【作者】 苗苗；

【导师】 宋微波；

【作者基本信息】 中国海洋大学 ， 遗传学， 2009， 博士

【摘要】 纤毛虫原生动物由于复杂特化的细胞器结构(如纤毛、皮膜结构)、分司不同功能的大小两种核型以及独特的接合生殖方式，在分子水平的细胞学、真核生物遗传学，尤其在细胞衰老及基因调控、核-质关系、细胞分化与反分化、细胞器的结构与功能、寄生共生生态位间关系、生命的起源与进化等研究领域具有重要的研究价值和意义。我国纤毛虫原生动物的研究，尤其是海洋类群纤毛虫原生动物系统发育学的研究，虽然有了二十多年的积累，但由于传统技术手段的限制以及相关信息的局限(包括细胞发生资料和分子生物学信息)，许多类群的分类地位及系统关系长期存疑(特别是那些罕见及处于关键演化地位的类群)，大量空缺以及前人所遗留下的不足、混乱和错误急需分子水平的修订和清理，同时许多迄今尚未涉及类群的分子生物学信息的空白有待填补。利用纤毛虫相对保守的小亚基单位核糖体基因(SSU rRNA)及相对变异较大的ITS1-5．8S-ITS2序列探讨其分子分类学特征、系统发生和演化地位，已成为本领域以形态学和细胞发生学为主的系统学研究不可或缺的重要组成。然而，由于国际间分子生物学研究起步较晚以及类群间工作进展的不平衡，导致了大量的地位不明种或关键类群相关信息的缺失，此又进一步地限制了对诸多相关研究(基因种的建库、纲目级阶元的系统定位等)的开展。尤其是在许多发生学资料缺乏并且长期系统学地位不明的类群，分子工作的开展将在很大程度上解决和完善许多关键类群在系统进化上的争议和不足。本工作在SSU rRNA、ITS区等序列测序、分析的基础上，首次完成了对纤毛门内系统发育地位长期不明的若干重要科属之系统学分析，涉及5纲、10目、16科、19属、25种纤毛虫的小亚基单位核糖体rRNA基因、13种纤毛虫的转录间隔区域(ITS1-5．8S-ITS2序列)分析、分子树构建、系统地位的确认，并对其分类学上的混乱和疑点进行讨论和澄清。主要创新成果如下：1．旋唇纲(涉及六个代表类群腹棘虫、心毛虫、盘头虫、拟盘头虫、伪小双虫及线双虫的7个SSU rRNA基因)(1)腹棘虫与拟游仆虫形成单源发生系即腹棘虫科，构成双眉虫-尾刺虫-游仆虫复合体(包括游仆虫，舍太虫，楣纤虫等典型的游仆类)的中间类群；(2)心毛虫和凯毛虫证实为旋唇类中一个祖先型，代表了一个处于外围的低等分支；(3)盘头类与伪小双虫类之单元发生分别被确认，且表现了较近的系统关系；分子信息表明，两类纤毛虫同为排毛亚纲和游仆亚纲的边缘类群，代表旋唇纲下的目级阶元，即盘头目，该目下辖两个亚目：盘头亚目和伪小双虫亚目；(4)对旋唇纲及其下亚纲的单系性进行多次讨论，并对该纲种类的分类学上的混乱和疑点进行部分澄清。2．寡膜纲(涉及2个代表类群阔口虫和污栖虫的SSU rRNA基因以及10个代表类群的ITS区序列)(1)根据其独特的形态特征建立了1新科(阔口虫科)、1新属(阔口虫属)、1新种(中华阔口虫)，其在分子系统树深位的分支显示，该阔口虫代表了一个与膜袋虫科平行的科级阶元，支持新科的成立；(2)嗜污虫科下辖的两属嗜污虫和污栖虫未表现出较近的亲缘关系，显示嗜污虫科可能为多源发生；(3)首次利用转录间隔区序列(ITS)二级结构信息并对比SSU rRNA基因数据全面讨论了盾纤亚纲内各主要代表类群的系统关系，在最新数据的采依基础上，构建了新的分子发育树，同时确认了二级结构信息在纤毛虫科级及以上阶元划分中的指标价值和作用。3．叶咽纲(涉及3个代表类群斑吸管虫、壳吸管虫和拟壳吸管虫的7个SSUrRNA基因)(1)吸管类纤毛虫为一单系类群；(2)在吸管虫进化枝内部，位于最底部是外生类，内生类位于进化枝的末端，外翻类处于两者之间，显示吸管虫的出芽生殖方式的进化与基因进化一致，其进化过程为外出芽生殖--外翻出芽生殖--内出芽生殖。4．异毛纲(涉及7个代表类群突喇叭虫、突口虫、蚕豆虫、瓶囊虫、爽口虫、环须虫和丽克虫的8个SSU rRNA基因)(1)广义的(经典的)异毛类纤毛虫为一多源发生系，包含现代意义的异毛纲，部分旋唇纲及其他种类；(2)首次预测并比较了广义的异毛类纤毛虫SSU rRNA基因二级结构；(3)在单源发生的异毛纲内，环须虫先于其他种类分支，推测可能是该类纤毛虫的祖先型；传统的爽口虫科实为一并系复合体；蚕豆虫-海水喇叭虫-平囊虫，突喇叭虫-突口虫分别显示了密切的亲缘关系。5．未明类群(涉及1个代表类群中缢虫的SSU rRNA基因)(1)中缢虫的SSU rRNA基因序列及二级结构较其他纤毛虫明显高变；(2)尽量减少分子系统树中长枝吸引的影响，中缢虫被认为是介于纤毛虫两大亚门之间的中间类群。另外，本研究分析GenBank数据库中纤毛虫5．8S rRNA基因，预测其二级结构，对比前人推测给出合理解释。更多还原

