【作者】 李康琴；

【导师】 李火根；

【作者基本信息】 南京林业大学 ， 林木遗传育种， 2013， 博士

【摘要】 作为被子植物中最原始的类群，木兰科植物对研究有花植物的起源、分布和系统发育有重要价值。木兰科（Magnoliaceae）鹅掌楸属（Liriodendron）为第三纪孑遗树种，现仅存两个种，即鹅掌楸（L. chinense Sarg.）和北美鹅掌楸（L. tulipifera Linn.）。鹅掌楸和北美鹅掌楸是典型的东亚-北美间断分布“种对”。是植物群体遗传学和分子系统发育地理学的理想材料。本研究以北美鹅掌楸、鹅掌楸31个自然群体及5个子代群体为研究对象，利用SSR分子标记检测鹅掌楸属树种天然群体的遗传结构及子代遗传多样性，分析种内不同群体的遗传分化及亲缘关系，比较鹅掌楸种间遗传多样性及遗传分化，以及鹅掌楸及其子代的遗传多样性和群体间分化。检测群体、家系及个体三层次的遗传变异分配程度。同时，通过对特定基因序列（cpDNA的psbA-trnH和trnT-trnL基因片段及nrDNA ITS序列）的克隆测序，推测鹅掌楸属植物第四纪冰期的避难所，以探讨鹅掌楸属群体地理分布形成的原因。主要研究结果如下：鹅掌楸天然群体遗传结构。利用14对SSR引物对12个鹅掌楸天然群体的318个个体的遗传多样性进行扩增检测，发现鹅掌楸天然群体有较高的遗传多样性（H_e=0.7385）。鹅掌楸群体之间存在中等的遗传分化（Fst=0.1956）以及低水平的基因流（N_m=1.0283）。此外，在其中6个群体中检测到显著的瓶颈效应。Mantel检验结果表明，鹅掌楸天然群体遗传距离和地理距离存在极显著相关性（r=0.5011, P=0.002）。据此，推测地理隔离和片段化分布导致了鹅掌楸群体结构的形成。且鉴于云南南部群体与其他群体在表型及基因变异上均存在较大差异，可将其视为鹅掌楸的一个变种。鹅掌楸、北美鹅掌楸遗传多样性比较。鹅掌楸属树种有较高的遗传多样性（H_e=0.7793），且无论是天然群体还是种源群体，均发现北美鹅掌楸的遗传多样性高于鹅掌楸。鹅掌楸23.28%的遗传变异存在群体间，且群体间的基因流N_m仅为0.8239。而北美鹅掌楸仅有8.75%的遗传变异存在群体间，但群体间基因流明显高于鹅掌楸，达N_m=2.6077。根据遗传距离，用UMPGA法构建聚类图，13个天然群体分为两个类群：云南的两个群体YN-JP和YN-MLP群体为一类群；其它11个群体为一类群。进一步证实鹅掌楸云南南部群体与鹅掌楸其他群体的遗传分化较大。鹅掌楸天然群体与子代群体遗传多样性比较。比较了5个鹅掌楸天然群体及其自由授粉子代群体的遗传多样性，发现鹅掌楸天然群体遗传多样性（N_e=4.14, H_e=0.74）高于子代群体（N_e=3.51, H_e=0.68）。天然群体中，12.54%的遗传变异存在群体间，而子代群体中，有17.22%变异存在于群体间，天然群体的基因流（N_m=1.7432）大于子代群体的基因流（N_m=1.2017）。表明鹅掌楸群体间遗传分化有增大的趋势。鹅掌楸群体、家系、个体三水平分子遗传变异。利用14个SSR分子标记分析鹅掌楸群体、家系及个体三个层次的遗传变异模式。结果显示，鹅掌楸遗传变异在各层次上表现不同：群体水平上，JX群体变异最大(N_ei=0.63)，XY群体最小(N_ei=0.41)；家系水平上，XY26家系变异最小(N_ei=0.2487)，ST27的N_ei最大（0.5642）；个体水平上，LP25-27遗传多样性最大(N_ei=0.4456)，XY3-2最小(N_ei=0)。分子变异分析（AMOVA）结果显示，鹅掌楸在群体间、群体内家系间以及家系内个体间这3个层次均存在极显著的遗传分化，分别占总变异的16.47%、20.77%、62.76%。表型数据分析结果显示，鹅掌楸苗高性状在群体、家系、个体三水平的变异模式与分子数据结果相似。可见，鹅掌楸大多数变异（表型、遗传）都存在于群体内家系间及个体间，而群体间变异较小。鹅掌楸属树种的分子系统地理学研究。利用2个叶绿体基因（psbA-trnH、trnT-trnL）及1个核基因（nrDNAITS）的序列变异信息研究了鹅掌楸属2个种的分子系统地理学。在2个叶绿体基因（psbA-trnH、trnT-trnL）中，鹅掌楸共检测到29个变异位点，多于北美鹅掌楸的19个。且鹅掌楸的核苷酸多态性（π=0.004135, θw=0.005305）也高于北美鹅掌楸（π=0.00204, θw=0.004125）。鹅掌楸有61.424%遗传变异存在于群体之间，北美鹅掌楸有13.855%的遗传变异存在于群体间。利用Tajima’s D，Fu and Li’s D*和F*中性检测分析两个基因座的进化模式，结果表明鹅掌楸属树种在历史上均经历过群体扩张。TCS分析、系统树分析和单倍型地理分布的结果显示，鹅掌楸和北美鹅掌楸在冰期后各自扩张。推测位于云贵高原及其周边地区的鹅掌楸群体在第四纪冰期不受冰期气候的影响，且位于武夷山南麓的FJ-WYS鹅掌楸群体及位于大娄山北坡的GZ-XS群体所处地区很有可能分别为鹅掌楸东、西部群体的第四纪冰期避难所。鹅掌楸属nrDNAITS的长度变化幅度（808～852bp）与被子植物一致。而在鹅掌楸中ITS的长度变化幅度大于北美鹅掌楸。鹅掌楸属在ITS区中总共检测到90个变异位点，其中信息位点为53个。其中北美鹅掌楸中检测到的变异位点（S=62）和信息位点数（I=51）都高于鹅掌楸（S=46，I=12）。同时，在ITS1、5.8s和ITS2区，北美鹅掌楸中检测到的变异位点和信息位点数也都高于鹅掌楸。另外，不管在ITS区还是在ITS1、5.8s和ITS2区，北美鹅掌楸的核苷酸多态性都比鹅掌楸高。对ITS基因座位的进化模式分别进行了Tajima’s D，Fu andLi’s D*和F*中性检测。结果显示，在鹅掌楸属的水平上，都表现为负值，且Fu and Li’s D*和F*中性检测达到显著负值，也支持鹅掌楸属树种群体扩张模式。以白玉兰为外类群，利用nrDNAITS序列变异信息对鹅掌楸属各群体进行了系统发育分析。鹅掌楸、北美鹅掌楸所有个体基本分为北美鹅掌楸和鹅掌楸两个分支。结果支持鹅掌楸属树种早期分东亚和北美两支，随后在这两个大陆上各自分布扩张，独立进化。更多还原

【Abstract】 As members of the class of “basal angiosperms” defined by plant phylogeny, plantsbelonging to the order Magnoliales have been keys to understanding the evolutionary history offlowering plants. The genus Liriodendron within the family Magnoliaceae, is primarilyrepresented by fossils from the late Cretaceous and early Tertiary of North American and CentralAsia. The only two extant members of Liriodendron are L. chinense Sarg. and L. tulipifera Linn.L. chinense and L. tulipifera are a relic species pair with a typical discontinuous distributionacross East Asia-North America. They have important scientific value for studying on plantpopulation genetics and molecular phylogeography. In this paper, microsatellite markers wereused to quantify the genetic variation, genetic structure, and genetic differentiation among naturalmature populations of L. chinense and L. tulipifera. The author also compared the level of geneticdiversity between natural mature populations of L. chinense