DNA Barcoding Of Mealybugs(Hemiptera:Pseudococcidae)and Cophylogenetic Study Between Mealybugs And Anagyrus Parasitoids (Hymenoptera:Encyrtidae)

Mealybugs(Hemiptera:Coccomorpha:Pseudococcidae)are an insect group that feeds on plant sap.They represent a great threat to the agroforestry production due to their small size,cryptic habitat and strong fertility.Pseudococcidae is the second largest family of Coccomorpha,currently comprising more than 270 genera and over 2000 species;many are cosmopolitan quarantine pests of ornamental plants and crops.The difficulty of morphological identification of mealybugs,especially the immature intercepted at ports,points to a need for a rapid and effective identification method,like DNA barcoding,to assist morphological taxonomy.Besides,having a grasp on the host specificity of parasitoids is vital for classic biocontrol programs,which often depend on host-specific natural enemy species.The endoparasitoids in the genus Anagyrus are the most successful parasitic enemies used in the biocontrol of mealybugs.However,cophylogeny between parasitoids(Anagyrus)and their insect hosts(mealybugs)has been rarely investigated.This study,firstly,accessed the efficacy of of DNA barcoding with COI(mitochondrial cytochrome c oxidase I)and 28SD2D3(large ribosomal subunit gene D2D3 region)in the identification of mealybugs and applied the integrative taxonomy to the species recognition of the Phenococcus aceris species-group;secondly,we performed cophylogenetic analysis between Anagyrus parasitoids and their hosts of scale insects with intention to explore their interaction relationship and understand the differentiation process of encyrtids.The results are as follows.(1)DNA barcoding of mealybugs in mainland China.This study employed distance-based method(best close match,BCM),tree-based method(neighbor-joining,NJ),and character-based method(Barcoding with LOGic formulas,BLOG)to test the efficiency of COI and 28S that could be used for species identification of 246 individuals of 54 mealybug species collected from 49 localities of China.In the COI data set,every species formed a distinct clade with>99 bootstrap support in NJ tree,and showed diagnostic character combination in BLOG.Distance-and character-based methods showed COI could provide 94.12-99.5%correct identification.In the 28S data set,species except for Planococcus citri,P.minor and Coccura suwakoensis formed well-supported clades in NJ tree.Diagnostic character combination was detected in every species.28S could provide 98.75-100%correct identification.The intraspecific and interspecific genetic distances were 0-3.54%,1.96-21.90%for COI and 0-3.55%,0.27%-33.36%for 28S.Although there was overlap between intraspecific and interspecific genetic distances,the distance to the nearest neighbor still exceeded the maximum intraspecific value,demonstrating that the two genes could achieve accurate identification of species.Species delimitation PTP(The Poisson Tree Process)analysis based on COI sequences generated more MOTUs in Antonina tesquorum,Atrococcus paludinus,and Formicococcus sp.than morphological identification.Among these three cases,the sequences of At.paludinus showed 3.55%variation at the 28S locus,possibly reflecting cryptic diversity in this taxon.This study corroborates the utility of the COI and 28S genes in the efficient identification of mealybugs and the rapid discovery of cryptic species,and the barcode library we provide will create an effective identification system for mealybug pest management in China.(2)Species recognition of P.aceris species-group using integrative taxonomy.The maple mealybug,Phenacoccus aceris(Signoret),is a widespread polyphagous pest.The taxonomic treatment of P.aceris and some congeners is controversial.This study utilized the molecular[one mitochondrial(cytochrome oxidase 1)and two nuclear(EF-1? and 28S D2D3)genes]and morphological(the number and width of circuli and the distribution of oral-collar tubular ducts on tergites of cephalothorax)data to recognize the P.aceris species-group based on 116 individuals collected from 17 localities in China.Analyses of nucleotide sequence data recovered four well-supported clades(referred to as PACE1,2,3,4 hereafter)that enabled us to reassess the taxonomic utility of the number of circuli used for species recognition in the P.aceris species-group.The polymorphism of circuli number was detected in PACE1(range 1-3),PACE2(range 3-4)and PACE4(range 3-5).Such results suggested that the number of circuli alone is unfit for the species separation.But all vouchers of PACE3 possessed five circuli with the first one large(the width exceeds one third of the circulus on abdominal segment ?),which represents a significant taxonomic feature.And the distribution of oral-collar tubular ducts on tergites of cephalothorax could be used for distinguishing PACE2 and PACE4.In addition,specimens that keyed morphologically to P.aceris contained three putative species(PACE2,3,4),which are not separated by host-plant preferences.These three putative species are genetically and morphologically different from P.aceris in France(type locality of P.aceris),thus representing new to science,they will be described elsewhere.The network analysis based on COI haplotypes showed that Fraxinus spp.and Zanthoxylum bungeanum in North China were attacked by both P.aceris and P.azaleae.Therefore,previous identifications of this group according to the identity of the plants they damaged is misleading.The taxonomic confusion of P.aceris species-group may be brought out by the co-occurrence of multiple putative species.(3)Cophylogenetic analysis of Anagyrus and their scale insect hosts.Specimens of Anagyrus were reared from 50 batches(?1.9%of the total of 2600 batches)of scale insects.Morphological examination resulted in 21 species of scale insects(20 are mealybugs,one is eriococcid)and 18 species of Anagyrus.COI and 28SD2 were obtained from 177 Anagyrus individuals and combined to reconstruct the phylogenetic tree.Species delimitation(GMYC,sympatry criterion,monophyly)generated 23 putative species and cryptic species occurred in five morphospecies,of which two A.dactylopii,and A.rugas were each split into two host-specific putative species,demonstrating the cryptic host specificity in these taxa.The tanglegram recovered from TreeMap 3.0? suggested that the tree of scales and Anagyrus did not perfectly match.The cophylogenetic analysis based on Jane4 found significant global cost in all the three cost models,with the number of sorting and host switching events highest,suggesting that host shift played an important role in the divergence of encyrtids.