【Abstract】 Because of special nuclear dualism,complicated morphogenetic process and uniquesexual reproduction (conjugation),ciliates play important roles in molecular biology,cell biology and researches on relationships between nucleus and cytoplasm,especiallyin the eukaryotic biological evolution.Systematics and phylogenetics on the ciliatedprotozoa have been carried out for over 20 years.However,its phylogeny still remainsconfusing as regards the evolutionary relationships and systematic positions of manywell-known groups.This is due to the high diversity of their morphology,the difficultyin recognizing which similarities are due to convergent evolution,the loss ofintermediate forms during the long period of time and insufficient molecularinformation.Molecular marker genes were chosen e.g.SSU rRNA gene and ITS 1-5.8S-ITS2 geneas to study systemetics,phylogenetic position or relationships and evolutionary historiesof some key and / or ambiguous taxa of ciliated protozoa.Based on the gene sequenceing and analyses,our work resolved some confusions ofmorphological taxonomy and phylogenetic positions of some key taxa within threecalsses at the genetical level,which included,SSU rRNA gene sequences for 25 species,and ITS1-5.8S-ITS2 gene sequences for 13 species were determined,and submitted tothe GenBank/EMBL database.We used multiple computer-assisitent algorithms inferredfrom combinations of molecular sequences and morphological and morphogeneticcharacterizations to supply important basis and parameters for molecular systematics,areconstruction of phylogenetic positions and evolutionary relationships of key taxaciliated protozoa.The conclusions were confirmed:1.Spirotrichea(1) Gastrocirrhus and Euplotidium form a monophyletic group,namely the familyGastrocirrhidae,and appear to be intermediate taxa bridging the evolution of theDiophrys-Uronychia and Euplotes-complexes (i.e.,Euplotes,Certesia and Aspidisca);(2) Caryotricha,together with Kiitricha,diverges at the deep level from all othermembers of the spirotrichs.Its branching position is between Phacodiniidia andLicnophoridia.It is strongly support the distinct separation of the Kiitricha-Caryotrichaclade,which always branches basal to the Stichotrichia-Hypotrichia-Oligotrichia -Choreotrichia assemblage;(3) The monophyly of pseudoamphisiellids and discocephalines was supported,bothof which were separated between the other stichotrichs and hypotrichs by a deep split.The order Discocephalida,representing an ancestral group,should contain twosuborders:Discocephalina and Pseudoamphisiellina.(4) Monophyly of the class Spirotrichea and its subclasses included were discussedseveral times,and some of questions were cleared.2.Oligotrichea(1) After morphological investigations and molecular analyses,an organism isbelieved as an undescribed family and a new genus:i.e.Eurystomatidae n.fam.,Eurystoma n.gen.According to phylogenetic analyses of SSU rRNA gene sequences,Eurystoma clusters with the genus Cyclidium,as a sister group to the familyPleuronematidae;(2) Philaster falls into the clearly outlined order Philasterida and it is most closelyrelated to Uronema-Paranophrys assemblage,which groups then with clustersconsisting of uronematids,parauronematids,entorhipidiids etc;the family Philasteridae,including Philaster and Philasterides,appears to be polyphyletic;(3) The secondary structure of ITS2 region in eleven representative scuticociliates andtwo ambiguously related genera are analyzed for the first time.It is found that thesecondary structure information can help to improve alignment and utilize appropriatelyphylogenetic strategies.Clearly,the variable ITS2 regions are more likely to accumulatemutations that could potentially record the divergence of the major (e.g.fmily level)lineages.3.Phyllopharyngea(1) It has a strong support for the monophyly of the class Phyllopharyngea andsubclass Phyllopharyngia;(2) Suctorian budding types are monophyletic,and exogenous budding appears to bebasal to evaginative and endogenous budding.4.Heterotrichea(1) The“traditional”heterotrichs (sensu Corliss,1979) consist of several paraphyleticgroups,including the current classes Heterotrichea,Armophorea and part of theSpirotrichea;(2) Secondary structures of SSU rRNA gene for“traditional”heterotrichs arepredicted and analysed; (3) The class Heterotrichea is confirmed as a monophyletic assemblage,and the genusPeritromus is demonstrated to be a peripheral group;Condylostentor is more closelyrelated to Condylostoma than to Stentor;Folliculina,Eufolliculina,and Maristentoralways cluster together;Climacostomum occupies a paraphyletic position distant fromFabrea,showing a close relationship with Condylostomatidae and Chattonidiidaedespite of modest support.5.Unknown species(1) The genus Mesodinium had numerous deletions and substitutions in its SSU rRNAgene,which is very divergent from all other ciliates;(2) Maximum likelihood analysis places Mesodinium on the basal subphylumIntramacronucleata,following removal of ambiguously aligned regions.In addition,secondary structure model of 5.8S rRNA gene for ciliated protozoa isrepredicted,compared with the previous model established before.更多还原