Vorticellid ciliates generally dwell in freshwater. In nature, the species have up until now been identified by comparison with previous descriptions. It is difficult to identify between species of the genus Vorticella, because the morphological markers of vorticellid ciliates described in reports are limited and variable. Unfortunately, culturing them has only succeeded with certain species such as Vorticella convallaria, but many others have been impossible to culture. To find out whether the sequence of a small subunit rRNA gene was an appropriate marker to identify vorticellid ciliates, the gene was aligned and compared. Finding a new convenient method will contribute to research on vorticellid ciliates. In strains of V. convallaria, classified morphologically, some varieties of the SSrRNA gene sequences were recognized, but there were large variations within the same species. According to the phylogenetic tree, these strains are closely related. However, the difference was not as big as between Vorticella and Carchesium. In addition, Carchesium constructed a distinct clade from the genus Vorticella and Epistylis. These results show the possibility that the SSrRNA gene is one of the important markers to identify species of Vorticella. This study is first to approach and clarify the complicated taxa in the genus Vorticella.
The vorticellid ciliate, Vorticella, adheres to the surface of materials such as water plants with stalks. Anyone who has had experience with the identification of free-living ciliates will understand the difficulty of distinguishing between species of Vorticella. Their various body shapes, variable size and highly contractile nature makes them among the most difficult of all ciliates to study and identify. This has resulted in the creation of numerous species and varieties, many of which are of doubtful taxonomic status.
With so many taxa, the descriptions of which can be found in other reports, the task of the ecologist in trying to identify individual isolates has become particularly onerous, even with the aid of identification keys such as those of Stokes (1885), Kahl (1935), Green (1974) and Warren (1986). Warren (1986) provided drawings and descriptions of the extant 82 species of Vorticella when he compiled the taxonomic status of the many species. But these 82 species did not include all Vorticella species. We have tried to identify species and constantly have had to refer to previous studies.
It is important that research at the molecular level should be based on a consistent species of organisms. One reason identification is quite difficult is because the morphological features are too vague. Vorticella have to be collected from a natural source because their culture is also difficult. Each classification of Vorticella is typically based on morphological features such as cell size and shape, cytostome, nuclear characteristics and cortical patterns. However, the morphological characteristics of V. convallaria are not distinguishable when the ambiguous form is found under certain culture conditions and among strains collected from nature. Many forms of V. convallaria from nature have been reported (Sladecek, 1971). Ciliate literature is confused with varieties of ciliates from V. convallaria. According to other reports, the two species used for this research were classified into the V. convallaria based on zooid size, inverted bell shape, situation of contractile vacuole, J-shaped macronuclear and characteristic stalk contraction (Warren, 1986).
As for Vorticella, mass cultivation has been reported (Vacchiano, 1991). However, long-term culture of the species collected from nature has not been possible. In most cases of molecular works, experiments should be with short-term cultures collected from nature. Therefore, we faced the difficulty whether sometimes the species was exactly the same as those reported in other works. Thus correct identification is important.
The purpose of this research was to evaluate the effectiveness of comparing partial SSrRNA gene sequencing to identify species of the genus Vorticella. The phylogeny of vorticellid ciliates is also discussed.
MATERIALS AND METHODS
Source of species and culturing
Stock cultures of the strains Vorticella convallaria Chicago, V. convallaria Tokyo and Vorticella sp. Tianjin were kept in our laboratory. Each cell of Carchesium polypinum Miyagi and Epistylis sp. Miyagi were isolated from water at a sewerage works and grown to be clones (Miyagi, Japan). The cells were cultured at 25°C in a solution mixed with natural mineral water (Volvic, France) and a lettuce medium that was inoculated with Klebsiella pneumoniae (Hiwatashi, 1968). Live cells were observed with Nomarski interference contrast.
It was difficult to collect a lot of cells efficiently because vorticellid ciliates are known to adhere to the bottom of glass vessels. Especially in the genus Vorticella, where individual cells grow at a distance from each other. Trying to collect many adhering cells from glass vessels was not an efficient method, so in the present research we mainly used the swimming form, “telotroch”, because it was easier to collect.
