Phylogenetic position of species in the genera Anoplophrya, Plagiotoma, and Nyctotheroides (Phylum Ciliophora), endosymbiotic ciliates of annelids and anurans

We have used small subunit rRNA gene sequences to determine the phylogenetic relationships of species in three genera of endosymbiotic ciliates. We have confirmed that the astome Anoplophrya marylandensis is related to ciliates in the Class Oligohymenophorea, supporting the view that astomes are derived from hymenostome-like ancestors. We confirmed that Plaglotoma lumbrici, formerly considered to be a heterotrich, is a stichotrich spirotrich ciliate most closely related to Paraurostyla weissei in this analysis. Thus, the somatic polykinetids of Plagiotoma can be concluded to be cirri. We report the details of our isolation of Nyctotheroides deslierresae and Nyctotheroides parvus and confirm previous reports that these clevelandellids are related to the metopid and caenomorphid ciliates, now placed in the Class Armophorea.     

旋唇纲纤毛虫系统发育分子树构建的影响因素

以36种旋唇类高等类群纤毛虫的核糖体小亚基核苷酸(Small subunit ribosomal RNA,SS rRNA)基因序列为素材,比较研究了不同条件(包括外类群、内类群的选择,同一基因不同序列长度的组合,不同建树方法和不同分析软件的使用)对纤毛虫分子系统树构建结果的影响。结果表明,上述因素均可不同程度地影响拓扑结构。结果同时提示,在利用有限数据进行相关研究,特别是在对未明类群的系统关系分析中,必须充分考虑因建树条件的不同所带来的影响。作者同时也建议,在当前可用的分子信息欠充分的前提下,对于纤毛虫任何类群的分子系统学探讨而言,慎重形成结论并尽可能地结合和参照形态学、发生学等资讯,仍是需优先考虑的工作路线。     

Multiple acquisition of methanogenic archaeal symbionts by anaerobic ciliates

Anaerobic heterotrichous ciliates (Armophoridae and Clevelandellidae) possess hydrogenosomes that generate molecular hydrogen and ATP. This intracellular source of hydrogen provides the basis for a stable endosymbiotic association with methanogenic archaea. We analyzed the SSU rRNA genes of 18 heterotrichous anaerobic ciliates and their methanogenic endosymbionts in order to unravel the evolution of this mutualistic association. Here, we show that the anaerobic heterotrichous ciliates constitute at least three evolutionary lines. One group consists predominantly of gut-dwelling ciliates, and two to three, potentially four, additional clades comprise ciliates that thrive in freshwater sediments. Their methanogenic endosymbionts belong to only two different taxa that are closely related to free-living methanogenic archaea from the particular ecological niches. The close phylogenetic relationships between the endosymbionts and free-living methanogenic archaea argue for multiple acquisitions from environmental sources, notwithstanding the strictly vertical transmission of the endosymbionts. Since phylogenetic analysis of the small-subunit (SSU) rRNA genes of the hydrogenosomes of these ciliates indicates a descent from the mitochondria of aerobic ciliates, it is likely that anaerobic heterotrichous ciliates hosted endosymbiotic methanogens prior to their radiation. Therefore, our data strongly suggest multiple acquisitions and replacements of endosymbiotic methanogenic archaea during their host's adaptation to the various ecological niches.     

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.     

A case study to estimate the applicability of secondary structures of SSU‐rRNA gene in taxonomy and phylogenetic analyses of ciliates

Phylogenetic studies of ciliates are mainly based on the primary structure information of the nuclear genes. Some regions of the small subunit ribosomal RNA (SSU‐rRNA) gene have distinctive secondary structures, which have demonstrated value as phylogenetic/taxonomic characters. In the current work, we predict the secondary structures of four variable regions (V2, V4, V7 and V9) in the SSU‐rRNA gene of 45 urostylids. Structure comparisons indicate that the V4 region is the most effective in revealing interspecific relationships, while the V9 region appears suitable at the family level or higher. The V2 region also offers some taxonomic information, but is too conserved to reflect phylogenetic relationships at the family or lower level, at least for urostylids. The V7 region is the least informative. We constructed several phylogenetic trees, based on the primary sequence alignment and based on an improved alignment according to the secondary structures. The results suggest that including secondary structure information in phylogenetic analyses provides additional insights into phylogenetic relationships. Using urostylid ciliates as an example, we show that secondary structure information results in a better understanding of their relationships, for example generic relationships within the family Pseudokeronopsidae.     

