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.     

Upper limit of the rate and per generation effects of deleterious genomic mutations

Unbiased or upper limit estimates of the rate (U) of genomic mutations to mildly deleterious alleles are crucial in genetic and conservation studies and in human health care. However, only a few estimates of the lower bounds of U are available. We present a fairly robust estimation that yields an upper limit of U and a nearly unbiased estimate of the per generation fitness decline due to new deleterious mutations. We applied the approach to three species of the freshwater microcrustacean Daphnia and revealed that the upper limit of U for egg survivorship is 0.73 (SD = 0.30) in 14 D. pulicaria populations. For the first four clutches, per generation decline in fecundity due to deleterious mutations ranged from 2.2% to 7.8% in 20 D. pulex populations and from 1.1% to 5.1% in 8 D. obtusa populations. These results indicate the mutation pressure is high in natural Daphnia populations. The approach investigated here provides a potential way to quickly and conveniently characterize U and per generation effects of deleterious genomic mutations on fitness or its important components such as fecundity.     

Deleterious Mutations, Apparent Stabilizing Selection and the Maintenance of Quantitative Variation

Apparent stabilizing selection on a quantitative trait that is not causally connected to fitness can result from the pleiotropic effects of unconditionally deleterious mutations, because as N. Barton noted, "...individuals with extreme values of the trait will tend to carry more deleterious alleles...." We use a simple model to investigate the dependence of this apparent selection on the genomic deleterious mutation rate, U; the equilibrium distribution of K, the number of deleterious mutations per genome; and the parameters describing directional selection against deleterious mutations. Unlike previous analyses, we allow for epistatic selection against deleterious alleles. For various selection functions and realistic parameter values, the distribution of K, the distribution of breeding values for a pleiotropically affected trait, and the apparent stabilizing selection function are all nearly Gaussian. The additive genetic variance for the quantitative trait is kQa2, where k is the average number of deleterious mutations per genome, Q is the proportion of deleterious mutations that affect the trait, and a2 is the variance of pleiotropic effects for individual mutations that do affect the trait. In contrast, when the trait is measured in units of its additive standard deviation, the apparent fitness function is essentially independent of Q and a2; and beta, the intensity of selection, measured as the ratio of additive genetic variance to the "variance" of the fitness curve, is very close to s = U/k, the selection coefficient against individual deleterious mutations at equilibrium. Therefore, this model predicts appreciable apparent stabilizing selection if s exceeds about 0.03, which is consistent with various data. However, the model also predicts that beta must equal Vm/VG, the ratio of new additive variance for the trait introduced each generation by mutation to the standing additive variance. Most, although not all, estimates of this ratio imply apparent stabilizing selection weaker than generally observed. A qualitative argument suggests that even when direct selection is responsible for most of the selection observed on a character, it may be essentially irrelevant to the maintenance of variation for the character by mutation-selection balance. Simple experiments can indicate the fraction of observed stabilizing selection attributable to the pleiotropic effects of deleterious mutations.     

Functional origins of fitness effect-sizes of compensatory mutations in the DNA bacteriophage phiX174

Abstract Epistasis is an important and poorly understood aspect of mutations and strongly influences the evolutionary impact of genetic variation on adaptation and fitness. Although recent studies have begun to characterize the distribution of epistatic effects between mutations affecting fitness, there is currently a lack of empirical information on the underlying biological causes of these epistatic interactions. What are the functional constraints that determine the effectiveness of a compensatory mutation at restoring fitness? We have measured the effect‐sizes of 52 compensatory mutations affecting nine different deleterious mutations in the major capsid and spike proteins of the DNA bacteriophage X174. On average, an experimentally detectable compensatory mutation recovers about two‐thirds of the fitness cost of the preceding deleterious mutation. Variation in fitness effect‐sizes is only weakly associated with measures of the distance separating the deleterious and compensatory mutations in the amino acid sequence or the folded protein structure. However, there is a strong association of fitness effect‐size with the correlation in the effects of the mutations on the biochemical properties of amino acids. A compensatory mutation has the largest effect‐size, on average, when both the compensatory and deleterious mutations have radical effects on the overall biochemical make‐up of the amino acids. By examining the relative contributions of specific biochemical properties to variation in fitness effect‐size, we find that the area and charge of amino acids have a major influence, which suggests that the complexity of the amino acid phenotype is simplified by selection into a reduced number of phenotypic components.     

