Class Spirotrichea Research Articles

Large-scale phylogenomic analysis reveals the phylogenetic position of the problematic taxon Protocruzia and unravels the deep phylogenetic affinities of the ciliate lineages

The Ciliophora is one of the most studied protist lineages because of its important ecological role in the microbial loop. While there is an abundance of molecular data for many ciliate groups, it is commonly limited to the 18S ribosomal RNA locus. There is a paucity of data when it comes to availability of protein-coding genes especially for taxa that do not belong to the class Oligohymenophorea. To address this gap, we have sequenced EST libraries for 11 ciliate species. A supermatrix was constructed for phylogenomic analysis based on 158 genes and 42,158 characters and included 16 ciliates, four dinoflagellates and nine apicomplexans. This is the first multigene-based analysis focusing on the phylum Ciliophora. Our analyses reveal two robust superclades within the Intramacronucleata; one composed of the classes Spirotrichea, Armophorea and Litostomatea (SAL) and another with Colpodea and Oligohymenophorea. Furthermore, we provide corroborative evidence for removing the ambiguous taxon Protocruzia from the class Spirotrichea and placing it as incertae sedis in the phylum Ciliophora.

Pyrosequencing for assessing diversity of eukaryotic microbes: analysis of data on marine planktonic ciliates and comparison with traditional methods

Assessing microbial diversity requires analysis of all three domains of life, including eukaryotic microbes. We examined the diversity of two ecologically important clades of microbial eukaryotes, ciliates in the subclasses Oligotrichia and Choreotrichia (class Spirotrichea), by comparing pyrosequencing to Sanger-sequenced clone libraries and microscopy. Using samples from a large temperate estuary (Long Island Sound, USA), we gained three major insights. First, richness estimates varied by up to one order of magnitude either using different criteria for pyrosequence processing or among pyrosequencing, cloning and microscopy, while taxon identification was almost always coherent. Error-correcting algorithms for pyrosequences ('denoising') reduced discrepancies in richness but also removed known morphospecies from the data. Second, although most of the pyrosequenced operational taxonomic units (OTUs) were distributed within known orders and families, we found evidence of a previously uncharacterized or unknown clade even in these ciliate lineages that have a rich history of morphological description. Third, pyrosequencing allowed the detection of OTUs that were either dominant or extremely rare in different samples. Our findings confirm the potential of pyrosequencing for quantifying microbial diversity, but also highlight the importance of careful evaluation of pyrosequence processing for using this method to address ecological questions.

Variations and evolution of polyubiquitin genes from ciliates

Polyubiquitin genes from seven ciliate species were amplified, cloned and sequenced. It is estimated that Strombidium sulcatum, Euplotes vannus, E. rariseta and Anteholosticha manca have a polyubiquitin gene of 3 repeats, and A. parawarreni, Paramecium caudatum and Pseudokeronopsis flava 4 repeats. The newly obtained ubiquitins mostly differ from that of humans by 1–5 residues in amino acid sequences. A neighbor-joining tree constructed based on monomeric ubiquitin genes supports the monophyly of an assemblage comprising the litostomateans and some oligohymenophoreans, but not the class Spirotrichea. The monomers from the same species are generally placed together and highly supported for the class Litostomatea, the genera Paramecium and Ichthyophthirius, but not for other species. The non-synonymous/synonymous rate ratio (dN/dS) at the protein level are less than 1, and the synonymous nucleotide differences per synonymous site (pS) from intraspecific comparisons are fairly high (0.02–0.72). These results indicate that ciliates have not only the conserved, but also some quite divergent, polyubiquitin genes and confirm that the polyubiquitin genes in ciliates evolve according to the birth-and-death mode of evolution under strong purifying selection.

Characterization and Taxonomic Validity of the Ciliate Oxytricha trifallax (Class Spirotrichea) Based on Multiple Gene Sequences: Limitations in Identifying Genera Solely by Morphology

Oxytricha trifallax - an established model organism for studying genome rearrangements, chromosome structure, scrambled genes, RNA-mediated epigenetic inheritance, and other phenomena - has been the subject of a nomenclature controversy for several years. Originally isolated as a sibling species of O. fallax, O. trifallax was reclassified in 1999 as Sterkiella histriomuscorum, a previously identified species, based on morphological similarity. The proper identification of O. trifallax is crucial to resolve in order to prevent confusion in both the comparative genomics and the general scientific communities. We analyzed nine conserved nuclear gene sequences between the two given species and several related ciliates. Phylogenetic analyses suggest that O. trifallax and a bona fide S. histriomuscorum have accumulated significant evolutionary divergence from each other relative to other ciliates such that they should be unequivocally classified as separate species. We also describe the original isolation of O. trifallax, including its comparison to O. fallax, and we provide criteria to identify future isolates of O. trifallax.

