The Species Diversity And DNA Barcoding Of Bamboo-feeding Leafhoppers From Southwest China

Bamboo-feeding leafhoppers are the general name for the leafhoppers that feed on bamboo,and many species have become important pests of bamboo.In this paper,the species diversity of bamboo-feeding leafhoppers in southwest China and DNA barcode system has been researched,the main contents include:collecting specimens of bamboo-feeding leafhoppers in southwest China,the classification and identification of bamboo-feeding leafhoppers species resources;discover new classification order,and disription,draw and photograph with them.On the basis of fixed point investigation,the species diversity and niche research of bamboo-feeding leafhoppers were carried out.By using modern molecular biology methods and techniques,the DNA barcode database of bamboo-feeding leafhoppers was preliminarily constructed based on the DNA barcode research of COI and other gene sequences.Based on the DNA barcode,the phylogenetic relationship and classification status of the important genus of bamboo leafhoppers were studied and analyzed.The main results are as follows:1.TaxonomyThrough field collection and the system of collection specimens,a total of identified 60 genera and 117 species of bamboo-feeding leafhoppers in southwest China,including 3 new genera,9 species,first discovered three species of bamboo-feedig leafhoppers.Lists the existing list of 117 species of bamboo-feeding leafhoppers in southwest,include the geographical distribution and host plants.The detail of new genera and new species for the first time repoeted feeding bamboos are illustrated,and the main morphological characteristics and digital image are provides to those new genus,new species,and first reported bamboo-feeding leafhoppers species.At the same time,the key to index the subfamily,genaus and species were prepared.The new taxa studied here as follows:New genera:Spiniplateus gen.nov.;Caudasubita gen.nov.;Parabambusana gen.nov.New species:Abrus furcatus sp.nov.;Bambusana gulinqingensis sp.nov.;Parabambusana caudatasubito sp.nov.;Caudasubita quatturmacula sp.nov.;Caudasubita geminus sp.nov.;Mukaria hainanensis sp.nov.;piniplateus yunnanensis sp.nov.;Spiniplateus kuankuoshuiensis sp.nov.;Nirvana flavoracoronam sp.nov.2.Biodiversity and niche research of bamboo-feeding leafhoppers in southwest China.From May to October,2017,six representative places in guizhou province(Kuankuoshui national nature reserve in northeast Guizhou;Guiyang and Longli in central Guizhou;Doupenshan in southern Guizhou;Leigong mountain national nature reserve in southeast guizhou;Weining in northwest Guizhou)where the species diversity and ecological position of the bamboo-feeding leafhoppers were investigated in period of six months,and more than 2,000 specimens were collected from bamboos.Species richness analysis results show that there are in total of 39 species in Deltocephalinae(54.16%),10 species in Evacanthinae(13.89%),8 species in Cicadellinae(11.11%),7 species in Typhlocybinae(9.72%),4 species in Coelidiinae(5.56%),3 species in Megophthalminae(4.17%)and 1 species of Iassinae(1.42%).In terms of quantity of these leafhoppers,the total number of Deltocephalinae was 50.09%,Typhlocybinae is 42.13%;Cicadellinae accounted for 3.88%,and these three sub-families accounted for more than 90%of the total insect population quantity,and the dominant group of bamboo-feeding leafhoppers in guizhou province was the Deltocephalinae.All kinds plots of insect community diversity index and Pielou index is:Kuankuoshui national nature reserve(diversity index:2.398;Pielou index:0.935)>leigong mountain national nature reserve(diversity index:2.189;Pielou index:0.913)>Doupengshan forest park(diversity index:2.158;Pielou index:0.899)>Huaxi of Guiyang(diversity index:1.549;Pielou index:0.673)>Longjiashan forest park(diversity index:1.440;Pielou index:0.804)>Qianlinshan of Guiyang(diversity index:1.332;Pielou index:0.744)>Guiyang forest park(longdongbao)(diversity index:1.290;Pielou index:0.719)>Changpolin of Guiyang(diversity index:0.765;Pielou index:0.393);the diversity index feature of various plots of insect communities was not consistent with the uniformity index and richness index.In the time series,the diversity index of insect community of Doupengshan forest park was the smallest.The results of principal component analysis showed that the insect community of bamboo-feeding leafhoppers in Guizhou province was mainly affected by monthly mean temperature,average monthly relative humidity and longitude.All insects have niche overlap between groups,some of the dominant the proportion of niche similarity and the niche overlap index is high,suggests that these groups of time and space resources utilization in partial addicted,it is likely to have compete between those species.The dominant insect communities niche breadth index of bamboo leaf hoppers in Guizhou province is higher,and there are also havs some differences.Deltocephalinae(Temporal-spatial of niche breadth is 12.121)has the highest niche breadth index among them,while the lowest was Megophthalminae(Temporal-spatial of niche breadth is 1).All insects have niche overlap between groups,some of the dominant the proportion of niche similarity and the niche overlap index is high,suggests that these groups of time and space resources utilization in partial addicted,is likely to compete.3.Study on DNA barcode of bamboo-feeding leafhoppers in southwest China.In this study,more than 2000 specimens for DNA barcode study were obtained,involving 40 genera and 72 species,and 46 species of 28 