DNA extraction and sequencing
Swimming form cells were collected by centrifugation. About 4×103 cells were collected from 50ml of culture medium and washed once in distilled water. After centrifugation, the pellet was transferred into an NDS solution (1% SDS, 50mmol/L Na_EDTA, 100mmol/L Tris-HCl pH9.5) and inoculated for 10 minutes at 65°C. Finally, DNA was extracted with phenol-chloroform and precipitated with ethanol.
The rRNA genes were amplified by a PCR method using the forward primer 60f (5′-CRGYGAAACTGCTGCGAATGGCTC-3′) and the reverse primer 1,160r (5′-CCMGTGTTGAGTCAAATTAAGCCGC-3′). The forward and reverse primers were created at 60bp and 1,160bp, respectively, from the 5′-ends of the SSrRNA gene sequence in V. convallaria (AF070700). PCR products were isolated by the electrophoresis and purified with Geneclean III Kit (Bio 101 CA, USA). The purified DNA was sequenced directly on an ABI PRISM 310 Genetic Analyzer (PE Biosystems, CA, U.S.A.).
The accession Nos. in GenBank/EMBL/DDBJ of the sequences of the nucleic acids used for this paper were: Epistylis plicatilis ( AF335517), E. urceolata ( AF335516), E. chrysemydis ( AF335514), E. hentscheli ( AF335513), E. wenrichi ( AF335515), Opisthonecta henneguyi ( X56531), Paramecium caudatum ( AF217655), Tetrahymena thermophila ( M10932), Vorticella campanula ( AF335518), V. convallaria ( AF070700), V. microstoma ( AF070701).
Sequence alignment was performed with CLUSTAL W through the WWW server of DNA Data Bank of Japan (DDBJ; Mishima, Japan). Similarity degrees between vorticellid ciliates were calculated using GeneDoc ver2.6.001. PUZZLE ver. 4.0.2 (Maximum likelihood method: Strimmer and von Haeseler, 1999) was used to construct maximum-likelihood trees with quartet puzzling support value for the internal branches and maximum likelihood branch lengths. Phylogenetic tree constructions were drawn using the program Tree View (Page, 1996).
Two strains of Vorticella convallaria for this research have different morphotypes (Fig.1A, B.). V. convallaria strain Chicago differs from strain Tokyo in cell length and the shape beneath the peristomial lip. Strain Tokyo was constricted beneath the peristomial lip more than strain Chicago. The cell body of V. sp. conformed to most of the characteristics of V. convallaria, except for the pattern of spasmoneme contraction. V. sp. had an awkward style of contraction. As a result, V. sp. strain Tianjin couldn't be classified into V. convallaria.
Amplification of vorticellid genomic DNA with primers complementary to the specific parts of eukaryotic SSrRNA coding regions yielded a product about 1,100 nucleotides long. Accession Nos. of EMBL/GenBank/DDBJ and the length of partial SSrRNA gene sequenced in this experiment were as follows: V. convallaria strain Chicago 1,001 nucleotides ( AB074082); V. convallaria strain Tokyo 991 nucleotides ( AB074081); V. sp. strain Tianjin 1,003 nucleotides ( AB074083); Carchesium polypinum strain Miyagi 962 nucleotides ( AB074079); Epistylis sp. strain Miyagi 986 nucleotides ( AB074080). The sequences of Vorticella species were aligned with V. convallaria ( AF070700), V. campanula ( AF335518) and V. microstoma ( AF070701), and compared (Fig.2).
The genus Vorticella has a low G+C content in partially fixed SSrRNA gene sequences (Chicago 43%; Tokyo 42%; Tianjin 43%), similar to that found in Carchesium and Epistylis (Carchesium 45%; Epistylis 42%). In the genus Vorticella, the SSrRNA gene sequences had differences among species. The sequences of the Vorticella SSrRNA gene had many variations near 5′-ends, while the sequences of Carchesium SSrRNA gene, especially at the middle position, had fixed sequences. There was a specific region for only V. sp. near 410-bp from 5′-ends(14-bp). Differences in the sequences came out noticeably among the three strains of V. convallaria. Comparisons of the SSrRNA gene sequences were made from some clones of each vorticellid ciliates: no differences were found (data not shown).
