Barcodes Reveal 48 New Species of Tetrahymena, Dexiostoma, and Glaucoma: Phylogeny, Ecology, and Biogeography of New and Established Species.

Tetrahymena mitochondrial cox1 barcodes and nuclear SSUrRNA sequences are particularly effective at distinguishing among its many cryptic species. In a project to learn more about Tetrahymena natural history, the majority of >1,000 Tetrahymena-like fresh water isolates were assigned to established Tetrahymena species with the remaining assigned to 37 new species of Tetrahymena, nine new species of Dexiostoma and 12 new species of Glaucoma. Phylogenetically, all but three Tetrahymena species belong to the well-established "australis" or "borealis" clades; the minority forms a divergent "paravorax" clade. Most Tetrahymena species are micronucleate, but others are exclusively amicronucleate. The self-splicing intron of the LSUrRNA precursor is absent in Dexiostoma and Glaucoma and was likely acquired subsequent to the "australis/borealis" split; in some instances, its sequence is diagnostic of species. Tetrahymena americanis, T. elliotti, T. gruchyi n. sp., and T. borealis, together accounted for >50% of isolates, consistent with previous findings for established species. The biogeographic range of species found previously in Austria, China, and Pakistan was extended to the Nearctic; some species show evidence of population structure consistent with endemism. Most species were most frequently collected from ponds or lakes, while others, particularly Dexiostoma species, were collected most often from streams or rivers. The results suggest that perhaps hundreds of species remain to be discovered, particularly if collecting is global and includes hosts of parasitic forms.

Agolohymena aspidocauda nov. gen., nov. spec., a histophagous freshwater tetrahymenid ciliate in the family Deltopylidae (Ciliophora, Hymenostomatia), from Idaho (northwest USA): morphology, ontogenesis and molecular phylogeny.

Morphology, ontogeny and the molecular phylogeny of Agolohymena aspidocauda nov. gen., nov. spec., a new freshwater tetrahymenid ciliate from Idaho, U.S.A, are described. The ontogeny and histophagous mode of nutrition are similar to those of Deltopylum rhabdoidesFauré-Fremiet and Mugard, 1946. The new genus is placed with Deltopylum in the resurrected family Deltopylidae Song and Wilbert, 1989. We emend the diagnostic features of the family to include division by polytomy, right and left somatic kineties extending into the preoral suture, crook-shaped or sigmoid adoral membranelles 1 and 2, markedly reduced adoral membranelle 3 and a tetrahymenid silverline pattern. The main diagnostic features of the new genus are a disc-shaped caudal ciliary array and formation of two types of resting cysts, one smooth and the other bearing tangled tubular or cylindrical lepidosomes. Nuclear small subunit ribosomal RNA gene and mitochondrial cytochrome oxidase subunit 1 gene sequences place the new genus basal within the order Tetrahymenida, well separated from members of the family Tetrahymenidae (Lambornella and Tetrahymena) and also from other tetrahymenids (Colpidium, Dexiostoma, Glaucoma). The genetic divergences between this species and other genera in Tetrahymenida are large enough to suggest placement of the new genus in a separate family. This corroborates the morphological data, since the elaborate caudal ciliary array and the lepidosome-covered resting cyst of this species are not found in other Tetrahymenidae.

Barcoding ciliates: a comprehensive study of 75 isolates of the genus Tetrahymena.

The mitochondrial cytochrome-c oxidase subunit 1 (cox1) gene has been proposed as a DNA barcode to identify animal species. To test the applicability of the cox1 gene in identifying ciliates, 75 isolates of the genus Tetrahymena and three non-Tetrahymena ciliates that are close relatives of Tetrahymena, Colpidium campylum, Colpidium colpoda and Glaucoma chattoni, were selected. All tetrahymenines of unproblematic species could be identified to the species level using 689 bp of the cox1 sequence, with about 11 % interspecific sequence divergence. Intraspecific isolates of Tetrahymena borealis, Tetrahymena lwoffi, Tetrahymena patula and Tetrahymena thermophila could be identified by their cox1 sequences, showing <0.65 % intraspecific sequence divergence. In addition, isolates of these species were clustered together on a cox1 neighbour-joining (NJ) tree. However, strains identified as Tetrahymena pyriformis and Tetrahymena tropicalis showed high intraspecific sequence divergence values of 5.01 and 9.07 %, respectively, and did not cluster together on a cox1 NJ tree. This may indicate the presence of cryptic species. The mean interspecific sequence divergence of Tetrahymena was about 11 times greater than the mean intraspecific sequence divergence, and this increased to 58 times when all isolates of species with high intraspecific sequence divergence were excluded. This result is similar to DNA barcoding studies on animals, indicating that congeneric sequence divergences are an order of magnitude greater than conspecific sequence divergences. Our analysis also demonstrated low sequence divergences of <1.0 % between some isolates of T. pyriformis and Tetrahymena setosa on the one hand and some isolates of Tetrahymena furgasoni and T. lwoffi on the other, suggesting that the latter species in each pair is a junior synonym of the former. Overall, our study demonstrates the feasibility of using the mitochondrial cox1 gene as a taxonomic marker for 'barcoding' and identifying Tetrahymena species and some other ciliated protists.