Size increase and form allometry during evolution of ciliate species in the genera Colpoda and Tillina (Ciliophora: Colpodida).

With the absence of a fossil record to provide direct evidence of evolutionary relationships, many researchers have inferred by circular reasoning that body size increase and form allometry have occurred during the evolution of ciliated protozoa. This study establishes ancestor-descendant relationships among five species of the related genera Colpoda and Tillina using ultrastructural characteristics which are not apparently dependent on size and form. This independent test suggests that phylogenetic size increase and phylogenetic form allometry have occurred during the evolution of these ciliates. Interrelationships of body size increase, increase in number of cortical organelles, and form allometry are discussed in reference to the divergence of these ciliate species.     

Evolution of Histone H4 and H3 Genes in Different Ciliate Lineages

The histones H4 are known as highly conserved proteins. However, in ciliates a high degree of variation was found compared both to other eukaryotes and between the ciliate species. To date, only H4 histones of species belonging to two distantly related classes have been investigated. In order to obtain more detailed information on histone H4 variation in ciliates we undertook a comprehensive sequence analysis of PCR-amplified internal H4 fragments from 12 species belonging to seven out of the nine currently recognized ciliate classes. In addition, we used PCR primers to amplify longer fragments of H3 and H4 genes including the intergenic region. The encoded amino acid sequences reveal a high number of differences when compared with those of other eukaryotes and the ciliate species investigated. Furthermore, in some species H4 gene variants were detected, which result in amino acid differences. The greatest number of substitutions and insertions found was in the amino terminal region of the H4 histones. However, all sequences possess a conserved region corresponding to those of all other eukaryotic H4 histones. The histone gene variations were used to reconstruct phylogenetic relationships. The tree from our data matches perfectly with the ribosomal RNA data: The heterotrichs, which were considered as a late branching lineage, diverge at the base of the ciliate tree and groups formerly thought to represent ancestral lineages now appear as highly derived ciliates. Received: 4 April 1997 / Accepted: 1 August 1997     

An ITS-based phylogenetic framework for the genus Vorticella: finding the molecular and morphological gaps in a taxonomically difficult group

Vorticella includes more than 100 currently recognized species and represents one of the most taxonomically challenging genera of ciliates. Molecular phylogenetic analysis of Vorticella has been performed so far with only sequences coding for small subunit ribosomal RNA (SSU rRNA); only a few of its species have been investigated using other genetic markers owing to a lack of similar sequences for comparison. Consequently, phylogenetic relationships within the genus remain unclear, and molecular discrimination between morphospecies is often difficult because most regions of the SSU rRNA gene are too highly conserved to be helpful. In this paper, we move molecular systematics for this group of ciliates to the infrageneric level by sequencing additional molecular markers—fast-evolving internal transcribed spacer (ITS) regions—in a broad sample of 66 individual samples of 28 morphospecies of Vorticella collected from Asia, North America and Europe. Our phylogenies all featured two strongly supported, highly divergent, paraphyletic clades (I, II) comprising the morphologically defined genus Vorticella. Three major lineages made up clade I, with a relatively well-resolved branching order in each one. The marked divergence of clade II from clade I confirms that the former should be recognized as a separate taxonomic unit as indicated by SSU rRNA phylogenies. We made the first attempt to elucidate relationships between species in clade II using both morphological and multi-gene approaches, and our data supported a close relationship between some morphospecies of Vorticella and Opisthonecta, indicating that relationships between species in the clade are far more complex than would be expected from their morphology. Different patterns of helix III of ITS2 secondary structure were clearly specific to clades and subclades of Vorticella and, therefore, may prove useful for resolving phylogenetic relationships in other groups of ciliates.     

Extrusomes in ciliates: diversification, distribution, and phylogenetic implications

Exocytosis is, in all likelihood, an important communication method among microbes. Ciliates are highly differentiated and specialized micro-organisms for which versatile and/or sophisticated exocytotic organelles may represent important adaptive tools. Thus, in ciliates, we find a broad range of different extrusomes, i.e ejectable membrane-bound organelles. Structurally simple extrusomes, like mucocysts and cortical granules, are widespread in different taxa within the phylum. They play the roles in each case required for the ecological needs of the organisms. Then, we find a number of more elaborate extrusomes, whose distribution within the phylum is more limited, and in some way related to phylogenetic affinities. Herein we provide a survey of literature and our data on selected extrusomes in ciliates. Their morphology, distribution, and possible function are discussed. The possible phylogenetic implications of their diversity are considered.     

