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题名: 十齿花谱系地理学和保护遗传学研究
作者: 袁庆军
学位类别: 博士
答辩日期: 2007-01-11
授予单位: 中国科学院昆明植物研究所
授予地点: 昆明植物研究所
导师: 彭华
关键词: 十齿花 ; 谱系地理学 ; 遗传多样性和遗传结构 ; 进化显著单元 ; 微卫星
学位专业: 植物学
中文摘要: 伴随着青藏高原的上升,在这一地区形成了极其复杂的地形结构和气候条件,使青藏高原东南部成为世界上植物进化历史最为复杂的地区,基于其物种的丰富、特有现象的明显和濒危程度的严重,青藏高原东南部及其毗邻的西南地区被列为世界生物多样性的热点之一。然而,目前从DNA分子水平探讨这一地区植物居群水平的遗传多样性和进化历史的研究还很缺乏。十齿花Dipentodon sinicus是主要分布在这一地区的东亚特有古老树种,在中国物种红色名录中被列为易危植物,通过对十齿花谱系地理学和保护遗传学研究,从居群水平揭示青藏高原东南部复杂的地理历史对植物遗传多样性和进化历史的影响,对保护这一地区特有物种遗传的完整性提供理论指导。 本文对十齿花进行了基本的生殖生态学研究,初步了解十齿花的传粉机制和种子传播方式;并进行了染色体初步观察;重点根据叶绿体DNA基因间序列数据,运用群体遗传学和谱系地理学方法,揭示了十齿花的遗传多样性和遗传结构及其影响因素,探讨了青藏高原东南部复杂的地理历史和气候条件对十齿花进化历史的影响;在此基础上,根据“进化显著单元(ESU)”的划分标准,提出了保护十齿花遗传完整性的具体策略和措施;为了进一步应用SSR分子标记进行核基因谱系地理学和保护遗传学研究,构建了十齿花微卫星文库。主要研究结果如下: 1.基本生殖生态学特性和染色体特征 在彝良居群进行了传粉生态学初步研究,控制授粉实验表明,十齿花的传粉几乎完全为虫媒传粉,存在一定的风媒传粉;昆虫访花实验表明,主要访花昆虫归属2科、5属、6种,即食蚜蝇科(Syrphidae)3属4种:长尾管蚜蝇(Eristalis tenax Linnaeus)、灰带管蚜蝇(Eristalis cerealis Fabricius)、黑带食蚜蝇(Episyrphus balteatus Geer.)、黑足食蚜蝇(Syrphus vitripennis Meigen);蜜蜂科(Apidae)2属2种:中华蜜蜂(Apis cerana Fabricius)和贞洁熊蜂(Bombus parthenius Richards)。对多个居群成熟果实观察的结果表明,十齿花的种子靠蒴果成熟时开裂的弹力传播。对彝良和云龙两居群染色体观察的结果表明,十齿花不同居群的染色体均为2n=34。 2.谱系地理分析 应用2个cpDNA基因间序列psbA–trnH和rps16–trnQ对十齿花的16个居群进行谱系地理分析,结果表明,十齿花具有高度的遗传多样性(hT=0.902)和遗传分化(NST=0.987和GST=0.948),有显著的谱系地理结构。漫长的进化历史和多变的生境引起十齿花高的遗传多样性,低效的种子传播方式和分布区内巨烈的地壳上升共同形成了它高度分化的遗传结构。嵌套分支分析(NCA)支持地形变化引起的异域片断化是形成高度分化遗传结构的原因。然而,三级分支反映的种内(或刘建生划分的种间)遗传分化和形态的分化是不一致的;同时,二级分支的遗传距离和地理距离也表现出不一致,地理上最接近的云贵高原东南边缘居群(分支2-1)和西北部居群(分支2-2)却有着最大的遗传分化。我们认为形成这种谱系地理模式的原因有两种可能的解释:一是青藏高原和云贵高原的上升引起祖先居群片断化而导致的谱系分选和遗传漂变使居群间的遗传分化不断增大;二是由于青藏高原东南部板块的平面移动将原先分布在一起的居群强行分离的结果。因此,我们的结论是伴随着青藏高原及毗邻地区的上升而显著增加的地理和生态多样性对这一地区生物多样性的进化有着非常深刻的影响。 3.“进化显著单元(ESU)”的划分及其保护 十齿花总的保护策略是加强就地保护,居群间的相互移植或混合不同居群个体的迁地保护都会导致远交压力和适应性特征的丧失,应该避免。利用最大简约树和UPGMA树,根据单倍型的地理分布、遗传距离和AMOVA分析,十齿花的16个居群可划分为4个ESUs (ESU A-D),保护的优先权从高到低为:ESU B、ESU A、ESU D和ESU C。祖先的ESU B(贡山、福贡1、福贡2)应给予最优先的保护,保护的重点放在FG2;其次是居群间分化最大的ESU A(元江、彝良、安龙),其中的三个居群必须同时加强保护,保护力度应在现有基础上有所加大;再次是分化最大的ESU D(越南、麻栗坡、惠水、雷山、从江、田林、融水),保护重点放在VN、LS和HS居群,可考虑重建1–2个VN居群,HS居群就地保护亟待加强;最后保护的是ESU C(腾冲、云龙、泸水),主要保护LUS居群,TC和YL居群保护二者之一即可。 4.微卫星文库的构建 采用Dynabeads和选择性杂交法分离微卫星位点。首先将基因组DNA用限制性内切酶Rsa I消化为300–1000 bp大小的片段,然后用生物素标记的简单重复寡核苷酸序列做探针与其杂交,杂交复合物用抗生链霉素蛋白包裹的磁珠吸附,经过一系列洗脱、沉淀,得到富含微卫星的DNA片段,最后进行克隆测序。结果显示,用于测序的96个克隆序列中88个(91.67%)含有微卫星片段,其中72个片段完全不相同,从中筛选出可用于引物设计的34个片段构建微卫星文库,为下一步应用微卫星(SSR)分子标记进行十齿花核基因谱系地理学和保护遗传学研究奠定了基础。
英文摘要: The evolutionary history of plants in southeast Tibetan Plateau might be the most complicated around the world because of the extremely complex topography and climate in this area induced by its uplift. High biodiversity, rich endemic plant and rapid population declines have led this area and adjacent region become a world biodiversity hotspot. However, so far the studies using molecular markers at the intra-specific level are lacking for plant taxa inhabiting the Tibetan Plateau area and adjacent areas. Dipentodon sinicus is the endemic archaic tree of Eastern Asia restricted to this area, which is listed as vulnerable species in the China Species Red List. We implemented a phylogeography and conservation genetics study using chloroplast sequences on Dipentodon sinicus with an attempt to test how tectonic activities shaped the plant population structure in this area. The results of this study will shed additional light on the evolution of biodiversity on Tibetan Plateau and adjacent areas and provide historical background for the conservation of biodiversity in this area, undoubtedly including D. sinicus. After investigating pollination mechanism, seed dispersal mechanism and chromosome numbers of D. sinicus, this study revealed genetic diversity and population genetic structure of this species based on chloroplast sequence, discussed how tectonic activities and complex climate in southeast Tibetan Plateau shaped the plant population structure, defined its evolutionarily significant units and suggested the conservation strategy, constructed microsatellite DNA library of D. sinicus for nuclear phylogeography and conservation genetics in next work. The main results are summarized as follows: 1. Reproduction ecology and cytology The results of anthecology in YIL population showed that D. sinicus was almost completely pollinated by insects, just a little by wind. The main insects recorded as flower-visitors were Eristalis tenax Linnaeus, Eristalis cerealis Fabricius, Episyrphus balteatus Geer., Syrphus vitripennis Meigen, Apis cerana Fabricius and Bombus parthenius Richards. The field observation for the ripe fruits indicated that this species dispersed its seeds through ejecting from capsules. The study of cytology showed that chromosome numbers of individual in YIL and YL populations are all 2n=34. 