【作者】 管毕财；

【导师】 邱英雄； 傅承新；

【作者基本信息】 浙江大学 ， 植物学， 2008， 博士

【摘要】 八角莲（Dysosma versipellis Hance M.Cheng）属小檗科（Berberidaceae）八角莲属植物,为我国特有二级濒危保护植物。本研究通过叶绿体DNA（cpDNA）基因间隔区序列变异、扩增片段长度多态性（Amplified Fragment LengthPolymorphism,AFLP）以及繁育系统的研究,对八角莲群体进化历史、群体遗传结构、遗传多样性水平以及濒危机制进行了探讨,同时,结合种群生态学调查资料,提出了就地保护和迁地保护策略。主要研究结果如下:1.繁育系统与传粉生物学特点八角莲花粉胚珠比（P/O）为20933.7±844.89,杂交指数（OCI）值为5。控制传粉实验表明,作为对照的自然条件下的结实率是11.8%,去雄不套袋的结实率是2.2%,不去雄而对整个花序套袋以及人工白花授粉均不产生果实,同株异花传粉的结实率为1.4%,异株异花传粉的结实率62.7%。以上实验表明,八角莲是自交不亲和的专性异花授粉植物,结实依赖传粉昆虫。有效传粉昆虫为长角甲虫（long hornbeetle）,其飞行距离较短,不超过两米,访花频率很低（0.086visits/flower/hour）。因此,八角莲不同的克隆或亚群体间缺乏有效的花粉传播,是其结实率低的主要原因,也是造成八角莲濒危的重要因素之一。2.cpDNA遗传多样性与群体谱系地理结构筛选了3对cpDNA的基因间隔区片段（trnL-trnF,trnL-ndhJ,trnS-trnfM）,对10个八角莲群体（共107个体）进行序列变异分析。共检测出了33个变异位点,可分辨出24种单倍型。以桃儿七（Sinopodophyllum hexandrum Ying）和北美足叶草（Podophyllum peltatum L.）作为外类群的最大简约树显示,八角莲24个cpDNA单倍型可分为中东部和西部两个主要的单倍型谱系。八角莲物种水平的核苷酸多样性（π）为0.236×10^-3,单倍型多样性（h）为0.394。中东部群体单倍型多样性（h=0.408）和核苷酸多样性（π=0.000264）要高于西部群体(h=0.329,π=0.125×10^-3)。N_ST（0.887）显著大于其相应的G_ST（0.600）,表明了现存的八角莲群体存在明显的谱系地理结构。失配分布（mismatch distribution）分析表明,八角莲西部谱系的避难所群体,没有经历扩张事件,而中东部八角莲谱系则经历了明显的扩张过程。遗传距离与地理距离的相关性分析表明,八角莲的整个分布区内群体间的遗传距离和地理距离之间没有显著相关性（r=0.145,P=0.1908）,同样,中东部地理组内群体间的遗传距离和地理距离之间也没有显著相关性（r=0.014,P=0.4735）。3.AFLP遗传多样性与群体遗传结构筛选了8对AFLP引物组合,对9个八角莲群体（共184个体）的遗传变异进行了分析。8对AFLP引物共扩增出了406条带峰值清晰、可重复、可判读的条带,其中364条（89.66%）为多态性条带。遗传变异水平最高的为西部地区的DJ群体（h=0.1537）,最低的为中东部地区的SX群体（h=0.0379）。对八角莲群体进行AMOVA分子变异分析,结果显示,八角莲群体的遗传变异主要存在于群体间（58.56%）。AMOVA的分层次分析表明,将所有群体分为西部部地区群体、中东部地区群体2个地理组进行进行分子变异分析,一共检测到67.71%的遗传变异,其中37.02%来自于地理组间变异(F_CT=0.370),低于cpDNA检测到的结果(F_CT=0.307)。UPGMA分析、PCoA分析以及Structure分析表明,西部地区群体和中东部地区群体呈现出两个独立进化的路线。4.八角莲群体的进化历史及其保护本研究揭示八角莲群体分化为西部和中东部两个谱系,由于气候变化和地理隔离,阻止了两个谱系分化后的基因交流。中东部群体在谱系分化后发生了群体扩张事件,另外,地理隔离使中东部八角莲群体间发生产生了一定的群体分化。长期的地理隔离可能造成了八角莲谱系间的生殖隔离,八角莲群体西部谱系和中东部谱系可能正处于物种形成进程中的地理种形成的初期。根据群体进化历史和群体遗传结构研究结果,我们提出了相应的保护策略:（1）对八角莲就地保护时,把西部地区和中东部地区群体确定为进化显著单元,对所有群体都进行就地保护,采取策略促进这些群体内的幼苗更新。将同一群体内不同分布点的个体进行人为的移栽换位,加强远距离个体之间的基因交流,提高结实率。（2）在迁地保护、取样以及培育幼苗时,应从所有群体中取样,建立迁地保护园,最大限度地保护八角莲的遗传多样性,同时避免远交衰退。更多还原

