【作者】 程金凤；

【导师】 高亚辉；

【作者基本信息】 厦门大学 ， 生态学， 2007， 博士

【摘要】 微型浮游硅藻是浮游植物中的重要类群,也是一类重要的海洋生物资源,但同时有些种类会引发赤潮,其中硅藻门的骨条藻属Skeletonema、海链藻属Thalassiosira、角毛藻属Chaetoceros等是比较常见的赤潮藻类。对这些重要的优势微型浮游硅藻种类的分类鉴定和遗传特性的研究,特别是分析这些典型种类的遗传地理差异,了解属内种间的遗传多样性,成为了海洋生态和赤潮研究的基础。本文以我国沿海典型微型浮游硅藻属一骨条藻、海链藻、角毛藻的常见种类为研究对象,先分离并建立各个种的无菌纯种培养;然后利用光学显微镜、透射电镜和扫描电镜对其进行形态学观察、特征描述,并进行分类研究;再选择核糖体SSU nrDNA和LSU nrDNA基因对其进行分子生物学分析,包括基因序列差异性分析、遗传多样性分析和系统进化分析。旨在对我国沿海几种典型微型浮游硅藻进行形态学分类和分子鉴定,并分析其种间、种内、不同地理株系的地理遗传差异性和系统进化关系,探明我国沿海主要微型浮游硅藻的种类和遗传多样性水平。本论文主要结果如下:1、四种骨条藻的形态学、遗传差异及系统进化分析分析了分离自深圳、香港、青岛、北黄海、厦门、长江口、广州和韩国等地的21株骨条藻。根据其形态学特征,鉴定为4个种:玛氏骨条藻Skeletonemamarinoi Sarno et Zingone,多恩骨条藻Skeletonema dohrnii Sarno et Kooistra,敏盐骨条藻Skeletonema subsalsum(Cleve)Bethge和热带骨条藻Skeletonema tropicumCleve。分析其nrDNA序列,并构建系统进化树,结果表明,热带骨条藻和敏盐骨条藻为单系的,而玛氏骨条藻和多恩骨条藻没有被区分开。因为玛氏骨条藻和多恩骨条藻种间差异非常小,SSU nrDNA序列的相似性非常高,整个序列相似性≥99.62%,而其它种类间的序列相似性在98.37%～98.93%之间。这两个种LSUnrDNA序列相似性≥99.11%,明显高于其它几种间的序列相似性(92.47%～96.3%)。2、八种海链藻的形态学、遗传差异及系统进化分析研究了分离自香港、海口、舟山、台湾海峡、胶州湾等地的20株海链藻。根据形态学特征鉴定为8个种,分别为极小海链藻Thalassiosira minima Gaarder、威氏海链藻Thalassiosira weissflogii(Grun.)Fryxell&Hasle、园海链藻Thalassiosira rotulaMeunier、细弱海链藻Thalassiosira subtilis(Ostenfele)Gran、双环海链藻Thalassiosiradiporocyclus Hasle、假微型海链藻Thalassiosira pseudonana Hasle et Heimdal、萎软海链藻Thalassiosira mala Takano和艾伦海链藻Thalassiosira allenii Takano。nrDNA序列和系统进化树分析结果表明,支持细弱海链藻、假微型海链藻、极小海链藻、艾伦海链藻、威氏海链藻、萎软海链藻和双环海链藻为单系起源。威氏海链藻是8种海链藻中最早分歧的谱系,这说明威氏海链藻与其它7种海链藻遗传距离较大、亲缘关系较远。细弱海链藻和假微型海链藻的亲缘关系比较近。两个序列分析结果都显示双环海链藻与其它7种海链藻之间的差异最大,种间存在差异最大的两个种是威氏海链藻和双环海链藻(SSU nrDNA,6.57%;LSU nrDNA,12.82%)。差异最小的两个种是圆海链和细弱海链藻(SSU nrDNA,1.52%;LSUnrDNA,1.71%)。两个序列结果都表明细弱海链藻与假微型海链藻序列差异不大(2.00%～2.60%)。SSU nrDNA基因序列分析中,海链藻种内序列差异从0.13%到2.53%,LSU nrDNA基因序列分析的海链藻种内序列差异与SSU nrDNA基因序列分析中的结果相一致。3、两种角毛藻的遗传差异及系统进化分析对分离自香港、海口、黄海和厦门的4株角毛藻(形态学鉴定为2个种,旋链角毛藻Chaetoceros curvisetius Cleve和短孢角毛藻Chaetoceros brevis Sch(u|¨)tt)进行遗传差异和系统进化分析。结果表明,SSU nrDNA基因序列分析中,旋链角毛藻与短孢角毛藻间的种间差异性为6.64%到10.77%,而旋链角毛藻种内的差异为0.18%到0.59%。角毛藻的SSU nrDNA基因组内存在多态性,旋链角毛藻的基因组内多态性平均为0.45%。LSU nrDNA基因序列分析中,短孢角毛藻与旋链角毛藻间的种间差异从17.15%～19.42%。而旋链角毛藻不同的地理株系也存在一定的遗传差异。更多还原

