The Numerical Taxonomy And Molecular Systematics Using DNA Barcode Of Phlebotomine Sandflies (Diptera: Psychodidae)

Objective:Explore the classification positions of Phlebotomine sandflies from China through the Numerical Taxonomy. Discuss the availability and effectiveness of Molecular Systematics study of sandflies using cytochrome oxidase subunit I (COI) in identification and investigation of phylogenetic relationships. To complement the systematics of sandflies through comprehensive analysis which combined fundamental Classic Taxonomy, Numerical Taxonomy and Molecular Systematics of DNA barcode.Methods:1. Cluster Analysis was carried out with 40 species of Phlebotomine sandflies from China as taxa and 68 morphological characters as index by SPSS 15.0 software. The research result was exported as a dendrogram.2. Molecular Systematics of DNA barcode used sandflies which is caught in different places. Samples were identified using classic classifications method followed by the PCR amplification and sequencing of COI. The sequences of COI were checked and revised in BioEdit software, assembled in Seqman of DNA star. The blast of the sequences in NCBI proved they are aim ones. Sequences alignment was carried out by Clustal W program in MEGA v4.1 followed by nucleic acid base statistics, genetic distances analysis and phylogeny reconstruction.Results:1. The result of cluster analysis is consistent with classic taxonomy in the genus classifications of Phlebotominae and subgenus classifications of genus Phlebotomus. It reveals the steady taxonomy. While it shows complex of the taxonomy in the subgenus classifications of genus Sergentomyia, giving inspiration to the position of some sandfly species. S.malayensis, S.nankingensis, S.lanzhouensis and S.turfanensis probably hold a position of multispecies-subgenus or group in subgenus level. S.koloshanensi, S.fanglianensis, S.yunnanensis and nicnic group may occupy a new genus.2. The sequences of COI of Chinius.junlianensis, P.lengi, P.yunshengensis, S.squamirostris are all 709 bp including primers. They have highly A+T bias in COI. The average TS/TV ratio (transition/transversion) is 39/34=1.14. Intraspecific distances means is 0.0094, far less than interspecific distances means which is 0.1590. Each species samples assemble in one monophyly in phylogenetic relationships inferred through Neighbor-Joining (NJ), Minimum Evolution (ME), Maximum Parsimony (MP) and Unweighted Pair-Group Method with Arithmetic Means (UPGMA) methods. These phylogenetic relationships show stable topology structure which supports Classic Taxonomy.Conclusion:The result of cluster analysis is consistent with Classic Taxonomy on the whole. It confirms the reliability of classic taxonomy and also reveals the uncertain classifications of some species in genus Sergentomyia. The genetic distance analysis and Molecular phylogenetic relationships support that COI can be used for identification and phylogeny exploring. The construction of sandfly DNA barcode system which contains multiaspect information is operable and significant.