Correlated responses to selection across diverse environments during experimental evolution of Tetrahymena thermophila.

Correlated responses to selection have long been observed and studied; however, it remains unclear when they will arise, and in what direction. To contribute to a growing understanding of correlated responses to selection, we used experimental evolution of the ciliate Tetrahymena thermophila to study direct and correlated responses in a variety of different environmental conditions. One experiment focused on adaptation to two different temperatures and the correlated responses across temperatures. Another experiment used inhibitory concentrations of a variety of compounds to test direct and correlated responses to selection. We found that all populations adapted to the environments in which they evolved. We also found many cases of correlated evolution across environments; few conditions resulted in trade-offs and many resulted in a positive correlated response. Surprisingly, in many instances, the correlated response was of a larger magnitude than the direct response. We find that ancestral fitness predicts the extent of adaptation, consistent with diminishing returns epistasis. Unexpectedly, we also find that this pattern of diminishing returns holds across environments regardless of the environment in which evolution occurs. We also found that the correlated response is asymmetric across environments, that is, the fitness of a population evolved in one environment and assayed in a second was inversely related to the fitness of a population evolved in the second environment and assayed in the first. These results support the notion that positive correlated responses to selection across environments are frequent, and worth further study.

Molecular basis of phenotypic plasticity in a marine ciliate.

Phenotypic plasticity, which involves phenotypic transformation in the absence of genetic change, may serve as a strategy for organisms to survive in complex and highly-fluctuating environments. However, its reaction norm, molecular basis, and evolution remain unclear in most organisms, especially microbial eukaryotes. In this study, we explored these questions by investigating the reaction norm, regulation, and evolution of phenotypic plasticity in the cosmopolitan marine free-living ciliates Glauconema spp., which undergo significant phenotypic changes in response to food shortages. This study led to the de novo assembly of macronuclear genomes using long-read sequencing, identified hundreds of differentially expressed genes associated with phenotypic plasticity in different life stages, validated the function of two of these genes, and revealed that the reaction norm of body shape in response to food density follows a power-law distribution. Purifying selection may be the dominant evolutionary force acting on the genes associated with phenotypic plasticity, and the overall data support the hypothesis that phenotypic plasticity is a trait maintained by natural selection. This study provides novel insight into the developmental genetics of phenotypic plasticity in non-model unicellular eukaryotes, and sheds light on the complexity and long evolutionary history of this important survival strategy.

Integrative analyses on the ciliates Colpoda illuminate the life history evolution of soil microorganisms.

Microorganisms play a central role in sustaining soil ecosystems and agriculture, and these functions are usually associated with their complex life history. Yet, the regulation and evolution of life history have remained enigmatic and poorly understood, especially in protozoa, the third most abundant group of organisms in the soil. Here, we explore the life history of a cosmopolitan species-Colpoda steinii. Our analysis has yielded a high-quality macronuclear genome for C. steinii, with size of 155 Mbp and 37,123 protein-coding genes, as well as mean intron length of ~93 bp, longer than most other studied ciliates. Notably, we identify two possible whole-genome duplication events in C. steinii, which may account for its genome being about twice the size of C. inflata's, another co-existing species. We further resolve the gene expression profiles in diverse life stages of C. steinii, which are also corroborated in C. inflata. During the resting cyst stage, genes associated with cell death and vacuole formation are upregulated, and translation-related genes are downregulated. While the translation-related genes are upregulated during the excystment of resting cysts. Reproductive cysts exhibit a significant reduction in cell adhesion. We also demonstrate that most genes expressed in specific life stages are under strong purifying selection. This study offers a deeper understanding of the life history evolution that underpins the extraordinary success and ecological functions of microorganisms in soil ecosystems.IMPORTANCEColpoda species, as a prominent group among the most widely distributed and abundant soil microorganisms, play a crucial role in sustaining soil ecosystems and promoting plant growth. This investigation reveals their exceptional macronuclear genomic features, including significantly large genome size, long introns, and numerous gene duplications. The gene expression profiles and the specific biological functions associated with the transitions between various life stages are also elucidated. The vast majority of genes linked to life stage transitions are subject to strong purifying selection, as inferred from multiple natural strains newly isolated and deeply sequenced. This substantiates the enduring and conservative nature of Colpoda's life history, which has persisted throughout the extensive evolutionary history of these highly successful protozoa in soil. These findings shed light on the evolutionary dynamics of microbial eukaryotes in the ever-fluctuating soil environments. This integrative research represents a significant advancement in understanding the life histories of these understudied single-celled eukaryotes.

