F. Paul Doerder

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.

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.

Ecological Genetics of Tetrahymena thermophila: Mating Types, i‐Antigens, Multiple Alleles and Epistasis

Until recently, Tetrahymena thermophila has rarely been isolated from nature. With improved sampling procedures, T. thermophila has been found in ponds in many northeastern states. The availability of resident populations makes possible both population and ecological genetic studies. All seven known mating types have been recovered; no eighth mating type has been found. Crosses among whole‐genome homozygotes derived from Pennsylvania isolates reveal a spectrum genotypes with mating type alleles resembling traditional A (IV‐ and VII‐) and B(I‐) categories. The genotypes differ significantly with respect to mating type frequency, both among themselves and from previously described genotypes. One A‐category genotype appears to lack mating type II, while one A‐category and all B‐category genotypes have low frequencies of mating type III, thus accounting for the low frequency of III in the pond. The low frequency of III in all five B‐category genotypes examined suggests that the founding allele in this region was low for III. These and other differences are discussed both in terms of mating type frequencies in the pond and in terms of the possible molecular structure of mat alleles. By contrast, numerous variants of the cell surface immobilization antigen are found in addition to the previously described i‐antigens. Variants of the known SerH alleles include those with restriction fragment length polymorphisms and temperature sensitivity as well as alleles with new antigenic specificity. Multiple alleles are present in single ponds. Genes exhibiting serially dominant epistasis over SerH genes also are found. In two instances (K and C), families of antigenically similar polypeptides are expressed in place of H i‐antigen. Molecular weight differences suggest that these paralogous i‐antigen genes evolve by gene duplication and unequal crossing over within central repeats. The existence of complex patterns of epistasis together with seasonal changes in i‐ag frequencies suggest that i‐ag play an important, but as yet unknown, ecological role related to the occurrence of frequent conjugation.

Genetic and Environmental Factors Affecting Mating Type Frequency in Natural Isolates of Tetrahymena thermophila

Abstract In Tetrahymena thermophila mating type alleles specify temperature sensitive frequency distributions of multiple mating types. A-like alleles specify mating types I, II, III, V and VI, whereas B-like alleles specify mating types II through VII. We have characterized the mating type distributions specified by several A- and B-like genotypes segregated by genomic exclusion from cells isolated from a pond in northwestern Pennsylvania. The B-like genotypes are alike in specifying very low frequencies of mating type III, but differ with respect to the frequencies of other mating types, particularly II and VII. An A-like genotype specifies a high frequency of mating type III and is unstable in successive generations for the expression of mating type II, suggesting a possible modifier. Inter se crosses performed at 18 °C, 28 °C and 34 °C showed that each genotype specifies a frequency distribution that is uniquely affected by temperature. No mating type was affected the same way by temperature in all genotypes. In A/B heterozygotes, the B-like genotype exhibited partial dominance. The genotypes described here differ significantly from previously described genotypes from the same pond, indicating that there are numerous mating type alleles. For frequency-dependent selection to equalize mating type frequencies, it must act not only on complex multiple alleles but also on the response of mating type alleles to temperature.

Immobilization Antigen Variation in Natural Isolates of Tetrahymena thermophila

In Tetrahymena thermophila alternative forms of the major cell surface glycoprotein (the immobilization antigen) are specified by both allelic and non-allelic genes. The differential expression of non-allelic genes is affected primarily by temperature and culture medium. This report describes expression and genetic variation of immobilization antigens among 2,600 clones isolated from natural populations. The temperature regulated L (<20 o ) and H (20 o C-36 o C) antigens and two new antigens J and K were present among ∼57% of isolates; the remaining 43% appear to have unknown antigens. Genetic and Southern analyses show that J and K are due to genes with dominant epistasis over H and that the gene for K is also epistatic over that for J. This is the first reported instance of naturally occurring epistasis involving immobilization antigen expression in T. thermophila. In ponds, the frequencies of J and H vary inversely in a manner consistent with dominant epistasis. The frequencies of J and H also show seasonal variation, with J more common in the late spring, and H more common in the late summer and fall. L and H (and J) also show seasonal variation, with L more common in the early spring and late fall. Allelic variation was also found among the H antigens. Immunodiffusion showed that the H3 protein of natural isolates is partially identical to H3 of inbred strain B. In addition, two Hind III restriction fragment length polymorphisms were found among the natural SerH3 genes. New SerH3 genes also appeared to segregate in crosses. The genetic and seasonal variation in i-antigen frequencies suggests an important biological role for these surface proteins