Properties of spontaneous mutations affecting quantitative traits

Recent mutation accumulation results from invertebrate species suggest that mild deleterious mutation is far less frequent than previously thought, implying smaller expressed mutational loads. Although the rate (lambda) and effect (s) of very slight deleterious mutation remain unknown, most mutational fitness decline would come from moderately deleterious mutation (s approximately 0.2, lambda approximately 0.03), and this situation would not qualitatively change in harsh environments. Estimates of the average coefficient of dominance (h) of non-severe deleterious mutations are controversial. The typical value of h = 0.4 can be questioned, and a lower estimate (about 0.1) is suggested. Estimated mutational parameters are remarkably alike for morphological and fitness component traits (excluding lethals), indicating low mutation rates and moderate mutational effects, with a distribution generally showing strong negative asymmetry and little leptokurtosis. New mutations showed considerable genotype-environment interaction. However, the mutational variance of fitness-component traits due to non-severe detrimental mutations did not increase with environmental harshness. For morphological traits, a class of predominantly additive mutations with no detectable effect on fitness and relatively small effect on the trait was identified. This should be close to that responsible for standing variation in natural populations.     

Rates and fitness consequences of new mutations in humans

The human mutation rate per nucleotide site per generation (μ) can be estimated from data on mutation rates at loci causing Mendelian genetic disease, by comparing putatively neutrally evolving nucleotide sequences between humans and chimpanzees and by comparing the genome sequences of relatives. Direct estimates from genome sequencing of relatives suggest that μ is about 1.1 × 10(-8), which is about twofold lower than estimates based on the human-chimp divergence. This implies that an average of ~70 new mutations arise in the human diploid genome per generation. Most of these mutations are paternal in origin, but the male:female mutation rate ratio is currently uncertain and might vary even among individuals within a population. On the basis of a method proposed by Kondrashov and Crow, the genome-wide deleterious mutation rate (U) can be estimated from the product of the number of nucleotide sites in the genome, μ, and the mean selective constraint per site. Although the presence of many weakly selected mutations in human noncoding DNA makes this approach somewhat problematic, estimates are U ≈ 2.2 for the whole diploid genome per generation and 0.35 for mutations that change an amino acid of a protein-coding gene. A genome-wide deleterious mutation rate of 2.2 seems higher than humans could tolerate if natural selection is "hard," but could be tolerated if selection acts on relative fitness differences between individuals or if there is synergistic epistasis. I argue that in the foreseeable future, an accumulation of new deleterious mutations is unlikely to lead to a detectable decline in fitness of human populations.     

Fitness change in relation to mutation number in spontaneous mutation accumulation lines of Chlamydomonas reinhardtii

Although all genetic variation ultimately stems from mutations, their properties are difficult to study directly. Here, we used multiple mutation accumulation (MA) lines derived from five genetic backgrounds of the green algae Chlamydomonas reinhardtii that have been previously subjected to whole genome sequencing to investigate the relationship between the number of spontaneous mutations and change in fitness from a nonevolved ancestor. MA lines were on average less fit than their ancestors and we detected a significantly negative correlation between the change in fitness and the total number of accumulated mutations in the genome. Likewise, the number of mutations located within coding regions significantly and negatively impacted MA line fitness. We used the fitness data to parameterize a maximum likelihood model to estimate discrete categories of mutational effects, and found that models containing one to two mutational effect categories (one neutral and one deleterious category) fitted the data best. However, the best‐fitting mutational effects models were highly dependent on the genetic background of the ancestral strain.     