【Abstract】 The marine nanoplanktonic diatoms are important component of marine phytoplankton and marine resources,and some species can cause red tide.In the diatoms,Skeletonema,Thalassiosira and Chaetoceros are relatively common red tide causative species.The study on the classification and genetic characteristic of these importantly dominant nanoplanktonic diatom species is the base of marine ecology and red tide research with emphasis on the analysis of genetic diversity of inter-species,intra-species,and geographical genetic differentiation of typical species.This dissertation focused on Skeletonema,Thalassiosira and Chaetoceros as the typical genus of nanoplanktonic diatoms along the coast of China.The single isolates of the diatoms were isolated and cultivated.And the morphological studies were carried out using LM,SEM,and TEM.These studies include the morphological observation under the electron microscope,the description of morphological characteristics and classification.And then the SSU nrDNA and LSU nrDNA were used as DNA marker for molecular studies.The main purpose of this dissertation centered on the morphological classification and molecular identification of these typical nanoplanktonic diatoms,and analyzed the interspecies difference,intraspecies difference,geographical genetic difference,and phylogenetic relation.The main results were showed as follows:1.The morphology,genetic difference and the phylogenetic analysis on four species of SkeletonemanrDNA sequences analysis were carried out on 21 strains of Skeletonema isolated from the coast of China including Shenzhen,Hongkong,Qingdao,North Huanghai sea,Xiamen,Yangtse River estuary,Guangzhou etc.And what should be mentioned is that one of the strains is from Korea.According to the morphological characteristics, four species were identified:Skeletonema marinoi Sarno et Zingone,Skeletonema dohrnii Sarno et Kooistra,Skeletonema subsalsum(Cleve) Bethge and Skeletonema tropicum Cleve.The analyses of the nrDNA sequences and the phylogenetic tree indicated that S.subsalsum and S.tropicum were monophyletic and the S.marinoi and S.dohrnii cannot be distinguished.The latter may be attributed to the very small morphological difference between S.marinoi and S.dohrnii,which is further confirmed by the high similarity of SSU nrDNA sequences between them(above 99.62%).Nevertheless,the similarity of SSU nrDNA between other species was about 98.37%～98.93%.The similarity of LSU nrDNA between the the S.marinoi and S.dohrnii was above 99.11%,markedly higher than the similarity,about 92.47%～96.3%,between other species.2.The morphology,genetic difference and the phylogenetic analysis on eight species of ThalassiosiraThe morphological study and nrDNA sequences analysis were carried out on 20 strains of Thalassiosira which were mainly isolated from Hongkong,Hainan, Zhoushan,Taiwan Strait,Jiaozhou Bay etc.Based on the traditionally morphological characteristics,eight Thalassiosira species were identified:Thalassiosira minima Gaarder,Thalassiosira weissflogii(Grun.)Fryxell & Hasle,Thalassiosira rotula Meunier,Thalassiosira subtilis(Ostenfele) Gran,Thalassiosira diporocyclus Hasle, Thalassiosira pseudonana Hasle et Heimdal,Thalassiosira mala Takano and Thalassiosira allenii Takano.On the molecular level,the analyses of the nrDNA sequences and the phylogeny showed that T.subtilis,T.pseudonana,T.weissflogii,T. allenii,T.minima,T.mala and T.diporocyclus were monophyletic.And T.weissflogii was the earliest divergence from other species,which suggested that the genetic distance between T.weissflogii and other seven species was comparatively large.SSU and LSU sequences analysis both revealed that the difference between the T. diporocyclus and other seven species was most distinct.The most distinct difference was between the T.weissflogii and the T.diporocyclus(SSU nrDNA,6.57%;LSU nrDNA,12.82%),while,the least was between the T.rotula and T.ubtilis(SSU nrDNA,1.52%;LSU nrDNA,1.71%).Both SSU and LSU sequence implied that difference between T.ubtilis and T.seudonana was not distinct(2.00%～2.60%). According to the SSU nrDNA gene analysis,the difference of Thalassiosira species under this study was from 0.13%to 2.53%,which was coherent to the LSU nrDNA gene analysis.3.The morphology,genetic difference and phylogenetic analysis of two species of ChaetocerosThe morphological study an nrDNA sequences analysis were carried out on four strains of Chaetoceros isolated from the Hongkong,Hainan,Xiamen,Huanghai etc.. According to the traditionally morphological characteristics,two species were identified:Chaetoceros curvisetius Cleve and Chaetoceros brevis Schütt.On the molecular level,the analysis results of the nrDNA sequences and the phylogenetic tree indicated that the difference between C curvisetius and C brevis,based on the nrDNA gene analysis,was from 6.64%to 10.77%,while within C curvisetius species, the difference was from 0.18%to 0.59%.The nucleotide polymorphism existed in the SSU nrDNA gene sequences of species of Chaetoceros.The average nucleotide polymorphism of C curvisetius approximated to 0.45%.According to the LSU nrDNA gene sequences,the difference between C curvisetius and C brevis was from 17.15%～19.42%and the difference also existed in the strains from different locations.更多还原