New considerations on the phylogeny of cyrtophorian ciliates (Protozoa,Ciliophora): expanded sampling to understand their evolutionary relationships

To rationalize the confusing relationships among the cyrtophorian ciliates, we expanded the taxon sampling by sequencing the small subunit ribosomal RNA (SSU rRNA) gene of representatives of 12 genera (20 species, 23 new sequences). The SSU rRNA sequences of Spirodysteria, Agnathodysteria, Brooklynella and Odontochlamys are reported for the first time. Phylogenetic trees were constructed, and secondary structures of variable region 4 (V4) of all genera for which SSU rRNA gene sequence data are available were predicted. The results indicate that (i) Brooklynella is likely an intermediate taxon between Dysteriidae and Hartmannulidae; (ii) the genus Dysteria is paraphyletic with Spirodysteria and Mirodysteria nested within it; (iii) the genus Agnathodysteria is well separated from Dysteria based on both molecular and morphological data; and (iv) Trithigmostoma is a basal genus of Chilodonellidae, based on both the morphological and molecular data.     

Characterization of Euplotes lynni nov. spec., E. indica nov. spec. and description of E. aediculatus and E. woodruffi (Ciliophora,Euplotidae) using an integrative approach

Four species belonging to the genus Euplotes have been investigated, namely: E. lynni nov. spec., E. indica nov. spec., E. aediculatus, and E. woodruffi. All populations are from India and were investigated using morphological and molecular markers. The phylogenetic relationships were inferred from small subunit ribosomal rRNA gene (SSU rRNA), internal transcribed spacer (ITS) region, and mitochondrial cytochrome c oxidase subunit I (COI) gene. Predicted secondary structure models for two new species using the hypervariable region of the SSU rRNA gene and ITS2 region support the distinctness of both species. Morphological characters were subjected to principal component analysis (PCA) and genetic variations were studied in-depth to analyze the relatedness of the two new species with their congeners. An integrative approach combining morphological features, molecular analysis, and ecological characteristics was carried out to understand the phylogenetic position of the reported species within the different clades of the genus Euplotes.     

Insights into the phylogeny of systematically controversial haptorian ciliates (Ciliophora, Litostomatea) based on multigene analyses

The ciliate subclass Haptoria is a diverse taxon that includes most of the free-living predators in the class Litostomatea. Phylogenetic study of this group was initially conducted using a single molecular marker small-subunit ribosomal RNA (SSU rRNA genes). Multi-gene analysis has been limited because very few other sequences were available. We performed phylogenetic analyses of Haptoria incorporating new SSU rRNA gene sequences from several debated members of the taxon, in particular, the first molecular data from Cyclotrichium. We also provided nine large-subunit ribosomal RNA (LSU rRNA) gene sequences and 10 alpha-tubulin sequences from diverse haptorians, and two possible relatives of controversial haptorians (Plagiopylea, Prostomatea). Phylogenies inferred from the different molecules showed the following: (i) Cyclotrichium and Paraspathidium were clearly separated from the haptorids and even from class Litostomatea, rejecting their high-level taxonomic assignments based on morphology. Both genera branch instead with the classes Plagiopylea, Prostomatea and Oligohymenophora. This raises the possibility that the well-known but phylogenetically problematic cyclotrichiids Mesodinium and Myrionecta may also have affinities here, rather than with litostomes; (ii) the transfer of Trachelotractus to Litostomatea is supported, especially by the analyses of SSU rRNA and LSU rRNA genes, however, Trachelotractus and Chaenea (more uncertainly) generally form the two deepest lineages within litostomes; and (iii) phylogenies of the new molecular markers are consistent with SSU rRNA gene information in recovering order Pleurostomatida as monophyletic. However, Pleurostomatida branches cladistically within order Haptorida, as does subclass Trichostomatia (on the basis of SSU rRNA phylogenies). Our results suggest that the class-level taxonomy of ciliates is still not resolved, and also that a systematic revision of litostomes is required, beginning at high taxonomic levels (taxa currently ranked as subclasses and orders).     

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.     