The effect of antagonistic pleiotropy on the estimation of the average coefficient of dominance of deleterious mutations

We investigate the impact of antagonistic pleiotropy on the most widely used methods of estimation of the average coefficient of dominance of deleterious mutations from segregating populations. A proportion of the deleterious mutations affecting a given studied fitness component are assumed to have an advantageous effect on another one, generating overdominance on global fitness. Using diffusion approximations and transition matrix methods, we obtain the distribution of gene frequencies for nonpleiotropic and pleiotropic mutations in populations at the mutation-selection-drift balance. From these distributions we build homozygous and heterozygous chromosomes and assess the behavior of the estimators of dominance. A very small number of deleterious mutations with antagonistic pleiotropy produces substantial increases on the estimate of the average degree of dominance of mutations affecting the fitness component under study. For example, estimates are increased three- to fivefold when 2% of segregating loci are over-dominant for fitness. In contrast, strengthening pleiotropy, where pleiotropic effects are assumed to be also deleterious, has little effect on the estimates of the average degree of dominance, supporting previous results. The antagonistic pleiotropy model considered, applied under mutational parameters described in the literature, produces patterns for the distribution of chromosomal viabilities, levels of genetic variance, and homozygous mutation load generally consistent with those observed empirically for viability in Drosophila melanogaster.     

Sex and U

Resolution of several unsettled problems in genetics depends on the genomic rate of deleterious mutation, U. Selection against mutations can be a major factor in evolution only if U > or =1. Recently, significant progress has been made in measuring U in multicellular eukaryotes. An indirect estimate, based on a human-chimpanzee pseudogene comparison, produced U>3 for hominoids. By contrast, an estimate for Drosophila based on comparison of synonymous protein-coding sites produced U<0.1. However, the Drosophila figure might be underestimated because of selection at synonymous sites. Perhaps, the best way to measure U is to observe mutations shortly after they appear. So far, this direct approach has been applied only to humans and Caenorhabditis elegans, yielding high estimates of mutation rates.     

Deleterious genomic mutation rate for viability in Drosophila melanogaster using concomitant sibling controls

New deleterious mutations may reduce health and fitness and are involved in the evolution and maintenance of numerous biological processes. Hence, it is important to estimate the deleterious genomic mutation rate (U) in representative higher organisms. However, these estimated rates vary widely, mainly because of inadequate experimental controls. Here we describe an experimental design (the Binscy assay) with concomitant sibling controls and estimate U for viability in Drosophila melanogaster to be 0.31. This estimate, like most published studies, focuses on viability mutations and the overall deleterious genomic mutation rate would therefore be higher.     

Selection on Accessible Chromatin Regions in Capsella grandiflora

Accurate estimates of genome-wide rates and fitness effects of new mutations are essential for an improved understanding of molecular evolutionary processes. Although eukaryotic genomes generally contain a large noncoding fraction, functional noncoding regions and fitness effects of mutations in such regions are still incompletely characterized. A promising approach to characterize functional noncoding regions relies on identifying accessible chromatin regions (ACRs) tightly associated with regulatory DNA. Here, we applied this approach to identify and estimate selection on ACRs in Capsella grandiflora, a crucifer species ideal for population genomic quantification of selection due to its favorable population demography. We describe a population-wide ACR distribution based on ATAC-seq data for leaf samples of 16 individuals from a natural population. We use population genomic methods to estimate fitness effects and proportions of positively selected fixations (α) in ACRs and find that intergenic ACRs harbor a considerable fraction of weakly deleterious new mutations, as well as a significantly higher proportion of strongly deleterious mutations than comparable inaccessible intergenic regions. ACRs are enriched for expression quantitative trait loci (eQTL) and depleted of transposable element insertions, as expected if intergenic ACRs are under selection because they harbor regulatory regions. By integrating empirical identification of intergenic ACRs with analyses of eQTL and population genomic analyses of selection, we demonstrate that intergenic regulatory regions are an important source of nearly neutral mutations. These results improve our understanding of selection on noncoding regions and the role of nearly neutral mutations for evolutionary processes in outcrossing Brassicaceae species.     