and their corresponding offspringpopulations. Furthermore, the genetic variation pattern of L.chinense was dissected into threelevels of populations, families within population, and individuals within family. In addition, thegeographical distribution and possible refugia area in history were deduced using DNAsequences of3genes as chloroplast DNA （cpDNA） psbA-trnH and trnT-trnL intergenic spacersand the nuclear ribosomal DNA ITS region.The main conclusions are as follows:The population genetic structure of L. chinense. To better understand the genetic structureand differentiation among remnant populations of L. chinense, the author determined thegenotypes of14simple sequence repeats （SSRs） loci across318individuals from12naturalpopulations. It showed that L. chinense maintained high genetic diversity （H_e=0.7385） withinpopulations but moderate genetic differentiation （Fst=0.1956） and low gene flow （N_m=1.0283）between populations. This indicates that L. chinense may have mechanisms to maintain itsgenetic diversity. Moreover, significant bottlenecks in six populations were detected in theapplication of two-phased model of mutation （TPM）. A Mantel test revealed a statisticallysignificant correlation between the geographic distances and genetic distances betweenpopulations （r=0.5011, P=0.002）. H_ence, the author presumes that geographical isolation andhabitat fragmentation might contribute jointly to current population structure of L. chinense. Theauthor also suggests that the populations from southern Yunnan can be regarded as a variatas of L.chinense, given their large deviation of phenotypic character and allelic variation from otherpopulations.The comparison on the level of genetic diversity between L. chinense and L. tulipifera. Theresults showed that plants in Liriodendron maintained a high level of geneticdiversity（H_e=0.7793）. The genetic diversities of natural population and provenances of L.tulipifera were higher than those of L. chinense. L. chinense maintained high levels of genetic differentiation （Fst=0.2328） and low levels of gene flow （N_m=0.8239） among populations,while L. tulipifera maintained low levels of genetic differentiation （Fst=0.0875） and high levelsof gene flow （N_m=2.6077） among populations. To elucidate the genetic relationships amongpopulations studied, the average genetic distances were used to generate a UPGMA tree. Thethirteen natural populations were grouped into two distinct clusters. Two populations （YN-JP andYN-MLP） from southern Yunnan formed a cluster, and the remaining eleven populations formedanother cluster. This supported that L. chinense from southern Yunnan have a large geneticdifferentiation to other populations.The comparison of genetic diversity between natural populations and their offspringpopulations in L. chinense. The genetic diversity of natural populations （N_e=4.14, H_e=0.74） washigher than that of their offspring populations （N_e=3.51, H_e=0.68）. The genetic differentiationcoefficient （Fst） among offspring populations was0.1722, which was significantly higher thanthat of natural populations （0.1254）. It indicated that the genetic variation of L. chinense mighttend to enlager. And, the level of gene flow （N_m=1.7432） among natural populations was largerthan that among offspring populations （N_m=1.2017）.The genetic variation pattern of L. chinense. To understand the genetic variation pattern of L.chinense, the overall genetic variation was dissected into three hierarchical levels of populations,families within population and individuals within family. In this section, five natural populationsof L. chinense were chosen as sample