【作者】 伊珍珍；

【导师】 宋微波；

【作者基本信息】 中国海洋大学 ， 水生生物， 2009， 博士

【摘要】 纤毛虫门隶属原生生物界,原生动物亚界,是形态上最复杂和最高等的单细胞真核生物,在进化上具有特殊的地位。然而,由于纤毛虫复杂的生活史和丰富的多样性,使得基于形态学/形态发生学特征构建系统受到很大限制,且不同系统之间存在很大的差异。而目前基于分子标记所涉及类群、基因均十分有限,越来越多的研究者已认识到这极易造成分子树与真实系统树之间的差别。因此,大量纤毛虫类群的系统关系仍处于未知、半知甚至空白状态,大量探索性研究等待深入,大量的争议、混乱和错误等待修订。基于上述问题,本工作于2004-2008年间采集了青岛沿岸为主的山东沿海多类生境（潮间带、岩礁、泥沙底质、自由开放水域、入海口、水产养殖/育苗水体等）中的多种旋唇纲、寡膜纲和前口纲纤毛虫,开展了以分子系统学为主的研究,通过多基因测序、大量增加测序物种/种群、改进数据分析方法等手段,来提高分子系统分析的可靠性。同时,在当前可用分子信息欠缺的前提下,我们结合本研究室在纤毛虫形态学/发生学研究中的优势,采用结合和参照形态学、发生学的资讯,讨论分子系统学的结果的工作路线,尽可能地避免研究技术所带来的缺陷。主要成果包括：1)首次构建了14属21种旋唇纲纤毛虫的转录间隔区2的二级结构。14个属分别为：异列虫属Anteholosticha,偏角毛虫属Apokeronopsis,博格氏虫属Bergerilla,双轴虫属Diaxonella,斜额虫属Epiclintes,全列虫属Holosticha,后尾柱虫属Metaurostylopsis,新列虫属Nothoholosticha,拟双棘虫属Parabirojimia,砂隙虫属Psammomitra,伪尾柱虫属Pseudourostyla,伪小双虫属Pseudoamphisiella,伪角毛虫属Pseudokeronopsis,趋角毛虫属Thigmokeronopsis)2)建立了尾柱目下1个新亚目：拟双棘虫亚目Parabirojimina Yi et al.2008,并提升了1个新阶元：砂隙虫科Psammomitridae。3)对旋唇纲纤毛虫国际间长期系统地位混乱以及未明的5个属9个种的系统位置做了调整和确认。9个种分别为：厚偏角毛虫Apokeronopsis crassa,波氏偏角毛虫Apokeronopsis bergeri,斯特后尾柱虫Metaurostylopsis struederkypkella,盐后尾柱虫Metaurostylopsis salina,中华后尾柱虫Metaurostylopsis sinica,斯太克趋角毛虫Thigmokeronopsis stoecki,拉氏伪小双虫Pseudoamphisiella lacazei,泡状伪小双虫Pseudoamphisiella alveolata,相似拟双棘虫Parabirojimia similis。4)首次从分子系统学的角度详细探讨了游仆目的内部亲缘关系：“典型”的游仆虫为多源发生,分为明显的两支；双眉虫复合属之间存在较深的分化；游仆虫科分为五个稳定的分支；五种盾纤虫之间的种间分化要远远高于其它近缘属内部的种间分化。此外,本工作以该类群为研究对象,探讨了多基因联合分析在纤毛虫系统分析中的应用前景：联合数据集因为包含更多地有效信息,因此结果具有更高的可信度5)对寡膜纲纤毛虫国际间长期系统地位混乱以及未明的6个种的系统位置做了调整和确认。6个种分别为：丝状小尾丝虫Uronemella filificum,查匹克帆口虫Pleuronema czapikae,中华帆口虫Pleuronema sinica,裂纱虫属未定种Schizocalyptra sp-WYG07060701,艾斯特裂纱虫Schizocalyptra aeschtae,拟四膜虫属未定种Paraterahymena sp.。6)首次构建了盾纤亚纲内20个属30个种的小核糖体亚基基因的V4区的二级结构：Helix E23₇末端环区的大小能够将嗜污目和帆口目明显区分开。20个属分别为：平腹虫虫属Homalogastra,伪康纤虫属Pseudocohnilembus,后阿脑虫属Metanophrys,异阿脑虫属Mesanophrys, Anophyroides,嗜污虫属Philasterides,迈阿密虫属Miamiensis,拟尾丝虫属Parauronema, Plagiopyliella, Thyrophylax,内扇虫属Entorhipidium,内盘虫属Entodiscus,尾丝虫属Uronema,小尾丝虫属Uronemella,拟阿脑虫属Paranophrys,康纤虫属Cohnilembus, Schizocaryum,帆口虫属Pleuronema,裂纱虫属Schizocalyptra,膜袋虫属Cyclidium。7)以盾纤亚纲为研究对象,首次探讨了形态学数据与基因序列联合建树在纤毛虫系统分析中的应用前景。8)对前口纲纤毛虫国际间长期系统地位混乱以及未明的5个种的系统位置做了调整和确认,并探讨了增加环境未定种序列对纤毛虫分子系统树构建的影响。5个种分别为：偏榴弹虫未定种Apocoleps sp-WYG07060702, Apocoleps sp-FXP07101005,巨大偏榴弹虫Apocoleps magnus,纺锤披巾虫Tiarina fusa,诺兰德虫属未定种Nolandia sp-WYG07050702。此外,本文完成了对旋唇纲、寡膜纲和前口纲内部44个种的小核糖体亚基基因、26个种的转录间隔区、15种旋唇纲纤毛虫的alpha微管蛋白基因、以5种旋唇纲纤毛虫的组蛋白H4基因的测序和提交。更多还原