Estimate of the mutation rate per nucleotide in humans

Many previous estimates of the mutation rate in humans have relied on screens of visible mutants. We investigated the rate and pattern of mutations at the nucleotide level by comparing pseudogenes in humans and chimpanzees to (i) provide an estimate of the average mutation rate per nucleotide, (ii) assess heterogeneity of mutation rate at different sites and for different types of mutations, (iii) test the hypothesis that the X chromosome has a lower mutation rate than autosomes, and (iv) estimate the deleterious mutation rate. Eighteen processed pseudogenes were sequenced, including 12 on autosomes and 6 on the X chromosome. The average mutation rate was estimated to be approximately 2.5 x 10(-8) mutations per nucleotide site or 175 mutations per diploid genome per generation. Rates of mutation for both transitions and transversions at CpG dinucleotides are one order of magnitude higher than mutation rates at other sites. Single nucleotide substitutions are 10 times more frequent than length mutations. Comparison of rates of evolution for X-linked and autosomal pseudogenes suggests that the male mutation rate is 4 times the female mutation rate, but provides no evidence for a reduction in mutation rate that is specific to the X chromosome. Using conservative calculations of the proportion of the genome subject to purifying selection, we estimate that the genomic deleterious mutation rate (U) is at least 3. This high rate is difficult to reconcile with multiplicative fitness effects of individual mutations and suggests that synergistic epistasis among harmful mutations may be common.     

Sexual selection on spontaneous mutations strengthens the between‐sex genetic correlation for fitness

A proposed benefit to sexual selection is that it promotes purging of deleterious mutations from populations. For this benefit to be realized, sexual selection, which is usually stronger on males, must purge mutations deleterious to both sexes. Here, we experimentally test the hypothesis that sexual selection on males purges deleterious mutations that affect both male and female fitness. We measured male and female fitness in two panels of spontaneous mutation‐accumulation lines of the fly, Drosophila serrata, each established from a common ancestor. One panel of mutation accumulation lines limited both natural and sexual selection (LS lines), whereas the other panel limited natural selection, but allowed sexual selection to operate (SS lines). Although mutation accumulation caused a significant reduction in male and female fitness in both the LS and SS lines, sexual selection had no detectable effect on the extent of the fitness reduction. Similarly, despite evidence of mutational variance for fitness in males and females of both treatments, sexual selection had no significant impact on the amount of mutational genetic variance for fitness. However, sexual selection did reshape the between‐sex correlation for fitness: significantly strengthening it in the SS lines. After 25 generations, the between‐sex correlation for fitness was positive but considerably less than one in the LS lines, suggesting that, although most mutations had sexually concordant fitness effects, sex‐limited, and/or sex‐biased mutations contributed substantially to the mutational variance. In the SS lines this correlation was strong and could not be distinguished from unity. Individual‐based simulations that mimick the experimental setup reveal two conditions that may drive our results: (1) a modest‐to‐large fraction of mutations have sex‐limited (or highly sex‐biased) fitness effects, and (2) the average fitness effect of sex‐limited mutations is larger than the average fitness effect of mutations that affect both sexes similarly.     

