基于线粒体基因组控制区序列的中国冰清绢蝶遗传分化和谱系生物地理研究

Genetic differentiation and phylogeography of the alpine butterfly Parnassius glacialis (Papilionidae: Parnassinae) in China: evidence from mitogenomic AT-rich region

【目的】分析中国冰清绢蝶Parnassius glacialis遗传多样性和遗传分化情况及其系统发生关系,推测其起源及分歧时间,并探讨其历史生物地理分布格局的成因。【方法】以2008-2016年采集于中国13个地区的冰清绢蝶的325头及绢蝶属其他11个种的11头个体样品,对其线粒体基因组的AT富集区(控制区)序列进行PCR扩增和序列测定,采用MEGA 6.0, Dna SP 5.1和Arlequin 3.5等软件分析其遗传多样性和遗传分化情况;以其他近缘绢蝶种类作为外类群,采用PhyML3.0, MrBayes 3.2和BEAST V1.8.3等软件重建冰清绢蝶的系统发生树,并利用宽松分子钟和校准方法推测中国冰清绢蝶的起源和分歧时间;同时结合现今冰清绢蝶的地理分布特点和第四纪以来的地球环境事件,分析其扩散路径,探讨其历史生物地理分布格局及成因。【结果】冰清绢蝶AT富集区序列长度介于487～495 bp之间,平均为491 bp,其长度的差异主要体现在Poly(T)或Poly(A)的数目差异,A+T平均含量为95.76%。供试冰清绢蝶13个地理居群325头个体中,共检测出基于线粒体基因组AT富集区序列的239个单倍型,单倍型多样性Hd为0.9971;核苷酸多态性指数π值为0.02948, Theta (per site)Eta值为0.06594。系统发生分析和分子定年显示,冰清绢蝶和白绢蝶P.stubbendorfii为近缘姊妹种,二者在距今7.52百万年(Ma)的中新世晚期开始分化。冰清绢蝶的祖先可能起源于我国西南地区(现今横断山脉-喜马拉雅附近的青藏高原东北缘)距今1.53 Ma的更新世时期,先由起源地先期扩散到达小陇山、秦岭一带;后来,由于第四纪的冰期-间冰期轮回事件和扩散路径的不同而分化出2大支系,但两个支系之间存在少量的单倍型相互混杂现象;随后,2大支系继续向低海拔、低纬度地区扩散,即通过伏牛山脉向东北方向扩散到嵩山、泰山、昆嵛山一带,经大别山系向东南方向扩散到琅琊山、紫金山和天目山一带。【结论】中国13个居群的冰清绢蝶没有形成明显的地理分化格局,其遗传变异主要来自居群内部,居群间的遗传分化现象尚不明显;冰清绢蝶起源于更新世时期的较高海拔地区,由于第四纪的冰期-间冰期轮回事件以及扩散路径的不同而形成2大支系并向中国东部和南部低海拔和低纬度地区扩散。

【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 in China and 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.