Species Delimitation Of Chironominae In China Using DNA Barcodes And Morphology

The subfamily Chironominae(Diptera: Chironomidae)was erected by Macquart in 1838 and occurred in all zoogeographical regions except The Antarctic.It is one of most species-rich genus in Chironominae(11 known subfamily),containing about 128 genus 2800 described species in the world and 383 species recorded in China.As Hebert(Canadian zoologist)developed DNA barcodes and it has been proven successful in species identification and taxonomy in flora and fauna.With the development of DNA barcodes,it has been increasingly useful in species delimitation and life stage association in species,which would deeply depend on the perfect DNA barcode reference library of the biological species.Compared to the diversity of species on the China region,especially for the Chironomid,there are relatively few records of DNA barcodes of this group in the public database.In this study,we aimed to explore the utility of the mitochondrial gene cytochrome c oxidase subunit 1(CO1)as the barcode region to deal with species delimitation of new species and species life stage matching in Chironominae.Meanwhile,it has a importance significance to perfecting the DNA barcode library of Chironominae in China.There are 363 individuals representing 112 morphological species.Partial mitochondrial gene cytochrome c oxidase subunit Ⅰ(COⅠ)as the barcode region was amplified,finally gaining the standard 658-bp sequences.Combined with the sequences from Barcode Of Life Data System(BOLD)and Genbank,a total of 560 involved sequences were analysed.Both Automatic Barcode Gap Discovery(ABGD)well support 174-184 molecular clusters,and 2%-4% was proposed as the threshold to delimit Chironominae species.In addition,intraspecies and interspecific divergences were calculated,with the intraspecific and interspecific divergences 0-0.097 and 0-0.288 respectively.Besides,the mean interspecific genetic distance(0.178)was larger 10.47 times than the mean intraspecific genetic distance(0.017),with existing a distinct “DNA barcoding gap”.Based on the result of the Neighbor joining tree,157 morphospecies are separated into 175 clades,with match rate to 89.71%.Taxonomic problems were discussed combined with molecular data.Our thesis explored the utility of DNA barcodes and morphology in species delimitation of new species in the subfamily Chironominae,with detected a new species Polypedilum.heberti sp.n and a new record species Sergentia.Kizakiensis.Result 1: In view of morphological study,the new species P.heberti sp.n was similar to known species of Polypedilum.exilicaudatum,and can be distinguished from P.exilicaudatum by the following combination of characters: wing pale with a black spot on the basal half area of wing.Based on the Neighbor joining tree of five species within the subgenus Polypedilum(Cerobregma)revealed P.heberti sp.n was the individual cluster that can be differentiated from P.exilicaudatum,and both existed biggish genetic distance(0.140).The above results proved that both were different species.Result 2: S.kizakiensis of China region was similar to this species of Japan region in morphology,and can be distinguished in the length of the antenna.According to the result of the NJ trees of 13 Sergentia DNA barcodes dataset showed that the newly recorded species in China S.kizakiensis was clustered with the known S.kizakiensis from Japan.The genetic distance of Chinese species was more than 0.048 divergence in partial COI sequences and slightly exceeded the threshold of Chironominae,that can be differentiated from Japanese specie.Thus this result combined with morphological analysis showed that both existed different,which may indicate the presence of geographical difference in species.Based on the data from this study,and explored the utility of DNA barcodes and morphology in species life stage matching in the subfamily Chironominae,while will provide an effective way to match adult larvae of Chironomidae and other completely abnormal insects in the future.The results showed that has been matched successfully to 6 pairs,including Polypedilum.asakawaense Sasa,Polypedilum.cultellatum Goetghebuer,Polypedilum.convexum Johannsen,Polypedilum.unifascium Tokunaga,Dicrotendipes.sinicus Qi & Lin and Xiaomyia.aequipedes S?ther et Wang.Meanwhile,two first described larvae species,P.unifascium and X.aequipedes.In this study,the 363 DNA barcodes sequence in Chironominae contained the more than 400 photos and the relative information of samples were uploaded to the BOLD system.Meanwhile,the more than 800 voucher specimens were deposited at the Laboratory of Genetic Breeding of Tianjin,College of Fishery.The study successfully confirmed DNA barcoding as an useful methods to delimit species in Chironominae,and match different life stage in chironomid insect.Our result was an important supplement for the international DNA barcode database of Chironomidae and fill the blankness in China,Meanwhile,it provided data support for studying the ecological environment and biodiversity assessment within China.