Mitogenomic architecture and evolution of the soil ciliates Colpoda.

Colpoda are cosmopolitan unicellular eukaryotes primarily inhabiting soil and benefiting plant growth, but they remain one of the least understood taxa in genetics and genomics within the realm of ciliated protozoa. Here, we investigate the architecture of de novo assembled mitogenomes of six Colpoda species, using long-read sequencing and involving 36 newly isolated natural strains in total. The mitogenome sizes span from 43 to 63 kbp and typically contain 28-33 protein-coding genes. They possess a linear structure with variable telomeres and central repeats, with one Colpoda elliotti strain isolated from Tibet harboring the longest telomeres among all studied ciliates. Phylogenomic analyses reveal that Colpoda species started to diverge more than 326 million years ago, eventually evolving into two distinct groups. Collinearity analyses also reveal significant genomic divergences and a lack of long collinear blocks. One of the most notable features is the exceptionally high level of gene rearrangements between mitochondrial genomes of different Colpoda species, dominated by gene loss events. Population-level mitogenomic analysis on natural strains also demonstrates high sequence divergence, regardless of geographic distance, but the gene order remains highly conserved within species, offering a new species identification criterion for Colpoda species. Furthermore, we identified underlying heteroplasmic sites in the majority of strains of three Colpoda species, albeit without a discernible recombination signal to account for this heteroplasmy. This comprehensive study systematically unveils the mitogenomic structure and evolution of these ancient and ecologically significant Colpoda ciliates, thus laying the groundwork for a deeper understanding of the evolution of unicellular eukaryotes.IMPORTANCEColpoda, one of the most widespread ciliated protozoa in soil, are poorly understood in regard to their genetics and evolution. Our research revealed extreme mitochondrial gene rearrangements dominated by gene loss events, potentially leading to the streamlining of Colpoda mitogenomes. Surprisingly, while interspecific rearrangements abound, our population-level mitogenomic study revealed a conserved gene order within species, offering a potential new identification criterion. Phylogenomic analysis traced their lineage over 326 million years, revealing two distinct groups. Substantial genomic divergence might be associated with the lack of extended collinear blocks and relaxed purifying selection. This study systematically reveals Colpoda ciliate mitogenome structures and evolution, providing insights into the survival and evolution of these vital soil microorganisms.

Genome-wide identification of ATP-binding cassette transporter B subfamily, focusing on its structure, evolution and rearrangement in ciliates.

ATP-binding cassette subfamily B (ABCB) has been implicated in various essential functions such as multidrug resistance, auxin transport and heavy metal tolerance in animals and plants. However, the functions, the genomic distribution and the evolutionary history have not been characterized systematically in lower eukaryotes. As a lineage of highly specialized unicellular eukaryotes, ciliates have extremely diverse genomic features including nuclear dimorphism. To further understand the genomic structure and evolutionary history of this gene family, we investigated the ABCB gene subfamily in 11 ciliates. The results demonstrate that there is evidence of substantial gene duplication, which has occurred by different mechanisms in different species. These gene duplicates show consistent purifying selection, suggesting functional constraint, in all but one species, where positive selection may be acting to generate novel function. We also compare the gene structures in the micronuclear and macronuclear genomes and find no gene scrambling during genome rearrangement, despite the abundance of such scrambling in two of our focal species. These results lay the foundation for future analyses of the function of these genes and the mechanisms responsible for their evolution across diverse eukaryotic lineages.

Sex, amitosis, and evolvability in the ciliate Tetrahymena thermophila.

