【作者】 孟祥锋；

【导师】 陈学新；

【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2008， 博士

【摘要】 二化螟Chilo suppressalis（Walker）属鳞翅目Lepidoptera,草螟蛾科Crambidae,其分布范围广、经济危害重,是水稻种植区的最主要害虫之一。由于其食性较杂,寄主较多,分布范围广泛,二化螟与众多寄主之间以及与复杂多变的环境之间的交互作用可能会带来其较强的遗传变异,从而增加对该种害虫防治工作的难度。因此,了解二化螟不同地理种群的遗传结构,分析各个种群间的亲缘关系,再现演化历史,探讨分化成因及预防机制就显得非常必要。本研究首次采用扩增二化螟4个微卫星位点（microsatellite loci）和4个线粒体DNA（mtDNA）基因片断等两种分子标记相结合的手段,运用系统发育分析方法、谱系生物地理学方法以及遗传变异参数分析等多种生物信息学的研究方法,对采自我国18个不同地区的二化螟种群进行系统而深入的比较研究,获得以下结果与结论:1)扩增了我国18个不同地区的二化螟种群中381个个体的4个微卫星位点（microsatellite loci）和4个线粒体DNA（mtDNA）基因片断。这18种群基本覆盖了二化螟在我国的分布区域。在4个微卫星位点上,共检测到39个等位基因。在4个线粒体基因中,共获得并向GenBank中注册了98个单倍型,其中包括:22个16S基因的单倍型,27个COⅠ基因的单倍型,24个COⅡ基因的单倍型,和25个ND1基因的单倍型。2)微卫星标记（microsatellite marker）和线粒体（mtDNA）基因序列数据均表明18个取样点的二化螟种群间出现了明显的遗传分化,只是基于前者获得的结果较后者的为小。以微卫星标记得出的种群间分化参数F_STS在-0.00011-0.12946之间,以16S基因序列数据求得的种群间分化参数F_STS在-0.00358-0.41001之间,COⅠ数据的F_STS在-0.01918-0.55016之间变化,COⅡ数据的F_STS在-0.02904-0.38614之间波动,ND1数据的F_STS在-0.03202-0.48485间变动。其中COⅠ和COⅡ基因具有很高的遗传变异度。平均核酸多样度分别为0.86%和0.69%,变异位点占序列长度的比例依次为13.9%和9.6%。3)基于微卫星标记数据构建的NJ树和依据线粒体基因序列数据生成的MP树以及ML树,以及4个基因的单倍型网状图分析均可得出如下结论:中国的二化螟C.suppressalis很可能分化为遗传上不同的、与地理位置相一致的3个分支,即华中分支、西南分支和华北东北分支。这3大分支在南北方向上被秦岭、伏牛山、长江、淮河等山脉和河流分隔开来;长江以南区域,则以罗霄山脉为界分为东西两支。而且,二化螟的遗传分化大致与其在3大地理区域内每年发生的代次相一致。在华中地区,二化螟一年发生3到4代,而在西南地区和华北东北地区,二化螟分别发生2到3代和1到2代。4)中性检验结果表明华中、西南和华北东北等3大地理区域间存在明显的遗传分化。当18个种群放在一起考虑时,4个基因的Tajima’s D均为显著性的负值（从-1.85到-1.47,P＜0.05）,Fu’s F值为显著性的负值或接近显著性的负值（-7.59--2.90,P=0.03-0.26）;当把华中、西南以及华北东北等3大地理区域的种群各自单独作为一组分析时,虽然Tajima’s D未达到显著性水平,但仍为负值（从-1.37到-0.42）,而Fu’s F值在不同的区域中出现负值甚至出现正值（-0.49-2.27）,表明3大地理区域间遗传分化明显。5)基于微卫星标记数据和mtDNA基因序列数据的遗传变异分析（AMOVA）结果均显示我国的二化螟种群遗传分化明显。种群分化参数F_ST为:微卫星标记数据计算结果是0.06004(P＜10^-4),16S、COⅠ、COⅡ和ND1等4个mtDNA基因序列数据计算结果分别为0.19485(P＜10^-4)、0.27607(P＜10^-4)、0.22949(P＜10^-4)和0.29285(P＜10^-4)。华中、西南和华北东北等3大地理区域间种群分化参数F_CT为:微卫星标记数据计算结果是0.03855(P＜10^-4),16S、COⅠ、COⅡ和ND1等4个基因序列数据计算的F_CT分别为0.10682(P＜10^-4)、0.21533(P＜10^-4)、0.16862(P＜10^-4)和0.23845(P＜10^-4)。再次证明了中国的二化螟种群已分化为华中、西南和华北东北等3个分支。各区域内种群分化参数F_SC为:微卫星标记数据计算结果是0.02236(P＜10^-4),16S、COⅠ、COⅡ和ND1等4个基因序列数据计算的F_SC分别为0.09855(P＜10^-4)、0.07741(P＜10^-4)、0.07322(P＜10^-4)和0.07143(P＜10^-4)。两种分子标记手段获得了相同的结果,即区域间种群分化参数F_CT是同一标记方法的区域内种群分化参数F_SC的二倍或更多,表明华中、西南和华北东北等3大区域间基因交流有限。6)二化螟两两种群间的基因交流水平较低（M＜100,81.7%N_em＜1.0）,即使在地理距离最近的种群间也是如此。这可能是由于地理隔离以及该害虫的非迁飞性特性引起的。在本研究涉及的整个取样范围内,没有发现与经度或纬度相一致的单向性基因流动。不过,当分析范围缩小至华中、西南或华北东北这些较小的区域时,基因流动的趋向变得明显起来。在华中地区,基因流倾向于从宁波流向西部,如衢州、南昌等地。在西南地区,基因流可能是从柳州向西流向贵阳、雅安等地。在华北东北地区,基因则可能流自武汉或驻马店,向北到沈阳、长春等地。在3个小范围内基因交流比较明显,而在全国整个取样点的范围内缺乏基因交流进一步确认了地理隔离作用,而且暗示着中国的二化螟可能起源于3个不同的避难所（refugia）,并经历了平行进化从而形成了目前的遗传结构。本研究所得的二化螟有效种群（effective populaion size）较小、距离最近的种群间基因交流有限以及很强的距离隔离关系（isolation-by-distance relationship）等结果表明二化螟处于遗传平衡状态（genetic equilibrium）,3个地区之间的遗传分化很可能是迁徙-漂变平衡的结果（migration-drift equilibrium）。7)二化螟4个mtDNA基因的错配分布（mismatch distribution）均为单峰（unimodal）。按照2.3%/1百万年的分子变异率,依据上述4个基因序列数据计算的合并时间（相当于群体扩张的启动时间）依次大约是65000或128800年,100000或282000年,100000或282000年,和100000或133000年。总的来说,在这4个基因中,每个基因在3大区域中的绝大多数τ值基本相近,各区域间τ值的95%置信值（95%C.I.）相互重叠,表明各区域的种群扩张时间大体相同。在华中地区,COⅠ,COⅡ,和ND1基因数据得出的合并时间早于60000年。在华北东北地区,16S,COⅠ,和ND1基因的分化时间大于120000年。在西南地区,16S,COⅠ,COⅡ,和ND1基因分化早于140000年前。只有华北东北地区的COⅡ基因和华中地区的16S基因例外。两者计算结果为种群分化时间晚于10000年前。考虑到昆虫分子钟的不精确性和缺乏必要的昆虫化石资料,3大分支分隔于3个避难所的时间大约开始于60000年以前,这个时间远早于水稻的人工栽培（domestication）时间。这可能表示二化螟的种群分化很可能是气候因素和/或地质事件（如,最后一次冰盛期）单独或共同作用的结果,二化螟在野生稻的人工栽培过程中经历了寄主转换。伴随着野生稻的人工栽培过程,栽培稻为蛀食于其植株体内的蛀虫提供了新的生境,进而强化了业已形成的遗传分化。更多还原

