Molecular characterization and revised systematics of Microdiaphanosoma arcuatum (Ciliophora, Colpodea)

Microdiaphanosoma arcuatum Wenzel, 1953 is a world-wide distributed ciliate, recorded mainly in soil samples, which we have also identified in ground water samples from South Africa. This ciliate has been frequently overlooked or not determined due to its small size, ～12 μm. The genus Microdiaphanosoma is nowadays included in the class Colpodea, order Bryometopida, family Kreyellidae. The first complete small subunit (SSU) rDNA gene sequence of this ciliate was obtained from a South African isolate. Phylogenetic analysis including available SSU rDNA sequences from another Colpodea species in the GenBank strongly supported the position of M. arcuatum within the order Cyrtolophosidida instead of the order Bryometopida. The analysis also suggested a sister relationship between this species and species from the family Cyrtolophosididae.     

Phylogeny and Systematic Position of Zosterodasys (Ciliophora, Synhymeniida): A Combined Analysis of Ciliate Relationships Using Morphological and Molecular Data

ABSTRACT. The Synhymeniida is characterized both by a band of somatic dikinetids, the synhymenium, extending across the surface of the cell and by a ventral cell mouth lacking specialized feeding cilia but subtended by a well-developed cyrtos. The synhymeniids have been hypothesized to be members of the class Nassophorea but our previous ultrastructural study of the synhymeniid genus Zosterodasys did not show any clear synapomorphies that would permit definitive placement in the Nassophorea or as a sister taxon to any of the other ciliate groups possessing a cyrtos. In the present study, simultaneous analysis of morphological and small subunit rDNA molecular data indicates that the Synhymeniida are sister to the class Phyllopharyngea and that this clade is, in turn, sister to the remaining Nassophorea, although this result is sensitive to dataset inclusion and alignment parameters. While this suggests that taxa with a ventral cyrtos might be united into a named taxon (e.g. resurrecting the Hypostomata), additional data are needed to reach a definitive conclusion.     

Molecular phylogenetic analysis of class Colpodea (phylum Ciliophora) using broad taxon sampling

The ciliate class Colpodea provides a powerful case in which a molecular genealogy can be compared to a detailed morphological taxonomy of a microbial group. Previous analyses of the class using the small-subunit rDNA are based on sparse taxon sampling, and are therefore of limited use in comparisons with morphologically-based classifications. Taxon sampling is increased here to include all orders within the class, and more species within previously sampled orders and in the species rich genus Colpoda. Results indicate that the Colpodea may be paraphyletic, although there is no support for deep nodes. The orders Bursariomorphida, Grossglockneriida, and Sorogenida are monophyletic. The orders Bryometopida, Colpodida, and Cyrtolophosidida, and the genus Colpoda, are not monophyletic. Although congruent in many aspects, the conflict between some nodes on this single gene genealogy and morphology-based taxonomy suggests the need for additional markers as well as a reassessment of the Colpodea taxonomy.     

Re-analysis of the 18S rRNA gene phylogeny of the ciliate class Colpodea

We critically re-analyzed the 18S rRNA gene phylogeny of the ciliate class Colpodea where four main lineages have been recognized: (1) Bursariomorphida including bryometopids, (2) Platyophryida including sorogenids, (3) Cyrtolophosidida, and (4) Colpodida including bryophryids and grossglockneriids. The Platyophryida branched off first and the Cyrtolophosidida and Colpodida were classified as sister groups. On basis of multiple statistical tests, we unraveled three problematic issues in colpodean phylogenies: the positions of the Bursariomorphida and Platyophryida are unstable and depend on alignment masking; a sister relationship of the Platyophryida and Cyrtolophosidida cannot be excluded by any statistical tree topology test; and clustering of bryophryids and grossglockneriids outside the Colpodida are also statistically valid possibilities. Natural classification of the highly diverse order Colpodida remains puzzling, possibly due to the lack of a phylogenetic signal and morphostasis of the oral ciliature in several Colpoda-like lineages. According to the “Ur-Colpoda” hypothesis, Colpoda represents the stem lineage from which both Colpoda-like and morphologically more derived taxa might have branched off. This evolutionary concept preserves not only information on morphology, ecology, and evolutionary processes of colpodid ciliates, but also aids practicability because the connection to the traditional literature is optimally maintained.     

