【作者】 邱英雄；

【导师】 傅承新； 洪德元；

【作者基本信息】 浙江大学 ， 植物学， 2003， 博士

【摘要】 明党参为我国特有的濒危植物，主产于华东地区。本研究通过广泛的资源调查，选择7个居群，从形态性状、核型、等位酶和DNA水平，分析了明党参居群的遗传多样性水平和遗传结构状况。从形态解剖学、种群生态学和繁育生物学角度对明党参濒危的机制进行了研究。结合形态性状和ITS序列分析，重建了川明参的系统位置。综合以上研究，得出如下结论： 1．明党参的生长对生境的要求极严，其适宜的生境为常绿落叶阔叶混交林，多生长在排水较好的山坡上。适宜生长的土壤为石灰岩发育的黄棕壤，土层深厚，有机质、N及K含量高，pH值约为6.5。种群的空间结构为集群型分布。 2．明党参种子生物学研究表明，明党参种子成熟时胚仍很小，处于球形或心形胚阶段，需经4～6个月的后熟过程才能萌芽。明党参种子在自然条件下的生理活性只有1年，属于非持续的种子库类型。水分和温度是限制胚的后熟过程、种子萌芽及幼苗生长的关键环境因子。野外居群的生境与种子空瘪率关系很大。开阔和上层盖度过大的生境不利于种子的发育和形成。传粉生物学研究表明，明党参在自然条件下的结实率为72.5％，套袋结实率为28.5％，在封闭的玻璃房内，植株的结实率也有13.7％，表明明党参在环境胁迫的条件下存在自花传粉现象。从明党参的花部结构特征、雄蕊发育时相和结实率可见，明党参是以异花传粉为主的植物，昆虫在明党参的生殖成功中起了重要的作用。主要的传粉昆虫——甲虫的近距离传粉活动导致了居群内植株间近交频繁。 3．双悬果性状在居群间的变异大，约83％的变异来源于居群间，17％的变异来源于居群内。从西南往东，果实的长度趋向于更长，背腹面加厚。果实的横切面观，合生面油管数多为3，少为4或5，棱槽油管数为3。明党参果实油管数与川明参相同。居群间子叶类型有变异，从研究的5个居群看，长江以南为单子叶类型，长江以北为双子叶类型。 4．核型分析结果表明：染色体数目恒定(2n=20)，核型类型均为2A型，居群间染色体结构的变异主要表现在同源染色体的杂合性和随体位置的变化。 5．等位酶分析的结果表明，明党参物种水平的多态性位点百分率(PPB)为73.68％。居群水平的遗传变异程度较低，多态性位点百分率(PPB)为33.35％，每个位点的等位基因平均数(A)为1.43，基因观测杂合度(Ho)为0.03，期望杂合度(He)为0.07。总的基因位点变异中，有58％的变异来自于居群间，42％的变异存在于居群内。居群杂合体过量的位点仅为13％。按照基因频率进行分类，41个等位基因中共有8个特异基因，杭州居群涵盖最多的特异基因(AAT-1c，DIA-2a，IDH-1a，IDH-1d，TPI-1a)。RAPD标记分析的结果表明，物种水平的多态性位点百分率(PPB)为94.91％，基因多样度(H_E)为0.3323。然而，明党参居群内则表现出相对较低的遗传多样性水平，多态性位点百分率(PPB)为44.38％，居群内的基因多样度(H_Epop)为0.172。ISSR分析的结果和RAPD基本一致，物种水平的多态性位点百分率(PPB)为84.69％，基因多样度(H_E)为0.2420。居群水平的多态性位点百分率(PPB)为32.58％，居群内的基因多样度(H_Epop)为0.114。通过与其他异交植物(包括一些濒危植物)的相同的RAPD遗传博士学位论文中国特有濒危植物明党参的保护生物学研究 参数进行比较发现，明党参物种水平具有较高的变异水平，居群水平的遗传变异处于偏低水平。 RAPD和isSR分析揭示了明党参居群间分化程度很高(RAPD:GsT=48.23%，巾sT二54.40%: IsSR:GsT=53.8%，。sT二54.31%)。就居群内的遗传多样性而言，RAPD和ISSR所分析的 结果基本一致，长江以北居群的遗传变异高于长江以南居群.根据明党参的的遗传多样性指标 分析，结合明党参的抱粉学和核型研究结果，作者认为明党参可能曾广布于华东地区，由于气 候的改变及生境破碎化，以及由此引起的种子适合度下降，使得居群分布范围缩小。目前，明 党参表现出显著的小居群遗传漂变，从而降低了居群内的遗传变异，减小了有效居群的面积， 而不同居群间分化程度则加大。6.RAPD和IsSR分析表明:明党参与川明参种间在全基因组水平己出现了明显的遗传分化。rrs 序列分析显示:明党参的6个不同居群间的rrs序列己稍有差异。rrs一1的GC含量在 58.3一59.2%，ITS一2的GC含量在54.3一56.6%。ITS一l的长度范围为215一219 bp，ITS一2的长度 范围为224一227 bp。川明参与明党参种间在ITs序列上己表现出一定程度的种间分化。对2 个中国特有种应该作为独立进化显著单元(ESU)分别进行保护。7.芹亚科APioideae6个族13个代表种的ITs序列分析结果，支持将明党参置于美味芹族 乱妙犷刀ieae的分类地位。在 rrs系统树上，川明参与明党参为姊妹种，并与美味芹族的其它种 形成单系类群。综合形态解剖性状、基因组遗传变异和rrs序列分析，我们认为川明参应置于 美味芹族(&即r”iea。)—明党参属(Cha矛啥lum)。提出了新组合:changi溯violaceum(sheh et Shan)丫X.Qiu et C.X.Fu，Comb.Nov’。8.从研究结果看，明党参濒危的原因有三点:第一、生境的破碎化及人为对资?更多还原