A molecular approach to the tangled intrageneric relationships underlying phylogeny in Euplotes (Ciliophora, Spirotrichea).

The cosmopolitan genus Euplotes is remarkable among ciliates for its species richness. To understand the still tangled taxonomy and phylogenetic relationships within the genus, small subunit rRNA sequences of 11 morphologically defined species colonizing different habitats were determined. Euplotes 18S rRNA is unique among ciliates for its anomalous length and high evolutionary rate. Phylogenetic reconstruction pointed to a high divergence between this genus and the other Spirotrichea, together with a high variability within the genus. Some of the relationships within the Euplotes group were also resolved. Data from the literature, based on morphological features, habitat, and symbiotic relationships, have been compared with our results and are critically discussed. In many cases, the molecular phylogenetic analysis disagreed with species relatedness established on morphological and ecological grounds. The occurrence of a radiation phenomenon in the evolution of the genus is postulated.     

游仆目纤毛虫(纤毛门,旋唇纲)的分子系统学研究

在最新GenBank分子信息的汇集、分析基础上,利用小亚基单位核糖体RNA基因序列分别构建了最大简约树、距离树、最大似然树及贝叶斯树,对长期存在争议的游仆目纤毛虫的系统关系进行了探讨和修订.工作显示:1)游仆目不是1个单源发生系;2)盘头虫类从游仆目中较早分化出来,呈现与排毛类、寡毛类平行的进化关系;3)腹棘虫科内的腹棘虫和拟游仆虫体现了密切的亲缘关系;4)游仆虫科、舍太虫科、檐纤虫科在所有分子树中均表现了十分稳定的拓扑结构;5)游仆虫属内可以细分为7个亚类群;6)尾刺虫科在游仆目内分化较早,支持前人有关其在系统演化时序中处于原始阶段的论断.     

A comparative study of ciliated protozoa communities in activated-sludge plants

Abstract: Ciliated protozoa present in ten activated sludge plants at Madrid (Spain) were identified. The abundance of key groups of ciliates was determined at each plant; attached ciliates made up the most abundant and representative group. Multivariate statistical analysis was employed to study relationships between ciliates and both the physico-chemical and operational parameters of the plants. Partial correlation analysis revealed: (1) The indicator value of attached ciliates in assessing management and performance of the activated sludge process, (2) the relationship of swimming ciliates with short-aged sludges and lower quality effluents and (3) the direct association between swimming-crawling ciliates and bad settlement conditions of the sludge. Factor analysis showed the associations of the most frequent species of ciliates with the operational parameters of the plants, suggesting the indicator value of some of the species: Vorticella striata was related with poor quality of effluent; Aspidisca cicada with stable plant conditions, and Litonotus lamella with a deficiently settling sludge.     

Re-examining alveolate evolution using multiple protein molecular phylogenies

Alveolates are a diverse group of protists that includes three major lineages: ciliates, apicomplexa, and dinoflagellates. Among these three, it is thought that the apicomplexa and dinoflagellates are more closely related to one another than to ciliates. However, this conclusion is based almost entirely on results from ribosomal RNA phylogeny because very few morphological characters address this issue and scant molecular data are available from dinoflagellates. To better examine the relationships between the three major alveolate groups, we have sequenced six genes from the non-photosynthetic dinoflagellate, Crypthecodinium cohnii: actin, beta-tubulin, hsp70, BiP, hsp90, and mitochondrial hsp10. Beta-tubulin, hsp70, BiP, and hsp90 were found to be useful for intra-alveolate phylogeny, and trees were inferred from these genes individually and in combination. Trees inferred from individual genes generally supported the apicomplexa-dinoflagellate grouping, as did a combined analysis of all four genes. However, it was also found that the outgroup had a significant effect on the topology within alveolates when using certain methods of phylogenetic reconstruction, and an alternative topology clustering dinoflagellates and ciliates could not be rejected by the combined data. Altogether, these results support the sisterhood of apicomplexa and dinoflagellates, but point out that the relationship is not as strong as is often assumed.     