Phylogenetic relationships within the genus Aspidisca (Protozoa, Ciliophora, Euplotida) revealed by ITS1-5.8S-ITS2 region sequences

The internal transcribed spacer regions (ITS1 and ITS2) and 5.8S rRNA genes were sequenced in six populations of four Aspidisca species, namely A. leptaspis, A. orthopogon, A. magna and A. aculeata. Phylogenetic trees were constructed by means of Bayesian inference (BI), Maximum Parsimony (MP), Neighbor-Joining (NJ), and Maximum Likelihood (ML) to assess the inter- and intra-species relationships within the genus Aspidisca. All trees show similar topologies with stable supports and indicate that: (1) four well known groups, i.e., Oligotrichia, Stichotrichia, Choreotrichia and Hypotrichia, are distinctly outlined within the class Spirotrichea, and all are monophyletic other than Hypotrichia; (2) members of Aspidisca can be distinguished well, based on the ITS1-5.8S-ITS2 region sequences, and A. leptaspis and A. magna shared a closer relationship than other species; (3) Aspidisca and Euplotes branch early in the subclass Hypotrichia. To compare the phylogenetic relationships based on different genes, SSU rRNA trees were also constructed with nearly the same species inclusion, which revealed different topologies of inter-species, inter-genera and inter-subclasses.

Alternative processing of scrambled genes generates protein diversity in the ciliate Chilodonella uncinata

In ciliates, chromosomal rearrangements occur during the development of the somatic macronuclear genome from the germline micronuclear genome. These rearrangements are extensive in three ciliate classes-Armophorea, Spirotrichea, and Phyllopharyngea-generating a macronucleus with up to 20,000,000 gene-sized chromosomes. Earlier, we have shown that these three classes also share elevated rates of protein evolution relative to other ciliates. To assess the evolution of germline-limited sequences in the class Phyllopharyngea, we used a combination of traditional and walking PCR to analyze micronuclear copies of multiple genes from two lines of the morphospecies Chilodonella uncinata for which we had previously characterized macronuclear sequences. Analyses of the resulting data yield three main results: (1) conserved macronuclear (somatic) regions are found within rapidly evolving micronuclear (germline) regions; (2) gene scrambling exists within this ciliate lineage; and (3) alternative processing of micronuclear regions yields diverse macronuclear beta-tubulin paralogs. To our knowledge, this is the first study to demonstrate gene scrambling outside the nonsister class Spirotrichea, and to show that alternative processing of scrambled genes generates diversity in gene families. Intriguingly, the Spirotrichea and Phyllopharyngea are also united in having transient "giant" polytene chromosomes, gene-sized somatic chromosomes, and elevated rates of protein evolution. We hypothesize that this suite of characters enables these ciliates to enjoy the benefits of asexuality while still maintaining the ability to go through sexual cycles. The data presented here add to the growing evidence of the dynamic nature of eukaryotic genomes within diverse lineages across the tree of life.

Protocruzia, a highly ambiguous ciliate (Protozoa; Ciliophora): Very likely an ancestral form for Heterotrichea, Colpodea or Spirotrichea? With reevaluation of its evolutionary position based on multigene analyses

The ciliate genus Protocruzia belongs to one of the most ambiguous taxa considering its systematic position, possible as a member of the classes Heterotrichea, Spirotrichea or Karyorelictea, which is tentatively placed into Spirotrichea in Lynn's 2008 system. To test these hypotheses, multigene trees (Bayesian inference, evolutionary distance, maximum parsimony, and maximum likelihood) were constructed using the small subunit rRNA (SSU rRNA) gene, internal transcribed spacer 2 (ITS2) and a protein coding gene (histone H4). All analyses agree that: (1) four morphotypes of Protocruzia from different geographical origins group together and form a monophyletic clade, which cannot be assigned to any of the eleven described ciliate classes; (2) it is invariably positioned on an isolated branch separated from the class Spirotrichea suggesting that this clade should be clearly removed from Spirotrichea; (3) this leads us to hypothesize that this taxon may indeed represent a lineage on a class rank. Based on the fact that it is, both morphologically and in molecular features, closely related to heterotrichs, Colpodea and Oligohymenophorea, Protocruziida might be an ancestral form for the subphylum Intramacronucleata in the evolutionary line from the class Heterotrichea (subphylum Postciliodesmatophora) to higher taxa.