Understanding the mechanisms that generate genetic variation, and thus contribute to the process of adaptation, is a major goal of evolutionary biology. Mutation and genetic exchange have been well studied as mechanisms to generate genetic variation. However, there are additional factors, such as genome architecture, that may also impact the amount of genetic variation in some populations, and the extent to which these variation generating mechanisms are themselves shaped by natural selection is still an open question. To test the effect of genome architecture on the generation of genetic variation, and hence evolvability, we studied Tetrahymena thermophila, a ciliate with an unusual genome structure and mechanism of nuclear division, called amitosis, whereby homologous chromosomes are randomly distributed to daughter cells. Amitosis leads to genetic variation among the asexual descendants of a newly produced sexual progeny because different progeny cells will contain different combinations of parental alleles. We hypothesize that amitosis thus increases the evolvability of newly produced sexual progeny relative to their unmated parents and species that undergo mitosis. To test this hypothesis, we used experimental evolution and simulations to compare the rate of adaptation in T. thermophila populations founded by a single sexual progeny to parental populations that had not had sex in many generations. The populations founded by a sexual progeny adapted more quickly than parental populations in both laboratory populations and simulated populations. This suggests that the additional genetic variation generated by amitosis of a heterozygote can increase the rate of adaptation following sex and may help explain the evolutionary success of the unusual genetic architecture of Tetrahymena and ciliates more generally.

Experimental Evolution in Tetrahymena.

Experimental evolution has provided novel insight into a wide array of biological processes. Species in the genus Tetrahymena are proving to be a highly useful system for studying a range of questions using experimental evolution. Their unusual genomic architecture, diversity of life history traits, importance as both predator and prey, and amenability to laboratory culture allow them to be studied in a variety of contexts. In this paper, we review what we are learning from experimental evolution with Tetrahymena about mutation, adaptation, and eco-evolutionary dynamics. We predict that future experimental evolution studies using Tetrahyemena will continue to shed new light on these processes.

Temperature affects the repeatability of evolution in the microbial eukaryote Tetrahymena thermophila.

Evolutionary biologists have long sought to understand what factors affect the repeatability of adaptive outcomes. To better understand the role of temperature in determining the repeatability of adaptive trajectories, we evolved populations of different genotypes of the ciliate Tetrahymena thermophila at low and high temperatures and followed changes in growth rate over 6,500 generations. As expected, growth rate increased with a decelerating rate for all populations; however, there were differences in the patterns of evolution at the two temperatures. The growth rates of the different genotypes tended to converge as evolution proceeded at both temperatures, but this convergence was quicker and more pronounced at the higher temperature. Additionally, over the first 4,000 generations we found greater repeatability of evolution, in terms of change in growth rate, among replicates of the same genotype at the higher temperature. Finally, we found limited evidence of trade-offs in fitness between temperatures, and an asymmetry in the correlated responses, whereby evolution in a high temperature increases growth rate at the lower temperature significantly more than the reverse. These results demonstrate the importance of temperature in determining the repeatability of evolutionary trajectories for the eukaryotic microbe Tetrahymena thermophila and may provide clues to how temperature affects evolution more generally.

Symposium on Ciliates in Memory of Denis Lynn.

Denis Lynn (1947-2018) was an outstanding protistologist, applying multiple techniques and data sources and thus pioneering an integrative approach in order to investigate ciliate biology. For example, he recognized the importance of the ultrastructure for inferring ciliate phylogeny, based on which he developed his widely accepted classification scheme for the phylum Ciliophora. In this paper, recent findings regarding the evolution and systematics of both peritrichs and the mainly marine planktonic oligotrichean spirotrichs are discussed and compared with the concepts and hypotheses formulated by Denis Lynn. Additionally, the state of knowledge concerning the diversity of ciliates in bromeliad phytotelmata and amitosis in ciliates is reviewed.

accuMUlate: a mutation caller designed for mutation accumulation experiments.

Summary: Mutation accumulation (MA) is the most widely used method for directly studying the effects of mutation. By sequencing whole genomes from MA lines, researchers can directly study the rate and molecular spectra of spontaneous mutations and use these results to understand how mutation contributes to biological processes. At present there is no software designed specifically for identifying mutations from MA lines. Here we describe accuMUlate, a probabilistic mutation caller that reflects the design of a typical MA experiment while being flexible enough to accommodate properties unique to any particular experiment. Availability and implementation accuMUlate is available from https://github.com/dwinter/accuMUlate. Supplementary information: Supplementary data are available at Bioinformatics online.

Meiotic Genes in Colpodean Ciliates Support Secretive Sexuality.