Ciliate food vacuole content and bacterial community composition in the warm-monomictic crater Lake Alchichica, México

To evaluate trophic relationships between ciliates and bacterioplankton during the stratification period in a lake, samples from three different layers [the upper part of the metalimnion (UM), the base of the metalimnion (BM) and the hypolimnion] were studied. The autotrophic picoplankton numbers, phylogenetic composition of heterotrophic picoplankton (HPP), and HPP composition in ciliates' food vacuoles were analyzed. Additionally, in situ incubations in dialysis bags were performed at the same selected depths to assess potential changes in picoplankton composition related to ciliates' feeding activity. Among the in situ HPP assemblage, no phylogenetic group dominated in the selected layers within the course of the study. The ciliate assemblage was dominated by scuticociliates, haptorids, and hypotrichs. Comparing ciliates' food vacuole content and in situ HPP composition, a high preference for Alphaproteobacteria was found at all three depths. Planctomycetes, and Delta- and Gammaproteobacteria were selected at BM and hypolimnion, respectively. However, selection of a given phylogenetic group in the time course of this study was observed only for Alphaproteobacteria in the UM. Similar trends were found in the incubations, but no relationship was found between the vacuole content of the ciliates and changes in HPP composition.     

Evolution of discocephalid ciliates: Molecular,morphological and ontogenetic data support a sister group of discocephalids and pseudoamphisiellids (Protozoa,Ciliophora) with establishment of a new suborder Pseudoamphisiellina subord. n.

Discocephalids and pseudoamphisiellids are possibly two of the most confused groups among hypotrichous/euplotid ciliates regarding their systematic position and phylogenetic relationships. The former were often regarded as related to euplotids while the latter, in the absence of molecular data, were mostly assigned to the urostylid-like hypotrichs. In the present work, the small subunit rRNA genes of several rarely observed discocephalid and pseudoamphisiellid genera were analyzed to obtain insights into the phylogenetic relationships of these highly ambiguous Spirotrichea. Four different tree reconstruction algorithms yielded nearly identical topologies, which indicated both groups belong to the same assemblage. This assemblage is clearly isolated as a deep-branching clade and invariably positioned between Euplotida and Hypotricha. The sister group relationship of the Pseudoamphisiellidae and Discocephalidae supports the previous suggestion that they might represent an ordinal taxon, the Discocephalida. Both morphological and morphogenetic features indicate that the pseudoamphisiellids should be placed in the order Discocephalida but as a sister group to other typical discocephalids. Thus we propose establishing a new suborder, Pseudoamphisiellina subord. n. The new taxon is diagnosed by the following characteristics: (i) two distantly separated midventral rows that are morphogenetically formed with an urostylid mode; (ii) absence of the “frontoterminal row”, which is formed from the posterior-most frontoventral-transverse cirral anlage in all other typical urostylids; (iii) numerous caudal cirri that derive from each of the dorsal kinety anlagen; (iv) right marginal row that has a unique de novo origin; and (v) inhabiting periphytic communities. The validity of the suborder Pseudoamphisiellina is firmly supported by molecular data.     

Phylogenetic relationships within the genus Tetrahymena inferred from the cytochrome c oxidase subunit 1 and the small subunit ribosomal RNA genes

Details of the phylogenetic relationships among tetrahymenine ciliates remain unresolved despite a rich history of investigation with nuclear gene sequences and other characters. We examined all available species of Tetrahymena and three other tetrahymenine ciliates, and inferred their phylogenetic relationships using nearly complete mitochondrial cytochrome c oxidase subunit 1 (cox1) and small subunit (SSU) rRNA gene sequences. The inferred phylogenies showed the genus Tetrahymena to be monophyletic. The three “classical” morphology-and-ecology-based groupings are paraphyletic. The SSUrRNA phylogeny confirmed the previously established australis and borealis groupings, and nine ribosets. However, these nine ribosets were not well supported. Using cox1 gene, the deduced phylogenies based on this gene revealed 12 well supported groupings, called coxisets, which mostly corresponded to the nine ribosets. This study demonstrated the utility of cox1 for resolving the recent phylogeny of Tetrahymena, whereas the SSU rRNA gene provided resolution of deeper phylogenetic relationships within the genus.     

