Phylogeography Of Diaphorina Citri Kuwayama(Hemiptera:Liviidae)and Its Primary Endosymbiont,"Candidatus Carsonella Ruddii"

Asian citrus psyllid(ACP,Diaphorina citri Kuwayama)is the most notorious pest of citrus because it transmits “Candidatus Liberibacter asiaticus”,an ?-proteobacterium associated with the most serious form of huanglongbing(HLB,citrus greening disease),which threatens the viability of citrus industry worldwide.As the research on eradication of the plant pathogen is hindered by the bottleneck of the unculturable characteristic of “Ca.L.spp.”,there is no known cure for the disease.Measures taken now to limit the impact of the disease include propagation and planting of pathogen-free trees and strategies for suppressing populations of D.citri.Studies on the phylogeography of D.citri may help us understand the evolutionary history and origins of the psyllid,and thus,facilitate the development of effective management strategies for both the inset and the disease.In this study,I used multiple marker systems(mitochondrial genome associated with nuclear genome-wide SNPs generated via whole-genome resequencing)to trace spread and differentiation of D.citri populations in Asia and the Americas.To determine whether the psyllid and its primary endosymbiont(P-endosymbiont)“Ca.Carsonella ruddii” exhibit parallel evolution,five mitochondrial fragments from D.citri and three housekeeping genes from “Ca.C.ruddii” were used combined with fine-scale sampling to reconstruct phylogenies,and then to measure congruence between the phylogenetic trees derived tor the two putative species.The main content and results were as follows:(1)Preliminary mitochondrial genome diversity of 74 adults from 23 D.citri populations was analysed based on whole-genome scanning via Next Generation Sequencing(NGS).Diversity of the mitochondrial genome could be detected among populations,and also within a population.Nevertheless,complete consistent mitochondrial genomes could be found in individuals from distinct populations.Based on the gene trees inferred by Neighbor-joining(NJ)method,D.citri phylogeny was identified into two major clades: individuals from the Caribbean,Pakistan,Bhutan(Phuentsholing),Viet Nam(Son La),and single individual from Myanmar clustered into a well-supported clade.The other clade included the samples from Myanmar,Bhutan(Tsirang),Viet Nam,Bangladesh and etc.Whereas,the mitochondrial genome is not an ideal marker to show the division of fine-scale groups,on account of too big gap between the two clades.In conclusion,screening of proper mitochondrial markers is particularly important.(2)Eight primer pairs were designed to obtain five proper mitochondrial fragments from D.citri and three fragment covering the single copy,housekeeping genes(argH,dnaQ and rpoC)form “Ca.C.ruddii”.Maximum likelihood,haplotype network,principal components and Bayesian clustering were combined to analyse codiversification between D.citri and its “Ca.C.ruddii” at the intraspecific level.From 249 individuals of 24 populations,the two sets of markers identified three lineages: a Western clade containing individuals from Pakistan,Bhutan(Phuentsholing),Viet Nam(Son La),the United States of America(USA),Myanmar and China(Ruili,Yunnan);a Central clade,with accessions originating from Southwest China,Bhutan(Tsirang)and Bangladesh;and an Eastern clade,the most diverse one,containing individuals from Southeast Asian countries,and East and South China.The PARAFIT analysis was used to test the parallel evolution of D.citri and its P-endosymbiont,and the result showed that the two sets of data were strongly congruent.Therefore,we suggested that the P-endosymbiont may facilitate investigations into the genealogy and migration history of the host psyllid.The biogeography of D.citri and its P-endosymbiont indicated that D.citri colonized and underwent a secondary dispersal from South Asia to East and Southeast Asia.(3)74 individuals were screened for nuclear SNP variation,and identifying 2 to 4 clades based on NJ analysis,ADMIXTURE analysis and principal component analysis.The individuals within each clade were similar with those identified by mitochondrial genome with the D.citri phylogeny is clustering into two clades,indicating the existence of genetic gap.When clustering into 4 clades,populations from Caribbean formed a well-supported clade;individuals from Bhutan,Bangladesh,Pakistan formed a West clade;individauls from Myanmar,China(Sichuan and Yunnan)form the Central clade,whereas ADMIXTURE analysis showed the similarity of Central clade with those transitivity individuals from Guangzhou,Guizhou,Jiangxi and Vietnam;individuals from south and east China,Viet Nam,Myanmar,Indonesia and Uruguay formed a clade,and individuals from Uruguay were very close to those from Indonesia.In conclusion D.citri of USA and Caribbean were close to the individuals from Pakistan,and those of Uruguay possibly source from Indonesia.