两种缘毛类纤毛虫胞质Hsp70基因序列的克隆及其系统发育分析

克隆得到2种缘毛类纤毛虫——钟形钟虫(Vorticella campanula)和螅状独缩虫(Carchesium polypinum)的胞质Hsp70基因部分序列,长度均为438bp,编码146个氨基酸。以细菌为外类群,利用最大似然法和邻接法构建包括其他5种纤毛虫在内的共26个物种的Hsp70基因氨基酸序列系统发育树,其拓扑结构显示：V．campanula和C．polypinum聚在一起,并与另2种寡膜纲的嗜热四膜虫(Tetrahymena thermophila)及草履虫(Paramecium tetraurelia)聚为姊妹枝,提示了缘毛类纤毛虫为单系,且隶属于寡膜纲的系统发育地位。     

五株纤毛虫遗传关系的ISSR分析

采用ISSR分子标记技术,尝试对四种五株纤毛虫(褶累枝虫(Epistylis plicatilis)、绿草履虫(Paramecium bursaria)、多态喇叭虫(Stentor polymorphus)、嗜热四膜虫BF1株(Tetrahymena thermophilaBF1)和嗜热四膜虫BF5株(T.ther-mophilaBF5))进行遗传关系研究。用13个ISSR引物对五株纤毛虫进行扩增,六个ISSR引物获得多态片段。根据Nei s遗传距离矩阵构建了五株纤毛虫的遗传关系树状图。UPGMA,NJ聚类图表明:两株嗜热四膜虫最先聚在一起;其次是褶累枝虫和多态喇叭虫聚在一起,然后再与嗜热四膜虫聚在一起;咽膜亚纲的绿草履虫形成独立的一枝。结果显示:①缘毛亚纲纤毛虫可能是寡膜纲中较独特的一个类群,建议提升缘毛亚纲纤毛虫的分类地位;②缘毛亚纲褶累枝虫与膜口亚纲嗜热四膜虫的亲缘关系近于咽膜亚纲绿草履虫,在寡膜纲中绿草履虫处于原始地位;③五株纤毛虫基因组中均含有微卫星DNA序列:(GTG)4(、GACA)4(、AG)8(、CAA)6和(GAA)6。     

五种缘毛类纤毛虫遗传关系的ISSR研究

应用ISSR分子标记揭示了5种缘毛类纤毛虫(Carchesium polypinum,Epistylis chrysemydis,E.plicatilis,E.urceolata和Vorticella campanula)的遗传关系.从34个引物中筛选到13个多态性高的引物进行研究.得到的遗传距离(0.666 7～1.000 0)显示ISSR技术具有较高的分辨率.依据构建的UPGMA聚类树,V.campanula首先和其他种类分开;C.polypinum和E.chrysemydis聚在了一起;在3种Epistylis纤毛虫中,E.plicatilis和E.urceolata聚在了一起.对比由核糖体小亚基RNA基因序列构建的系统发育树,发现:1)ISSR引物在缘毛类纤毛虫基因组中可以得到多态扩增;2)C.polypinum与Epistylis的关系近于V.campanula,在缘毛类纤毛虫分类中,单生或群居是重要的系统发育特征;3)E.plicatilis和E.urceolata的关系近于E.chrysemydis.在Epistylis属纤毛虫中,柄的中空与否是一个有用的系统发育和分类特征.本研究表明ISSR方法在纤毛虫相近和相似种遗传关系研究中是一种新的有用方法.     

盘头类和伪小双虫类纤毛虫的进化关系：小核糖体rRNA基因信息支持建立新亚目Pseudoamphisiellinasubord.nov.