Abandoning sex: multiple origins of asexuality in the ciliate Tetrahymena

BackgroundBy 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.ResultsAmicronucleates 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.ConclusionsNaturally 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.

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.

In Memoriam: David L. Nanney (1925-2016)

CILIATE geneticist and Tetrahymena advocate David L. Nanney passed away on June 4, 2016 at the age of 90. A student of pioneer ciliate geneticist Tracy M. Sonneborn, Nanney domesticated Tetrahymena thermophila and described many of the phenomena that led to its becoming a model research organism. He is also remembered for his theoretical contributions regarding epigenetics and the role of the cytoplasm in heredity. David Ledbetter Nanney was born on October 10, 1925 in the Blue Ridge Mountains of Virginia and grew up in Wewoka, Oklahoma, the small town capital of the Seminole Nation. As a child he was an avid reader and contrarian student. Exempted from WWII service due to complications from childhood illness, he graduated from Oklahoma Baptist University in 1946 with a degree in English. When funding for graduate studies in literature fell through, Nanney, prompted by his biology professor, took advantage of the better postwar funding in zoology and, after rejecting one offer, accepted the more lucrative assistantship at Indiana University at Bloomington; his duty there was to assist teaching comparative anatomy to war veteran premeds. At Indiana it was Hermann J. Muller’s course that drew him to genetics, but it was Sonneborn with whom he earned a Ph.D. in zoology in 1951. Sonneborn would continue to be a mentor until his death 30 years later. After graduation Nanney married Jean Kelly, a graduate student in piano, who had her own career first as a public school music teacher and second as a minister to the elderly. In 1951 Nanney joined the faculty of the University of Michigan in Ann Arbor where he began lifelong work on Tetrahymena. In 1958 he spent a year at the California Institute of Technology, and in 1959 he joined the faculty at the University of Illinois in UrbanaChampaign as a Professor of Genetics and Development. He retired from Illinois in 1991, though he continued to publish until 2008. A tribute to David Nanney was published in 1992 (Allen and Orias 1992). Nanney’s career with Tetrahymena began at Michigan when matings among the Woods Hole Tetrahymenas in Alfred Elliott’s collection produced viable progeny; these strains would later become known as T. thermophila. Nanney discovered that, as in Paramecium, the cells belonged to specific mating types. He spent the next few years working out the genetics of mating type heredity (Nanney et al. 1955) and laboriously establishing the inbred strains still used today. He found that a single mating type allele specifies five or six of the seven mating types and that (usually) only one mating type is chosen for expression by a developing macronucleus; not all mating types are chosen with equal frequency, and the frequency with which a given mating type is chosen is inherited with the allele. For a given pair of conjugants, each of which develops two new macronuclei, there could be four different mating types among otherwise genetically identical progeny. A mating type gene pair specific for every mating type has now been sequenced, and the programmed DNA editing necessary to assemble a given gene pair from noncontiguous DNA segments is now known (Cervantes et al. 2013). However, it is still unknown, for example, how mating types are chosen in development in their hereditary frequency. This is typical of much of Nanney’s work: the biological phenomena he described in intriguing detail still challenge researchers to find their molecular mechanisms. David L. Nanney with first printing of Experimental Ciliatology (1980).

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.

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.