrews have been proposed to be alternative experimental animals to primate in biomedical research.However,despite the extensive number of population studies that examined their distribution and phenotypic morphology and microsatellites,knowledge of population genomics and adaptation within T.belangeri populations is limited because of the lack of the studies at the population genomic level.In this study,134 individuals from 12 populations of 6 subspecies of T.belangeri were selected as subjects.Simple genome sequencing and resequencing were firstly used to clarify the subspecies classification,which plays an important role in the scientific utilization of T.belangeri.Moreover,the genetic diversity and population dynamics of six subspecies were explored to provide important genetic data for the development of superior T.belangeri strains and human disease models;Additionally,to elucidate the driving factors of genetic differentiation,the level of genetic differentiation among populations was evaluated and the relationship between environmental factors and genetic differentiation level was analyzed;To elucidate the correlation between phenotypic differentiation and environment,the level of phenotypic differentiation among populations was evaluated and the relationship between environmental factors and phenotypic differentiation was analyzed;Finally,we integrate the genetic,environmental and phenotypic data to reveal the effect of natural selection and genetic drift on phenotypic differentiation,and to explore the survival adaptation mechanism and evolutionary significance of T.belangeri to changing environment.The experimental results mainly include the following four parts:1.Firstly,the population structure analysis,principal component analysis and phylogenetic tree construction showed that T.b.chinensis,T.b.yunalis and T.b.yaoshanensis should be the synonyms and combined into new T.b.chinensis,so tree shrews should be divided four effective subspecies,including new T.b.chinensis,T.b.tonquinia,T.b.modesta,and T.b.gaoligongensis.Moreover the results of genetic divergence time of different populations showed that T.b.chinensis,T.b.yunalis and T.b.yaoshanensis did not separate in the genetic level.In addition,differentiation occurred between the following populations over the last 25 Ka: T.b.tonquinia and T.b.gaoligongensis,T.b.tonquinia and new T.b.chinensis,T.b.gaoligongensis and new T.b.chinensis,T.b.gaoligongensis and T.b.modesta,and T.b.modesta and new T.b.chinensis.Alternatively,the differentiation between T.b.modesta and T.b.tonquinia occurred relatively recently among 10 Ka.Additionally,the demographic histories of T.belangeri subspecies was reconstructed,and the results showed that the effective population of T.belangeri had similar trends among different subspecies,but had different fluctuations.Our analyses highlight the role of historical global climates in the T.belangeri population dynamics and indicate that the decrease of the T.belangeri population size may be due to the low temperature.Finally,considerable genetic variations were found in different subspecies which exhibit high genetic diversity.2.Subspecies exhibited different degrees of genetic differentiation among different populations,with the range of Fst ranging from 0.028 to 0.914（average0.499）.T.b.modesta,T.b.tonquinia and T.b.gaoligongensis have high levels of genetic differentiation with other populations,while between the populations of new T.b.chinensis has low or medium levels of genetic differentiation.Mantel test showed that there was no significant correlation between pairwise Fst as well as Fst/（1-Fst）and geographic distance（IBD）,suggesting that T.belangeri not support the isolation-by-distance model,and support isolation-by-island model.There was significant correlation between pairwise Fst and Fst/（1-Fst）as well as environment distance（IBE）,altitude distance（IBA）,temperature distance and precipitation distance（IBP）,except between Fst/（1-Fst）and IBP.Moreover,IBA has the greatest influence on genetic differentiation.Interestingly,our data revealed that the genes associated with olfactory function were eliminated in high-altitude populations when compared with low-altitude populations;this indicates that the olfaction-associated gene may involved in the environmental adaptation of tree shrews.3.One-way ANOVA analysis of phenotypic traits showed that the morphological indexes of different populations showed obvious differentiation,and the skull indexes were more stable than the body indexes.Secondly,inter-population variation was greater than intra-population variation,indicating that inter-population variation was the main factor of phenotypic variation,and