The rate of compensatory mutation in the DNA bacteriophage phiX174

A compensatory mutation occurs when the fitness loss caused by one mutation is remedied by its epistatic interaction with a second mutation at a different site in the genome. This poorly understood biological phenomenon has important implications, not only for the evolutionary consequences of mutation, but also for the genetic complexity of adaptation. We have carried out the first direct experimental measurement of the average rate of compensatory mutation. An arbitrary selection of 21 missense substitutions with deleterious effects on fitness was introduced by site-directed mutagenesis into the bacteriophage phiX174. For each deleterious mutation, we evolved 8-16 replicate populations to determine the frequency at which a compensatory mutation, instead of the back mutation, was acquired to recover fitness. The overall frequency of compensatory mutation was approximately 70%. Deleterious mutations that were more severe were significantly more likely to be compensated for. Furthermore, experimental reversion of deleterious mutations revealed that compensatory mutations have deleterious effects in a wild-type background. A large diversity of intragenic compensatory mutations was identified from sequencing fitness-recovering genotypes. Subsequent analyses of intragenic mutation diversity revealed a significant degree of clustering around the deleterious mutation in the linear sequence and also within folded protein structures. Moreover, a likelihood analysis of mutation diversity predicts that, on average, a deleterious mutation can be compensated by about nine different intragenic compensatory mutations. We estimate that about half of all compensatory mutations are located extragenically in this organism.     

Fixation of clonal lineages under Muller's ratchet

For clonal lineages of finite size that differ in their deleterious mutational effects, the probability of fixation is investigated by mathematical theory and Monte Carlo simulations. If these fitness effects are sufficiently small in one or both lineages, then the lineage with the less deleterious effects will become fixed with high probability. If, however, in both lineages the deleterious effects are larger than a threshold s(c), then the probability of fixation is independent of the fitness effects and depends only on the initial frequencies of the lineages. This threshold decreases with decreasing genomic mutation rate U and increases with population size N. (For N = 10(5), we have s(c) approximately = 0.1 if U = 1, and s(c) approximately = 0.015 if U = 0.1). Above the threshold, the competition is not driven by the ratio of mean fitnesses of the lineages, but by the relative sizes of the zero-mutation classes, which are independent of the fitness effects of the mutations. After the loss of the zero-mutation class of a lineage, the other lineage will spread to fixation with high probability and within a short time span. If the mutation rates of the lineages differ substantially, the lineage with the lower mutation rate is fixed with very high probability unless the lineage with the larger mutation rate has very slightly deleterious mutational effects. If the mutation rates differ by not more than a few percent, then the lineage with the higher mutation rate and the more deleterious effects can become fixed with appreciable probability for a certain range of parameters. The independence of the fixation probability on the fitness effects in a single population leads to dramatic effects in metapopulations: lineages with more deleterious effects have a much higher fixation probability. The critical value s(c), above which this phenomenon occurs, decreases as the migration rate between the subpopulations decreases.     

Estimates of the rate and distribution of fitness effects of spontaneous mutation in Saccharomyces cerevisiae

The per-genome, per-generation rate of spontaneous mutation affecting fitness (U) and the mean fitness cost per mutation (s) are important parameters in evolutionary genetics, but have been estimated for few species. We estimated U and sh (the heterozygous effect of mutations) for two diploid yeast strains differing only in the DNA mismatch-repair deficiency used to elevate the mutation rate in one (mutator) strain. Mutations were allowed to accumulate in 50 replicate lines of each strain, during 36 transfers of randomly chosen single colonies (approximately 600 generations). Among wild-type lines, fitnesses were bimodal, with one mode showing no change in mean fitness. The other mode showed a mean 29.6% fitness decline and the petite phenotype, usually caused by partial deletion of the mitochondrial genome. Excluding petites, maximum-likelihood estimates adjusted for the effect of selection were U = 9.5 x 10(-5) and sh = 0.217 for the wild type. Among the mutator lines, the best fit was obtained with 0.005 < or = U < or = 0.94 and 0.049 > or = sh > or = 0.0003. Like other recently tested model organisms, wild-type yeast have low mutation rates, with high mean fitness costs per mutation. Inactivation of mismatch repair increases the frequency of slightly deleterious mutations by approximately two orders of magnitude.     

