The Complete Mitochondrial Genomes Of Six Parnassius Butterfly Species And The Relevant Phylogenetic And Phylochronological Analysis

The Genus Parnassius are a group of rare butterflies which is subordinate to the subfamily Parnassiinae of Papilionidae.Most of them are distributed in high mountains of north of Palaearctic region,while a few of species are distributed in the west of Neoarctic region.Their larvae use Crassulaceae and Violaceae as their host plants,and their audlts usually harbor thin,transparent,the silk-like wings.So far,about 55 species of Parnassius have been reported around the world,and about 33 species have been found in China.Up to the present,only a few studies have been carried out about the Parnassius phylogeny and evolution,and the results have not reached a high state of consensus,and additionally,the phylogenetic analyses of Parnassius with other Parnassiinae groups are also limited,and some relevant issues are standing unresolved as well,awaiting further investigations.Owing to the characteristics of moderate size,maternal inheritance,and no gene recombination,the insect mitogenome have been often used as the powerful tool in the fields of molecular phylogenetic and evolutionary studies.In this paper,the mitogenome sequences of six Parnassius butterfly species were newly determined,and compared with other known Parnassius species with detail;in addition,the Maximum likelihood and Bayesian trees of total 44 Parnassius species were reconstructed based on the mitochondrial COI,ND1,ND5 and lrRNA combined gene sequence data,in order to further clarify the phylogeny of Parnassius butterflies with the incorporation of morphological characters.Furthermore,the origin and divergence times of Papilionidae groups,including the Parnassius species were estimated using the relaxed molecular clock method based on the selected fossil records,in order to enrich the phylochronolgical data of butterflies.The mitogenomes of six Parnassius species are 15.3 kb in length,similar to other Papilionidae species.They are all composed of 13 protein coding genes(PCGs),2 ribosome RNA(lrRNA and srRNA)genes,22 transfer RNA(tRNA)genes,and a non-coding AT-rich region(D-loop).Some intergenic spacers and overlapping sequences are scattered throughout the whole mitogenomes.The intergenic spacers are 12 to 15 in number,and their length are ranged from1 bp to 52 bp.The longest spacer is located between tRNAGln and ND2 genes,with a relatively high level of similarity with ND2 gene,indicating its probable ND2 origination.The overlapping sequences are 11 to 12 in number,ranged from 1 bp to 8 bp.The 7 bp ATGATAA sequence located between ATP8 and ATP6 is present in all the six species determined,functioning in posttranscriptional modification.The AT contents of the whole genomes are about 81.5%,which is higher than that of GC,and for the the D-loop regions,the values are about 90%.The maximum likelihood(ML)and bayesian inference(BI)phylogenetic trees of Parnassius species based on four mt DNA gene sequences(COI?ND1?ND5?lrRNA)showed that the 44 Parnassius species belonging to eight subgenera can be divided into five clades,and the phylogenetic relationship is shown as((((Kreizbergia+Driopa)+(Lingamius+Tadumia)+Parnassius)+(Kailasius+Kora mius))+Sachaia).These results are in remarkably uncongruent with those obtained from other former studies.Meanwhile,the Parnassius choui is certified as a new valid species instead of a subspecies of Parnassius szechenyii according to its status in phylogenetic tree and its morphological features.The phylogenetic trees of Papilionidae were reconstructed based on mitogenomic 13 PCG sequence data and the origin and divergence times of Papilionidae exemplar species were estimated via relaxed molecular clock method based on the selected fossil records.The results showed that Papilionidae was originated at around 80 millions years ago(Ma)in the period of late Cretaceous,which was coordinated with the period of the angiosperm flourishing;the divergence of the genus Parnassius occurred at around 20 Ma in the early of Miocene,which was synchronous with the early uplift of the Qinghai-Tibetan Plateau,providing them the ecological environments of cooling and anoxia with the latitude increasing.