A Molecular Phylogenetic Investigation of Bakuella,Anteholosticha, and Caudiholosticha (Protista,Ciliophora, Hypotrichia) Based on Three‐Gene Sequences

Traditionally classifications of the Urostyloida have been mainly based on morphology and morphogenesis. Recent molecular phylogenetic analyses have been largely based on single‐gene data for a limited number of taxa. Consequently, incongruence has arisen between the morphological/morphogenetic and the molecular data. In this study, the three phylogenetic markers (SSU rDNA, ITS1‐5.8S‐ITS2 region, and LSU‐rDNA) of three urostyloid genera represented by four species (Bakuella granulifera, Anteholosticha monilata, Caudiholosticha sylvatica, and C. tetracirra) were sequenced to investigate their phylogeny. The results show that: (1) all three genera should be regarded as the members of the order Urostyloida within the subclass Hypotrichia, as indicated by morphological characters; (2) phylogenetic analyses and sequence similarities both indicate that neither Anteholosticha nor Caudiholosticha are monophyletic and the systematic assignment of both genera awaits further evaluation; and (3) Bakuella has a closer relationship with Urostyla than with bakuellids (e.g. Apobakuella and Metaurostylopsis), suggesting Bakuella may belong to the family Urostylidae rather than the family Bakuellidae.     

Molecular phylogeny of phyllopharyngean ciliates and their group I introns

We analyzed small subunit ribosomal DNA (ssu-rDNA) sequences to evaluate both the monophyly of the ciliate class Phyllopharyngea de Puytorac et al. (1974), and relationships among subclasses. Classifications based on morphology and ultrastructure divide the Phyllopharyngea into four subclasses, the Phyllopharyngia, Chonotrichia, Rhynchodia, and Suctoria. Our analyses of ssu-rDNA genealogies derived from sequence data collected from diverse members representing three of the four subclasses of Phyllopharyngea (Suctoria: Ephelota spp., Prodiscophyra collini, Acineta sp.; Phyllopharyngia: Chlamydodon exocellatus, Chlamydodon triquetrus, Dysteria sp.; and Chonotrichia: Isochona sp.) provide strong support for the monophyly of the Phyllopharyngea, and show that the Chonotrichia emerge from within the Phyllopharyngia. Based on this initial sampling, suctorian budding types are monophyletic, and exogenous budding appears to be basal to evaginative and endogenous budding. Further, we report the discovery of a group I intron at position 891 in the Suctoria Acineta sp. and Tokophrya lemnarum, and a second group I intron at position 1506 in T. lemnarum. These introns represent only the second examples of group I introns in a ciliate ribosomal gene, since the discovery of ribozymes in the LSU rRNA gene of Tetrahymena thermophila. Phylogenetic analyses of Group I introns suggest a complex evolutionary history involving either multiple loses or gains of introns within endogenously budding Suctoria.     

Description of a new epibiotic relationship (Suctorian–Copepoda) in NE Atlantic waters: from morphological to phylogenetic analyses

Paraeuchaeta hebes is one of the most important carnivorous copepods in the coastal upwelling system off Galician waters (Ría de Vigo, NE Atlantic). A suctorian epibiont of the genus Pelagacineta was found attached to the surface of these copepods. The abundance and distribution on the copepod surface were analysed, taking into account the sex of the crustacean, revealing some preference for females and also a different attachment point in both sexes. The morphological study allowed us to identify a new species of this Suctoria epibiont as Pelagacineta hebensis. Moreover, the 18S rRNA gene was partially sequenced to inspect the phylogenetic position of Pelagacineta hebensis within the subclass Phyllopharyngea. The maximum‐likelihood (ML) tree obtained was consistent with the morphological and with previous molecular studies and showed that P. hebensis belongs to the order Endogenina, as a sister clade of the few taxa sequenced within this order. Including new genetic data to the Endogenina will allow building new hypothesis about the evolution of the most derived clade of suctorians.     