Genetic linkage and natural selection

The prevalence of recombination in eukaryotes poses one of the most puzzling questions in biology. The most compelling general explanation is that recombination facilitates selection by breaking down the negative associations generated by random drift (i.e. Hill–Robertson interference, HRI). I classify the effects of HRI owing to: deleterious mutation, balancing selection and selective sweeps on: neutral diversity, rates of adaptation and the mutation load. These effects are mediated primarily by the density of deleterious mutations and of selective sweeps. Sequence polymorphism and divergence suggest that these rates may be high enough to cause significant interference even in genomic regions of high recombination. However, neither seems able to generate enough variance in fitness to select strongly for high rates of recombination. It is plausible that spatial and temporal fluctuations in selection generate much more fitness variance, and hence selection for recombination, than can be explained by uniformly deleterious mutations or species-wide selective sweeps.     

Rate of deleterious mutation and the distribution of its effects on fitness in vesicular stomatitis virus

Despite their importance, the parameters describing the spontaneous deleterious mutation process have not been well described in many organisms. If mutations are important for the evolution of every living organism, their importance becomes critical in the case of RNA-based viruses, in which the frequency of mutation is orders of magnitude larger than in DNA-based organisms. The present work reports minimum estimates of the deleterious mutation rate, as well as the characterization of the distribution of deleterious mutational effects on the total fitness of the vesicular stomatitis virus (VSV). The estimates are based on mutation-accumulation experiments in which selection against deleterious mutations was minimized by recurrently imposing genetic bottlenecks of size one. The estimated deleterious mutation rate was 1.2 mutations per genome and generation, with a mean fitness effect of –0.39% per generation. At the end of the mutation-accumulation experiment, the average reduction in fitness was 38% and the distribution of accumulated deleterious effects was, on average, left-skewed. The magnitude of the skewness depends on the initial fitness of the clone analysed. The implications of our findings for the evolutionary biology of RNA viruses are discussed.     

The effect of spontaneous mutations on competitive ability

Understanding the impact of spontaneous mutations on fitness has many theoretical and practical applications in biology. Although mutational effects on individual morphological or life‐history characters have been measured in several classic genetic model systems, there are few estimates of the rate of decline due to mutation for complex fitness traits. Here, we estimate the effects of mutation on competitive ability, an important complex fitness trait, in a model system for ecological and evolutionary genomics, Daphnia. Competition assays were performed to compare fitness between mutation‐accumulation (MA) lines and control lines from eight different genotypes from two populations of Daphnia pulicaria after 30 and 65 generations of mutation accumulation. Our results show a fitness decline among MA lines relative to controls as expected, but highlight the influence of genomic background on this effect. In addition, in some assays, MA lines outperform controls providing insight into the frequency of beneficial mutations.     

Mutation accumulation and the effect of copia insertions in Drosophila melanogaster

Repeated efforts to estimate the genomic deleterious mutation rate per generation (U) in Drosophila melanogaster have yielded inconsistent estimates ranging from 0.01 to nearly 1. We carried out a mutation-accumulation experiment with a cryopreserved control population in hopes of resolving some of the uncertainties raised by these estimates. Mutation accumulation (MA) was carried out by brother sister mating of 150 sublines derived from two inbred lines. Fitness was measured under conditions chosen to mimic the ancestral laboratory environment of these genotypes. We monitored the insertions of a transposable element, copia, that proved to accumulate at the unusually high rate of 0.24 per genome per generation in one of our MA lines. Mutational variance in fitness increased at a rate consistent with previous studies, yielding a mutational coefficient of variation greater than 3%. The performance of the cryopreserved control relative to the MA lines was inconsistent, so estimates of mutation rate by the Bateman-Mukai method are suspect. Taken at face value, these data suggest a modest decline in fitness of about 0.3% per generation. The element number of copia was a significant predictor of fitness within generations; on average, insertions caused a 0.76% loss in fitness, although the confidence limits on this estimate are wide.