Recent phylogenetic analyses of the peritrich genus Vorticella have suggested that it might be paraphyletic, with one Vorticella species - Vorticella microstoma grouping with the swimming peritrichs Astylozoon and Opisthonecta in a distant clade. These results were based on very limited taxon sampling and thus could not be accepted as conclusive evidence for revising the generic classification. We tested paraphyly of the genus Vorticella by making a new analysis with a broad range of samples from three continents that yielded 52 new sequences of the gene coding for small subunit rRNA. Our results, together with the available sequences in Genbank, form a comprehensive set of data for the genus Vorticella. Analyses of these data showed that Vorticella microstoma morphotypes, Astylozoon, and Opisthonecta form a well-supported, monophyletic clade, that is distinct from and basal to the family Vorticellidae containing other species of Vorticella. Paraphyly of the genus Vorticella and family Vorticellidae was strongly confirmed by these results. Furthermore, the two clades of Vorticella identified by the SSU rRNA gene are so genetically diverse whereas the genetic distances within the one containing Vorticella microstoma morphotypes, Astylozoon, and Opisthonecta were so slight, which marked it as a separate family that must be defined by molecular characters in the absence of unifying morphological and morphogenetic characters. An emended characterization and status of the genus Vorticella, the families Vorticellidae and Astylozoidae are presented and discussed.

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

The stalked, ciliated protozoan Vorticella convallaria possesses a highly contractile cytoskeleton consisting of spasmonemes and myonemes. The major component of these contractile organelles is the calcium-binding protein(s) called spasmin. Cloning and characterization of spasmin would help elucidate this contractile system. Therefore, enriched spasmoneme protein preparations from these contractile stalks were used to produce a monoclonal antibody to spasmin. A monoclonal antibody, 1F5, was obtained that immunolocalized specifically to the spasmonemes and the myonemes and recognized a 20-kD calcium-binding protein in spasmoneme protein preparations. A putative spasmin cDNA was obtained from a V. convallaria cDNA library and the derived amino acid sequence of this cDNA revealed an acidic, 20-kD protein with calcium-binding helix-loop-helix domains. The physical properties of the putative spasmin were assessed by characterization of a recombinantly-produced spasmin protein. The recombinant spasmin protein was shown to bind calcium using calcium gel-shift assays and was recognized by the anti-spasmin antibody. Therefore, a V. convallaria spasmin was cloned and shown to be a member of the EF-hand superfamily of calcium-binding proteins.

The peritrichs have been recognized as a higher taxon of ciliates since 1968. However, the phylogenetic relationships among them are still unsettled, and their placement within the class Oligohymenophorea has only been supported by the analysis of the small subunit rRNA gene sequence of Opisthonecta henneguyi. DNA was isolated directly from field-sampled species for PCR, and was used to resolve relationships within the genus Epistylis and to confirm the stability of the placement of peritrichs. Small subunit rRNA gene sequences of Epistylis plicatilis, Epistylis urceolata, Epistylis chrysemydis, Epistylis hentscheli, Epistylis wenrichi, and Vorticella campanula were sequenced and analyzed using both distance-matrix and maximum-parsimony methods. In phylogenetic trees, the monophyly of both the genus Episrylis and the subclass Peritrichia was strongly supported, while V. campanula clustered with Vorticella microstoma. The topology in which E. plicatilis and E. hentscheli formed a strongly supported sister clade to E. urceolata, E. chrysemydis, and E. wenrichi was consistent with variations in the thickness of the peristomial lip. We concluded that the peristomial area, especially the peristomial lip, might be the important phylogenetic character within the genus Epistylis.

Eight isolates of Miamiensis avidus (scuticociliates) were collected from olive flounder Paralichthys olivaceus with symptoms of severe ulcers and haemorrhages at several culture farms in 1999 and 2003. Cloned strains were produced and the complete small subunit ribosomal RNA gene (SSU rRNA) of each strain was sequenced for classification and phylogenic study. The SSU rRNA is 1759 bp in length and the sequence was deposited in the GenBank under accession number AY550080. All 8 strains exhibited the same sequence, but this sequence did not match any previously deposited scuticociliate SSU rRNA sequence. Phylogenetic analysis placed Miamiensis avidus in a sister lineage to Cohnilembus verminus, Pseudocohnilembus hargisi and P. marinus.