populations, in each population seeds from five maturetrees were harversted, thus five half-sib families each population were obtained.29-30seedlingseach family were sampled for the detection of14SSR loci and measurement of tree height.Population JX maintained the highest genetic diversity (N_ei=0.63), while population XY had thelowest genetic diversity (N_ei=0.41). To the class of families, XY26had the lowest geneticdiversity (N_ei=0.2487), while ST27the highest (N_ei=0.5642). As to the level of individuals,LP25-27had the highest genetic diversity (N_ei=0.4456), XY3-2has the lowest (N_ei=0). Most ofgenetic variations were contained within population. AMOVA analysis revealed that there wassignificant genetic differentiation existing among three hierarchical levels （populations, familieswithin population and individuals within family） in L. chinense, indicating limited gene flowamong populations. Among the overall genetic variations of L. chinense,16.47%,20.27%and62.76%variations were explained by populations, families within population and individualswithin family respectively. Moreover, the variation pattern on tree height was also analyzed, anda pattern similar to the AMOVA result was acquired.The molecular phylogeography of Liriodendron. The molecular phylogeography ofLiriodendron was investigated using allelic variation of three genes, two from chloroplast DNA（cpDNA）（psbA-trnH and trnT-trnL） intergenic spacers and one from the nuclear ribosomalDNA ITS region.Totally,148individuals from31populations with of Liriodendron were analyzed withcpDNA psbA-trnH and trnT-trnL intergenic spacer. The variation loci and nucleotide diversityof L. chinense （S=29; π=0.004135, θw=0.005305） were more than that of L. tulipifera （S=19;π=0.00204, θw=0.004125）.61.424%of the total genetic variation was distributed among populations of L. chinense, indicating that L. chinense contained a high level of genetic variation.While in L. tulipifera,13.855%of the total genetic variation was distributed among populations.Results of neutrality tests （Tajima’s D, Fu and Li’s D*and F*） supported the idea thatpopulations in Liriodendron underwent a process of expansion in histery. Analysis on TCS,phylogenetic and the distribution of cpDNA haplotypes revealed that both L. tulipifera and L.chinense underwent a process of population expansion, though their expansion routes werediverse. In the glacial period, two possible refugia of L. chinense were Ta-lou Mountains for thewestern populations, and south slope of Mount Wuyi for the eastern populations. The presenteastern populations of L. chinense might origin from the two populations of HB-EX andJX-WYS.Similar to most angiosperms plants, the length of ITS region in Liriodendron ranged from808bp to852bp, with larger variation ranges in L. chinense. A total of90allelic mutaions and53informative loci were detected in Liriodendron. Both the number of allelic mutaions andinformative loci of L. tulipifera （S=62, I=51） were higher than that of L. chinense（S=46, I=12）,similar to the nucleotide diversity. N_eutrality tests （Tajima’s D, Fu and Li’s D*and F*） supportedthe viewpoint that both species in Liriodendron had undergone population expansion in history.Taking Magnolia denudata as outgroup plants, the phylogeography of Liriodendron was alsoinvestigated using nrDNA ITS sequences. All individuals in Liriodendron were basicallyclassified into two branches of L. chinense and L. tulipifera. The results suggested that the twospecies in Liriodendron, the North American species and the East Asia species, might haveundergone population expansion and evolved independently.更多还原