The putatively asexual Colpodean ciliates potentially pose a problem to macro-organismic theories of evolution. They are extremely ancient (although asexuality is thought to hasten extinction), and yet there is one apparently derived sexual species (implying an unlikely regain of a complex trait). If macro-organismic theories of evolution also broadly apply to microbial eukaryotes, though, then most or all of the colpodean ciliates should merely be secretively sexual. Here we show using de novo genome sequencing, that colpodean ciliates have the meiotic genes required for sex and these genes are under functional constraint. Along with these genomic data, we argue that these ciliates are sexual given the cytological observations of both micronuclei and macronuclei within their cells, and the behavioral observations of brief fusions as if the cells were mating. The challenge that colpodean ciliates pose is therefore not to evolutionary theory, but to our ability to induce microbial eukaryotic sex in the laboratory.

Low Base-Substitution Mutation Rate in the Germline Genome of the Ciliate Tetrahymena thermophil.

Mutation is the ultimate source of all genetic variation and is, therefore, central to evolutionary change. Previous work on Paramecium tetraurelia found an unusually low germline base-substitution mutation rate in this ciliate. Here, we tested the generality of this result among ciliates using Tetrahymena thermophila. We sequenced the genomes of 10 lines of T. thermophila that had each undergone approximately 1,000 generations of mutation accumulation (MA). We applied an existing mutation-calling pipeline and developed a new probabilistic mutation detection approach that directly models the design of an MA experiment and accommodates the noise introduced by mismapped reads. Our probabilistic mutation-calling method provides a straightforward way of estimating the number of sites at which a mutation could have been called if one was present, providing the denominator for our mutation rate calculations. From these methods, we find that T. thermophila has a germline base-substitution mutation rate of 7.61 × 10 - 12 per-site, per cell division, which is consistent with the low base-substitution mutation rate in P. tetraurelia. Over the course of the evolution experiment, genomic exclusion lines derived from the MA lines experienced a fitness decline that cannot be accounted for by germline base-substitution mutations alone, suggesting that other genetic or epigenetic factors must be involved. Because selection can only operate to reduce mutation rates based upon the "visible" mutational load, asexual reproduction with a transcriptionally silent germline may allow ciliates to evolve extremely low germline mutation rates.

Mutational robustness of morphological traits in the ciliate Tetrahymena thermophila.

Ciliate nuclear architecture, in particular the sequestration of a transcriptionally silent germline genome, allows for the accumulation of mutations that are "hidden" from selection during many rounds of asexual reproduction. After sexual conjugation, these mutations are expressed, potentially resulting in highly variable phenotypes. Morphological traits are widely used in ciliate taxonomy, however, the extent to which the values of these traits are robust to change in the face of mutation remains largely unknown. In this study, we examine the effects of mutations accumulated in the germline genome to test the mutational robustness of four traits commonly used in ciliate morphological taxonomy (number of somatic kineties, number of postoral kineties, macronuclear size, and cell size). We found that the number of postoral kineties is robust to mutation, confirming that it should be preferentially used in taxonomy. By contrast, we found that, as in other unicellular and multicellular species, cell and macronucleus sizes change in response to mutation. Thus, we argue that cell and macronucleus sizes, which are widely used in taxonomy, should be treated cautiously for species identification. Finally, we found evidence of correlations between cell and macronucleus sizes and fitness, suggesting possible mutational pleiotropy. This study demonstrates the importance of, and methods for, determining mutational robustness to guide morphological taxonomy in ciliates.

Genetic background alters dominance relationships between mat alleles in the ciliate Tetrahymena thermophila.

The pattern of inheritance and mechanism of sex determination can have important evolutionary consequences. We studied probabilistic sex determination in the ciliate Tetrahymena thermophila, which was previously shown to cause evolution of skewed sex ratios. We find that the genetic background alters the sex determination patterns of mat alleles in heterozygotes and that allelic interaction can differentially influence the expression probability of the 7 sexes. We quantify the dominance relationships between several mat alleles and find that A-type alleles, which specify sex I, are indeed recessive to B-type alleles, which are unable to specify that sex. Our results provide additional support for the presence of modifier loci and raise implications for the dynamics of sex ratios in populations of T. thermophila.

