Taxonomy And Phylogeny Of Simuliidae (Diptera:Nematocera) Based On DNA Barcodes From Irtysh River Basin In Xinjiang

The family Simuliidae belongs to suborder Nematocerina(Insecta:Diptera).It distributes worldwide and includes about 2204 described species in 26 genera.Simuliidae is an important groups of arthropod vectors.Irtysh River basin is suitable for Simuliidae survival with rich water resources and developed animal husbandry.A huge number of Simuliidae were rampant,causing great harm to people's life and health,in summer and autumn.Accurate and rapid identification of Simuliidae is of great significance to Basic information collection of Simuliidae?biology,ecologyc as well as prevention and control measures.Black flies are a challenge for traditional taxonomy because of their small size and structural homogeneity.DNA barcoding has the characteristics of rapid and accurate identification of species.Previous studies have proved that DNA barcoding can accurately identify Simuliidae and reveal cryptic diversity.In this study,We identificated 17 species in 5 subgenus and 3 genus,besides 2 uncertain species based on morphological characteristics,and sequenced the COI fragmentes of 260 individuals of Simuliidae.We also downloaded 32 sequences of 10 species from BOLD and GenBank.According to the existing morphological basis and 292 sequences,we assessed the effectiveness of the three kinds of barcode methods.We tested the feasibility of DNA Barcoding technique in Simuliidae taxonomy,discussed the phylogenetic relationships of Simuliidae in Irtysh river basin and explored applications of DNA Barcoding in identificates Simuliidae by three methods.The results are as follows:1.The character-based method(BLOG)can accurately identify 88.24% of all species,found sequence characteristics of the logical relationship of 18 species(total 19 species)and the BLOG were efficient on all studied cases.Traditional method distance-based method did not find "Barcoding gap".New distance-based method(ABGD)can distinguish more species compared to other methods.Simulium lineatum was divided into two different species.It may be due to the bigger Intraspecific differences or cryptic species exists.The performance of the new proposed tree-based method(GMYC approach)is poor and dividing 18 MOUTS,and the division method is not consistent with the morphology-based method.TAXONDNA showed that precise identification rate of Best match was 96.81% and best close match was 95.54%.We preliminarily identified 17 kinds of morphology species correctly.2.Phylogenetic analysis based on COI gene fragments show that 3 genus(Simulium,Prosimulium,Metacnephiaare)are all monophyletic group,3 subgenus(Wilhelmia,Boophthora,Nevermannia)are all monophyletic group.Eusimulium is also a monophyletic group,but Eusimulium is incorporated into the Simulium,forming a paraphyletic group.Wilhelmia and Nevermannia are sister groups.Simulium and Metacnephia are sister groups.Two haplotypes of uncertain species S.OUT1 respectively belong to different subgenus.While,the haplotypes of uncertain species S.OUT1 all belong to Eusimulium.The rest species had different haplotype formed none-overlapping clusters.3.Phylogeny of Simuliidae revealed by 3 methodes: The traditional distance-based method shows that the K2 P distances(0.0789)between subgenus simulium and Nevermannia was the smallest in genus simulium.The K2 P distances(0.1390)between Boophthora and Eusimulium is the biggest in genus simulium.Simulium and Nevermannia are closely related to each other,which is consistent with previous studies.The GMYC method is not applicable to the phylogenetic analysis,but traditional tree-based method is suitable for phylogenetic analysis.The character-based method(BLOG)shows relationship between Simulium kiritshenkoi and Simulium intermedium is relatively close based on mitochondrial COI gene,sharing pos 414=T.BLOG method is not applied to phylogenetic analysis but suitable for species identification.With comprehensive analysis of morphology and BARCODING taken into consideration,the uncertain species S.OUT1 was identificated as Simulium(Simulium)bezzii(Corti,1914)and S.OUT2 was Simulium(Wilhelmia)takahasii(Rubtsov,1962).Thus,with COI,all species can be differented.The characteristic method is the best choice for species identification,and more appropriate,but other methods can do for the evidence to evaluate the accuracy of the barcode identification of species.It will be more reliable by adding more genetic information for phylogenetic analysis than only based on COI gene.