Reconsideration of the Phylogenetic Positions of Five Peritrich Genera,Vorticella, Pseudovorticella,Zoothamnopsis, Zoothamnium,and Epicarchesium (Ciliophora,Peritrichia, Sessilida), Based on Small Subunit rRNA Gene Sequences

ABSTRACT. In order to re‐evaluate the systematics of sessilid peritrich ciliates, small subunit (SSU) rRNA gene sequences were determined for 12 species belonging to five genera: Vorticella, Pseudovorticella, Epicarchesium, Zoothamnium, and Zoothamnopsis. Phylogenetic trees were deduced using Bayesian inference, maximum parsimony, and maximum likelihood methods. The phylogenetic analyses suggest that (1) sessilids which have stalks with continuous myonemes that contract in a zig‐zag fashion form a separate clade from those which have stalks that contract independently and in a spiral fashion, supporting the separation of the family Zoothamniidae from the family Vorticellidae and (2) Epicarchesium and Pseudovorticella, both of which have reticulate silverline systems, are more closely related to each other than to other vorticellids, suggesting that differences in the silverline system (i.e. transverse vs. reticulate) may be the result of genuine evolutionary divergence among sessilid peritrichs. However, the newly sequenced Zoothamnopsis sinica, which has a reticulate silverline pattern, nests within the unresolved Zoothamnium species that have transverse silverline patterns. Thus, there were at least two evolutions of the reticulate silverline pattern character state from a plesiomorphic transverse state in the peritrichid ciliates. The molecular work demonstrates the genus Zoothamnium to be paraphyletic in relation to morphological studies, and suggests that Astylozoon, Opisthonecta, and Vorticella microstoma possibly share a SSU rRNA secondary structure in the helix E10‐1 region.     

Molecular evolution of rDNA in early diverging Metazoa: First comparative analysis and phylogenetic application of complete SSU rRNA secondary structures in Porifera

Background  

The cytoplasmic ribosomal small subunit (SSU, 18S) ribosomal RNA (rRNA) is the most frequently-used gene for molecular phylogenetic studies. However, information regarding its secondary structure is neglected in most phylogenetic analyses. Incorporation of this information is essential in order to apply specific rRNA evolutionary models to overcome the problem of co-evolution of paired sites, which violates the basic assumption of the independent evolution of sites made by most phylogenetic methods. Information about secondary structure also supports the process of aligning rRNA sequences across taxa. Both aspects have been shown to increase the accuracy of phylogenetic reconstructions within various taxa.     

124 Evidence for Lateral Transfer of a IE Intron between Fungal and Red Algal Small Subunit Ribosomal RNA Genes

In a recent study of the North American biogeography of the red algae genus Hildenbrandia, the presence of group I introns were noted in the nuclear SSU rRNA gene of the marine species H. rubra (Hildenbrandiales). Group I introns in the nuclear encoded rRNAs have been previously reported in the Hildenbrandiales as well as the Bangiales. All reported introns within the red algae have been identified as belonging to the IC1 subclass and occur at two insertion sites in the nuclear small subunit rRNA (516 and 1506). However, an unclassified intron was discovered at position 989 in the nuclear SSU rRNA gene of a collection of H. rubra from British Columbia, Canada. We have determined that the intron is a member of the IE subclass and this is the first report of an IE intron and an intron in position 989 in the red algae. Phylogenetic analyses of the intron sequences reveal a close relationship between this group IE intron and similar ascomycete and basidiomycete fungal IE introns in the nuclear SSU rRNA genes at positions 989 and 1199. In addition, a common unique helix (structural signature) in the P13 domain of the Hildenbrandia intron and those of the fungi at the 989 and 1199 IE positions in the nuclear SSU rRNA gene also indicates a close relationship. Hence, this study provides evidence for a possible lateral transfer of the IE intron in position 989 between fungal and red algal nuclear SSU rRNA genes.     

