青藏高原地区两种鲜卑花属植物的分子谱系地理学与基因流研究

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	青藏高原地区两种鲜卑花属植物的分子谱系地理学与基因流研究
	付鹏程
学位类型	博士
导师	陈世龙
	2014-05
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	生态学
关键词	窄叶鲜卑花与鲜卑花比较谱系地理学微卫星基因流物种形成青藏高原
摘要	青藏高原是全球生物多样性的热点地区之一，蕴藏着丰富的植物资源；而第四纪冰期与间冰期的地质与环境剧变对青藏高原地区的物种分布与遗传分化造成了重要影响。本研究通过青藏高原地区蔷薇科（Rosacea）鲜卑花属（Sibiraea）的两种常见多年生落叶灌木，窄叶鲜卑花（Sibiraea angustata）和鲜卑花(Sibiraea laevigata)，采用叶绿体基因间区（trnS-trnG, rpl20-rps12, rpl16, rps15-ycf1）、核基因片段（nrITS）和低拷贝基因查尔酮合酶（CHS）相结合的分子标记，基于窄叶鲜卑花42个居群（637个个体）和鲜卑花13个居群（139个个体），通过遗传分化、种群动态历史、分歧时间估算等谱系地理学的研究，探究鲜卑花属在青藏高原的进化历史。主要研究结果与结论如下：（1）将叶绿体片段（cpDNA）串联排列，联合序列长度为2679-2707bp，在窄叶鲜卑花中共鉴定cpDNA单倍型21种（A1-A21），鲜卑花中共6种（L1-L6），其中L1-L6均出现在窄叶鲜卑花中。nrITS片段在两个种的长度均为656bp，在窄叶鲜卑花中鉴定单倍型13种（M1-M13）,鲜卑花中6种（N1-N6），其中N1-N6均出现在窄叶鲜卑花中。遗传多样性分析表明窄叶鲜卑花比鲜卑花拥有更高的基因多态性与核苷酸多态性。（2）根据系统发育关系与地理遗传结构分组，青藏高原的鲜卑花属植物可以分为北部、中部和南部3大支系。北部一支为两个种同域分布的区域，中部主要是窄叶鲜卑花，南部全部为窄叶鲜卑花。北部与中部两支的遗传组成相似，遗传交流频繁，但南部一支与北部和中部两支没有共享单倍型，表现出与其他两支明显的隔离。（3）根据冰期避难所假说，在窄叶鲜卑花中确定冰期避难所3处，即青藏高原东南边缘、玉树-囊谦地区和松潘高原，鲜卑花中鉴定出后2处冰期避难所。鲜卑花属植物在冰期历史中部分保存于高原台面，与其他多个物种类似，说明青藏高原各个区域在第四纪冰期历史中环境变化复杂，且不存在连续的大冰盖。两个种共享2处冰期避难所，使两个种遗传交流的机会大大增加，造成现今两个种的遗传组成高度相似。地理遗传样式与中央网络图表明，两个种在间冰期从冰期避难所往外扩张过程中均经历了明显的瓶颈效应与奠基者效应，形成了现今的遗传分布格局。（4）以分子钟模型为前提，基于cpDNA序列估算鲜卑花属的分歧时间，结果表明鲜卑花属的分化发生在3.70Ma以后；基于nrITS序列的结果表明鲜卑花属的分化发生在1.87Ma以后；基于CHS序列的结果表明鲜卑花属的分化始于1.81Ma。虽然基于不同序列估算的分歧时间并非完全一致，但均与青藏高原的整体快速隆升同时或更晚，说明青藏高原的快速隆升与第四纪冰期的环境剧变促进了鲜卑花属的遗传分化。（5）歧点分布与中性检验的结果均表明，无论是在种整体水平还是三大分支水平，窄叶鲜卑花与鲜卑花的种群动态历史十分模糊。因此，本研究继而通过低拷贝基因查尔酮合酶（CHS）,探讨鲜卑花属的种群动态历史。歧点分布与中性检验的结果均表明，鲜卑花和窄叶鲜卑花均经历了明显的近期居群扩张，估算扩张时间分别约为55.8Ka和56.9Ka，处在末次冰期间冰期。（6）以高山绣线菊和华北珍珠梅为外类群，基于cpDNA和nrITS单倍型的系统发育关系重建的结果均表明，两个种并不能相互形成单系，而是一起形成一支单系群。由于两个种的分布区完全重叠，遗传上高度相似，形态上差异不显著，我们推测窄叶鲜卑花和鲜卑花作为两个独立的种并不成立，分类地位有待进一步明确。由于两个种共同的第四纪冰期进化历史，所用的分子标记不足以探讨物种间的遗传交流。因此，本研究通过FIASIO磁珠富集法开发有多态性的鲜卑花属特异性微卫星（SSR）引物21对，并挑选8对SSR引物对两种鲜卑花的所有个体进行扩增，通过分析其遗传结构、种群历史和基因流的大小与方向，推测两个种的遗传分化及物种形成。主要结果与结论如下：（7）窄叶鲜卑花和鲜卑花均具有很高的遗传多样性，种间差异很小。窄叶鲜卑花每个居群的平均等位基因位点数为10.09，高于鲜卑花的8.56，但其观测杂合度为0.769，低于鲜卑花的0.883。AMOVA的分析结果表明，鲜卑花和窄叶鲜卑花的主要遗传变异均存在于每个种的居群内（92.17% vs 89.06%），两个种主要的遗传变异也是在居群内（86.3%）而不是种间（3.65%）。（8）窄叶鲜卑花的遗传多样性较高的4个种群在21.5ka左右由一个古老种群发生种群分化演变而来。（9）STRUCTURE遗传分组的结果显示，窄叶鲜卑花和鲜卑花独立分析时，分别可以分为6组和4组；当两个种一起分析时则分为2组，但并非两个种各成一组，而是部分混合。GeneClass的结果也表明两个种不能完全分开，鲜卑花中7.19%的个体被归入窄叶鲜卑花，后者中0.63%的个体被归入鲜卑花，第一代迁移个体数占总数的9.66%。因此两个种的界限并不显著，存在明显的渗入。（10）两个种间存在从鲜卑花到窄叶鲜卑花的不对称基因流。
其他摘要	Qinghai-Tibetan Plateau (QTP) is one of the world's biodiversity hot spots with abundant plant resources. The geological events and environmental fluctuations during the Quaternary glacitial and interglacial repeats had significant effects on biological distribution and genetic diversity of plants in this region. Sibiraea angustata and S. laevigata, two closely related shrub species belong to Sibiraea (Rosacea), are usually treated as dominant species with a wide distribution range in QTP. Here, we collected 42 populations (637 individuals) of S. angustata and 13 (139 individuals) of S. laevigata from QTP, employing four chloroplast intergenic spaces (cpDNA: trnS-trnG, rpl20-rps12, rpl16, rps15-ycf1) and two nuclear gene (ITS and low-copy-gene CHS), to study the genetic diversity, population demography history and divergence time for exploring the evolution history of Sibiraea in QTP. The main results and conclusions are as follows: (1) Concatenated cpDNA sequence has a length of 2679-2707bp. Twenty-one cpDNA haplotypes were detected in S. angustata (A1-A21) and six in S. laevigata (L1-L6), however, L1-L6 were shared in S. angustata. The nrITS was 656bp in both species, detected 13 haplotypes in S. angustata (M1-M21) and six in S. laevigata (N1-N6) which were all shared in S. angustata. Sibiraea angustata had higher gene diversity and nucleotide diversity than S. laevigata. (2) Sibiraea in QTP could be divided into three clades: northern, central and southern clade, based on phyogenetic relationship and geographic genetic structure. Northern clade includes two species, central clade mainly containes S. angustata and southern clade only includes S. angustata. The northern and central clades had similar genetic composition, but southern clade shared none haplotype with the other clades, indicating frequent genetic exchange between northern and central clades, whereas, southern clade was isolated from the others. (3) According to refuge hypothesis, three refugia were confirmed in S. angustata: south-east edge of QTP (SEQTP), Yushu-nangqian area (YNA) and Song-pan plateau (SPP), the last two refugia were also confirmed in S. laevigata. Similar to other species, Sibiraea survived in QTP platform during glaciations, which could the envirenment heterogenous among different areas of QTP and no large ice sheet developed in QTP. Two refugia were shared by both species, which can greatly increase the chance of genetic exchange led to the similar genetic composition of the two investigated species. Geographic genetic structure and median-joining network showed that the current genetic distribution was formed due to significant bottleneck and founder effect when two species expanded from refugia. (4) Divergence times of Sibiraea were estimated based on different data sets: cpDNA dataset suggested a divergence time of 3.7Ma, nrITS data of 1.87Ma and CHS data of 1.81Ma. Divergence times from the two nuclear data sets were extremely similar, but inconsistent with cpDNA data because of different heredity patterns of nuclear and cpDNA data and inaccuracy of substitution rate. Although the inconsistence, all the divergence times were contemporary with the most recent extensive uplift (5-1.8 Mya), indicating that the environmental changes during the fast uplift of QTP and the Quaternary Glaciaion triggered genetic divergence in Sibiraea. (5) Results of