DNA Barcoding Of Pteromalidae In Beijing-Tianjin-Hebei Area

Scientific and accurate identification of species is the basis and premise of deeper biology research.The morphological identification of species need taxonomists invest enough time and energy,more in need of the accumulation of years of experience.Pteromalidae is a kind of important natural enemies and plays an important role in controlling the population density of agricultural pests.However,morphological diversity and the small body made Pteromalidae as the most difficult family of Chalcidoidea to identify.Since Hebert proposed to employ mitochondrial cytochrome c oxidase subunit I(COI)genes as a DNA barcode of species identification in 2003,the ability of the technology has been demonstrated in more and more groups.Along with the development of research,some researchers found that COI genes as a DNA barcode has some problems itself may influence the accuracy of species identification,such as heteroplasmy,infection of Wolbachia,nuclear mitochondrial pseudogene and so on.For those reasons,some researchers tried to employ ribosomal internal transcribed spacer 2(ITS2)as a supplementary of DNA barcode.In the study,we choosed COI gene and ITS2 as DNA barcodes to identify the specimens of Pteromalidae which were collected in Beijing-Tianjin-Hebei area in 2015 and 2016.After examined all the specimens,we had totally 568 specimens of Pteromalidae.Specimens were divided into 325 groups according to the collection information and morphological identification.To establish the database of Pteromalidae DNA barcoding of Beijing-Tianjin-Hebei area,we recorded the original number of specimens,collection information,sequence information,photos of each group.According to the morphological identification and molecular identification,at least 8 genera are newly recorded in BeijingTianjin-Hebei area.All specimens extracted genomic DNA,amplified COI and ITS2 fragments.Then the obtained sequences were checked,spliced,aligned,cut into the same length,constructed the neighbor-joining tree based on the Kimura 2-parameter model and then computed the pairwise distances.ABGD and j MOTU were also used to define MOTU.Finally,we had totally 285 groups of specimens amplified COI or ITS2 fragments.In order to enrich some genera,we also chose 15 specimens collected in other places.166 specimens amplified COI and ITS2 fragments.According to the molecular identification,166 specimens were divided into 54 MOTUs.We also analysed specimens which only amplified COI or ITS2 fragments.Totally,302 molecular were divided into 96 MOTUs.And we also had several results as follows.(1)227 specimens which suffessfully amplified COI fragment were divided into 72 MOTUs.241 specimens which suffessfully amplified ITS2 fragment were divided into 83 MOTUs.(2)According to the results of divided MOTUs,some genera were not valid include Arthrolytus?Callimerismus?Chlorocytus?Eurydinota?Sphaeripalpus.Some specimens of Pteromalus?Gastrancistrus ?Trichomalopsis which can not affirmed by morphological identification were affirmed to be valid.We also suggest that some specimens of Cyrtoptyx?Nasonia?Propicrocytus?Pteromalus should be identified again since they were divided into other genera in our study.(3)According to the results of divided MOTUs,some species which can not affirmed by morphological identification or wrongly identified should be corrected.And we also identified 9 specimens which were not identified by morphological identification.(4)In our study,we found 5 Chinese newly recorded genera include Dinarmus?Dinotoides ? Heteroprymna ? Sympotomus ? Synedrus.We also found 8 newly recorded genera of Beijing-Tianjin-Hebei and they are Ablaxia ? Agiommatus ? Ammeia ?Cerocephala?Gastrancistrus?Hyperimerus?Norbanus?Zdenekiana.In conclusion,we choosed different ways to study the proper divided way of MOTU by COI and ITS2.COI as a DNA barcoding marker successfully solved our problems of species identification of Pteromalidae and 98% specimens were successfully divided.But some specimens with COI distance difficult to define.ITS2 was found a nice marker to solves the problem.And we also found all Spalangia specimens failed to amplified COI fragment but successfully amplified ITS2 fragment.So the two molecular markers COI gene and ITS2 not only increased the reliable of molecular identifition and eliminated some problems of using COI gene alone,but also avoided the problems of intraspecific distance limit.In the study,some species were not able to confirm their genera,because molecular identification can only separated them as different species and these species were not the same as any other species which had been identified.So morphological identification and molecular identification are both important and should work together.To make DNA barcoding works better,it's important to enrich species of Pteromalidae which can never be apart from morphological identification.