Pseudoknot structures with conserved base triples in telomerase RNAs of ciliates.

Telomerase maintains the integrity of telomeres, the ends of linear chromosomes, by adding G-rich repeats to their 3'-ends. Telomerase RNA is an integral component of telomerase. It contains a template for the synthesis of the telomeric repeats by the telomerase reverse transcriptase. Although telomerase RNAs of different organisms are very diverse in their sequences, a functional non-template element, a pseudoknot, was predicted in all of them. Pseudoknot elements in human and the budding yeast Kluyveromyces lactis telomerase RNAs contain unusual triple-helical segments with AUU base triples, which are critical for telomerase function. Such base triples in ciliates have not been previously reported. We analyzed the pseudoknot sequences in 28 ciliate species and classified them in six different groups based on the lengths of the stems and loops composing the pseudoknot. Using miniCarlo, a helical parameter-based modeling program, we calculated 3D models for a representative of each morphological group. In all cases, the predicted structure contains at least one AUU base triple in stem 2, except for that of Colpidium colpoda, which contains unconventional GCG and AUA triples. These results suggest that base triples in a pseudoknot element are a conserved feature of all telomerases.

Phylogenetic relationships amongst tetrahymenine ciliates inferred by a comparison of telomerase RNAs.

The phylogenetic relationships between ciliate species in the suborder Tetrahymenina, order Hymenostomata, was investigated by comparing their telomerase RNA (TER) sequences. This relatively small RNA is an integral part of telomerase, the ribonucleoprotein enzyme that catalyses the synthesis of telomeric DNA. Despite a relatively rapid rate of primary sequence divergence, conserved functional and structural elements within TERs facilitate the accurate alignment of truly homologous nucleotides. The tetrahymenine phylogeny derived from distance analysis of TER sequences is largely consistent with those based on rRNA and histone sequences.

Parallel evolution of histophagy in ciliates of the genus Tetrahymena.

BACKGROUND: Species of Tetrahymena were grouped into three complexes based on morphological and life history traits: the pyriformis complex of microstomatous forms; the patula complex of microstome-macrostome transformers; and the rostrata complex of facultative and obligate histophages. We tested whether these three complexes are paraphyletic using the complete sequence of the small subunit rDNA (SSrDNA). RESULTS: In addition to the 16 species of Tetrahymena whose SSrDNA sequences are known, we sequenced the complete SSrDNA from the following histophagous Tetrahymena species; Tetrahymena bergeri, Tetrahymena mobilis, Tetrahymena rostrata, and Tetrahymena setosa as well as the macrostome species Tetrahymena vorax. We also included a ciliate tentatively identified as Lambornella sp., a parasite of the mosquito Aedes sp. We confirmed earlier results using SSrDNA, which showed two distinct clusters of Tetrahymena species: the australis group and borealis group. The genetic distances among Tetrahymena are in general very small. However, all nodes were supported by high bootstrap values. With the exception of T. bergeri and T. corlissi, which are both histophagous and group as sister species, all other histophagous Tetrahymena species are most closely related to a bacterivorous species. Furthermore, Lambornella sp. and T. empidokyrea, both mosquito parasites, are sister species, although there is a considerable genetic distance between them. CONCLUSIONS: There has been parallel evolution of histophagy in the genus Tetrahymena and the three classical species complexes are paraphyletic. As the genus Lambornella arises within the Tetrahymena clade, it is not likely a defensible one.

Identification of an essential proximal sequence element in the promoter of the telomerase RNA gene of Tetrahymena thermophila.

Telomerase is a ribonucleoprotein reverse transcriptase that synthesizes and maintains telomeric DNA. Studies of telomeres and telomerase are facilitated by the large number of linear DNA molecules found in ciliated protozoa, such as Tetrahymena thermophila. To examine the expression of telomerase, we investigated the transcription of the RNA polymerase III-directed gene encoding the RNA subunit (TER1) of this enzyme. A chimeric gene containing the Glaucoma chattoni TER1 transcribed region flanked by 5' and 3' Tetrahymena regions was used to identify promoter elements following transformation of Tetrahymena cells. Disruption of a conserved proximal sequence element (PSE) located at -55 in the Tetrahymena TER1 5' flanking region eliminated expression of the chimeric gene. In addition, mutation of an A/T-rich element at -25 decreased expression markedly. A gel mobility shift assay and protein-DNA cross-linking identified a PSE-binding polypeptide of 50-60 kDa in Tetrahymena extracts. Gel filtration analysis revealed a native molecular mass of approximately 160 kDa for this binding activity. Our results point to a similar architecture between ciliate telomerase RNA and metazoan U6 small nuclear RNA promoters.