【Abstract】 Ciliated protozoa is the most morphorlogically complex and highly differentiated taxon among single-celled organisms, and plays an important role in eukaryotic evolutionary investigation. Based on the morphology, infraciliature and ontogenetic characters, some classical systems of ciliate classification were proposed. However, the significance of various structures and infraciliatures is rather different according to different investigators,, which caused different systems of classification. And these systems needed careful reconstruction. Recently, the molecular phylogenic analyses have thrown new light on the taxonomy and systematics of ciliates, which reveals phylogenetic relationships at the genic level. However, only one gene marker was chosen for most of the molecular phylogenetic investigations, which caused differences between phylogenetic trees and the "real" ones. Therefore, more investigations are needed to reveal the relatinships among Ciliophora.With molecular biological methods, this work resolved some confusions of morphological taxonomy and systematology of lower taxa within three ciliated classes, viz., Spirotrichea, Oligohymenophorea, Prostomatea, at the genetical level. In order to minimize the differences between molecular phylogenetic trees and "real" trees, we included more species, sequenced more gene marks, and improved the methods of data analyses.The following conclusions could be drawed:1) ITS2 secondary structures of 14 spirotrichean genera and 41 species were predicted. These 14 genera are:Anteholosticha, Apokeronopsis, Bergerilla, Diaxonella, Epiclintes, Holosticha, Metaurostylopsis, Nothoholosticha, Parabirojimia, Psammomitra, Pseudourostyla, Pseudoamphisiella, Pseudokeronopsis, Thigmokeronopsis2) A new suborder was established within the order Urostylida:Parabirojimina Yi et al. 2008; a new improved taxon:Psammomitridae。3) The phylogenetic positions of nine spirotrichean species, whose assignments were historically unclear, have been classified. These nine species are:Apokeronopsis crassa, A. bergeri, Metaurostylopsis struederkypkella, M. salina, M. sinica, Thigmokeronopsis stoecki, Pseudoamphisiella lacazei, P. alveolata, Parabirojimia similis.4) The molecular phylogenetic relationships among the order Euplotida were analysed for the first time. Result showed:(i) the ’typical’ euplotids comprised a paraphyletic assemblage composed of two divergent clades (family Uronychiidae and families Euplotidae-Certesiidae-Aspidiscidae-Gastrocirrhidae); (ii) in the family Uronychiidae, the genera Uronychia and Paradiophrys formed a clearly outlined, well-supported clade that seemed to be rather divergent from Diophrys and Diophryopsis, suggesting that the Diophrys-complex may have had a longer and more separate evolutionary history than previously supposed; (iii) inclusion of 12 new SSrRNA sequences in analyses of Euplotidae revealed two new clades of species within the family and cast additional doubt on the present classification of genera within the family; (iv) the intraspecific divergence among five species of Aspidisca was far greater than those of closely related genera. In addition, the ITS1-5.8S-ITS2 coding regions and partial histone H4 genes of six morphospecies in the Diophrys-complex were sequenced along with their SSrRNA genes and used to compare phylogenies constructed from single data sets to those constructed from combined sets. Results indicated that combined analyses could be used to construct more reliable, less ambiguous phylogenies of complex groups like the order Euplotida, because they provide a greater amount and diversity of information.5) The phylogenetic positions of six oligohymenophorean species, whose assignments were historically unclear, have been classified. These six species are:Uronemella filificum, Pleuronema czapikae, P. sinica, Schizocalyptra sp-WYG07060701, S. aeschtae, Paraterahymena sp.6) The variable region 4 secondary structures of SSrRNA gene of twenty oligohymenophorean genera and thirty species were predicted. There are several minor differences among ITS2 secondary structures of four species, while three P. carnea populations are identical. The size of the terminal bulge in Helix E23-7 is probably different for the orders Philasterida and Pleuronematida. These twenty genera are:Homalogastra, Pseudocohnilembus, Metanophrys, Mesanophrys, Anophyroides, Philasterides, Miamiensis, Parauronema, Plagiopyliella, Thyrophylax, Entorhipidium, Entodiscus, Uronema, Uronemella, Paranophrys, Cohnilembus, Schizocaryum, Pleuronema, Schizocalyptra, Cyclidium.7) The possibility of tree construction using combination data constaining morphological characters and gene sequences is discussed.8) The phylogenetic positions of five prostomatean species, whose assignments were historically unclear, have been classified. These five species are:Apocoleps sp-WYG07060702, Apocoleps sp-FXP07101005, Apocoleps magnus, Tiarina fusa, Nolandia sp-WYG07050702. And accuracy improvement of the phylogenetic estimates caused by increased environmental taxa sampling is also proved.In addition, SSrRNA gene of 44 species, ITS-5.8S-ITS2 region of 26 species, alpha-tubulin gene of 15 species, and histon H4 gene of five species are sequenced and submitted.更多还原