| Chilo suppressalis(Walker)(Lepidoptera:Crambidae),the striped stem borer(SSB) or Asian rice borer,is a widespread insect species,extending from Asia and Oceania into the Middle East and Europe.It is regarded as one of the most important rice pests in rice growing regions.The wide range distributions,large amount of its host plants and the complex interactions between the borer and the diverse environments may bring to its high genetic variation,which makes it difficult to manage this pest.It is reasonable to find out the population genetic structure of the C.suppressalis populations hosting on rice in China,analyze its phylogeography and infer the demographic history of this species as well as its differentiation mechanism.This dissertation deals with the phylogenetic relationships among 18 C.suppressalis populations hosting on rice in China,examines geographical pattern of C.suppressalis microsatellites and haplotypes,and infers the demographic history and differentiation mechanism of this species,using 4 microsatellite markers and the sequences of 4 mtDNA genes.The principle achievements of this dissertation are as follows:1.We amplified the sequences of 4 microsatellite loci and 4 mtDNA genes of 381 individuals from 18 populations(covering the potential distribution range of C suppressalis in China).We observed 39 alleles in 4 microsatellite loci,obtained and submitted to GenBank 98 different haplotypes,including 22 haplotypes of the 16S gene, 27 haplotypes of the COâ… gene,24 haplotypes of the COâ…¡gene,and 25 haplotypes of the ND1 gene.2.The data of microsatellite marker and mtDNA gene sequences show that SSB C. suppressalis is highly differentiated,though microsatellite markers display reduced differentiation among samples compared with mtDNA sequence data.The population pairwise FSTS from the former marker range -0.00011-0.12946 and those from the latter one are -0.00358 -0.41001 for 16S,-0.01918 -0.55016 for COâ… ,-0.02904 -0.38614 for COâ…¡,-0.03202 -0.48485 for ND1.In addition,our results reveal high genetic variability of COâ… and COâ…¡genes in C.suppressalis,meanÏ€=0.86%and 0.69%.respectively;variable sites are 13.9%and 9.6%,respectively.3.Based on whether NJ tree reconstructed from the microsatellite data or MP and ML tree from mtDNA sequences,or the statistical parsimony networks analysis of the 4 genes,the conclusion can be drawn that C.suppressalis probably has three genetically diverse and geographically localized clades in China,that is,CC(central China)clade, SW(southwestern China)clade and NN(northern and northeastern China)clade,which are isolated by Qinling Mountain,Funiu Mountain,Yangtze River and Huaihe River along latitude and by Luoxiao Mountains in the vast area south to Yangtze River.Moreover,the genetic differentiation of C.suppressalis is roughly in accordance with the observed number of generations of this species in a year.In CC clade,in which rice is planted in two seasons,C suppressalis has three to four generations per year. Whereas in SW clade and NN clade,in which large number of diverse rice varieties grow in one season,the striped stem borers develop in two to three generations in the former region,and they appear in one to two generations in the latter one.4.Neutrality tests support the idea that C.suppressalis is highly differentiated as well.All Tajima's D of the 4 genes are significant negative(ranging-1.85 to-1.47,P＜0.05)in all C.suppressalis populations pooled in one group,and negative(ranging-1.37 to-0.42),though not significant,in CC,NN,SW regions.For each of the 4 genes,all populations pooled in one group,Fu's F statistic was(nearly)significantly negative (-7.59- -2.90,P=0.03-0.26);Fu's F statistic was negative or even positive for various subgroups of samples.Thus,strong population subdivision was evident.5.AMOVA both from microsatellite data and mtDNA gene sequences indicate that significant genetic structure of C.suppressalis exists at various hierarchical levels (among regions,among populations within regions,and within populations).The genetic differentiation of C.suppressalis among samples is highly significant.FSTfrom microsatellite data is 0.06004(P＜10-4).FSTfrom 16S,COâ… ,COâ…¡,and ND1 gene is 0.19485(P＜10-4),0.27607(P＜10-4),0.22949(P＜10-4),and 0.29285(P＜1014), respectively.The fixation index among CC,SW and NN clusters is highly significant. FCTis 0.03855(P＜10-4)for microsatellite data,FCTfor 16S,COâ… ,COâ…¡,and ND1 is 0.10682(P＜10-4),0.21533(P＜10-4),0.16862(P＜10-4),and 0.23845(P＜10-4), respectively,indicating that C.suppressalis in China is subdivided into three groups (CC,SW,and NN).The fixation index among samples within groups is significant.FSC is 0.02236(P＜10-4)for microsatellite data.FSCis 0.09855(P＜10-4),0.07741(P＜10-4),0.07322(P＜10-4),and 0.07143(P＜10-4)for 16S,COâ… ,COâ…¡,and ND1, respectively The fixation index among three regions is almost twice or even more than twice the one among populations within them,an indicator that there is limited gene flow between regions.6.The estimates indicate small amounts of gene flow(M＜100,81.7%Nem＜1.0), between pairwise populations,even between most adjacent populations of C. suppressalis.This may be due to the geographic isolation and the borers' nonmigratory character.There are no apparent unidirectional gene flows among the populations along latitude or longitude over China.However,when analysis is confined in different separate regions the trends are clearer.In CC region,the gene flow tends to be from Ningbo to western parts like Quzhou,Nangchang.In SW region,it is likely to be from Liuzhou to the places western to it,like Guiyang and Yaan;while in NN region,it seems to start in Wuhan or Zhumadian,northward till Changchun.The unidirectional gene flow in the three specific regions and the absence of gene flow at larger scales over China confirm the strong isolation-by-distance relationships of this species and may imply that C.suppressalis in the three different regions have arisen in separate refuges and experienced parallel evolutions.These findings that C.suppressalis populations have small long-term effective population sizes,limited gene flow between adjacent populations,and strong isolation-by-distance relationships,support the idea that populations of this species are at or very close to genetic equilibrium,and the observed differentiation among three regions is likely the result of migration-drift equilibrium7.The unimodal mismatch distributions of each of 16S,COâ… ,COâ…¡,and ND1 gene within C.suppressalis reflect the main coalescence depth of the haplotypes,ultimately tracing back to a single ancestor.Based on 2.3%/a million years(Myr),the coalescence time(equated with the onset of demographic or range expansion)for 16S,COâ… ,COâ…¡, and ND1 is about 65 000 or 128 800 years,100 000 or 282 000 years,approximately 100 000 or 282 000 years,and 100 000 or 133 000 years,respectively.In general,most ofÏ„-values at each of the 4 gene segments were similar among the three regions and all had overlapping 95%C.I.(confidence interval),indicating that the population expansions date back to roughly the same time period.The coalescence time of CC clade at COâ… ,COâ…¡,and ND1 is earlier than 60 000 years.The divergence time of NN clade at COâ… ,16S,and ND1 is more than 120 000 years,and the separation date of SW clade at COâ… ,COâ…¡,16S,and ND1 dataset is over 140 000 years ago.The only two exceptions are NN clade at COâ…¡and CC clade at 16S,which are less than 10 000 years. Considering the approximate nature of molecular clock in insects,and the absence of fossil data,the three major clades may have started their divergence in three refugia isolated at one period of approximately at least 60 000 years ago,which is long before rice domestication.This implies that the differentiations of C.suppressalis are probably due to climatic and /or geological events(e.g.the last glacial maximum),and C. suppressalis switched from wild to cultivated rice during the process of domestication. The domesticated rice has been providing a new ecotope for the herbivore and subsequently strengthened the genetic differentiations of the borer along with rice domestication process.
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