Preliminary Study On The Genetic Diversity And Phylogeography Of The Two Colias Butterfly Species(Colias Fieldii And C.erate)in China(Pieridae:Coliadinae)

Colias fieldii and C.erate are the two commonly found butterfly species which are categorized into the subfamily Coliadinae of family Pieridae,mostly inhabiting at areas of grassy,shrubs,and forest edges in the majority of areas of China.Both of the two species have a distinctly wide range of morphological and ecological differences,and are frequently used as model organisms in the fields of ecology and evolutionary biology,meanwhile,they also serves as important insect pollinators,playing a critical role in maintaining the ecosystem balance and regulating plant reproduction.However,their genetic diversities,genetic structures as well as their origin and diversification patterns are still unclear,and thus further investigations of this issue are especially needed.In this study,firstly we collected 115 C.fieldii individuals from 23 localities and 165 C.erate individuals from 31 localities of China respectively,and newly determined the four mitochondrial gene sequence data(COI,Cytb,NDI,and ND5).Secondly,upon these datasets,their genetic differentiation,phylogeographic structure and demographic history were analysed using a variety of genetic diversity analysis methods,such as the mismatch distribution and neutral test analysis methods,and so on.In addition,their phylogenetic trees and the divergence times of their main clades were constructed and estimated,respectively,through phylogenetic and relaxed molecular dating analysis methods,aiming to clarify their spatiotemporal differentiation patterns and the underlying geological and environmental events in the evolutionary history.The main results are as following: 1.Genetic differentiation analysis1)Colias fieldii: The aligned sizes of mitochondrial COI,Cytb,NDI and ND5 gene segments are 648 bp,699 bp,393 bp and 777 bp,respectively,and the concatenated sequence of the four genes is 2517 bp,which is shown to be significantly AT biased.A total of 18 haplotypes of the concatenated mitochondrial gene sequence were detected from the 115 C.fieldii individuals of the whole population,with the total haplotype diversity and nucleotide diversity being 0.677(±0.048)and 0.00066(±0.00007),respectively.The phylogenetic analysis showed that 18 C.fieldii haplotypes are distinctly categorized into two large populations(clades,clade A and B): clade A includes 13 haplotypes from Shannxi,Henan,Gansu,Anhui,Hubei,Sichuan,Qinghai,and partial Yunnan regions;clade B consists of 5 haplotypes from some Yunnan and Tibet regions of the Qinghai-Tibet Plateau;the result of reconstructed haplotype median-joining network showed that their haplotype distributions are generally consistent with those in the phylogenetic tree;the AMOVA analysis indicated that a larger level of population differentiation was occured between the two C.fieldii clades(64.36%)and a subtle differentiation existed within clade(35.64%);the mismatch distribution and neutrality tests indicated that clade A did not experience a population expansion event,whereas clade B probably experienced a sudden demographic expansion at about 0.085 Ma in the late Pleistocene,a little earlier than the Last Glacial Maximum event,which is likely to be caused by the increased warmth and humidity of the climate,the expansion of forest and grassland,as well as the decreased heavy rainfall,during this interglacial period.2)Colias erate: The aligned sizes of mitochondrial COI,Cytb,NDI and ND5 gene segments are 645 bp,702 bp,393 bp and 726 bp,respectively,and the concatenated sequence of the four genes is 2466 bp,which shows obvious AT biased.A total of 14 haplotypes were detected from the 165 C.erate individuals of the whole population with its total haplotype diversity and nucleotide diversity are 0.483(±0.050)and 0.00029(±0.00004),respectively.The phylogenetic analysis showed that 14 C.erate haplotypes were distinctly clustered into two clades(I and II): clade II includes 3 haplotypes from Tianshan regions of China,clade I consisted of 11 haplotypes in the central and eastern China;the analysis of reconstructed haplotype median-joining network showed that their haplotypes distributions were consistent with those in the phylogenetic tree;the AMOVA analysis indicated that a relatively higher level of population differentiation was occured between I and II clades(80.08 %),whereas a relatively small differentiation was detected within population(11.09 %);the mismatch distribution and neutrality tests indicated that C.erate total population did not show signs of population expansion,which might be due to the fact that most C.erate small populations of this study are plain-area located with a little of geographical barriers,and thus frequent gene flows are continuously happened among them.2.Phylogeographic analysisOur molecular dating result indicated that the Colias species began to diverge at about 10.01 million years ago(Ma)in the late Miocene,apparently coinciding with the rapid uplifting of Qinghai Tibet Plateau(13-8 Ma).The C.fieldii early diversification happened at about 1.02 Ma during the early Pleistocene,and this event caused the C.fieldii two clades to be high-and low-altitude distributed.During this period,the Kunlun-Huanghe Tectonic Movement(1.2-0.6 Ma)occurred in the Qinghai Tibet Plateau and initiated a wide range of mountain glaciers,this geological event and the corresponding drastic climate oscillations caused the gene flows of C.fieldii to be reduced dramatically,shaping their current geographical distribution patterns.The two C.erate clades began to diverge at about 0.81 Ma,which coincides with the upliftting of the Tianshan Mountains and climate-induced habitat fluctuations in the middle Pleistocene,this geological events and climate changes effectively prevented the gene flow between the main clades of C.erate,resulted in their obvious geographical isolations;additionally,during the Shishapangma Glaciation,the declined temperature and widespread glaciers,the decreased forests and grasslands caused the the C.erate high-latitude population to disperse into the eastern and southern areas of China.In summary,the habitat fragmentations,the complex mountains barriers,as well as the climate changes caused by the geological events and glacial-interglacial cycles in the Quaternary Period also remarkably reduced the gene flow among the clades of Colia butterflies in this study,resulting in their highly differentiated and significant phylogeographic structures.