Similarity values for vorticellid SSrRNA sequences showed apparent differences in the same species (Table 1). For example, the maximum and minimum similarity values were respectively 96% between the strain Tokyo and the strain Chicago, and 88% between the strain Chicago and AF070700. There were many variations of SSrRNA gene sequences in the same species. Similarity values between all of the V. convallaria strains and V. microstoma strain was from 82% to 85%. V. campanula showed a higher similarity to V. convallaria species rather than V. microstoma. Carchesium had more deletions or insertions than other vorticellid ciliates (the gap per cent; 5–6%). Furthermore there was little similarity between Carchesium and other vorticellid ciliates (70–76%). Epistylis had a higher similarity to the genus Vorticella rather than Carchesium (83–88%).
Similarity values for vorticellid SSrRNA sequences. The upper triangle shows the rate of similarity. The lower triangle shows the rate of deletions or insertions for all pairs.
The SSrRNA sequences of vorticellid and other ciliates aligned with gaps were 1065 nucleotides and were used to generate a phylogenetic tree. These sequences corresponded to positions 121–1109 of the V. convallaria (AF070700) SSrRNA gene. The maximum-likelihood analysis and the constructed tree showed inferred phylogenetic relationships of vorticellid ciliates. Paramecium tetrauleria was chosen as the out-group species. The species that belongs to Peritrichia formed one cluster (Fig.3). Three different strains of V. convallaria, V. campanula and V. sp. formed a strongly monophyletic group (73–100%). However V. microstoma were grouped with Opisthonecta henneguyi rather than the other Vorticella species. In this study, Carchesium polypinum separated and revealed a considerable distance from the other vorticellid ciliate clusters.
For a long time, the taxonomy of the genus Vorticella has been based on macro-morphological descriptions such as the shape and size of the cell, cilia, and the form of the macronuclei. It has been difficult to choose a morphological character as a phylogenetic marker, the macro-morphological information being insufficient to identify the species, especially in the genus Vorticella. The vorticellid ciliates inhabit various environments, but culture has been restricted to a few strains of vorticellid ciliates. According to our experience, most of the cells collected from nature were initially able to grow but eventually stopped when they grew into a certain quantity. Therefore, we had to repeat the isolation from nature for some experiments. If the gene sequence data, like the small subunit rRNA gene sequences, were effective for identification of Vorticella species, it would be possible to study them using collected cells without misidentifying the species.
The taxonomy of a subclass in the Oligohymenophorea class has been established by previous research. For example, the phylogenetic relationships within the genus Paramecium were confirmed by molecular data of SSrRNA gene sequences (Struder-Kypke, 2000a, b). The phylogenies among the genus Tetrahymena were also defined by inferring from SSrRNA gene sequences and histone H4 proteins (Brunck, 1990; Sadler, 1992; Wright, 1995). But information about the gene of the vorticellid ciliates has been limited, as against many extant species. Additionally SSrRNA gene sequences of the genus Vorticella are restricted to three species though there are descriptions of many species in previous studies (Vorticella convallaria, V. microstoma and V. campanula). Therefore, additional species within Peritrichia are needed to give a strong confirmation of the relationships of the class Oligohymenophorea (Greenwood, 1991). Miao et al. (2001) showed that within the subclass Peritrichia, species were separated into two clades: one including colonial Epistylis, and the other solitary Vorticella and O. henneguyi. Both Carchesium and Vorticella have been classified into the same family Vorticellidae morphologically (Ehrenberg, 1838). Carchesium was separated from the genus Vorticella in the phylogenetic tree (Fig.3). The present result suggests the possibility that clarifying a relationship between Carchesium and Vorticella is more remote than the prospect of morphological classification. Our results suggested that the subclass Peritrichia were separated into three clades according to the phylogenetic tree based on partial SSrRNA gene sequences.