genera,28 genera,and 46 species of bamboo leafhopper were selected for DNA extraction and PCR amplification.A total of 2 gene fragments were selected:CO I and 16S R DNA.The CO I sequence 64,16S r sequences 75,were selected.Using the DNA barcoding gap analysis of distance depends on method,the barcode gap automatic retrieval methods(ABGD)and Klle-diagram vector analysis method.Such as the taxa level of species in bamboo-feeding leafhoppers were evaluated,preliminarily determine DNA barcode for bamboo leaf hoppers that the segments of COI gene sequences proved its feasibility is high than 16S rRNA gene fragment.The results showed that the genetic distance between the genus was 0.161-0.341,and the genetic distance between species was 0.063-0.136,and the genetic distance between poputions was 0.00-0.013.The bar code of COI gene sequence to bamboo-feeding leafhoppers is long 567 bp,its base of sequence detected 206 conservative sites of the COI gene sequence,363 mutation loci,339 simple information site,24 Single mutation site,eight insertions and deletions loci.The contents of the bases T,C,A and G in the COI gene sequence were 38.0%,17.5%,30.2%and 14.3%respectively,with obvious A+T bias.This study obtained 64 COI gene sequences(upload 20 to GenBank)and preliminarily constructed the DNA barcode database of 28 genus,46 species of bamboo-feeding leafhoppers in southwest China.4.Phylogenetic analysis of part of bamboo leafhoppers based on DNA barcode.Based on the DNA barcode of COI gene sequence,the genus of Malaxella,which is the planthopper,was used as outgroup to analyze the phylogenetic relationship among the 28 genera of the bamboo-feeding leafhoppers.The results showed that 28 genera are divided into big four branchs:the((((Mukaria+Neomohunia)+ Bambusimukaria)+ Abrus + Spiniplateus)+((Flatfronta + Xenovarta)+ Phlogotettix))gathered for a together,constitute the most evolutional branch of the tree,(Mukaria + Neomohunia)and Bambusimukaria is closest relative group,and beceme a sister group form each other,this branch is Deltocephalinae;The Bumizana and Batracomorphus were combina to a large branch,which became the second larger branch of the tree,it was the Hecalinae and Iassinae.((Atkinsoniella)+ Erragonalia)+(Scaphoideus+ Bambusana)is one of the branch.Anaka,is a single branch,reprent the Typhlocybinae,and Located at the root of phylogenetic tree.Between bamboo leafhoppers belong to most of the clustering results according to the form of the subfamily branch,but also has some not gathered by subfamily,the result suggest that bamboo leafhoppers COI gene fragment of the bar code belongs to the phylogenetic relationships among the reconstruction effect is not too ideal.5.Based on DNA barcode,the genus and species are determined.the outer groups was the Malaxella tetracantha and Malaxella flava of the planthopper family,together with Mohunia introspina,Flatfronta pronga,and Tiaobeinia emeiensis relationship species of the Mukaria.There are 7 species of Mukaria,a total of 14 samples,14 sequences were preliminary used to phylogenetic analysis.The result revealed that the Mukaria genus was divided into two major branches:the most evolutive one was the((M.Bambusana)+ M.pallipes).The more original ones is(M.albinotata+(M.maculate + M.nigra)).This indicates that the genus is not a monophyletic group,and the two branches of phylogenetic tree are just two types of different male genital gonopore:single-genital pore and bigenital pore,the first one branch is type of single-genital pore,and the latter one is the typr of bigenital pore.The relationship between the two branches is the sister-group relationship between each other,and the male genitalia with bigenital pore were more primitive species.In view of the branch form of the molecular phylogeny of the genus,it is suggested that the taxonomic status of some species in Mukaria should be adjusted in a certain way,establish a new genus with type of single-genital pore in Mukaria.Flatfronta pronga was selected as outer group in the determination of the genus Abrus,and Abrus anlongensis;A.brecis;A.coneus;A.xishuiensis,A.daozhenensis 5 species,a total of 6 samples,6 sequences were analyzed for phylogenetic analysis.In the results of the Abrus phylogenetic tree,Abrus daozhenensis and Abrus xishuiensis were recently gathered together.And Abrus brevies and Abrus coneus is located in the root,which reveal that(A.daozhenensis + A.xishuiensis)and A.anlongensis are sister group relationship formation,and((A.daozhenensis + A.xishuiensis)+ A.anlongensis)gathered together as one branch,with far away relatives to the branch of(A.coneus + A.longer).In combination with the similarity of its morphology characters and small genetic distances between the two species,it shold be identified as the same species.In the determination of the genus of Bambusana,Bambusananus bipunctatus,Bambusananus Lii,Bambusananus maculipennis and Flatfronta pronga are joined together for the outer group.A total of 13 samples,6 species,13 sequences of Bambusana bambusae,B.fopingensis,B.biflaka and outgroup were selected and were used by phylogenetic analysis.Bambusana phylogenetic result shows that B.bambusae and B.fopingensis more primitive,and B.fopingensis more primitive than B.bambusae,but B.biflaka has a closer relatives to Bambusananus maculipennis,which is the related genera species.To integrated its morphological features,genetic distance and closer relationship to another genus,the monophyly of current Bambusana is still not clear,needs further research to understand.It is recommended that the B.biflaka shulod be removed from Bambusana.