【Abstract】 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 COⅠgene,24 haplotypes of the COⅡgene,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 F_STS 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 COⅠ,-0.02904 -0.38614 for COⅡ,-0.03202 -0.48485 for ND1.In addition,our results reveal high genetic variability of COⅠand COⅡgenes in C.suppressalis,meanπ=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.F_STfrom microsatellite data is 0.06004(P＜10^-4).F_STfrom 16S,COⅠ,COⅡ,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. F_CTis 0.03855(P＜10^-4)for microsatellite data,F_CTfor 16S,COⅠ,COⅡ,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.F_SC is 0.02236(P＜10^-4)for microsatellite data.F_SCis 0.09855(P＜10^-4),0.07741(P＜10^-4),0.07322(P＜10^-4),and 0.07143(P＜10^-4)for 16S,COⅠ,COⅡ,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%N_em＜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,COⅠ,COⅡ,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,COⅠ,COⅡ, 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 ofτ-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 COⅠ,COⅡ,and ND1 is earlier than 60 000 years.The divergence time of NN clade at COⅠ,16S,and ND1 is more than 120 000 years,and the separation date of SW clade at COⅠ,COⅡ,16S,and ND1 dataset is over 140 000 years ago.The only two exceptions are NN clade at COⅡand 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.更多还原