Vorticellid ciliates and the genus Tetrahymena belong to the Oligohymenophorea class. Tetrahymena has been considered to be a genus in which morphological stability has been maintained (Nanney, 1998), while there has been considerable variation in molecular constituents of the cells (Borden, 1977; Williams, 1986). However, a few differences in the sequence have been found among closely related Tetrahymena species in small subunit rRNAs (Jerome, 1996; Sogin, 1986; Wright, 1995) and in large subunit rRNAs (Nanney, 1998). Both Tetrahymena and Paramecium can show all the mating crosses required for species identification. In this study, SSrRNA gene sequences revealed the existence of a lot of differences among the same species of V. convallaria. Specifically, there were more insertions and deletions in SSrRNA gene sequences of the genus V. convallaria than in one of the genus Tetrahymena. The similarity value of the genus Vorticella was lower than one between the genus Tetrahymena (over 98%; Wright, 1995). The variation existed in both SSrRNA gene sequences as well as the forms of V. convallaria. Accordingly, the genus Vorticella did not seem to maintain morphological stability. However, it was possible to identify the species on the phylogenetic tree based on SSrRNA gene sequences, because strains having the main morphological characteristic of V. convallaria formed the monophyletic group. In this research, the sequence regions compared may have been too short and the compared species too small to resolve relationships within the genus Vorticella. There is only one report about the genus Epistylis and no information about the degree of the SSrRNA gene sequence variation among the different stocks in the same species of other ciliates. Relationships within the genus Vorticella are poorly resolved on the available sequence information.
In the cells morphologically classified into Vorticella convallaria, there are variations in SSrRNA gene sequences. The variation in the same species is as large as the one among the different species. The morphological cell type of V. convallaria is different from those of V. campanula and V. microstoma. According to the phylogenetic tree V. convallaria formed a monophyletic group with V. sp. having the morphological characteristic of V. convallaria. The contractile pattern of spasmoneme was not an important phylo-genetic character. Our two subgroups in the genus Vorticella were related by the size of the peristomial lip in relation to the maximum body width; V. convallaria and V. campanula have a peristomial lip diameter equal to, or greater than, respectively, the maximum body width while the one of V. microstoma was distinctly less than the greatest body width. In the genus Epistylis, subgroups were related based on the thickness of the peristomial lip (Miao et al., 2001). The peristomial area may thus be an important phylogenetic character within vorticellid ciliates.
However, our results don't support a monophyly of the genus Vorticella. This was also reported by Miao et al. (2001), that is that V. convallaria grouped with O. henneguyi, and not the other Vorticella species in the phylogenetic tree using SSrRNA complete sequences. Because of this, we considered, for example, the following: that the current classification was unsuitable, that identification erred, that data for analysis was unsuitable, and that the differentiation of the gene did not reflect the diversification of the taxa. If either O. henneguyi or V. microstoma were mis-identified, the genus Vorticella formed the monophyly. Therefore, the classification of the genus Vorticella is more complicated than that it was thought, and. it is necessary to acquire molecular sequence information to clarify the relationships between Vorticella and O. henneguyi.
These results show the possibility that the SSrRNA gene is one of the important markers for identifying species of Vorticella. The analysis of the SSrRNA gene sequence may be able to be used to distinguish V. convallaria collected from nature, and the variations of the SSrRNA gene sequence suggest a possibility that there are a number of variations in the gene of 20kDa calcium-binding protein (Spasmin) in the same species. The identification of species that can be seen to have been based on unsuitable markers of morphological characteristics may have caused this difficulty in species identification. Morphological diversification using outdated descriptions and phylogenetic complications within the genus Vorticella have also helped give rise to these difficulties. The analysis of other gene information and additional species may reveal important elements of morphological characteristics as identified markers.
We thank Dr. Che Ning (Tianjin University of Light Industry) for providing us with strains of Vorticella sp. strain Tianjin.