Accumulation of spontaneous mutations in the ciliate Tetrahymena thermophila.

Knowledge of the rate and fitness effects of mutations is essential for understanding the process of evolution. Mutations are inherently difficult to study because they are rare and are frequently eliminated by natural selection. In the ciliate Tetrahymena thermophila, mutations can accumulate in the germline genome without being exposed to selection. We have conducted a mutation accumulation (MA) experiment in this species. Assuming that all mutations are deleterious and have the same effect, we estimate that the deleterious mutation rate per haploid germline genome per generation is U=0.0047 (95% credible interval: 0.0015, 0.0125), and that germline mutations decrease fitness by s=11% when expressed in a homozygous state (95% CI: 4.4%, 27%). We also estimate that deleterious mutations are partially recessive on average (h=0.26; 95% CI: -0.022, 0.62) and that the rate of lethal mutations is <10% of the deleterious mutation rate. Comparisons between the observed evolutionary responses in the germline and somatic genomes and the results from individual-based simulations of MA suggest that the two genomes have similar mutational parameters. These are the first estimates of the deleterious mutation rate and fitness effects from the eukaryotic supergroup Chromalveolata and are within the range of those of other eukaryotes.

Chromosome copy number variation and control in the ciliate Chilodonella uncinata.

Copy number variations are widespread in eukaryotes. The unusual genome architecture of ciliates, in particular, with its process of amitosis in macronuclear division, provides a valuable model in which to study copy number variation. The current model of amitosis envisions stochastic distribution of macronuclear chromosomes during asexual reproduction. This suggests that amitosis is likely to result in high levels of copy number variation in ciliates, as dividing daughter cells can have variable copy numbers of chromosomes if chromosomal distribution during amitosis is a stochastic process. We examined chromosomal distribution during amitosis in Chilodonella uncinata, a ciliate with gene-size macronuclear chromosomes. We quantified 4 chromosomes in evolving populations of C. uncinata and modeled the amitotic distribution process. We found that macronuclear chromosomes differ in copy number from one another but that copy number does not change as expected under a stochastic process. The chromosome carrying SSU increased in copy number, which is consistent with selection to increase abundance; however, two other studied chromosomes displayed much lower than expected among-line variance. Our models suggest that balancing selection is sufficient to explain the observed maintenance of chromosome copy during asexual reproduction.

Chilodonella uncinata is not pathogenic to Culex pipiens mosquitoes.

Chilodonella uncinata was previously reported to be pathogenic to Culex and other mosquitoes based on the observation of high densities of ciliates inside of dead mosquito larvae. Here, we tested whether co-culturing Cx. pipiens with Ch. uncinata resulted in decreased survival of the mosquito larvae. We find no evidence that Ch. uncinata is pathogenic to Cx. pipiens. We suggest that Ch. uncinata may instead feed on already deceased larvae.

On the immortality of television sets: "function" in the human genome according to the evolution-free gospel of ENCODE.

A recent slew of ENCyclopedia Of DNA Elements (ENCODE) Consortium publications, specifically the article signed by all Consortium members, put forward the idea that more than 80% of the human genome is functional. This claim flies in the face of current estimates according to which the fraction of the genome that is evolutionarily conserved through purifying selection is less than 10%. Thus, according to the ENCODE Consortium, a biological function can be maintained indefinitely without selection, which implies that at least 80 - 10 = 70% of the genome is perfectly invulnerable to deleterious mutations, either because no mutation can ever occur in these "functional" regions or because no mutation in these regions can ever be deleterious. This absurd conclusion was reached through various means, chiefly by employing the seldom used "causal role" definition of biological function and then applying it inconsistently to different biochemical properties, by committing a logical fallacy known as "affirming the consequent," by failing to appreciate the crucial difference between "junk DNA" and "garbage DNA," by using analytical methods that yield biased errors and inflate estimates of functionality, by favoring statistical sensitivity over specificity, and by emphasizing statistical significance rather than the magnitude of the effect. Here, we detail the many logical and methodological transgressions involved in assigning functionality to almost every nucleotide in the human genome. The ENCODE results were predicted by one of its authors to necessitate the rewriting of textbooks. We agree, many textbooks dealing with marketing, mass-media hype, and public relations may well have to be rewritten.

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.