Molecular phylogenetic analysis places Percolomonas cosmopolitus within Heterolobosea: evolutionary implications

Percolomonas cosmopolitus is a common free-living flagellate of uncertain phylogenetic position that was placed within the Heterolobosea on the basis of ultrastructure studies. To test the relationship between Percolomonas and Heterolobosea, we analysed the primary structure of the actin and small-subunit ribosomal RNA (SSU rRNA) genes of P. cosmopolitus as well as the predicted secondary structure of the SSU rRNA. Percolomonas shares common secondary structure patterns of the SSU rRNA with heterolobosean taxa, which, together with the results of actin gene analysis, confirms that it is closely related to Heterolobosea. Phylogenetic reconstructions based on the sequences of the SSU rRNA gene suggest Percolomonas belongs to the family Vahlkampfiidae. The first Bayesian analysis of a large taxon sampling of heterolobosean SSU rRNA genes clarifies the phylogenetic relationships within this group.     

Phylogenetic relationships among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> (Bacillaceae: Bacillales) strains based on a comparison of SSU rRNA sequences and genome profiling

Bacillus thuringiensis Berliner has previously been classified via the serological identification of flagellar antigens. However, the phylogenetic relationships among strains of B. thuringiensis cannot be investigated by serotyping. Furthermore, high levels of homology have been found in gene sequences among various strains, complicating the determination of their evolutionary relationships. In order to elucidate the phylogenetic relationships within B. thuringiensis, we analyzed 40 strains belonging to typical serotypes using two approaches: an analysis of small subunit (SSU) rRNA sequences and genome profiling (GP) based on temperature gradient gel electrophoresis of random PCR products. The SSU rRNA analysis resulted in all 40 strains forming a single cluster with Bacillus cereus Frankland & Frankland. The distances among the subclusters were too small to further classify the strains. On the other hand, the phylogenetic analysis based on GP resulted in three clusters of B. thuringiensis strains. These results suggest that GP is a better method for the determination of phylogenetic relationships within B. thuringiensis.     

Structure of ribosomal RNA gene and phytogeny ofNosema isolates in Korea

The ribosomal RNA (rRNA) gene region of the fourNosema sp. isolates (C01, C02, C03 and C04) fromPieris rapae in Korea has been examined. Complete DNA sequence data (3779 bp) of The rRNA gene ofNosema sp. C01 are presented for the small subunit gene (SSU rRNA: 1236 bp), the internal transcribed spacer (ITS: 37 bp), and the large subunit gene (LSU rRNA 2506 bp). The secondary structures ofNosema sp. COI SSU and LSU rRNA genes are constructed and described. The SSU rRNA showed a hypervariable V4 region identified four additional stems including a pseudoknot. Phylogenetic analysis based on the SSU rRNA suggests that the four isolates belong to the ‘true’Nosema group. In contrast to theNosema/Vairimorpha clade, the members of the group are highly divergent.     

Description of a New Freshwater Ciliate Epistylis wuhanensis n. sp. (Ciliophora,Peritrichia) from China,with a Focus on Phylogenetic Relationships within Family Epistylididae

Two populations of Epistylis wuhanensis n. sp., a new freshwater peritrich ciliate, were isolated from different freshwater ponds located in Hubei, China. Their morphological characteristics were investigated using live observation, protargol impregnation, and scanning electron microscopy (SEM). Specimens from the two populations showed identical arrangement of the infraciliature and identical small subunit ribosomal RNA (SSU rRNA) gene and ITS1‐5.8S‐ITS2 sequences. The zooids present bell‐shaped and 90–175 × 27–54 μm in vivo. Macronucleus is variable in shape and located in the middle of cell. Pellicle is usually smooth with 139–154 and 97–105 striations above and below the trochal band, respectively. SSU rRNA gene and ITS1‐5.8S‐ITS2 sequences of E. wuhanensis n. sp. did not match any available sequences in GenBank. Phylogenetically, E. wuhanensis n. sp. clusters with the other Epistylis within the family Epistylididae, but is distinct from the major clades of Epistylis. Above all, the morphological characteristics and molecular analyses support that the present Epistylis is a new species. Expanded phylogenetic analyses of sessilids based on both SSU rRNA gene sequences and ITS1‐5.8S‐ITS2 sequences reveal that the genus Epistylis consists of Epistylis morphospecies and taxonomic revision of the genus is needed.     