盘头类和伪小双虫类是旋唇纲（Sprirotrichea）纤毛虫中系统进化地位长期存疑的两大类纤毛虫,前者经常被认为与游仆类相关,后者通常被归入腹毛类.本文分析了Discocephalus,Leptoamphisiella和Paradiscocephalus3属纤毛虫及2种Pseudoamphisiella的SSUrRNA基因序列,并在GenBank选取游仆亚纲（Hypotrichia）、寡毛亚纲（Oligotridria）、散毛亚纲（Choreotrichia）、排毛亚纲（Stichotrichia）下属56种纤毛虫的SSUrRNA序列,利用贝叶斯法、最大似然法、邻接法和最大简约法4种方法构建系统关系树.分析结果表明,盘头类与伪小双虫类均为单源发生,且表现出较近的系统关系;两类纤毛虫应为排毛亚纲和游仆亚纲的边缘类群,不支持Lynn将其作为游仆类姐妹群的安排.结合形态学及细胞发生学资料,本研究结论如下：两类纤毛虫代表旋唇纲下的目级阶元,即盘头目（Discocephalida Wicklow,1982）,下辖2个亚目Discocephalina和Pseudoamphisiellinan.subord..新亚目Pseudoamphisiellina特征如下：（1）具有两列明显分离的中腹棘毛列,以尾柱类模式发生;（2）无迁移棘毛,此结构在其他所有典型的尾柱类中,来源于最后一列额腹横棘毛原基;（3）多根尾棘毛,来自于每列背触毛原基;（4）右缘棘毛为特殊的独立发生;（5）周丛生活.     

西藏温泉两种中国新记录纤毛虫第一双小核草履虫和明布雷斯四膜虫的形态学和系统发育学研究

研究利用活体观察、蛋白银和氨银染色技术对采自西藏温泉的寡膜纲咽膜类纤毛虫第一双小核草履虫(Paramecium primaurelia)和膜口类纤毛虫明布雷斯四膜虫(Tetrahymena mimbres)进行了形态学研究, 首次描述了这两种纤毛虫细胞核器的形态和位置、纤毛图式和口膜的排布模式; 并且测定了SSU rDNA和COXⅠ标记基因序列, 基于这两种基因的系统发育树均支持P. primaurelia聚在草履虫P. aurelia复合种中, T. mimbres聚在四膜虫T. borealis类群中。两种纤毛虫均为中国新记录种, 且首次在高原温泉中发现, 不仅为西藏地区温泉原生动物生物资源的发掘提供新的方法和思路, 也为原生动物环境适应性研究提供基础资料。     

海洋喇叭虫rRNA基因18S-ITS1序列及其系统发育分析

海洋喇叭虫Maristentor dinoferus 1996年在关岛(Guam)的珊瑚暗礁上被发现,至今尚未阐明其确切的系统发育地位.克隆到的海洋喇叭虫的18S-ITS1-5.8S rDNA序列包括222 bp的18S序列,77 bp的ITS1序列和22 bp的5.8S序列.比较分析了纤毛虫主要类群的ITS1序列后得出:短的ITS1序列可能是异毛类纤毛虫的特征.根据18S序列,利用邻接法构建,最大简约法和最大似然法构建系统发育树.其拓扑结构显示海洋喇叭虫属于异毛纲纤毛虫,但并不隶属喇叭虫科,应予以新的分类地位.     

四种淡水缘毛类纤毛虫的形态学研究

通过活体显微观察及银染法对采自武汉东湖的4种固着缘毛类纤毛虫: 粗茎睫纤虫Ophrydium crassicaule Penard, 1922, 念珠伪钟虫Pseudovorticella monilata (Tatem, 1870) Foissner & Schiffmann, 1974, 垂盖虫Opercularia nutans (Ehrenberg, 1831) Stein, 1854和一个鞘居虫未定种Vaginicola sp.的活体、纤毛图式、银线系以及细胞核的形态学特征进行详细的描述, 补充和完善了缘毛类纤毛虫的形态学资料, 为纤毛虫分类和系统发育学的研究提供重要信息。此外, 首次在伪钟虫中观察到有性生殖现象, 为纤毛虫生殖进化的研究提供了基础资料。     