2. Phylogeography Based on psbA–trnH and rps16–trnQ sequences data in 16 populations of D. sinicus, the study of phylogeography revealed that D. sinicus had high genetic diversity (hT=0.902) and high genetic differentiation (NST=0.987 and GST=0.948). The high genetic diversity may be attributed to its long evolutionary history and highly diverse habitats. The ineffective mode of seed dispersal and dramatic tectonic elevation in the distribution range of D. sinicus could conspire to cause the high genetic structure in D. sinicus. Nested clade analysis supported that allopatric fragmentation induced by orogenesis could explain the highly differentiated structure. However, the intraspecific relationships of 3-step clades were not consistent with the morphology distinction of D. sinicus. And the haplotype network did not reflect the geographical distribution of 2-step clades, as geographically approximate southeast populations (clade 2-1) and northwest populations (clade 2-2) in Yungui Plateau were the most strongly differentiated. We supposed that a fragmentation of an ancestral population caused by the uplift of Tibet and Yungui Plateaus, resulting in lineage sorting of ancient clades and a loss of alleles through drift may be the function of this pattern. Tectonic movement of northwest Yunnan might be an alternative reason for this phylogeographic pattern. The results of this study demonstrated that significant increases in geological and ecological diversity that accompanied the uplift of Tibet Plateau and adjacent areas have very important impacts on the evolution of biodiversity in this area. 3. Evolutionarily significant units and conservation strategy For conservation management of D. sinicus, in situ strategy should be emphasized. Ex situ to enhance diversity through transplant or reintroduction from each other is not advisable, in order to avoid loss of critical adaptive features and the possibility of compromised long-term viability due to outbreeding depression. Based on most parsimonious and UPGMA trees, the geographical distribution of genetic variation, genetic distance and AMOVA, four ESUs (ESUA-D) were designated. The gradation of conservation priority is ESU B, ESU A, ESU D and ESU C. Ancestral ESU B (GS,FG1,FG2) should be given the highest priority to its conservation. The second priority should be given ESU A (YJ,YIL,AL) because cpDNA variation in the ESU was highest structured, its three populations must be separately managed in situ. For the most strongly differentiated ESU D (VN,MLP,HS,LS,CJ,TL,RS), it should gotten the third priority, population VN, HS and LS is emphases in the ESU. Finally, the last divergent ESU C (TC,YL,LUS) would be of the lowest conservation priority. 4. Microsatellite DNA library The genomic DNA was converted into the fragments and hybridized to biotinylated microsatellite oligonucleotides and captured on streptavidin-coated paramagnetic beads (Dynal). Unwanted DNA was washed away and captured’ DNA was cloned and sequenced, and then the largely enriched fragments containing SSR were obtained. The result showed that 91.67% (88) sequences containing the repeat motifs was found in 96 clones, 34 sequences selected from them composed microsatellite DNA library of D. sinicus. This library would establish a foundation for nuclear phylogeography and conservation genetics of D. sinicus in the future.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/82
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十齿花谱系地理学和保护遗传学研究.袁庆军[d].中国科学院昆明植物研究所,2007.20-25
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