Small Subunit rRNA Phylogenies Show that the Class Nassophorea is Not Monophyletic (Phylum Ciliophora)

ABSTRACT. The hypostome ciliates have been generally classified into two classes, Phyllopharyngea and Nassophorea. The status of Nassophorea and its relationship with Phyllopharyngea is one of the most controversial issues in ciliate systematics. Here we focus on the phylogenetic interrelationships of Nassophorea and Phyllopharyngea based on small subunit ribosomal RNA gene sequences. The three nassophorean subgroups, synhymeniids, microthoracids, and nassulids, each emerged as monophyletic, with synhymeniids as a sister group of Phyllopharyngea, and microthoracids as a sister of the synhymeniids+Phyllopharyngea clade in all phylogenies. The exact placement of the nassulids, however, remains uncertain. Following a detailed analysis of phenotypic characters, we hypothesize that: (1) the Phyllopharyngea could have evolved from synhymeniids, with the further development of their subkinetal microtubules as one of the major events; and (2) the development of monokinetid structures, as well as the reduction and specialization of the cyrtos and cortex, might have occurred during the diversifications of the microthoracids, synhymeniids, and Phyllopharyngea from a common ancestor. Expanding the class Phyllopharyngea to include the synhymeniids as a subclass, and designating a new subclass Subkinetalia n. subcl. for the group comprising cyrtophorians, chonotrichians, rhynchodians, and suctorians, are proposed.     

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,possibly 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 the 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.     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.     

Phylogenetic analyses of various euglenoid taxa (euglenozoa) based on 18s rdna sequence data

18S rRNA genes (SSU rDNA) of five newly sequenced species were used as molecular markers to infer phylogenetic relationships within the euglenoids. Two members of the order Euglenales ( Lepocinclis ovata Playfair , Phacus similis Christen), two of the order Eutreptiales ( Distigma proteus Ehrenberg, , D. curvata Pringsheim) and Gyropaigne lefévrei Bourelly et Georges of the order Rhabdomonadales were used in parsimony, maximum likelihood, and distance analyses. All trees derived from SSU rRNA data strongly supported the monophyletic origin of the Euglenozoa, with kinetoplastids as sister clade to the euglenoids and Petalomonas cantuscygni Cann et Pennick diverging at the base of the monophyletic euglenoid lineage. The data also supported the theory that phagotrophic euglenoids arose prior to osmotrophs and phototrophs. A lineage of Peranema trichophorum Ehrenberg and all sequenced Euglenales formed a sister clade to the osmotrophs. This suggests that the evolution of phototrophy within the euglenoids radiated from a single event.     

Polymorphism and Ultrastructure of a Colpodean Ciliate of the Genus Platyophryides Foissner, 1987

ABSTRACT. A ciliate isolated from a pond in Brazil, transformed to a giant form when its food was shifted from a bacterial prey to a ciliate prey. This polymorphism is immediately reversible when the prey ciliates, either Tetrahymena or Colpidium , disappear from the culture medium. By its life cycle, morphology, and ultrastructure, this ciliate belongs to the Class Colpodea. it could belong to the genus Platyophryides Foissner, 1987, except that its micronucleus is not enveloped by the macronuclear membrane. The systematic position of the genus Platyophryides , the validity of the three species in this genus, and the characteristics of the Cyrtolophosidida are discussed.     

Phylogenetic relationships among Syndermata inferred from nuclear and mitochondrial gene sequences

Phylogenetic relationships among Syndermata have been extensively debated, mainly because the sister-group of the Acanthocephala has not yet been clearly identified from analyses of morphological and molecular data. Here we conduct phylogenetic analyses on samples from the 4 classes of Acanthocephala (Archiacanthocephala, Eoacanthocephala, Polyacanthocephala, and Palaeacanthocephala) and the 3 Rotifera classes (Bdelloidea, Monogononta, and Seisonidea). We do so using small-subunit (SSU) and large-subunit (LSU) ribosomal DNA and cytochrome c oxidase subunit 1 (cox 1) sequences. These nuclear and mitochondrial DNA sequences were obtained for 27 acanthocephalans, 9 rotifers, and representatives of 6 phyla that were used as outgroups. Maximum parsimony (MP), maximum likelihood (ML), and Bayesian analyses were conducted on the nuclear rDNA(SSU+LSU) and the combined sequence dataset(SSU+LSU+cox 1 genes). Phylogenetic analyses of the combined rDNA and cox 1 data uniformly provided strong support for a clade including rotifers plus acanthocephalans (Syndermata). Strong support was also found for monophyly of Acanthocephala in analyses of the combined dataset or rDNA sequences alone. Within the Acanthocephala the monophyletic grouping of the representatives of each class was strongly supported. Our results depicted Archiacanthocephala as the sister-group to the remaining acanthocephalans. Analyses of the combined dataset recovered a sister-group relationship between Acanthocephala and Bdelloidea by parsimony, likelihood, and Bayesian methods. Support for this clade was generally strong. Alternative topologies that depicted a different rotifer sister-group of Acanthocephala (or monophyly of Rotifera) were significantly worse. In this paraphyletic assemblage of rotifers, the relative positions of Seisonidea and Monogononta to the clade Bdelloidea+Acanthocephala were inconsistent among trees based on different inference methods. These results indicate that Bdelloidea is the free-living sister-group to acanthocephalans, which should prove key for comparative investigations of the morphological, molecular, and ecological changes accompanying the evolution of parasitism.     

