【作者】 孔航辉；

【导师】 傅承新；

【作者基本信息】 浙江大学 ， 植物学， 2006， 博士

【摘要】 本研究通过形态特征分析，RAPD分析及nrDNA ITS，cpDNA的matK、trnT-L、trnL-F和rpl16等序列分析，以及等位酶分析，结合细胞学和形态学研究结果，对菝葜属菝葜组菝葜复合种和草本菝葜组的系统发育进行了研究，获得了如下主要结果：1．菝葜复合种的系统发育关系1．1菝葜复合种的形态特征分析用9个性状特征或导出值对菝葜复合种的60份材料进行了形态数量统计。研究表明，不同类群之间存在着一定的形状性状差异，但菝葜复合种的形态变异很大，其种间不同倍性群体存在明显的重叠。1．2基于RAPD的遗传分化分析用15个引物对21个类群进行RAPD扩增，共得到275条清晰的条带，平均每个引物产生18.22条带。其中多态性条带为246条，多态位点百分率为89.47％，与圆锥菝葜组的多态性相似，存在着丰富的遗传多样性。UPGMA聚类树显示，21个分类群分成A、B、C三大组。A组主要包括除广布种菝葜外的8个近缘种(不包括长托菝葜)，B组包括除广布种菝葜广西居群外的所有菝葜复合种，C组由长托菝葜和广布种菝葜的广西居群组成。RAPD分析结果表明，广布种菝葜是一个稳定的类群，与复合种内的各近缘种存在着较小的分化。1．3基于matk、ITS和trnT-L序列的系统发育分析nrDNA ITS序列和cpDNA matk以及trnT-L序列分析强烈支持菝葜复合种(包括广布种菝葜的不同居群、小果菝葜、三脉菝葜、红果菝葜、粗糙菝葜和S．biflora)为单系类群。二倍体的菝葜湖北居群、小果菝葜和三脉菝葜组成的一支与另一由广布种菝葜的大多数多倍体居群组成的分支有着十分密切的亲缘关系，揭示了二倍体类群在菝葜复合种中的祖先种地位。菝葜的崂山居群和韩国居群形成独立的分支，表明它们有共同的起源。4X的广布种菝葜的金佛山居群、红果菝葜和6X的粗糙菝葜形成另一独立的分支，揭示了这一分支与菝葜复合种核心类群有了一定的分化或起源于不同的二倍体亲本。联配矩阵显示，ITS序列存在两种类型，菝葜的金佛山居群、红果菝葜和粗糙菝葜三个类群呈现同一类型，与其他类群存在20多个位点的差异；菝葜的广西居群在系统树中形成单独的一支，与其它类群序列比对的结果发现，它的ITS、trnT-L和matK序列中都有很多特异性的插入／缺失；另外，小果菝葜与广布种菝葜的湖北居群聚在一起，推测小果菝葜可能是广布种菝葜的祖先种之一；认为果实的大小类型是一个可变的生态类型。1．4基于等位酶的多倍体化分析等位酶分析确认了菝葜的二倍体居群、小果菝葜和三脉菝葜之间的亲缘关系，确认它们是菝葜四倍体、六倍体的亲本之一；菝葜的二个四倍体居群——广西和贵州居群是异源多倍体；支持菝葜复合种内的多倍体是多次起源的；发现菝葜复合种是一个古老又在活跃分化着的类群。综合分析数据表明：1)菝葜复合种是一个由核心种菝葜(S．china)和小果菝葜、三脉菝葜、S．biflora、长托菝葜、红果菝葜和粗糙菝葜组成的单系类群；2)广布种菝葜的二倍体居群和二倍体小果菝葜、三脉菝葜及S．biflora是菝葜复合种多倍体的祖先类群；3)从广布种菝葜的四倍体居群和六倍体居群的分布情况，结合分子系统树，推测在更新世中期台湾岛屿、日本列岛与亚洲大陆分离之前，菝葜的4X居群已广泛分布，而现在亚洲大陆广泛分布的6X，应是在台湾、日本岛屿与亚洲大陆分离后才形成的，时间在新第三纪至更新世后；4)菝葜的四倍体金佛山居群、红果菝葜、长托菝葜和粗糙菝葜可能有另外的二倍体祖先种；5)菝葜复合种的起源中心可能在华中至西南一带，多倍体的起源应该是多次起源，即在一个以上地域的不同的群体间由未减数配子杂交并加倍发生；6)菝葜的果实大小并非稳定的遗传性状。2．草本菝葜组的系统发育关系利用三个片段所构建的分子系统树表明草本菝葜组是一稳定的单系类群，支持地理分布在该组的演化中起了重要作用，而不是习性的变化。分子证据表明草本菝葜组北美种与亚洲种是姐妹群关系，北美东部与西部的草本菝葜种也是姐妹群关系。采自东亚的牛尾菜和白背牛尾菜形成明显的两个分支，即存在两个种，但已出现一定的分化，即变种尖叶牛尾菜S．riparia var．acuminata。化石、地理及遗传学的证据表明，草本菝葜组起源于东亚。在北美洲和亚洲气温相似的时期，草本菝葜种从亚洲大陆的东北部，通过白令陆桥进入北美的西南部，然后向东部和南部扩张。由于地理隔离事件的发生，阻断了北美和亚洲草本菝葜种之间的基因交流：同时，北美居群片断化，形成美国西北部和东南部两个分布区域。随着冰川融化、气温回升，北美东部的草本菝葜种继续北移至分布的最北限。但是分布于北美西部的草本菝葜居群，由于存在着地理阻断，仅在加利福尼亚北部的克拉马斯山分布着S．jamesii。因此，北美的草本菝葜种可以分为两个类群，一个是来自加利福尼亚的S．jamesii，另一个是以S．herbacea为核心的东部复合群。结合形态和细胞学研究，我们认为S．herbacea复合群可以分成三组，即S．ecirrhata组(包括S．biltmoreana，S．hugeri和S．illinoensis)，S．herbacea组(包括S．lasioneuron和S．pulverulenta)和S．pseudo-china组。更多还原