Morphogenesis of Kiitricha marina Nozawa, 1941 (Ciliophora, Spirotrichea), a possible model for the ancestor of hypotrichs s. l.

Recent studies suggested that the marine ciliate Kiitricha Nozawa, 1941 represents a basal branch in the spirotrichs. The present study reflects the morphogenetical investigation of Kiitricha marina Nozawa, 1941. The results listed below show an extremely unusual developmental pattern compared with all other known forms in the class Spirotrichea or hypotrichs s. l. (i) The differentiation of the ciliary structures from the primordia occurs after cell division. This makes Kiitricha marina Nozawa, 1941 unique among hypotrichs s. l. (ii) Since the anlage of the undulating membranes in the opisthe derives from the division of the dedifferentiated parental structure and thus is not formed independently, Kiitricha marina Nozawa, 1941 is also unique among spirotrichs. (iii) Most somatic cirral structures appear in a post-anlage pattern, i.e. all cirri exhibit a primary arrangement in the sense that they are distributed as though they had just been segregated from the anlagen. (iv) Unlike most other known hypotrichs s. l., the undulating membrane-anlage does not contribute a frontal cirrus. (v) The dorsal kineties are formed in two ways: (a) from the posterior portion of the cirral anlagen which later generate the new dorsal kinety segments; and (b) by the irregular proliferation of dikinetids in the long dorsal kineties. (vi) All cirral anlagen are formed intrakinetally from the dedifferentiated parental cirri and develop in a primary mode. Taken collectively, these findings suggest that Kiitricha is a basal branching hypotrich and therefore a possible model for the ancestor of the hypotrichs s. l.

Does Kiitricha (Protista, Ciliophora, Spirotrichea) belong to Euplotida or represent a primordial spirotrichous taxon? With suggestion to establish a new subclass Protohypotrichia

The genus Kiitricha was long assumed to be the most primordial taxon in the Stichotrichia [hypotrichs sensu lato (s. l.)] based on its morphological features and was considered to be an intermediate between heterotrichs and the traditional hypotrichous assemblage. In order to evaluate the phylogenetic position of Kiitricha within the Hypotrichia, we sequenced the small-subunit rRNA gene and the alpha-tubulin gene for a Qingdao population of Kiitricha marina. Phylogenetic trees were constructed and compared to morphological and morphogenetic data. The results show that (i) Kiitricha is positioned near Phacodinium, both of which always form a sister clade to the assemblage including Stichotrichia, Hypotrichia, Oligotrichia and Choreotrichia, (ii) Kiitricha, which may represent an intermediate between heterotrichs (s. l.) and the Stichotrichia-Hypotrichia complex, is probably an ancestor-like form of the latter group and (iii) in contrast to morphological characters, both molecular and ontogenetic data support the separation of Kiitricha from the hypotrichs (s. l.). Thus, Kiitricha might be placed in the class Spirotrichea at about subclass level, next to Phaconidiidia, Hypotrichia and Stichotrichia, which supports the establishment of a new subclass Protohypotrichia n. subclass within the class Spirotrichea, with characterizations including slightly differentiated somatic ciliature (i.e. cirri on the ventral side generally uniform and non-grouped, no clearly defined marginal cirral rows, ciliature on the dorsal side mixed with cirri and dikinetids, no clearly differentiated dorsal kineties) and a unique but intermediate morphogenetic pattern of cortical structures between Hypotrichia and Stichotrichia.

First records of soil ciliates (Protozoa, Ciliophora) from classes Prostomea, Nassophorea, Spirotrichea, and Colpodea in Slovakia

In total, seven ciliate species were recorded in leaf-litter, moss and soil from a variety of sites in Slovakia for the first time: Chilophrya terricola Foissner, 1984; Holostichides typicus (Song et Wilbert, 1988) Eigner, 1994; Keronella gracilis Wiackowski, 1985; Notoxoma parabryophryides Foissner, 1993; Parafurgasonia sorex (Penard, 1922) Foissner et Adam, 1981; Paragonostomum multinucleatum Foissner, Agatha et Berger, 2002, and Territricha stramenticola Berger et Foissner, 1988. The paper deals with their distribution, ecology, and comparison with similar species. The shape and nuclear variants of Paragonostomum multinucleatum are presented and populations of P. multinucleatum and T. stramenticola are morphometrically characterized.