mismatch distribution and neutrality test based on cpDNA and nrITS could not give clear demographic history of either the two species or the three clades. Therefore, we explored demographic history through low-copy gene CHS for a clear conclusion. Results of mismatch distribution and neutrality test based on CHS indicated that both S. laevigata and S. angustata experienced population expansion at about 55.8ka and 56.9ka which coincide with the interstage of the last glaciations. (6) Phylogenetic relationships based on cpDNA and nrITS with Spiraea alpina and Sorbaria kirilowii as outgroups showed that S. laevigata and S. angustata were not reciprocally monophyletic but monophyletic when treated them together. Combined this with distribution area overlapping and morphological and genetic similarity, treat S. laevigata and S. angustata as one species may be more applausible than treated as two species. Because of the same Quaternary evolution history and genetic similarity, we tried to search better molecular markers for detect the genetic diversity between these two species. Therefore, 21 pairs of Sibiraea SSR primers were developed by FIASIO method. All individuals of S. laevigata and S. angustata were amplified by eigth pairs of SSR primers for exploring the genetic divergence and spciation of these two speices. The main results are as follows: (7) Both Sibiraea laevigata and S. angustata had high genetic diversity but genetic difference between speices was small. Sibiraea laevigata had higher observed heterozygosity than S. angustata (0.883 vs 0.769) but lower average alleles per population (8.56 vs 10.09). Results of AMOVA showed that most variation of two the species were within population rather than among species (86.3% vs 3.65%). Source of variation of S. laevigata and S. angustata respectively were also mainly within population (92.17% vs 89.06%). (8) Estimation of population differentiation history indicated that four high-diversity populations of S. angustata were differentiated from one ancient population at about 21.5ka. (9) Sibiraea laevigata and S. angustata were grouped into four and six groups in STRUCTURE, respectively. Two groups were divided when mixed two species together, S. laevigata and S. angustata each form a seperate group but with partial admixture between species. GeneClass also could not distinguish two species completely, 7.19% of S. laevigata were treated as S. angustata, 0.63% of S. angustata were treated as S. laevigata. Therefore, the delimitation of S. laevigata and S. angustata were not significant. (10) In Sibiraea, asymmetric gene flow occurred, from S. laevigata into S. angustata.
文献类型	学位论文
条目标识符	http://210.75.249.4/handle/363003/4010
专题	中国科学院西北高原生物研究所
推荐引用方式 GB/T 7714	付鹏程. 青藏高原地区两种鲜卑花属植物的分子谱系地理学与基因流研究[D]. 北京. 中国科学院研究生院,2014.

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