【Abstract】 Dysosma versipellis is an endemic endangered plant in China.The determination of genetic diversity,population structure,glacial refugia and historical processes which D.versipellis has undergone are of crucial importance in order to put forward feasible conservation strategies.Our present work investigated the factor induced rarity of D.versipellis.The analysis was carried out based on cpDNA trnL-trnF, trnL-ndhJ,trnS-trnfM intergenic spacer,Amplified Fragment Length Polymorphism （AFLP） and breeding system.Genetic diversity and population structure were detected in combination with the ecological investigations,population evolutionary history.Consequently,conservation strategies are proposed.There are 4 main conclusions of our research:1.Breeding system and pollination biologyD.versipellis is in flower from early April to July with a peak in May.The process of flower anthesis in D.versipellis lasts about 10-12 days.The number of pollen grains and ovules per flower is averaging 1060008±44184.13 and 50.7±1.94, respectively.The Pollen-ovule ratio（P/O） calculated is high,averaging 20933.7±844.89.Although Chrysomya megacephala（Calliphoridae） is the most common visitors,pollen transfer in this species is not highly effective and principally occurs by Longhorned beetle（Cerambycidae）.Breeding system experiments showed that the mean（±s.e.） fruit set（%） under natural condition（11.8±2.4%） is less than that resulting from manual cross-pollination（62.7±4.1%）.It is concluded that D. versipellis is self-incompatible,and pollinator-dependent for sexual reproduction,and lack of xenogamous pollination limits fruit formation in populations of D.versipellis. Thus,the identification and translocation of compatible mating types to create reproductively viable populations is essential for the recovery of D.versipellis.2.cpDNA genetic diversity and phylogeographic structureTen populations were analyzed using cpDNA trnL-trnF,trnL-ndhJ,trnS-trnfM intergenic spacer.A total of 2873 bp and 33 variation sites were detected,which gave rise to a total of 24 cpDNA haplotypes.Nucleotide diversity and haplotype diversity areπ=0.236×10^-3 and h=0.394 respectively.In wersten China,Nucleotide diversity and haplotype diversity areπ=0.000125 and h=0.329,respectively.In central/eastern China,17 haplotypes were found,Nucleotide diversity and haplotype diversity areπ=0.000264 and h=0.408.AMOVA revealed 86.8%variation exist among populations.High genetic differentiation was detected(F_ST=0.868,G_ST=0.60) among populations.The genetic differentiation was lower than that of cpDNA.When populations were grouped according to the major phylogeographic break identified by BARRIER,hierarchical AMOVA found 59%of the total cpDNA sequence variation distributed among western vs.central+eastern groups,33%was explained among populations within each group,and only 8%within populations.Neutrality test and mismatch distribution support significant postglacial population expansion of D. versipellis in China.The cpDNA data presented showed the near complete fixation of two major haplotype lineages in western vs.central/eastern populations.Irrespective of the outcome of such prospective studies,the two cpDNA lineages identified in D. versipellis should be taken into consideration as ’Evolutionarily Significant Units’ in any program aimed at conserving genetic resources of this rare and endangered species.3.AFLP genetic diversity and population genetic structureA total of 184 individuals from 9 populations were investigated.Eight primer combinations produced 406 fragments,among which 89.66%are polymorphic.High genetic diversity was detected in western population DJ（h=0.1537）,EM（h=0.1408）. Low genetic diversity was detected in the central/eastern populations of SX （0.0379）,TT（h=0.0388）.High genetic differentiation（58.56%） among populations was detected with F_ST=0.586.The genetic differentiation was lower than that of cpDNA.Hierarchical AMOVA found 37.02%of the total cpDNA sequence variation distributed among western vs.central+eastern groups,30.69%was explained among populations within each group,and 32.29%within populations.UPGMA,principal coordinate analysis and misture analysis revealed two major phylogeographical groups:western China,central/eastern China.The result of AFLP indicated that western and central/eastern populations should be taken into consideration as ’Evolutionarily Significant Units’.4.Population history and implication on conservationIn terms of speciation processes,we propose that the deep cpDNA divergence of western and central/eastern lineages of D.versipellis renders this species at an incipient stage of allopatric speciation,whereby climate-induced eco-geographic isolation through（a） biotic displacement of temperate-deciduous forested habitats （almost entirely） prevented gene flow during and after the separation of these lineages. At smaller spatial-temporal scales,the same vicariance factor seems to have promoted population isolation and divergence in allopatry in the central/east,albeit to a lesser extent.Overall,the molecular data for D.versipellis accord with the premise that Late Quaternary refugial isolation is likely to have enhanced allopatric（incipient） species formation of East Asian temperate plants.Based on cpDNA and AFLP marker,high genetic differentiation among populations was detected.High genetic diversity was found in western China and central/eastern SN and HS populations.The genetic structure detected in this study is critical for the establishment of the conservation strategies.In order to conserve the genetic resources of D.versipellis,The western and central/eastern population should be taken into consideration as ’Evolutionarily Significant Units’ in program aimed at conserving genetic resources of this rare and endangered species.We propose that the most desirable populations for the in situ conservation are those in western population（EM/DJ） and central /eastern HS populations.To enhance the adjustability and heterozygous of D.versipellis,gene flow and size of population should be increase.So,to retain as far as possible the genetic diversity of D.versipellis,most of population should be sample in ex situ conservation.And translocation of compatible mating types to create reproductively viable populations is essential for the recovery of D.versipellis.更多还原