【Abstract】 Changium smyrnioides Wolff, a species of monotypic genus in the family Apiaceae, is an endangered herbaceous perennial plant. It is endemic to China, and is distributed only in fragments in Zhejiang, Anhui, Jiangsu, Jiangxi, and Hubei Provinces. Seven populations throughout the entire distribution of the species were chosen to measure the genetic diversity and genetic structure by morphological and karyotypic and allozyme and RAPD and ISSR analysis. The pollination biology, plant anatomy and seed biology were investigated. The phylogenetic relationship of Changium smyrnioides and Chuanminshen was reconstructed by ITS and morphological analysis. Based on these results , the endangered mechanism and protection strategies were discussed. The results could be summarized as follows.1. Changium smyrnioides is inclined to grow in a range of habitats, for example along rocky hillside in subtropical broadleaf forest where soil is deeper and moister than in the other habitats. The content of N, P and K is high and the pH was about 6.5 in soil. The study of population biology indicated the species in spatial pattern was of clump distribution. We think that the deforestation and habitat fragmentation are the important endangered reasons of Changium smyrnioides.2, It is found that in the natural populations of Changium smyrnioide the recruitment of young seedlings is rare, although seed set is high. The anatomy experiment indicated that the embryo within mature seeds were at a primary stage. Seed germination experiment showed that the afterripening process of seeds ended after 4~6 months and seeds could successfully germinate in appropriate condition. In nature environment, the seeds of Changium smyrnioide can’t retain the physiological activity over a year.The seed banks of Changium smyrnioide are non-persistent. The study also revealed the incoordination between the afterripening of seed and the raining season of East China resulted in low ratio of seed germination.Thus, water and temperature are the important factors to restrict the afterripening and germination of seeds and seedling growth. The study of pollination biology indicated that the average ratio of seed set was about 72.5% in natural populations and only 28.5% by sheathing inflorescence test, and 13. 7% in the close greenhouse. So we can draw the conclusion that self-pollination maybe exisisted in the breeding system of Changium smyrnioides in stress. Insects played an important role in sexual reproduction. Although the breeding system of Changium smyrnioides was dominantly xenogamous. However,some species of beetles often visitedflowers of the same individual and frequent occurrence of pollination within a short distance would result in high inbreeding coefficient.3. Nested analysis of variance indicated that about 83% of the morphological variability of carpadelium was attributed to the differentiation among the populations and the remaining 17% resided within the populations. The shape of fruit tended to become longer, thicker and rounder from southwestern area to eastern area in the distribution. The number of vjttae was 3 in each groove and 4 ~5 in commissura. The type of cotyledon varied among populations. The seedlings of populations in the south of the Yangtze River were monocotyledonous, but the seedlings of populations in the north of the Yangtze River were dicotyledonous. The selection forces are. strong on the morphological characters.4. The karyotypic analysis of Changium smyrnioides indicated that the chromosomal number was stable (2n=20) in all populations. Karyotypes of the populations were all "2A" type. Karyotypical polymorphism was showed as the chromosomal heterozygosity, and position shift of the satellite.5. Genetic diversity of 4 populations was analysed with starch gel electrophoresis. The proportion of polymorphic loci (PPB) is 73.68% at species level. Genetic diversity within 4 populations was relatively low with the proportion of polymorphic loci (PPB) ranging from 19% to 42.9%, average number of alleles per locus (更多还原