Taxonomic Study On Orthocladiinae From Wuling Mountain Area (Diptera:Chironomidae)

Orthocladiinae is an early founded and largest subfamily of the family Chironomidae.It has recorded 174 valid genera and 2,275 valid species.The phylogenetic relationships in this subfamily are the most complex.It is relatively difficult to identify species by only relying on traditional morphological classifications.It is necessary to use molecular methods for validation.Wuling Mountain Area is a most popular region for biodiversity research in China and even the world.However,the research on Chironomidae in this area is relatively poor.In this study,the species of subfamily Orthocladiinae in Wuling Mountain region were described by morphological method,and then molecular identification was carried out based on COI and COII gene fragments,and finally,the material were classified and discussed by combining morphological classification with molecular identification.In this study,20 genera and 53 species of subfamily Orthocladiinae had been recorded in Wuling Mountain Area,and the old records had been identified and described again,and the distribution of some species had been expanded.Five new genus records were added,including: Bryophaenocladius,Cricotopus(Isocladius),Krenosmittia,Rheocricotopus,and Zalutschia.Two new recoreded species were added,including: Limnophyes palleocestus Wang & S?ther,and Cricotopus(Paratrichocladius)hamatus Wang & Zheng.Seven new species were founded,including Corynoneura cooperio sp.n.Fu & Fang,Corynoneura sesquipedalis sp.n.Fu & Fang,Corynoneura tumula sp.n.Fu & Fang,Eukiefferiella ovalis.sp.n.Liu,Limnophyes minerus sp.n.Kong,Pseudosmittia undata sp.n.Li,Psectrocladius(Mesopsectrocladius)rotundus sp.n Sun.In this experiment,the mitochondrial cytochrome c oxidase subunit 1 gene(COI)was obtained from 49 individuals of 20 morphological species and the mitochondrial cytochrome c oxidase subunit 2 gene(COII)from 68 individuals from 17 morphological species,117 sequences were obtained totally.Based on the distance matrix,the neighbor-joining tree and the maximum likelihood tree were constructed to obtain the molecular species.The successful matching rate of the molecular species and the morphological species is higher.At the same time,the intraspecific and interspecific genetic distances of some subfamilies of Orthocladiinae were analyzed in the experiment.The minimum genetic distance between COI was 12.59%,the maximum genetic distance within the species was 13.09%;and the minimum genetic distance of COII was 0,the maximum intraspecific genetic distance was 19.92%.In addition,the molecular analysis of the results showed that the intraspecific genetic distance of Limnophyes verpus is greater,and there might be cryptic species in this morphological species.This study investigated the diversity of subfamily Orthocladiinae in Wuling mountain area and verified the role of DNA barcoding in species identification.The results of the study filled the blank in the systematic research of subfamily Orthocladiinae in the region.The acquisition of molecular data has expanded the barcoding database of subfamily Orthocladiinae and provided a scientific basis for the phylogenetic analysis of Chironomidae.