DNA Analysis of Algal Endosymbionts of Ciliates Reveals the State of Algal Integration and the Surprising Specificity of the Symbiosis.

Many freshwater protists harbor unicellular green algae within their cells, but little is known of their degree of integration and specificity. Using algae-targeted PCR of whole ciliate cells collected at irregular intervals over 15 months from Lake Biwa, Japan, we explored the SSU-ITS rDNA of the endosymbiotic algae and its changes over time, obtaining sequences of algal rDNA fragments from four ciliate species. A high proportion of clonal algae was evident within the ciliate cells. The differences observed in those sequences from the SSU through to the ITS region were less than 1%. The name 'Chlorb' is proposed for these algae, with the implication that they represent a single 'species.' The sequences of the algal DNA fragments were identical for any given host species throughout the collection period, thus we conclude that these four ciliates stably retain their algae over long term. In contrast, algal DNA fragments obtained from Didinium sp. were variable within each sample, which indicates that this ciliate only temporarily holds its algal cells. The ITS1 sequences of Chlorb populations are close (at intraspecific level) to those of algae isolated from ciliates in Austria, which raises the possibility that Chlorb algae are universally shared as symbionts among various ciliates.

Vorticella Linnaeus, 1767 (Ciliophora, Oligohymenophora, Peritrichia) is a grade not a clade: redefinition of Vorticella and the families Vorticellidae and Astylozoidae using molecular characters derived from the gene coding for small subunit ribosomal RNA.

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.

Molecular phylogeny of the family Vorticellidae (Ciliophora, Peritrichia) using combined datasets with a special emphasis on the three morphologically similar genera Carchesium, Epicarchesium and Apocarchesium.

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.

Morphology, morphogenesis and gene sequence of a freshwater ciliate, Pseudourostyla cristata (Ciliophora, Urostyloidea) from the ancient Lake Biwa, Japan.

The urostyloid freshwater ciliate Pseudourostyla cristata was recorded for the first time from Lake Biwa, a 4-million-year-old lake located in Shiga Prefecture, Japan. Its morphology and morphogenesis were investigated using live observation and protargol impregnation, and the SSU ribosomal RNA gene was sequenced. Based on the current observations and previous descriptions, this species is readily recognized mainly by the following characters: body slender or broadly oval to elliptical, and dark grey in color; size in vivo about 170-400 x 40-150 microm; pellicle flexible and contractile, with extrusomes forming a hyaline seam underneath; ciliature comprising about 60-130 adoral membranelles, usually 1 buccal cirrus, 20-24 frontal, 2 frontoterminal, 17-26 pairs of midventral, and 5-16 transverse cirri, 4-6 left and 4-5 right marginal rows, and 8-10 dorsal kineties; 15-83 macronuclear nodules and 2-9 micronuclei; freshwater habitat. The main morphogenetic developments are: (1) the oral primordium for the proter originates de novo on the dorsal wall of the buccal cavity, and the dedifferentiated undulating membranes and some parental proximal membranelles join in the primordial development; the old adoral zone will be partly replaced by new structures; (2) the oral primordium for the opisthe occurs epiapokinetally left of the midventral complex between the adoral zone and the transverse cirri; (3) the fronto-midventral transverse cirral (FVT) anlagen develop separately in both dividers by dedifferentiation of most of the midventral cirri; (4) the single buccal cirrus is generated from the posterior end of FVT anlage II; (5) the leftmost frontal cirrus is derived from the anterior end of the undulating membranes anlage (FVT anlage I); (6) the marginal rows of each side are formed from a single anlage which arises within the rightmost row; (7) the dorsal kineties develop by intrakinetal basal body proliferation; and (8) the most posterior FVT anlage contributes the two fronto-terminal cirri at its anterior end. The present observations indicate that P. cristata has a wide geographic distribution, and possesses constant morphological and morphogenetic traits. Phylogenetic trees inferred from SSU rRNA gene sequences suggest paraphyly of the genus Pseudourostyla.