三亚珊瑚礁水域纤毛虫种类组成和数量分布及与环境因子的关系

2004年8月通过对三亚珊瑚礁水域纤毛虫进行表、底层采集分析,共鉴定6纲14目33属58种浮游纤毛虫,其中旋毛纲41种,叶口纲6种,寡膜纲5种,叶咽纲4种,前口纲和伪纤毛纲各1种。在14个目中,以旋毛纲环毛亚纲砂壳目纤毛虫种类最多,达到31种。主要优势种为布氏拟铃虫 Tintinnopsis bütschlii、小拟铃虫 Tintinnopsis minuta、丁丁急游虫Strombidium tintinnodes和具沟急游虫Strombidium sulcatum等,底层纤毛虫数量和种类都较表层高,底、表层平均丰度分别为375个/L和346 个/L,表、底层纤毛虫种类差异明显,表层主要由浮游纤毛虫组成,以旋毛纲环毛亚纲砂壳目纤毛虫为主,急游目纤毛虫次之;而底层主要由缘毛目组成,如长圆靴纤虫 Cothurnia oblonga、透明鞘居虫Vaginicola crystalline、钟虫Vorticella sp.以及吸管目的壳吸管虫 Acineta sp. 等。结果显示,在活珊瑚覆盖率高的站位（S4,S7和S9）纤毛虫数量明显低于珊瑚覆盖率低或没有珊瑚覆盖的站位,这暗示了珊瑚对纤毛虫的摄食作用。典型对应分析结果表明,影响三亚湾海域纤毛虫分布的主要因素有Chla、颗粒悬浮物SS和活性磷,以及水体溶解有机碳含量。通过对纤毛虫数量与环境因子关系的分析表明,三亚湾珊瑚礁水域纤毛虫的数量与叶绿素呈明显的正相关性。     

缘毛类纤毛虫伸缩泡收缩频率的研究

纤毛虫伸缩泡的主要功能是排出进入体内的多余水分及部分代谢废物(Kudo,1966)。Kitching(1948),Tsukuda et al.(1984)等曾对纤毛虫伸缩泡的收缩频率进行研究,但国内尚未见这方面的报道。鉴于缘毛类纤毛虫对环境的生物监测及污染水体的生物治理中具有重要作用,为探索有关规律,我们测定了53种缘毛类纤毛虫伸缩泡的收缩频率以及盐度和温度对伸缩泡收缩频率的影响。材料和方法缘毛类纤毛虫(简称缘纤虫)多固着在水生动植物及杂质上,可通过采集水生动植物、水中杂质或水中挂片得到。将采得的被固着物直接放于显微镜下观察,鉴定种类,以秒表记录每…     

长江口低氧区沉积物表层纤毛虫多样性及其时空变化

利用PCR-DGGE(变性梯度凝胶电泳)指纹图谱和测序技术,以及Ludox-QPS(密度梯度离心-定量蛋白银染色)方法,研究了2011年4月和8月长江口低氧区3个站位表层沉积物中纤毛虫多样性及季节变化.结果表明:不同站位之间纤毛虫分子多样性存在显著差异(ANOSIM分析:R=0.896,P=0.0001),但季节变化不显著(R=0.043,P=0.207).序列数最多的类群为旋唇纲中的寡毛类和舞毛类纤毛虫.Ludox-QPS法研究的纤毛虫活动虫体的种类数及丰度可维持在较高的水平,且在夏季增高2～5倍.Ludox-QPS法与DGGE技术检测到的不同站位间纤毛虫多样性变化趋势基本一致.但Ludox-QPS法检测到纤毛虫活动虫种类数及丰度随季节更替变化显著,该法检获的纤毛虫种类数高于DGGE条带数.长江口低氧区的纤毛虫丰度及多样性较高,可为潜在的水母水螅体提供食物支撑.     

