Abstract: 【Aim】 This study aims to analyze the genetic diversity, genetic differentiation, and phylogenetic relationships of populations of the alpine butterfly Parnassius glacialis in China, to infer their origin and divergence time, and to explore their spatiotemporally evolutionary history. 【Methods】 The mtDNA AT-rich sequences of 325 individuals from 13 geographic populations of P. glacialis inChinaand 11 individuals of other 11 Parnassius species were amplified and sequenced, and their sequence variation and genetic differentiation were analyzed using MEGA 6.0, Dna SP 5.1, Arlequin 3.5 and other genetics analysis software. Using the closest relatives as the outgroups, the P. glacialis phylogenetic trees were reconstructed with such analytical software as PhyML3.0, MrBayes 3.2, and BEAST V1.8.3, and the origin and divergence time of the P. glacialis lineages were estimated by using relaxed molecular dating method and calibrations of the previous studies. Based on the present biogeographic distribution of P. glacialis and the main earth environmental events since the Quaternary Period, its dispersal route and spatio-temporal pattern and the underlying earth environmental factors were tentatively inferred. 【Results】 The AT-rich sequences of 325 P. glacialis individuals from 13 geographic populations ranged from 487 to 495 bp in length with the mean of 491 bp, being relatively conserved in length. The main length difference of the AT-rich region lies in the Poly-T or Ploy-A copy number, and the mean A+T content of the region is up to 95.76%. Totally 239 halotypes based on mtDNA AT-rich sequences were found in the 325 individuals of the 13 populations of P. glacialis, with its haplotype diversity (Hd) value of 0.9971, the nucleotide polymorphism index π of 0.02948, and the Theta (per site) Eta value of 0.06594. The reconstructed phylogenetic trees and molecular dating showed that P. glacialis is most closely related to P. stubbendorfii, and these two sister species began to differentiate at about 7.49 million years (Ma) ago in the late Miocene. The ancestors of P. glacialis were originated in the southwest of China (near the Hengduan Mountain-Himalaya Mountain of the Qinghai-Tibet northeastern edge nowadays) at about 1.53 Ma ago; then, they dispersed from their originating site into Xiaolongshan and Qinling Mountains; later, they diverged into two main lineages with some inter-mingled halotypes along the mountainous routes, accompanied by the Quaternary glacial-interglacial changes; lastly, the one lineage continuously spread northeastward to Funiu, Songshan, Taishan, Kunyushan Mountains, and the other lineage spread step-by-step to Dabie, Langyashan, Zijinshan and Tianmushan Mountains. 【Conclusion】 The results of our phylogenetic analysis of P. glacialis indicate that no pronounced correlations exist between genetic differentiation and geographical distance judged from higher genetic variance within populations than between populations. In addition, we propose that the divergence of the two main lineages of P. glacialis occurred in the mountainous areas with relatively higher latitude in the Middle Pleistocene and then spread to the mountainous areas with relatively lower latitude in South and East China due to the Quaternary glacial-interglacial cycles and different dispersal routes.

Key words: Parnassius, Parnassius glacialis, mitochondrial genome, AT-rich region, phylochronology, genetic differentiation, phylogeography