Molecular phylogeny of the cyrtophorid ciliates (Protozoa, Ciliophora, Phyllopharyngea)

Evolutionary relationships of cyrtophorian ciliates are poorly known because molecular data of most groups within this subclass are lacking. In the present work, the SS rRNA genes belonging to 17 genera, 7 families of Cyrtophoria were sequenced and phylogenetic trees were constructed to assess their inter-generic relationships. The results indicated: (1) the assignment of cyrtophorians into two orders is consistently confirmed in all topologies; (2) the order Dysteriida is an outlined monophyletic assemblage while Chlamydodontida is paraphyletic with three separate monophyletic families; (3) Microxysma, which is currently assigned within the family Hartmannulidae, should be transferred to the family Dysteriidae; (4) the systematic position of Plesiotrichopidae remains unclear, yet the two genera that were placed in this family before, Pithites and Trochochilodon, should be transferred to Chlamydodontida; (5) a new family, Pithitidae n. fam., based on the type genus Pithites was suggested; and (6) the sequence of Isochona sp., the only available data of Chonotrichia so far, is probably from a misidentified species. In addition, three group I introns of SS rRNA gene were discovered in Aegyriana oliva, among which Aol.S516 is the first IE group intron reported in ciliates.     

Molecular evolution of the 5'-terminal domain of large-subunit rRNA from lower eukaryotes. A broad phylogeny covering photosynthetic and non-photosynthetic protists

This paper summarizes the present status of an analysis of protist phylogeny using rapid partial sequencing of 28S rRNA. Data from 12 protistan phyla are now available and have been used to construct a tentative dendrogram based on a distance matrix method. The tree is robust and has considerable internal consistency. The following salient points are observed: a number of flagellate groups (particularly Euglenozoa) emerge very early among eukaryotes, whereas ciliates and dinoflagellates emerge late, suggesting that some characteristics that had been considered as primitive may in fact be derived. Both chlorophytic and chromophytic photosynthetic protists emerge very late in the tree, close to the Metazoa-Metaphyta-Fungi radiation, suggesting relatively late occurrence of the photosynthetic symbiosis. Taxonomic and phylogenetic information is also obtained within a phylum where rRNA of enough species are sequenced. A deep trichotomy is thus observed within the ciliates. The data are discussed with respect to classical protist phylogenies.     

Phylogenetic Relationships of the Nassulida Within the Phylum Ciliophora Inferred from the Complete Small Subunit rRNA Gene Sequences of Furgasonia blochmanni, Obertrumia georgiana, and Pseudomicrothorax dubius

ABSTRACT. Using comparisons of complete small subunit rRNA sequences from the ciliated protozoans Furgasonia blochmanni, Obertrumia georgiana , and Pseudomicrothorax dubius we inferred the phylogenetic position of the Nassulida (Class Nassophorea) within the Ciliophora. In distance matrix analyses the Nassulida share a common ancestry with the colpodean ciliate Colpoda inflata. Distance matrix and parsimony methods convincingly demonstrate that the Nassulida plus Colpodida are members of a complex ciliate assemblage that also includes the oligohymenophorans and phyllopharyngeans. These phylogenetic inferences are largely congruent with recent analyses of 23S-like rRNA gene sequences and morphogenetic features. Groups traditionally thought to represent ancestral lineages now appear as highly derived ciliates. In contrast, heterotrichs which were considered to represent a highly evolved group, diverge at the base of the ciliates.     

Relationship between the flagellates and the ciliates.

The flagellates and the ciliates have long been considered to be closely related because of their unicellular nature and the similarity in the structures of the axoneme of the flagella and cilia in both groups. Most protozoologists believe that the ciliates arose from a flagellate. The flagellates that are most similar in structure to the ciliates are the dinoflagellates and two genera of uncertain taxonomic position, Colponema and Katablepharis. Structurally, dinoflagellates have a number of similarities with ciliates. These include the similarity of the cortical alveoli in the ciliates to the thecal vesicles in the dinoflagellates, the possession of tubular cristae, the similarity of the parasomal sac of the ciliates to the pusule of the dinoflagellates, the possession of similar trichocysts and mucocysts, and some similarity in the feeding apparatus. Colponema spp. are probably related to the dinoflagellates and have many of the same similarities with the ciliates. Katablepharis spp. are very similar in structure to the swarmer (embryo) of the suctorian ciliates. Indeed, reduction in the number of cilia to two in the suctorian swarmer and elimination of the macronucleus would result in a cell that is very similar to the Katablepharis cell. The feeding apparatus of Katablepharis spp. and the rest of the ciliates consists of two concentric microtubular arrays associated with vesicles. Information available from nucleotide sequencing of rRNA places the dinoflagellates in an ancestral position to the ciliates. The rRNA of Colponema and Katablepharis spp. has not yet been investigated. The use of stop codons in mRNA is discussed in relation to phylogeny.