Cultivation-independent analysis reveals a shift in ciliate 18S rRNA gene diversity in a polycyclic aromatic hydrocarbon-polluted soil

Using cultivation-independent methods the ciliate communities of a clay-rich soil with a 90-year record of pollution by polycyclic aromatic hydrocarbons (PAH) (4.5 g kg(-1) PAH) were compared with that of a nonpolluted soil collected in its vicinity and with similar properties. A ciliate-specific set of 18S rRNA gene targeting primers was designed and used to amplify DNA extracted from both soils (surface and 20 cm depth). Four clone libraries were generated with PCR products that covered an 18S rRNA gene fragment of up to 670 bp. Comparative sequence analysis of representative clones proved that the primer set was highly specific for ciliates. Calculation of similarity indices based on operational taxonomic units after amplified ribosomal DNA restriction analysis of the clones showed that the community from the nonpolluted surface soil was highly dissimilar to the other communities. The presence of several taxa, namely sequences affiliated to the orders Phyllopharyngia, Haptoria, Nassophorea, Peniculida and Scuticociliatia in samples from nonpolluted soil, points to the existence of various trophic functional groups. In contrast, the 18S rRNA gene diversity was much lower in the clone libraries from the polluted soil. More than 90% of these sequences belonged to the class Colpodea, a well-known clade of mainly bacterivorous and r-selected species, thus potentially also indicating a lower functional diversity.     

Phylogenetic utility of protein (RPB2, β-tubulin) and ribosomal (LSU,SSU) gene sequences in the systematics of Sordariomycetes (Ascomycota,Fungi)

The Sordariomycetes is an important group of fungi whose taxonomic relationships and classification is obscure. There is presently no multi-gene molecular phylogeny that addresses evolutionary relationships among different classes and orders. In this study, phylogenetic analyses with a broad taxon sampling of the Sordariomycetes were conducted to evaluate the utility of four gene regions (LSU rDNA, SSU rDNA, beta-tubulin and RPB2) for inferring evolutionary relationships at different taxonomic ranks. Single and multi-gene genealogies inferred from Bayesian and Maximum Parsimony analyses were compared in individual and combined datasets. At the subclass level, SSU rDNA phylogenies demonstrate their utility as a marker to infer phylogenetic relationships at higher levels. All analyses with SSU rDNA alone, combined LSU rDNA and SSU rDNA, and the combined 28 S rDNA, SSU rDNA and RPB2 datasets resulted in three subclasses: Hypocreomycetidae, Sordariomycetidae and Xylariomycetidae, which correspond well to established morphological classification schemes. At the ordinal level, the best resolved phylogeny was obtained from the combined LSU rDNA and SSU rDNA datasets. Individually, the RPB2 gene dataset resulted in significantly higher number of parsimony informative characters. Our results supported the recent separation of Boliniaceae, Chaetosphaeriaceae and Coniochaetaceae from Sordariales and placement of Coronophorales in Hypocreomycetidae. Microascales was found to be paraphyletic and Ceratocystis is phylogenetically associated to Faurelina, while Microascus and Petriella formed another clade and basal to other members of Halosphaeriales. In addition, the order Lulworthiales does not appear to fit in any of the three subclasses. Congruence between morphological and molecular classification schemes is discussed.     

