Ellen M. Simon

Comparison of Sequence Differences in a Variable 23S rRNA Domain among Sets of Cryptic Species of Ciliated Protozoa

ABSTRACT Studies were undertaken to discover the relative molecular distances separating some familiar forms of ciliated protozoa, and the genetic species they include. Sequences of 190 bases of the D2 domain of the large ribosomal nucleic acid molecule were obtained by polymerase chain reaction from protists of three distinctive groups of ciliated protozoa‐Colpoda, Paramecium and Tetrahymena. Evolutionary trees were constructed for each set of sequences using the PHYLOGEN 1.0 string programs. All three groups of ciliates manifested large molecular diversity among strains difficult or impossible to distinguish morphologically. The largest single evolutionary distance within a group was the 75 differences separating Tetrahymena paravorax from the other tetrahymenids. The largest mean distance for a group was the 21.2 for the colpodids. In all the protist groups the large molecular diversity is obscured by morphological conservatism associated with constraints of ancient designs. The molecular diversity within morphotypes argues for long evolutionary coexistence of species differentiated from each other in significant physiological, ecological, or nutritional ways.

Ciliate evolution: The ribosomal phylogenies of the tetrahymenine ciliates

SummaryWe have assembled and analyzed nucleotide sequences for several different rRNA components from tetrahymenine ciliates. These include previously published and some new 5S and 5.8S rRNAs for a total of 18 species. We also report sequences for some 30 species obtained by primer extension analysis of a region near the 5′ end of the 23S rRNAs (region 580). Phylogenetic trees have been constructed for these species, utilizing heuristics (shifting ditypic site analysis) described in a companion paper. The trees based on these sequences are consistent with each other and with those based on longer sequences of the 17S rRNA. They show the tetrahymenines to consist of a number of distinctive clusters of species. The clusters (ribosets) are homogeneous with respect to certain life history characteristics, especially the mode of mating type determination, but are inhomogeneous with respect to some morphological and life history features, such as cyst formation and adaptations to parasitism or carnivory. Using the same molecular data, we also begin to explore the relationships of the tetrahymenines to some other ciliate taxa and to some other protists.

Genetic control of maturity in Tetrahymena pyriformis

A new mutation, Em , is described for T. pyriformis , syngen 1. This dominant mutation eliminates the normal immature period in heterozygotes but is lethal in the homozygous state. This paper is dedicated to Dr. Ralph Cleland in honor of his 75th birthday.

Tetrahymena: effect of freezing and subsequent thawing on breeding performance.

Tetrahymena pyriformis, syngen 1, frozen in 10 percent dimethyl sulfoxide, stored for 2 months, and then thawed could conjugate normally. More significantly, they were viable, in normal numbers, through two sex ual reorganizations. The strains ap parently did not sustain genetic dam age during the treatment. The tech niques offer considerable promise for the maintenance of breeding stocks in ciliated protozoans.

Shifting ditypic site analysis: Heuristics for expanding the phylogenetic range of nucleotide sequences in Sankoff analyses

SummaryWe describe and illustrate a simple heuristic approach to the Sankoff methods for construction of parsimonious evolutionary trees from nucleotide sequence data. The procedure is intended to permit more valid inferences, particularly from relatively short sequences, concerning relationships among taxa separated for long time intervals. The procedure is based on the freat variability of evolutionary plasticity among sites in the molecules and removes from consideration the more highly variable sites. Editing is accomplished after classifying sites in carefully aligned arrays of sequences. Only “ditypic sites,” i.e., sites observed in only two evolutionary states within the array, are used in making phylogenetic inferences. This strategy makes possible the construction of good approximations to the most parsimonious Steiner strees, by means of efficient programs that require “dense species arrays,” i.e., species sets that differ from each other by relatively small numbers of differences in conservative sites. The technique is illustrated with 5S and 5.8S rRNA sequence data from published catalogs.

