The Molecular Phylogenetic Study Of Polytremis

Polytremis is a genus of the tribe Baorini, which belongs to subfamilyHesperiinae of the family Hesperiidae. Genus Polytremis was established by Mabillein1904, the holotype of it is Goiloba lubricans. It is recorded that there are18species all over the world,11of which are found only in China. The morphology ofspecies in Polytremis is similar. We need to examine the structure of male genitalia.Although the research of Baorini is relatively early, it is mainly concentrated inmorphological classification, the fauna distribution and new species reports, etc.Conbining the technique methods of molecular and the traditional classification, weconstructed molecular phylogenetic trees on Polytremis at the first time. It is our aimto provide some references on molecular phylogenetic research of Polytremis.We used the function of two-step cluster analysis in Spss13.0to confirmthe importance of variable, according to the18relatively steady characteristics inPolytremis. We used the mitochondrial genes Cytochrome Oxidase I (COI) andnuclear ribosomal DNA (rDNA) including D3region of28S rRNA, V4and V7region of18S rDNA to sequence a total of67specimens of15species, representing all majorspecies groups. All the sequences are aligned by BioEdit v.7.0.5.3and ClustalX1.83software. Sequences tatistics were performed using Mega4.1software. Weconstructed the phylogenetic trees under the methods of Neighbor joining (NJ),Maximum parsimony (MP) and Bayesian inference(BI). Then we chose36specimensof22species belonged to8genus in Baorini, trying to use DNA barcode to identifythe species in Baorini, with10relative species sequences from Genbank. It is our aimto explore whether we can use the fragment of mitochondrial genes COI as a DNAbarcode to identify the species in Baorini. The main conclusions of my study aresummed as below:（1）As it was showed by two-step cluster analysis, species of Polytremiswere divided into three clusters. In cluster I, the character of coecum penis is themost important feature. In cluster II, the character of forewing upperside with cellspot is the most important feature. In cluster III, the character of colour of spots on upperside of wings is the most important feature（2）The results of the different phylogenetic analyses (Neighbour-joining,Maximum parsimony and Bayesian inference) for COI data set were largely congruent.All analyses were largely congruent and revealed five main clades. Two haplotypes ofP. jigongi were formed strongly supported lineages and clearly separated from theother species.（3）The incongruencies of phylogenetic analyses between NJ tree basedon COI and rDNA occured: it was not possible for rDNA NJ tree to discriminate P.gotama from P. caerulescens. Except for P. theca, no intragenomic and intraspecificvariations were found within the analysed nuclear rDNA markers. COI tree revealedtwo distinct haplotype lineages without intermediates and K2P distances of4.2%washigher than the lowest interspecific K2P distances of1.7%. Additionally, bothsubspecies were also identified by the rDNA, giving evidence for a possible existenceof a sibling species pair with sympatric distribution. Additional specimens have to bestudied to evaluate these first results more in detail. P. theca fukia and P. theca thecahave been separated by Qingling mountain. It possibly emerged interspecificdifferentiation between them.（4）We successfully identified22species in Baorini. The NJ tree showedthat the specimens of same species had got together. It proved that we could use thefragment of mitochondrial genes COI we amplified as a DNA barcode to identify thespecies in Baorini.（5）Genus Pseudoborbo and Borbo have a close relationship. K2Pdistances of the two genus was3.7%, which was much lower than the averageintergeneric K2P distances of11.28%and even less than the interspecific K2Pdistances of4.13%. Pseudoborbo bevani may belong to Genus Borbo. Theeffectiveness of genus Pseudoborbo should be discussed in the future research.