Molecular identification of Tetrahymena species.

Mitochondrial cox1 689 bp barcodes are routinely used for identification of Tetrahymena species. Here, we examine whether two shorter nuclear sequences, the 5.8S rRNA gene region and the intergenic region between H3 and H4 histone genes, might also be useful either singly or in combination with each other or cox1. We obtained sequences from ~300 wild isolates deposited at the Tetrahymena Stock Center and analyzed additional sequences obtained from GenBank. The 5.8S rRNA gene and portions of its transcribed flanks identify isolates as to their major clade and uniquely identify some, but not all, species. The ~330 bp H3/H4 intergenic region possesses low intraspecific variability and is unique for most species. However, it fails to distinguish between two pairs of common species and their rarer counterparts, and its use is complicated by the presence of duplicate genes in some species. The results show that while the cox1 sequence is the best single marker for Tetrahymena species identification, 5.8S rRNA, and the H3/H4 intergenic regions sequences are useful, singly or in combination, to confirm cox1 species assignments or as part of a preliminary survey of newly collected Tetrahymena. From our newly collected isolates, the results extend the biogeographical range of T. shanghaiensis and T. malaccensis and identify a new species, Tetrahymena arleneae n. sp. herein described.

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.

Molecular Polymorphism in the MTA and MTB Mating Type Genes of Tetrahymena thermophila and Related Asexual Species.

Each of the seven mating types of Tetrahymena thermophila is determined by a pair of large genes, MTA and MTB, whose expression peaks at early conjugation. Each protein consists of a mating-type specific domain and a common transmembrane domain. To assess variation in natural populations, regions of both domains from wild isolates expressing mating types V and VII were analyzed. Corresponding regions of amicronucleates incapable of mating also were examined. MTA and MTB showed high haplotype diversity, with greater sequence variation in MTB. Mating type VII was less variable than mating type V, suggesting more recent origin. No polymorphism distinguished between mat1- and mat2-like alleles encoding different arrays of mating types, nor did polymorphisms give evidence of population structure. MTA and MTB variants have different phylogenies, suggesting independent rather than concerted evolution, and are under weak purifying selection. Codon usage is less biased than for housekeeping genes, and reassigned glutamine encoding stop codons are preferentially used. Amicronucleate T. thermophila and closely related nsp15 and nsp25 have higher levels of nucleotide and amino acid substitution, consistent with cox1 distances. The results suggest that complete sequencing of mating type genes of wild isolates coupled with functional analysis will be informative.

Abandoning sex: multiple origins of asexuality in the ciliate Tetrahymena.

BACKGROUND: By segregating somatic and germinal functions into large, compound macronuclei and small diploid micronuclei, respectively, ciliates can explore sexuality in ways other eukaryotes cannot. Sex, for instance, is not for reproduction but for nuclear replacement in the two cells temporarily joined in conjugation. With equal contributions from both conjugants, there is no cost of sex which theory predicts should favor asexuality. Yet ciliate asexuality is rare. The exceptional Tetrahymena has abandoned sex through loss of the micronucleus; its amicronucleates are abundant in nature where they reproduce by binary fission but never form conjugating pairs. A possible reason for their abundance is that the Tetrahymena macronucleus does not accumulate mutations as proposed by Muller's ratchet. As such, Tetrahymena amicronucleates have the potential to be very old. This study used cytochrome oxidase-1 barcodes to determine the phylogenetic origin and relative age of amicronucleates isolated from nature. RESULTS: Amicronucleates constituted 25% of Tetrahymena-like wild isolates. Of the 244 amicronucleates examined for cox1 barcodes, 237 belonged to Tetrahymena, seven to other genera. Sixty percent originated from 12 named species or barcoded strains, including the model Tetrahymena thermophila, while the remaining 40% represent 19 putative new species, eight of which have micronucleate counterparts and 11 of which are known only as amicronucleates. In some instances, cox1 haplotypes were shared among micronucleate and amicronucleates collected from the same source. Phylogenetic analysis showed that most amicronucleates belong to the "borealis" clade in which mating type is determined by gene rearrangement. Some amicronucleate species were clustered on the SSU phylogenetic tree and had longer branch lengths, indicating more ancient origin. CONCLUSIONS: Naturally occurring Tetrahymena amicronucleates have multiple origins, arising from numerous species. Likely many more new species remain to be discovered. Shared haplotypes indicate that some are of contemporary origin, while phylogeny indicates that others may be millions of years old. The apparent success of amicronucleate Tetrahymena may be because macronuclear assortment and recombination allow them to avoid Muller's ratchet, incorporate beneficial mutations, and evolve independently of sex. The inability of amicronucleates to mate may be the result of error(s) in mating type gene rearrangement.

Restricted distribution and limited gene flow in the model ciliate Tetrahymena thermophila.

The biogeography of microbial eukaryotes has long been debated, but few phylogeographic data have been available to assess whether protists tend to have ubiquitous or endemic distributions. We addressed this issue in the ciliate Tetrahymena thermophila, a highly successful model system in cell and molecular biology. We found that this species has a distribution that is restricted to the Eastern United States, with high diversity in the northeast and low diversity across the rest of its distribution. We find high levels of population subdivision, low rates of migration and significant isolation by distance, supporting the moderate endemicity model of protist biogeography. This restricted gene flow may be a result of small population size, which would reduce the probability of migration events, or the inability to establish after migration. This work lays the foundation for T. thermophila to become a valuable model system for studying population biology.