Crypthecodinium and Tetrahymena: An exercise in comparative evolution

SummaryNucleotide sequences have been determined for the highly variable D2 region of the large rRNA molecule for over 60 strains of dinoflagellates. These strains were selected from a worldwide collection that represents all the known sibling species (compatibility groups, Mendelian species) in the sibling swarm referred to as Crypthecodinium cohnii. A phylogenetic tree has been constructed from an analysis of the variations in a length of about 180 bases, using PHYLOGEN string analysis programs. The Crypthecodinium tree is compared with the previously published but here augmented tree constructed upon the same rRNA region for the sibling species of a worldwide collection of ciliated protozoa related to the genus Tetrahymena. The first reported sequence of Lambornella clarki, the parasite of tree-hole mosquitoes, is included.The dinoflagellate species complex is much more homogeneous with respect to ribosomal variation. The mean number of differences among sequences from different Crypthecodinium species is about 7, in comparison with 22 differences among the ciliate species examined. Moreover, all the diversity in the dinoflagellates can be explained by base substitutions, whereas insertions and deletions are common in the ciliates. The dinoflagellates are also much more uniform with respect to nutritional and genetic economies.The two complexes differ also in the relationship between molecular variations and breeding compatibility. All tetrahymenine sibling species thus far examined are monomorphic in the D2 region, but several dinoflagellate species are polymorphic. Several different dinoflagellate species, moreover, have identical D2 regions. This kind of ribosomal identity of incompatible strains is found in these ciliates only in one tight cluster of species—Group C.The tetrahymenine swarm is apparently much older than the Crypthecodinium swarm, and the dinoflagellate species produce incompatible progeny species much more readily than do the ciliates, perhaps by the acquisition of mutations that potentiate incompatibility in sympatric populations.

Freezing and storage in liquid nitrogen of axenically and monoxenically cultivated Tetrahymena pyriformis

Abstract One-hundred-thirty genetically competent strains of Tetrahymena pyriformis representing three syngens (genetically isolated species) cultivated axenically or monoxenically are stored in liquid nitrogen. All strains stored for longer than 1 year (85) have been recovered. Representative lines of nine other known syngens grown axenically and monoxenically have also been recovered from −196 °C. The effects of some of the variables involved in the procedures are summarized and discussed. Some strains are apparently most resistant to cryoinjury during early stationary phase; for one other strain resistance was just as high after 3 days in the stationary phase. The rate of cooling below the freezing point was more critical than that above. Survival in ampules left unsealed was greater, at least during the first few days, than in duplicate samples which were sealed.

Germinal aging in Tetrahymena thermophila

The manifestations of germinal aging in the ciliate Tetrahymena thermophila include death of the cells at conjugation and macronuclear retention in which the normal replacement of the old macronucleus by a new one fails to occur. Available data suggest that methods of routine maintenance that reduce the number of fissions may delay aging. Differences in breeding performance following maintenance for 1-5 years in axenic peptone broth vs. bacterized Cerophyl were not significant; those following maintenance at different temperatures were significant. The analysis of several hundred crosses is consistent with a random mutational basis for aging in the micronucleus and does not support the hypothesis of an age-correlated program in which the rate of deterioration increases with time. Following routine cultural maintenance for as long as 9-11 years, some lines show no deficiencies in their ability to produce viable progeny, and sublines of the same clone frequently differ significantly in their breeding performance. Moreover, breeding degeneration occurs at constant, but different, rates in different inbred strains.

Eukaryotic origins: String analysis of 5S ribosomal RNA sequences from some relevant organisms

SummaryUsing the PHYLOGEN tree-forming programs, we evaluate the published 5S rRNA sequences in certain of the files in the Berlin DataBank in an attempt to identify the connection between archaebacteria and the eukaryotic protists. These programs are based on methods of string analysis developed by Sankoff and others. Their discriminatory power is derived from their continuous realignment of sequences through repeated assessment of insertions and deletions as well as substitutions. The programs demonstrate that even these small molecules (ca. 120 bases) retain substantial records of evolutionary events that occurred over a billion years ago. The eukaryotes seem to have been derived from ancestors near the common origins of the halobacterial and Methanococcales groups. Identifying what might have been a primordial eukaryote is more difficult because several of the species considered as early derivatives from the common root are isolated species with large genetic differences from each other and from all other extant forms that have been sequenced. The ameboid, flagellated, and ciliated protists seem to have emerged nearly simulataneously from an ancient cluster, but the sarcodinid protozoa have preference as the group of most ancient origin. The euglenozoa and the ciliates are of later derivation. Our ability to tease plausible trees from such small molecules suggests that the mode of analysis rather than the size of the molecule is often a major limitation in the reconstruction of acceptable ancient phylogenies. The residual uncertainty with respect to the conclusions of the 5S analysis may indicate a real limit on the informational content of such small molecules; the period of evolutionary time during which the primary eukaryotic radiation occurred may have been very short relative to the rate of fixation of changes in this highly conserved molecule. Much of even this limitation may be resolved, however, when a sufficiently dense sample of the problematic taxa is examined.

Paramecium aurelia: Recovery from −196 ° C☆

Abstract Representative stocks of four syngens of Paramecium aurelia grown in bacterized Cerophyl survived exposure to −196 °C in 7.5% DMSO when cooled at 5 °C/min to freezing (−6 to −8 °C) following supercooling to between −9 and −15 °C, at 1 °C/min from the heat of fusion zone to −50 °C, then plunged into liquid nitrogen. Other critical factors included the age of the culture and the method of diluting thawed samples. A very small percentage of the cells was recovered even in the best samples. However, these cells gave rise to normal healthy cultures.