there was phenotypic variation among same subspecies.Moreover,the results of cluster analysis showed that the T.b.modesta and were clustered together,and other species were clustered into one branch.The two populations（TC population and PM population）of T.b.gaoligongensis were further grouped into one clade,and the DL population,ML population and LQ species of T.b.chinensis were clustered into one clade.The HK and XY populations of T.b.yunalis,KM and XC populations of T.b.chinensis,and LY species of T.b.yaoshanensis were clustered into a clade.Which including that KM and XC populations of T.b.chinensis,T.b.yunalis as well as T.b.yaoshanensis were clustered together based on phenotypic data.Environmental factors including altitude,longitude,latitude,precipitation and atmospheric pressure were significantly correlated with phenotypic traits of T.belangeri.4.The comparation between pairwise Pst and pairwise Fst showed that most or all pairwise Pst of phenotypic traits in new T.b.chinensis were larger than pairwise Fst.The results suggesting that the phenotypic traits of the new T.b.chinensis populations were under the pressure of natural selection,which indicates that natural selection played a major driving role in phenotypic differentiation.However,more or all pairwise Pst of phenotypic traits between four effective subspecies were lower than pairwise Fst,indicating that phenotypes were favored and stable selection occurred in different populations.IBD has no correlation with pairwise Pst,while IBA have greater influence on pairwise Pst.In conclusion,this study showed that: 1)T.belangeri has four effective subspecies,mainly including new T.b.chinensis which combined the T.b.chinensis,T.b.yunalis and T.b.yaoshanensis,T.b.tonquinia,T.b.modesta,and T.b.gaoligongensis.Moreover,T.b.chinensis,T.b.yunalis and T.b.yaoshanensis did not separate in the genetic level;Considerable genetic variations were found in different subspecies which exhibit high genetic diversity.New T.b.chinensis may be suitable to be selected as the field initial parent of cultivated strains,due to it has the relatively stable level of genetic diversity;2)Subspecies exhibited different degrees of genetic differentiation among different populations,T.b.modesta,T.b.tonquinia and T.b.gaoligongensis have high levels of genetic differentiation with other populations,while between the populations of new T.b.chinensis has medium levels of genetic differentiation.The results of Mantel test indicated that T.belangeri not support the isolation-by-distance model,and support isolation-by-island model,and IBA has the greatest influence on genetic differentiation;3)There were significant differences in phenotypic traits among different subspecies,which skull traits were more stable than body traits,and inter-population variation was the main factor.KM and XC populations of T.b.chinensis,T.b.yunalis as well as T.b.yaoshanensis were clustered together based on phenotypic data.In addition,environmental factors were significantly correlated with phenotypic traits of T.belangeri.The phenotypic differentiation of the new T.b.chinensis populations were under the pressure of natural selection,while phenotypes were favored and stable selection occurred between four effective subspecies;4)Our analyses highlight the role of historical global climates in the T.belangeri population dynamics and indicate that the decrease of the T.belangeri population size may be due to the low temperature.Moreover,we found that olfactory-related genes of high-altitude populations were selectively eliminated.Our data provide theoretical data for subsequent evolutionary ecological studies and the effects of global warming on the distribution of tree shrews.Our data proved for the first time that tree shrew was divided into four effective subspecies at the genetic level,and its genetic and phenotypic differentiation occurred to adapt to the environmental differences,provided a theoretical basis for exploring the adaptation mechanism of tropical small mammals to different environments.In brief,simple genome sequencing and resequencing were firstly used to clarify that T.belangeri should be four effective subspecies in genetic level;Subspecies exhibited different degrees of genetic differentiation among different populations,and T.belangeri not support the isolation-by-distance model,and support isolation-by-island model,suggesting that T.belangeri may be an important model for studying the differentiation of sympatonal species.This study provides a theoretical basis for exploring the adaptation mechanism of tropical small mammals to different environments. |
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