The life and times of Tetrahymena.

The genus Tetrahymena is defined on the basis of a four-part oral structure composed of an undulating membrane and three membranelles. It is a monophyletic genus with 41 named species and numerous unnamed species, many of which are morphologically indistinguishable. Nuclear small subunit rRNA and mitochondrial cytochrome c oxidase subunit 1 sequences indicate two major clades, a "borealis" clade of less closely related species and an "australis" clade of more closely related species that correlate to differences in mating-type determination and frequency of amicronucleates. Members of both clades show convergence for histophagy (primarily facultative), macrostome transformation, and (rare) cyst formation. Life cycle parameters of species are presented and problematic species discussed.

Natural populations and inbred strains of Tetrahymena.

Tetrahymena typically is found in freshwater lakes, ponds, and streams in association with submerged or emergent vegetation. The genus consists of numerous breeding species with micronuclei and many asexual species without micronuclei. In summer months when most populations are at their peak, 30-50% of water samples may yield one or more species of Tetrahymena. This chapter describes both bulk and trapping procedures for collecting Tetrahymena and also evaluates barcode methods for species identification. The history and inbreeding of the laboratory model Tetrahymena thermophila is also discussed. There are numerous unresolved questions about Tetrahymena evolution and biogeography that may be solved by additional collecting.

Barcoding Tetrahymena: discriminating species and identifying unknowns using the cytochrome c oxidase subunit I (cox-1) barcode.

DNA barcoding using the mitochondrial cytochromecoxidase subunit I (cox-1) gene has recently gained popularity as a tool for species identification of a variety of taxa. The primary objective of our research was to explore the efficacy of using cox-1 barcoding for species identification within the genusTetrahymena. We first increased intraspecific sampling forTetrahymena canadensis, Tetrahymena hegewischi, Tetrahymena pyriformis, Tetrahymena rostrata, Tetrahymena thermophila, and Tetrahymena tropicalis. Increased sampling efforts show that intraspecific sequence divergence is typically less than 1%, though it may be more in some species. The barcoding also showed that some strains might be misidentified or mislabeled. We also used cox-1 barcodes to provide species identifications for 51 unidentified environmental isolates, with a success rate of 98%. Thus, cox-1 barcoding is an invaluable tool for protistologists, especially when used in conjunction with morphological studies.

Patterns of protein evolution in Tetrahymena thermophila: implications for estimates of effective population size.

High levels of synonymous substitutions among alleles of the surface antigen SerH led to the hypothesis that Tetrahymena thermophila has a tremendously large effective population size, one that is greater than estimated for many prokaryotes (Lynch, M., and J. S. Conery. 2003. Science 302:1401-1404.). Here we show that SerH is unusual as there are substantially lower levels of synonymous variation at five additional loci (four nuclear and one mitochondrial) characterized from T. thermophila populations. Hence, the effective population size of T. thermophila, a model single-celled eukaryote, is lower and more consistent with estimates from other microbial eukaryotes. Moreover, reanalysis of SerH polymorphism data indicates that this protein evolves through a combination of vertical transmission of alleles and concerted evolution of repeat units within alleles. SerH may be under balancing selection due to a mechanism analogous to the maintenance of antigenic variation in vertebrate immune systems. Finally, the dual nature of ciliate genomes and particularly the amitotic divisions of processed macronuclear genomes may make it difficult to estimate accurately effective population size from synonymous polymorphisms. This is because selection and drift operate on processed chromosomes in macronuclei, where assortment of alleles, disruption of linkage groups, and recombination can alter the genetic landscape relative to more canonical eukaryotic genomes.

Polymorphism and selection at the SerH immobilization antigen locus in natural populations of Tetrahymena thermophila.

The SerH locus of Tetrahymena thermophila is one of several paralogous loci with genes encoding variants of the major cell surface protein known as the immobilization antigen (i-ag). The locus is highly polymorphic, raising questions concerning functional equivalency and selective forces acting on its multiple alleles. Here, we compare the sequences and expression of SerH1, SerH3, SerH4, SerH5, and SerH6. The precursor i-ags are highly similar. They are rich in alanine, serine, threonine, and cysteine and they share nearly identical ER translocation and GPI addition signals. The locations of the 39 cysteines are highly conserved, particularly in the 3.5 central, imperfect tandem repeats in which 8 periodic cysteines punctuate alternating short and long stretches of amino acids. Hydrophobicity patterns are also conserved. Nevertheless, amino acid sequence identity is low, ranging from 60.7 to 82.9%. At the nucleotide level, from 9.7 to 26.7% of nucleotide sites are polymorphic in pairwise comparisons. Expression of each allele is regulated by temperature-sensitive mRNA stability. H mRNAs are stable at <36 degrees but are unstable at >36 degrees. The H5 mRNA, which is less affected by temperature, has a different arrangement of the putative mRNA destabilization motif AUUUA. Statistical analysis of SerH genes indicates that the multiple alleles are neutral. Significantly low ratios of the rates of nonsynonymous to synonymous amino acid substitutions suggest that the multiple alleles are subject to purifying (negative) selection enforcing constraints on structure.