Taxonomy, molecular phylogeny, and ultrastructural morphology of Olpidiopsis porphyrae sp. nov. (Oomycetes, straminipiles), a unicellular obligate endoparasite of Bangia and Porphyra spp. (Bangiales, Rhodophyta)

Olpidiopsis porphyrae sp. nov., a marine oomycete endoparasite that infects the commercially cultivated red alga Porphyra yezoensis, is described and its phylogenetic position based on molecular data and ultrastructural morphology is discussed. O. porphyrae infects the host Porphyra by means of encysted zoospores. Spherical-shaped holocarpic thalli develop within the cytoplasm of its algal host, which produce monoplanetic, subapically biflagellate zoospores. The characteristic features of this isolate are the ellipsoidal, unicellular thallus and simple holocarpic zoosporangial development, which show morphological similarity with the genus Olpidiopsis. Laboratory infection experiments with a wide range of green, brown, and red algae revealed that O. porphyrae infects several stages of the bangialean red algae (the genera Bangia and Porphyra). Molecular phylogenetic analyses inferred from both SSU rRNA and cox2 genes showed O. porphyrae branched before the main saprolegnian and peronosporalean lineages within the monophyletic oomycete clade, indicating its phylogenetic separation from them. A single or double K-body-like organelle, which contains tubular inclusions, is found located to one side of the zoospore nucleus and shows similarities to homologous organelles previously described in O. saprolegniae. The ultrastructural morphology of O. porphyrae with zoospore initials containing K-bodies and tubular mitochondrial cristae is characteristic of oomycetes. Group I intron-like multiple insertions were found in the SSU rRNA gene of O. porphyrae. This is the first report of SSU group I introns in the class Oomycetes.     

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.     

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.     

Complete sequence and structure of ribosomal RNA gene of Heterosporis anguillarum

The ribosomal RNA (rRNA) gene region of the microsporidium Heterosporis anguillarum has been examined. Complete DNA sequence data (4060 bp, GenBank Accession No. AF402839) of the rRNA gene of H. anguillarum are presented for the small subunit gene (SSU rRNA: 1359 bp), the internal transcribed spacer (ITS: 37 bp), and the large subunit gene (LSU rRNA: 2664 bp). The secondary structures of the H. anguillarum SSU and LSU rRNA genes are constructed and described. This is the first complete sequence of an rRNA gene published for a fish-infecting microsporidian species. In the phylogenetic analysis, the sequences, including partial SSU rRNA, ITS, and partial LSU rRNA sequences of the fish-infecting microsporidia, were aligned and analysed. The taxonomic position of H. anguillarum as suggested by Lom et al. (2000; Dis Aquat Org 43:225-231) is confirmed in this paper.     

Phylogeny and Establishment of a New Ciliate Family,Wilbertomorphidae fam. nov. (Ciliophora,Karyorelictea), a Highly Specialized Taxon Represented by Wilbertomorpha colpoda gen. nov., spec. nov.

The morphology and molecular phylogeny of a new marine interstitial karyorelictean ciliate, Wilbertomorpha colpoda gen. nov., spec. nov., isolated from a sandy beach at Qingdao, China, are investigated. The ciliature is mainly on the right surface with the left surface barren except for a single marginal kinety, which is typical of the order Loxodida. Nevertheless, other morphological features, such as the highly reduced buccal structure, the absence of a buccal cavity and the presence of extremely specialized cortical granules, prevent its assignation to any known family. Furthermore, the secondary structure of the variable region 2 of the SSU rRNA gene is unique among loxodids. Thus, we establish a new family, Wilbertomorphidae fam. nov. for the genus Wilbertomorpha. Phylogenetic analyses based on SSU rRNA gene sequence data suggest that this taxon is a sister group to the family Geleiidae which belongs to the order Protoheterotrichida. However, morphologically Wilbertomorpha bears a much stronger resemblance to Loxodidae which belongs to the order Loxodida. Based on these findings, we tentatively place Wilbertomorphidae nov. fam. incertae sedis within Karyorelictea.