Phylogenetic relationships of the subclass Peritrichia (Oligohymenophorea, Ciliophora) inferred from small subunit rRNA gene sequences

The phylogenetic relationships among peritrichs remain unresolved. In this study, the complete small subunit rRNA (SSrRNA) gene sequences of seven species (Epistylis galea, Campanella umbellaria, Carchesium polypinum, Zoothamnium arbuscula, Vaginicola crystallina, Ophrydium versatile, and Opercularia microdiscum) were determined. Trees were constructed using distance-matrix, maximum-likelihood and maximum-parsimony methods, all of which strongly supported the monophyly of the subclass Peritrichia. Within the peritrichs, 1) E. galea grouped with Opercularia microdiscum and Campanella umbellaria but not the other Epistylis species, which indicates that the genus Epistylis might not be monophyletic; 2) the topological position of Carchesium and Campanella suggested that Carchesium should be placed in the family Zoothamniidae, or be elevated to a higher taxonomic rank, and that Campanella should be independent of the family Epistylididae, and probably be given a new rank; and 3) Opisthonecta grouped strongly with Astylozoon, which suggested that Opisthonecta species were not the ancestors of the stalked peritrichs.     

Phylogenetic relationships within the class Oligohymenophorea,phylum Cilophora,inferred from the complete small subunit rRNA gene sequences ofColpidium campylum,Glaucoma chattoni,andOpisthonecta henneguyi

Summary Phylogenetic relationships within the class Oligohymenophorea, phylum Ciliophora, were investigated by determining the complete small subunit rRNA (SSrRNA) gene sequences for the hymenostomesColpidium campylum, Glaucoma chattoni, and the peritrichOpisthonecta henneguyi. The affiliations of the oligohymenophoreans were assessed using both distance matrix (DM) and maximum parsimony (MP) analyses. Variations do exist in the phylogenies created by the two methods. However, the basic tree topologies are consistent. In both the DM and MP analyses the hymenostomes (C. campylum, G. chattoni, and the tetrahymenas) all form a very tight group associated with the peritrichO. henneguyi. TheTetrahymena lineage was monophyletic whereasColpidium andGlaucoma were more closely related to each other than either was to the tetrahymenas. The monophyly of the genusTetrahymena in the present analysis supports the phylogenies determined from morphological data and molecular sequence data from the histone H3II/H4II region of the genome. The perplexing and controversial phylogenetic position of the peritrichs is once again depicted in the present analysis. The distinctiveness of the peritrichOpisthonecta from both hymenostome and nassophorean ciliates based on evolutionary distances suggests that the elevation of the peritrichs to a higher taxonomic rank should be reconsidered.     

Alpha‐Tubulin and Small Subunit rRNA Phylogenies of Peritrichs Are Congruent and Do Not Support the Clustering of Mobilids and Sessilids (Ciliophora,Oligohymenophorea)

ABSTRACT. Peritrich ciliates have been traditionally subdivided into two orders, Sessilida and Mobilida within the subclass Peritrichia. However, all the existing small subunit (SSU) rRNA phylogenetic trees showed that the sessilids and mobilids did not branch together. To shed some light on this disagreement, we tested whether or not the classic Peritrichia is a monophyletic group by assessing the reliability of the SSU rRNA phylogeny in terms of congruency with α‐tubulin phylogeny. For this purpose, we obtained 10 partial α‐tubulin sequences from peritrichs and built phylogenetic trees based on α‐tubulin nucleotide and amino acid data. A phylogenetic tree from the α‐tubulin and SSU rRNA genes in combination was also constructed and compared with that from the SSU rRNA gene using a similar species sampling. Our results show that the mobilids and sessilids are consistently separated in all trees, which reinforces the idea that the peritrichs do not constitute a monophyletic group. However, in all α‐tubulin gene trees, the urceolariids and trichodiniids do not group together, suggested mobilids may not be a monophyletic group.     

Reevaluation of the phylogenetic relationship between mobilid and sessilid peritrichs (Ciliophora, Oligohymenophorea) based on small subunit rRNA genes sequences

Based on morphological characters, peritrich ciliates (Class Olygohymenophorea, Subclass Peritrichia) have been subdivided into the Orders Sessilida and Mobilida. Molecular phylogenetic studies on peritrichs have been restricted to members of the Order Sessilida. In order to shed more light into the evolutionary relationships within peritrichs, the complete small subunit rRNA (SSU rRNA) sequences of four mobilid species, Trichodina nobilis, Trichodina heterodentata, Trichodina reticulata, and Trichodinella myakkae were used to construct phylogenetic trees using maximum parsimony, neighbor joining, and Bayesian analyses. Whatever phylogenetic method used, the peritrichs did not constitute a monophyletic group: mobilid and sessilid species did not cluster together. Similarity in morphology but difference in molecular data led us to suggest that the oral structures of peritrichs are the result of evolutionary convergence. In addition, Trichodina reticulata, a Trichodina species with granules in the center of the adhesive disc, branched separately from its congeners, Trichodina nobilis and Trichodina heterodentata, trichodinids without such granules. This indicates that granules in the adhesive disc might be a phylogenetic character of high importance within the Family Trichodinidae.     