Phylogenetic Relationships of Orders Within the Class Colpodea (Phylum Ciliophora) Inferred from Small Subunit rRNA Gene Sequences

Molecular analyses have been used recently to refine our knowledge of phylogenetic relationships within the ciliated protozoa (phylum Ciliophora). A current Hennigian phylogeny of the orders in the class Colpodea, based on light and electron microscopic analyses, makes three important assumptions with regard to apomorphic character states, namely, (1) that the kreyellid silver line evolved early in colpodean phylogeny, separating bryometopids, such as Bryometopus, from all other colpodeans; (2) that the macro–micronuclear complex is an autapomorphy of the cyrtolophosidids, such as Platyophrya; and (3) that merotelokinetal stomatogenesis is an apomorphic character of colpodids, such as Colpoda, Bresslaua, and Pseudoplatyophrya. These predictions of relationships within the class Colpodea were investigated by determining the complete small subunit rRNA gene sequences for the colpodid Bresslaua vorax, the grossglockneriid Pseudoplatyophrya nana, and the cyrtolophosidid Platyophrya vorax and a partial sequence for the bryometopid Bryometopus sphagni. These sequences were combined with the previously published complete SSrRNA sequences for the colpodid Colpoda inflata and the bursariomorphid Bursaria truncatella. The affiliations were assessed using both distance matrix and maximum-parsimony analyses. The tree topologies for the class Colpodea were identical in all analyses, with bootstrap support for bifurcations always exceeding 60%. The results suggest the following. (1) Since the clade including Bryometopus and its sister taxon, Bursaria, is never basal, the kreyellid silver-line system evolved later in colpodean phylogeny and does not separate bryometopids from all other colpodeans. (2) Since Platyophrya is always the sister taxon to the other five genera, there is a fundamental phylogenetic significance for its macro–micronuclear complex. (3) Since the colpodids, Colpoda, Bresslaua, and Pseudoplatyophrya, always group in one clade, merotelokinetal stomatogenesis appears to be a derived character state. Received: 30 June 1998 / Accepted: 3 December 1998     

Phylogenetic analysis of ribosomal RNA operons from uncultivated coastal marine bacterioplankton

Analyses of small subunit ribosomal RNA genes (SSU rDNAs) have significantly influenced our understanding of the composition of aquatic microbial assemblages. Unfortunately, SSU rDNA sequences often do not have sufficient resolving power to differentiate closely related species. To address this general problem for uncultivated bacterioplankton taxa, we analysed and compared sequences of polymerase chain reaction (PCR)-generated and bacterial artificial chromosome (BAC)-derived clones that contained most of the SSU rDNAs, the internal transcribed spacer (ITS) and the large subunit ribosomal RNA gene (LSU rDNA). The phylogenetic representation in the rRNA operon PCR library was similar to that reported previously in coastal bacterioplankton SSU rDNA libraries. We observed good concordance between the phylogenetic relationships among coastal bacterioplankton inferred from SSU or LSU rDNA sequences. ITS sequences confirmed the close intragroup relationships among members of the SAR11, SAR116 and SAR86 clades that were predicted by SSU and LSU rDNA sequence analyses. We also found strong support for homologous recombination between the ITS regions of operons from the SAR11 clade.     

Protein coding gene trees in ciliates: comparison with rRNA-based phylogenies

We have reexamined the phylogeny of the ciliates using alpha-tubulin and phosphoglycerate kinase gene sequences. For alpha-tubulin, we have compared the amino acid and nucleotide sequences of 20 species representing seven of the nine classes of the phylum (Karyorelictea, Heterotrichea, Hypotrichea, Oligohymenophorea, Colpodea, Nassophorea, and Litostomatea). The phylogenetic tree resembles a bush from which three monophyletic lineages can be distinguished which correspond to the three classes Hypotrichea, Oligohymenophorea, and Litostomatea. For phosphoglycerate kinase, we have compared the amino acid sequences from 7 species representing three classes (Heterotrichea, Hypotrichea, and Oligohymenophorea). The branching pattern is resolved in three deeply separated branches with an early emergence of the heterotrich. Our comparative analysis shows that if alpha-tubulin phylogeny is not informative at the interclass level, the preliminary data from the phosphoglycerate kinase molecule appear more promising. Nevertheless, at low taxonomic level and at the class level, the resolved phylogenetic relationships inferred from both protein and rRNA sequence data are congruent.     