The morphology, infraciliature, and silverline system of two new peritrichous ciliates, Pseudovorticella clampi n. sp. and Zoothamnium pararbuscula n. sp., have been investigated based on both living and silver-impregnated specimens. Partial sequence of 18S-ITS1-5.8S rDNA of Z. pararbuscula is also determined in order to compare it with the closely related congener, Zoothamnium arbuscula. Zoothamnium pararbuscula can be distinguished from its close form Z. arbuscula by the different habitats, the appearance of the main stalk, the position of the contractile vacuole, and the information derived from 18S-ITS1-5.8S rDNA sequence analysis. Pseudovorticella clampi n. sp. is distinguished from its congeners by its body shape and size, pellicle granules, habitat, and number of transverse silverlines.

Little is known about the phylogeny of the family Vorticellidae at the generic level because few comprehensive analyses of molecular phylogenetic relationships between members of this group have, so far, been done. As a result, the phylogenetic positions of some genera that were based originally on morphological analyses remain controversial. In the present study, we performed phylogenetic analyses of vorticellids based on the sequence of the small-subunit (SSU) rRNA gene, including one species of the genus Apocarchesium, for which no sequence has previously been reported. Phylogenetic trees were reconstructed with SSU rRNA gene sequences by using four different methods (Bayesian analysis, maximum-likelihood, neighbour-joining and maximum-parsimony) and had a consistent branching pattern. Members of the genera Vorticella (except V. microstoma) and Carchesium formed a clearly defined, well supported clade that was divergent from the clade comprising members of the genera Pseudovorticella and Epicarchesium, suggesting that the differences in the silverline system (transverse vs reticulate) among vorticellids may be the result of genuine evolutionary divergence. Members of the newly established genus Apocarchesium clustered within the family Vorticellidae basal to the clade containing members of the genera Pseudovorticella and Epicarchesium and were distinct from members of the genus Carchesium, supporting the validity of Apocarchesium as a novel genus. Additional phylogenetic analyses of 21 strains representing seven genera from the families Vorticellidae and Zoothamniidae were performed with single datasets (ITS1-5.8S-ITS2, ITS2 alone) and combined datasets (SSU rRNA+ITS1-5.8S-ITS2, SSU rRNA+ITS2) to explore further the phylogenetic relationship between the three morphologically similar genera Carchesium, Epicarchesium and Apocarchesium, using characteristics not included in previous analyses. The phylogenetic trees reconstructed with combined datasets were more robust and therefore more reliable than those based on single datasets and supported the results of trees based on SSU rRNA sequences.

Zoothamnium arbuscula strain Kawagoe is a giant sessile peritrich ciliated protozoa that possesses a contractile organelle called a spasmoneme. We report here on the molecular characterization and provide an opportunity to discuss the evolutionary relationships of the Z. arbuscula spasmin; spasmins belong to the calmodulin superfamily and are the major components of spasmoneme filaments. We analysed and obtained the whole sequence of the spasmin 1 gene and a partial sequence of the spasmin 2 gene. It is surprising that the sequence of spasmin 1 does not contain introns and encodes an open reading frame of 531 bp. It predicts a product of 177 amino acids with a calculated molecular mass of 19659 Da and a pI of 4.68. The amino acid sequence has two putative calcium-binding domains. One of them is a functional domain, as defined by the EF-hand consensus. The varieties of spasmins were revealed by comparison with amino acid components and molecular relationships of spasmin 1 protein and other spasmins. A comparison of the amino acid sequence between the Z. arbuscula spasmin and known centrins indicates that spasmins have a one residue deletion in the EF-hand domain-2 and four residue insertions in domain-4, as does the Vorticella spasmin. However, there are large variations in the amino acid sequence at domain-4 within spasmin 1, spasmin 2 and the Vorticella spasmin.