【Abstract】 Phlogenetic reconstruction of the Smilax china complex (Smilacaceae) and the species of Smilax sect. Nemexia (Smilacaceae) is presented based on the analysis of morphological analysis, random amplified polymorphic DNA (RAPD), allozyme and sequence data from the nrDNA ITS region and four fragments of cpDNA including matK gene, trnT-L, trnL-F and rp116 intergenic spacers. In combination with the previous morphological and karyological data, conclusions were made as the followings:1. Phlogenetic reconstruction of the Smilax china complex (Smilacaceae)1.1 Morphological analysisA clatistic analysis of 60 samples of S. china complex was carried out based on nine morphological characters.The result showed that some variations were found among the different taxon, but they are overlapped due to high varibal morphologies.1.2 Genetic differentiation based on RAPD analysisRAPD analysis was conducted on 21 groups. Out of 36 RAPD primers, 15 generated highly reproducible and stable DNA fragments. Using these primers, 275 discernible DNA fragments were produced, among which 246 (PPB=89.47%) were polymorphic, which indicated high levels of genetic variation. A UPGMA dendrogram was constructed based on the genetic distance and showed three clusters, namely A, B and C. Cluster A included 8 outgroups, cluster B included all species of the complex except S. china in GX population, and cluster C included S. ferox and S. china in GX population. The results indicated that S. china is a stable species and little differentiation was found between S. china and its related species in the complex.1. 3 Phylogenetic analysis based on the sequence of nrDNA ITS, cpDNA matK and trnT-LSequence data of nrDNA ITS region and two fragments of cpDNA including matK gene and trnT-L intergenic spacer strongly supported the monophly of S. china complex (different populations of 5. china, S. davidiana, S. trinervula, S. polycolea, S. lebrunii, and S. biflora). Diploid population of S. china in Hubei, S. davidiana and S. trinervula formed a clade, which was closely related to the main clade composed of the major polyploidy complex. This indicated the diploid populations were possibly the ancestors of S. china complex. The populations in Mt. Lao and Korea formed a unique clade, which indicated the common origination of the two populations. Another clade comprised of 4X JF population, S. lebrunii and S. polycolea, showing the divergence or the different derivation from the main clade. As revealed in the aligned ITS sequences, the same sequence type was found in JF population, S. polycolea and S. lebrunii due to more than 20 different loci from others. GX population formed a unique clade and high frequency of fragment indels were found. Additionally, S. davidiana was supposed to be one of the ancestors of S. china complex because it was clustered with HB population.1. 4 Allozyme analysisVariation in allozyme patterns of 8 enzyme systems with 25 alleles in 11 populations of S. china and 3 affinitive species were investigated. The result supported that diploid populations of S. china complex, S. davidiana and S. trinervula were ancestor species of 4X and 6X species, and the S. china complex populations of GL and GZ are allotetraploid. Suggestions can also be made that S. china arose from multiple origins and it is an old, but being active differentiation group.In combination with the morphological, karyological and molecular data, inference can be made: 1) S. china complex is a monophly, including the key species of S. china, and other species of S. davidiana, S. trinervula, S. biflora, tetraploids S. polycolea and hexaploids S. ferox, S. lebrunii. 2) The diploid populations of S. china complex and diploid species of S. davidiana, S. trinervula and S. biflora are the ancestors of the polyploidy complex. 3) From the distribution of tetraploid and hexaploid populations of S. china and the gene tree, suggestions were given that before Taiwan, Ryukyus and Japan were seperated from Asia continent in the middle of Pleistocene, the tetraploid populations of S. china had distributed widely, however, the hexaploids populations derived lately from hybridization. 4) The tetraploid Jinfo population and S. polycolea, S. ferox and S. lebrunii may have other diploid ancestors. 5) Origination center of the complex is hypothesized to be the zone from central to southwestern China and the polyploidy has originated from the hybridyzation and reduplication of non-miosis gametes in multi places and populations. 6) The size of the fruits are not supposed to be stable genetic character.2. Phylogeny reconstruction of Smilax sect. NemexiaPhylogeny tree based on three molecular markers supported that the species of Smilax sect. Nemexia is a stable monophly and the geographical distribution has played an important role in the differentiation of this group, but not morphological character. Smilax sect. Nemexia exhibits an East Asian-North American phytogeographic disjunction. The common ancestor of the group is assumed to have originated in Asia and spread northeastward to North America through the Bering Land Bridge. Catastrophic geological activities of Pleistocene glaciation obstructed the geneflow between Asia and North-America, and fragmentated the continuous distribution of the group in North America into eastern and western parts of North-America. As revealed by the phylogeny tree, the North American species form a clade, with S. jamesii, the only species from western North America, as sister to the "5. herbacea complex" from eastern North America.更多还原