Reconsideration of systematic relationships within the order Euplotida (Protista, Ciliophora) using new sequences of the gene coding for small-subunit rRNA and testing the use of combined data sets to construct phylogenies of the Diophrys-complex

Comprehensive molecular analyses of phylogenetic relationships within euplotid ciliates are relatively rare, and the relationships among some families remain questionable. We performed phylogenetic analyses of the order Euplotida based on new sequences of the gene coding for small-subunit RNA (SSrRNA) from a variety of taxa across the entire order as well as sequences from some of these taxa of other genes (ITS1-5.8S-ITS2 region and histone H4) that have not been included in previous analyses. Phylogenetic trees based on SSrRNA gene sequences constructed with four different methods had a consistent branching pattern that included the following features: (1) the “typical” euplotids comprised a paraphyletic assemblage composed of two divergent clades (family Uronychiidae and families Euplotidae–Certesiidae–Aspidiscidae–Gastrocirrhidae), (2) 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, (3) 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, and (4) the intraspecific divergence among five species of Aspidisca was far greater than those of closely related genera. 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.     

Large-scale phylogenetic analysis provides insights into the diversification and evolution of sessilid peritrich ciliates (Protista: Ciliophora)

The Peritrichia is a speciose and morphologically distinctive assemblage of ciliated protists that was first observed by Antonie van Leeuwenhoek over 340 years ago. In the last two decades, the phylogenetic relationships of this group have been increasingly debated as morphological and molecular analyses have generated contrasting conclusions, mainly owing to limited sampling. In the present study, we performed expanded phylogenetic analyses of 152 sessilid peritrichs collected from 14 different provinces of China and 141 SSU rDNA peritrich sequences from GenBank. The results of the analyses revealed new divergent relationships between and within major clades that challenge the morphological classification of this group including, (1) the recovery of four major phylogenetically divergent clades in the monophyletic order Sessilida, (2) aboral structures such as the stalk and spasmoneme were evolutionary labile, (3) the stalk or/and spasmoneme was lost in each divergent clade indicating that parallel evolution occurred in sessilid peritrichs and (4) the life cycle and habit drive the diversity of aboral structures as well as diversification and evolution in peritrichs.     

A molecular phylogenetic investigation of Opisthonecta and related genera (Ciliophora, Peritrichia, Sessilida)

The gene encoding 18S small subunit ribosomal RNA (ssu rRNA) was sequenced in the sessiline peritrichs Opisthonecta minima and Opisthonecta matiensis, whose free-swimming, paedomorphic trophonts resemble telotrochs. Using these new sequences, phylogenetic trees were constructed with four different methods to test a previously published association between Opisthonecta henneguyi and members of the families Vorticellidae and Astylozoidae. All trees had similar topologies, with O. minima, O. henneguyi, Vorticella microstoma, and Astylozoon enriquesi forming a well-supported, certainly monophyletic clade. On the basis of genetic evidence, genera of the families Opisthonectidae and Astylozoidae are assigned to the family Vorticellidae, which already includes some species with free-swimming morphotypes. The ssu rRNA sequence of O. matiensis places it in the family Epistylididae; its taxonomic revision will be left to another group of authors. A close association of Ophrydium versatile with members of the family Vorticellidae was confirmed, casting doubt on the validity of the family Ophrydiidae. Epistylis galea, Campanella umbellaria, and Opercularia microdiscum are confirmed as comprising an extremely distinct, monophyletic, but morphologically heterogeneous clade that is basal to other clades of sessiline peritrichs.