Species Delimitation And Phylogeography Of Grassland Caterpillars(Lepidoptera:Lymantriinae:Gynaephora) Endemic To The Qinghai-tibetan Plateau

The Qinghai-Tibetan Plateau（QTP）Gynaephora（Lepidoptera:Erebidae:Lymantriinae）are major pests that severely damage the alpine grasslands in China.Currently,all eight species of Gynaephora reported in China are distributed in the QTP.However,molecular species delimitation based on mitochondrial genes does not support the current eight morphological species.Therefore,it is necessary to further evaluate and construct morphometry taxonomic characteristics and use more populations and genes to delimit species of the QTP Gynaephora,and to explore process of speciation.In this study,main morphological characteristics of male adults from six geographic populations of QTP Gynaephora were analyzed and measurement system of quantitative morphometry and geometric morphometry of Gynaephora was constructed.Based on 2 mitochondrial genes（cox1 and nad5）and restriction site-associated（RAD）single nucleotide polymorphisms（SNP）loci data,species delimitation and phylogeographic analysis for QTP Gynaephora was conducted.The aim is to clarify the species status and population differentiation of QTP Gynaephora and to provide evidence for the outbreak mechanism and effective control of QTP Gynaephora.The main results are listed as follows:1.In this study,eight morphological characteristics of male adults of QTP Gynaephora were selected and analyzed based on the clarification of morphological characteristics of eggs,larvae,female pupae,male pupae,female adults and male adults of G.menyuanenis.The results show that a total of 7 quantitative characteristics can be used to delimit the Mengyuan population and Nierong population based on the principal component analysis,including antennal length,body length,forewing length,hindwing length,length-height ratio of hamulus,length-height ratio of harpes,and length ratio of basiphallus-distiphallus.The results of the canonical variable analysis for five geometric morphological characteristics,including forewing,hindwing,hamulus,harpes,and penis,show that hamulus can delimit the Menyuan,Zeku,Ruoergai,Gande,Nierong and Qumalai populations,while both hindwing and harpes can delimit the Mengyuan population and Nierong population.Therefore,quantitative morphological characteristics are not effective for species delimitation of different Gynaephora,while geometric morphometric characteristics can provide effective species delimitation in Gynaephora.2.A total of 40 mitochondrial haplotypes were identified from two mitochondrial genes（cox1 and nad5）of 39 populations of QTP Gynaephora by PCR amplification and sequencing.The haplotype diversity（h）is 0.913±0.007,and the nucleotide diversity（π）is 2.288±0.033%.Phylogenetic analysis,haplotype network analysis and population structure analysis all support the delimitation of the 39 geographic populations into four clades:Clade A（including G.qinghaiensis,G.jiuzhiensis,and G.qumalaiensis）,Clade B（corresponding to G.alpherakii）,Clade C（corresponding to G.menyuanensis）,and Clade D（including G.aureata,G.rouergensis,and G.minora）.The vast majority of pairwise F_ST values between the 39 populations are significantly greater than 0.25.Mantel test results support a significant positive correlation between genetic distance and geographic distance（r=0.169-0.517,P<0.01）.Analysis of molecular variance（AMOVA）shows that the F statistic between clades are significantly positive（P<0.01）,whether grouped by the four clades,or by altitude.It is speculated that QTP Gynaephora exhibits a significant phylogeographical structure,and that geographic isolation and altitudinal environment are important drivers of population differentiation and speciation for QTP Gynaephora.QTP Gynaephora share a common ancestor and have been gradually differentiated into different species since their spread from abroad in the late Miocene（about 4.65 Ma）by the combined effects of the Pleistocene（2.08-0.1 Ma）uplift of the QTP and climate change.Biogeographic analysis supports the earliest arrival of QTP Gynaephora in the plateau hinterland（Clade A）,and the Sanjiangyuan region may have been a glacial refugium of QTP Gynaephora during the Pleistocene.The results of the gene flow analysis show that the direction of gene flow between the four clades is Clade A→Clade B→Clade C,and there is bidirectional gene flow between Clade C and Clade D.3.A total of 5074 SNP loci were identified by RAD-seq of 245 individuals from25 populations of QTP Gynaephora.The results of phylogenetic analysis,principal component analysis and population structure analysis support the delimitation of 245individuals from 25 populations into three clades:Clade I corresponding to G.menyuanensis,Clade II including G.alpherakii and G.qumalaiensis,and Clade III including other species of QTP Gynaephora.The best likelihood tree inferred by Tree Mix contains three migration events,which are the migration from Clade I to Clade II,the migration from NG to QML in Clade II,and the migration from YS to NG in Clade II.Thus,the non-monophyly of the two species of QTP Gynaephora in Clade II may originate from the gene flow between populations.The effective population size of QTP Gynaephora overall experienced increase,after which the effective population size tend to stabilize.Among the 25 geographic populations,nine populations experience significant expansion in history,including AB,REG,KS,MQ,NG,NQ,NR,TD,and ZK;while the EB population experience significant population contraction.