| The white-toothed shrews(genus Crocidura)belongs to the shrew family(Soricidae)of order EULIPOPHILA in Mammalia,and is widely distributed in Asia,Europe and Africa,with the highest species diversity among genera in mammalia in the world.There are many problems in species identification and taxonomy in Crocidura because of its conservative morphological characteristics.China has a complex topographies,which is easy to form some discontinuous ice age shelters or geographical diffusion barriers in the climate fluctuations in the geological history,resulting in the species differentiation and formation of small mammals.Because the high species diversity and rapid radiation of Crocidura species,the high species diversity and recent species formation,its taxonomy and systematic evolution have not been well solved for a long time.Based on the phylogenetic evolution and species delimitation analysis using polygenic loci,this study revealed the phylogenetic relationship,species differentiation and diversity of Chinese Crocidura,and studied the taxonomy and phylogeny of the Crocidura attenuata species complex,and discussed the genetic differentiation and biogeographic origin of the species complex.This study dealed with the species classification and systematic evolution of species complex from the three different levels of morphology,karyotype and molecule evidences,and explores to solve the problem of cryptic species of small mammals by using integrated taxonomy methods.In the use of molecular data,this study tried to combine mitochondrial genes and nuclear genes,and used different data strategies to calculate the species phylogenetic relationship,divergence time,species delimitation and biogeographic relationship of the species among the C.attenuata species complex,so as to achieve innovation in data quantity,data representation and research methods.In this study,two mitochondrial genes(2,685 bp),13 coding protein genes(11,347 bp)and 12 nuclear gene fragments(7,611 bp)of Chinese white-toothed shrews(Crocidura)were amplified and sequenced.Combined with the molecular data in GenBank database,Bayesian inference(BI)and maximum likelihood(ML)hylogenetic trees were constructed for the existing species of Chinese Crocidura species.The species delimitation was analyzed based on Automatic Barcode Gap Discovery(ABGD)and Bayesian Phylogenetics and Phylogeography(BPP).The genetic differentiation,population structure,divergence time,population dynamics and niche simulation of C.attenuata species complex were analyzed to infer its phylogeography.The following results were obtained:(1)The phylogenetic relationship of Chinese Crocidura species has been basically solved.The Chinese Crocidura is divided into North and south clades,of which the South clade has undergone rapid radiation evolution,ancient gene hybridization and mitochondrial gene introgression hybridization,with a complex evolutionary history.The uplift of the Qinghai Tibet Plateau and the global climate turbulence caused by the replacement in the late Pliocene and early Pleistocene jointly affected the diffusion,differentiation and species formation of the genus Crocidura in China;(2)The species diversity of Crocidura in China is seriously underestimated,and there are still many doubtful species need further delimitation.The C.attenuata species complex includes four species:C.attenuata,C.anhuiensis,C.tanakae and C.dongyangjiangensis,our results also determined that C.huangshanensis is a synonym of C.dongyangjiangensis;(3)Under the influence of geological and climatic changes,the C.attenuata species complex formed the existing species compositions and geographical distribution pattern after several population migrations and genetic differentiations in the southeast and East direction.It is speculated that this process was related to the complex mountain and river landforms in China caused by a series of geological uplift events in Southwest China,the three-level ladder terrain high in the West and low in the East,and the climate difference between warm and humid in eastern China and cold and dry in Western China caused by monsoon circulation;(4)The four species of C.attenuata species complex are distributed in the south of China.Among them,the distribution of C.attenuata is limited to the central and western regions of China.Around the western,northern and eastern fringe of Sichuan Basin,it is only sympatric with C.tanakae;The distribution of C.dongyangjiangensis and C.anhuiensis basically overlap,and they are all distributed in the southeast of China,including southern Anhui,Zhejiang,Jiangxi and Guangdong.C.tanakae is the most widely distributed species in southern China,and has the same domain distribution with other three species;(5)The four species of the C.attenuata species complex have a high level of genetic diversity and genetic differentiation,and there is obvious geographical population structure.It is revealed that the factors of genetic differentiation among populations are mainly due to the geographical isolation caused by the selection of different ice age shelters in the cyclones of recent ice age and interglacial age;(6)C.tanakae had many population expansion events,even after the last ice age,there was rapid genetic differentiation and population expansion,and ancient hybridization as well as mitochondrial introgression hybridization occurred in Guangdong and Yunnan respectively.The karyotypes of C.attenuata species complex were analyzed under the condition of correct species identification of by use of molecular taxonomic techniques.The results showed that:(1)the karyotypes of C.attenuata species complex showed two types:one was the single karyotype of C.attenuata,C.anhuiensis and C.dongyangjiangensis,that is,2n=40,FN=54,and the karyotype formula was 2n=40,6(m+sm)+6st+26t+X(sm)+Y(t);The other is the polymorphic karyotype of C.tanakae,that is,2n=24-40,FN=45-56.The karyotype formula is 2n=24?40,6?20(m+sm)+0?8st+0?24t+X(t,st,sm,m)+Y(t);(2)Although C.tanakae has karyotype polymorphism,the same karyotype was not observed among the other three species,so C.tanakae can be identified according to its karyotypes;(3)The polymorphism and karyotype variations of C.tanakae have obvious rules.According to 2n?FN and chromosome group,they can be divided into two chromosome groups,A and B groups,with obvious differences and there are obvious differences in their geographical distribution.The two chromosomal groups A and B are isolated by a series of mountains in southern China,including Mt.Wuyi?Mt.Nanling and Mt.Xuefeng.Mt.Nanling in Guangdong is the only sampling site with both chromosomal groups A and B,which indicates that this area is the hybrid band of the two chromosomal groups or the original region of karyotype polymorphism of C.tanakae,which also supports C.tanakae has a complex evolutionary history.Under the guarantee of correct species identification of the specimens of C.attenuata species complex by molecular procedures,the morphology of four species of C.attenuata species complex was analyzed.The results show that:(1)in terms of measurement indices,skull measurements are more stable than external measurements,and have better species identification effect.Both principal component analysis(PCA)and discriminant analysis(DA)can basically distinguish the four species.The measurement indices of C.dongyangjiangensis were significantly smaller than those of the other three species,while thats of C.attenuata,C.anhuiensis and C.tanakae overlapped seriously;(2)morphological characteristics only assure species identification for C.dongyangjiangensis,while the differences in morphological characteristics among C.tanakae,C.attenuata and C.anhuiensis are not significant and can not be used for their species identification.In this study,the C.attenuata species complex was classified and studied at the three levels of molecular,karyotype,and morphology based on integrated taxonomy,which not only revealed the underestimation of the species diversity of Crocidura genus in China,but also explored the role and relationship of evidence at three different levels.The research on the interrelationships between animal chromosome karyotype polymorphsim and cryptic species diversity in China has been filled up.It also provided theoretical and practical guidance for the health and epidemic prevention of Crocidura genus specie,agriculture and forestry and other related fields. |
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