Identity and systematic position of Paradinium poucheti and other Paradinium-like parasites of marine copepods based on morphology and nuclear-encoded SSU rDNA

Paradinium and Paradinium-like parasites were detected in various copepod hosts collected in the NW Mediterranean Sea, the North Atlantic Ocean, and the Godth?bsfjord (Greenland). The identity and systematic position of the parasitic, plasmodial protist Paradinium was investigated on the basis of SSU rDNA and morphology. SSU rDNA sequences were obtained from 3 specimens of Paradinium poucheti isolated from their cyclopoid copepod host, Oithona similis. In addition, a comparable sequence was obtained from a hitherto undescribed species of Paradinium from the harpactacoid copepod Euterpina acutifrons. Finally, SSU rDNA sequences were acquired from 2 specimens of a red plasmodial parasite (RP parasite) isolated from Clausocalanus sp. Both morphological and SSU rDNA sequence data supported that P. poucheti and Paradinium sp. are closely related organisms. In phylogenetic analyses based on SSU rDNA sequences, Paradinium spp. clustered with sequences from an uncultured eukaryote clone from the Pacific Ocean and two sequences from haplosporidian-like parasites of shrimps, Pandalus spp. This Paradinium clade branched as a sister group to a clade comprising the Haplosporidia and the Foraminifera. The RP parasite had a superficial morphological resemblance to Paradinium and has previously been interpreted as a member of this genus. However, several morphological characters contradict this and SSU rDNA sequence data disagree with the RP parasite and Paradinium being related. The phylogenetic analyses suggested that the RP parasite is a fast-evolved alveolate and a member of the so-called marine alveolate Group I (MAGI) and emerging data now suggest that this enigmatic group may, like the syndinian dinoflagellates, consist of heterotrophic parasites.     

Comparative analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene in ciliates (Alveolata,Ciliophora) and evaluation of its suitability as a biodiversity marker

The mitochondrial cytochrome c oxidase subunit 1 (COI) gene of ciliates was first successfully sequenced in species of the genera Tetrahymena and Paramecium (Class Oligohymenophorea). The sequence of the COI gene is extremely divergent from other eukaryotes and includes an insert, which is over 300 nucleotides long. In this study, we designed a primer pair that successfully amplified the COI gene of ciliates from five different classes: Heterotrichea, Spirotrichea, Oligohymenophorea, Nassophorea and Colpodea. These classes represent the diversity of the phylum Ciliophora very well, since they are widely distributed on the ciliate small subunit rRNA tree. The amplified region is approximately 850 nucleotides long and corresponds to the general barcoding region; it also includes the insert region. In this study, 58 new COI sequences from over 38 species in 13 orders are analysed and compared, and distance trees are constructed. While the COI gene shows high divergence within ciliates, the insert region, which is present in all classes, is even more divergent. Genetic distances calculated with and without the insert region remain in the same range at the intraspecific level, but they differ considerably at or above genus level. This suggests that the entire barcoding region is under similar selective constraints and that the evolutionary rate of the ciliate COI is extremely high and shows unequal rate variation. Although many problems still remain regarding standardization of barcoding methods in ciliates, the development of a universal or almost universal primer combination for the Phylum Ciliophora represents important progress. As shown in four examples, the resolution of COI at the intraspecific level is much greater than that of any nuclear genes and shows great potential to (1) identify species based on molecular data if a reliable database exists, and (2) resolve the relationships of closely related ciliate taxa and uncover cryptic species.     

Phylogenetic relationships among 28 spirotrichous ciliates documented by rDNA

The contiguous sequence of the SSU rDNA, ITS 1, 5.8S, ITS 2, and approximately 1370 bp at the 5(') end of the LSU rDNA was determined in 25 stichotrichs, one oligotrich, and two hypotrichs. Maximum parsimony, neighbor-joining, and quartet-puzzling analyses were used to construct individual phylogenetic trees for SSU rDNA, for LSU rDNA, and ITS 1+5.8S+ITS 2, as well as for all these components combined. All trees were similar, with the greatest resolution obtained with the combined components. Phylogenetic relationships were largely consistent with classical taxonomy, with notable disagreements. DNA sequences indicate that Oxytricha granulifera and Oxytricha longa are rather distantly related. The oligotrich, Halteria grandinella, is placed well within the order Stichotrichida. Uroleptus pisces and Uroleptus gallina probably belong to different genera. Holosticha polystylata (family Holostichidae) and Urostyla grandis (family Urostylidae) are rather closely related. These rDNA sequence analyses imply the need for some modifications of classical taxonomic schemes.