Unusual tubulins with deviant genetic code in the hypotrich ciliate Euplotidium itoi

We characterized nine macronuclear tubulin gene minichromosomes in the hypotrich ciliate Euplotidium itoi: two α‐tubulin genes, five β‐tubulin genes, and two γ‐tubulin genes. In particular, one peculiar β‐tubulin gene was identified. It shows only 81.5% amino acid similarity to the other characterized β‐tubulin types and an UAG stop codon in frame. Based on this result we started to analyse stop codon usage in Euplotidium itoi macronuclear tubulin genes and some unrelated macronuclear minichromosomes. Most of the ciliates independently evolved diverse non‐canonical genetic codes. Differences to the standard code consist in the reassignment of one or two of the three standard stop codons to encode a particular amino acid. Class Spirotrichea represents a particular case comprising two groups of organisms that do not share any stop codon. In oxytrichids, UAR is translated as glutamine and UGA is the single stop codon. This is in contrast to the genus Euplotes, close relatives of Euplotidium, in which UAR encodes a stop signal and UGA is translated as cysteine. Fourteen complete genes were characterized in Euplotidium itoi; in all cases UAA revealed as the single triplet used to code a stop, UGA was never and UAG rarely observed in frame. The UAG stop codon was observed in the peculiar type 3 β‐tubulin gene, in two dynein partial sequences and in some other putative genes. We could confirm the active expression of three of these genes, the unusual β‐tubulin, a putative lipase and a possible steroid‐binding protein, by RT‐PCR. Finally, a detailed analysis on the structure of the macronuclear tubulin genes, including nucleotide and amino acid differences among tubulin gene paralogs, polyadenylation sites, putative chromosome fragmentation sites, ATGC‐content in coding and non‐coding regions, telomere sequence and length will be presented.

Polymorphism, recombination and alternative unscrambling in the DNA polymerase alpha gene of the ciliate Stylonychia lemnae (Alveolata; class Spirotrichea).

DNA polymerase alpha is the most highly scrambled gene known in stichotrichous ciliates. In its hereditary micronuclear form, it is broken into >40 pieces on two loci at least 3 kb apart. Scrambled genes must be reassembled through developmental DNA rearrangements to yield functioning macronuclear genes, but the mechanism and accuracy of this process are unknown. We describe the first analysis of DNA polymorphism in the macronuclear version of any scrambled gene. Six functional haplotypes obtained from five Eurasian strains of Stylonychia lemnae were highly polymorphic compared to Drosophila genes. Another incompletely unscrambled haplotype was interrupted by frameshift and nonsense mutations but contained more silent mutations than expected by allelic inactivation. In our sample, nucleotide diversity and recombination signals were unexpectedly high within a region encompassing the boundary of the two micronuclear loci. From this and other evidence we infer that both members of a long repeat at the ends of the loci provide alternative substrates for unscrambling in this region. Incongruent genealogies and recombination patterns were also consistent with separation of the two loci by a large genetic distance. Our results suggest that ciliate developmental DNA rearrangements may be more probabilistic and error prone than previously appreciated and constitute a potential source of macronuclear variation. From this perspective we introduce the nonsense-suppression hypothesis for the evolution of ciliate altered genetic codes. We also introduce methods and software to calculate the likelihood of hemizygosity in ciliate haplotype samples and to correct for multiple comparisons in sliding-window analyses of Tajima's D.

Phylogenetic Relationships within the Class Spirotrichea (Ciliophora) Inferred from Small Subunit rRNA Gene Sequences

The small subunit rDNAs of five species belonging to the Euplotidae and eight species of the Oxytrichidae were sequenced to obtain a more detailed picture of the phylogenetic relationships within the Spirotrichea (Ciliophora). Various tree reconstruction algorhythms yielded nearly identical topologies. All Euplotidae were separated from the other Spirotrichea by a deep split. Further, a large genetic distance between the marine genus Moneuplotes and the freshwater species of Euplotoides was found. Differences between the methods used occurred only within the Oxytrichidae. Whereas the monophyly of the Stylonychinae was supported in all trees, the monophyly of the Oxytrichinae was not. However, the molecular data support the morphological and ontogenetic evidence that the pattern of 18 frontal–ventral–transversal cirri evolved in the stemline of the Oxytrichidae and was modified several times independently. Our results are also in agreement with taxonomic revisions: the separation of both Sterkiella nova from Oxytricha and Tetmemena pustulata from Stylonychia.