Morphological and molecular characterization of Histiobalantium natans viridis (Ciliophora, Scuticociliatia).

We investigated a Histiobalantium natans viridis population from the ancient Lake Biwa in Japan, using live observation, silver impregnation, and the small subunit rRNA gene sequence. The morphological and molecular data show, with high support, a close relationship of Histiobalantium, Schizocalyptra and Pleuronema, supporting the family Pleuronematidae Kent, whose nearest relatives are the Cyclidiidae Ehrenberg. A family Histiobalantiidae Puytorac and Corliss is not supported, either by the nucleotide sequences or the morphologic data, except for the curious dorsal location of the cytopyge. Likewise, the data do not support inclusion of Histiobalantium in the family Sulcigeridae Gajewskaja, as very recently suggested by Jankowski, whose classification is based on a misidentified Sulcigera comosa. Further, there are good reasons to synonymize the genus Gajewskiella Obolkina with Histiobalantium. The European and Asian populations of H. natans differ significantly in the structure of adoral membranelle 1, suggesting that they are different species. However, there is some indication that the differences are caused by deficient data on the European H. natans, which is thus in need of detailed redescription. This applies also to the North American Histiobalantium agile Stokes which is probably not conspecific with the Eurasian H. natans. The status of the forma H. natans viridis and H. natans nigricans is not known because detailed data from nigricans are lacking. We prefer subspecific rank at the present state of knowledge.

A description of Apocarchesium rosettum n. gen., n. sp. and a redescription of Ophrydium eichornii Ehrenberg, 1838, two freshwater peritrichous ciliates from Japan.

This paper includes the proposal of a new genus for a new species of peritrichous ciliate, Apocarchesium rosettum n. gen., n. sp., as well as the first description by modern techniques of another peritrich, Ophrydium eichornii Ehrenberg, 1838. The genus Apocarchesium is separated from the related genus Carchesium by the appearance of the stalk and the arrangement of zooids, and diagnosed as follows: colonial vorticellid, with all zooids attached in rosette fashion to the tip of their common stalk, which is unbranched and contracts spirally; pellicle with parallel silverlines. The morphology, infraciliature, and silverline system of both species were investigated by observation in vivo and silver-staining methods.

Morphology and gene sequence of Levicoleps biwae n. gen., n. sp. (Ciliophora, Prostomatida), a proposed endemic from the ancient Lake Biwa, Japan.

Levicoleps biwae n. gen., n. sp. was discovered in organic mud on the shore of Lake Biwa, Japan. Its morphology and small subunit rRNA gene sequence were studied with standard methods. Further, we established a terminology for the colepid armour and selected four features for genus recognition: the number of armour tiers, the structure of the tier plates, the presence/absence of armour spines, and the number of adoral organelles (three or five). The Japanese colepid, a barrel-shaped ciliate with an average size of 75 x 45 microm, has six armour tiers and hirtus-type tier plates, but lacks armour spines, both in the environment and in laboratory culture. Thus, it is considered to represent a new genus. This rank is supported by the considerable genetic distance (7%) from the common Coleps hirtus. Although L. biwae looks quite similar to C. hirtus in vivo, it is very likely most closely related to Coleps amphacanthus, a species with conspicuous armour spines, as indicated by body size, the number of ciliary rows and, especially, the multiple caudal cilia. Lake Biwa is about four million years old and inhabited by many endemic organisms, ranging from algae to large fish. Thus, we suspect that L. biwae is restricted to Lake Biwa or, at least, to Asia. Based on literature data and the generic features established, we also propose the new genus Reticoleps for Coleps remanei Kahl, 1933, and resurrect the genus Pinacocoleps Diesing, 1865 to include Coleps incurvus Ehrenberg, 1833, Coleps pulcher Spiegel, 1926, Coleps tessalatus Kahl, 1930 and, probably, Baikalocoleps quadratus Obolkina, 1995a. Nine colepid genera are diagnosed and dichotomously keyed.