Widespread distribution of extensive chromosomal fragmentation in ciliates.

Ciliates are a diverse group of eukaryotes characterized by their division of nuclear function into a "germ line" micronucleus and a "somatic" macronucleus. After conjugation, chromosomes in the transcriptionally active macronucleus develop by fragmentation, elimination, and amplification of germ line chromosomes. Extensive chromosomal processing that generates a macronucleus with gene-sized fragments has thus far been well documented in members of only one class of ciliates, the Spirotrichea. Here we establish the broad distribution of extensive fragmentation among members of the class Phyllopharyngea and the genera Metopus (order Armophorida) and Nyctotherus (order Clevelandellida). Moreover, analyses of small-subunit rDNA genealogies indicate that gene-sized chromosomes occur in members of the three separate clades: (1) the class Spirotrichea, (2) the class Phyllopharyngea, and (3) the two orders Clevelandellida and Armophorida. Together, these data indicate that the generation of gene-sized chromosomes is widespread and demonstrate multiple origins of extensive fragmentation within ciliates.

Phylogenetic position of the ciliates Phacodinium (Order Phacodiniida) and Protocruzia (Subclass Protocruziidia) and systematics of the spirotrich ciliates examined by small subunit ribosomal RNA gene sequences

The small subunit rRNA (SSrRNA) genes were sequenced for Protocruzia sp2, Phacodinium metchnicoffi, Holosticha multistylata, and Halteria grandinella. All four genera are placed within the Class Spirotrichea with strong bootstrap support in both distance matrix and parsimony tree construction methods and by maximum likelihood analysis using quartet puzzling. Protocruzia sp2 groups with Protocruzia sp1 with 100% bootstrap support, and the 5.6% genetic difference between them strongly argues that they are different species although they are morphologically quite similar. The Protocruzia species branch first in the spirotrich clade at a deep level, supporting their recognition as a Subclass Protocruziidia. Phacodinium branches after Euplotes at a deep level, confirming the conclusion that others have reached that this genus is related to the hypotrich and stichotrich spirotrichs. Phacodinium is assigned to its own family and order, and we conclude that the deep branching within the spirotrichs argues for its own subclass, the Subclass Phacodiniidia. Consistent with the partial SSrRNA sequences and with the sequence of a polymerase gene, Halteria groups within the stichotrich clade, supporting the argument that the oligotrichs are not monophyletic as currently conceived. Finally, Holosticha, which has been assigned to the Order Urostylida, groups outside the stichotrichs with parsimony analysis, which is consistent with this ordinal assignment. However, it is associated with Halteria and Oxytricha granulifera in the other analyses. Additional stichotrich sequences obviously are required before we can confidently begin revision of the Subclass Stichotrichia.

The Salient Features of Five Marine Ciliates in the Class Spirotrichea (Oligotrichia), with Notes on their Culturing and Behaviour

ABSTRACTDescriptive information is provided for five ciliates collected from marine coastal British Columbian surface waters. In this paper we identify three new species, Strobilidium neptuni n. sp., Strobilidium veniliae n. sp., and Strombidinopsis multiauris n. sp.; provide a new name and description for one previously described species, Strombidium siculum nom. nov. and provide more taxonomic data for Strombidinopsis cheshiri Snyder and Ohman, 1991.

Metopus, Cyclidium and Sonderia: ciliates enriched and cultured from sulfureta of a microbial mat community

Metopus contortus (Class Spirotrichea), Cyclidium borrori, Cyclidium sp., Sonderia tubigula, and two other species of Sonderia (Class Oligohymenophorea) were enriched from the sulfureta of microbial mat communities at Laguna Figueroa near San Quintin, Baja California, Mexico. The ability of some of these species to grow under anaerobic, high-sulfide conditions was demonstrated. The ultrastructural morphology of Metopus and Cyclidium revealed adaptations to anaerobic, high sulfide niches. Metopus lacks typical mitochondria and has structures which may be intracellular bacterial symbionts. Cyclidium borrori, when grown aerobically, has mitochondria with tubular cristae. When it is grown anaerobically in the presence of high amounts of hydrogen sulfide the number of mitochondria remains the same but the mitochondria dedifferentiate, becoming smaller with less well-defined cristae.