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Understanding how genes interact is a central challenge in biology. Experimental evolution provides a useful, but underutilized, tool for identifying genetic interactions, particularly those that involve non-loss-of-function mutations or mutations in essential genes. We previously identified a strong positive genetic interaction between specific mutations in KEL1 (P344T) and HSL7 (A695fs) that arose in an experimentally-evolved Saccharomyces cerevisiae population. Because this genetic interaction is not phenocopied by gene deletion, it was previously unknown. Using “evolutionary replay” experiments we identified additional mutations that have positive genetic interactions with the kel1-P344T mutation. We replayed the evolution of this population 672 times from six timepoints. We identified 30 populations where the kel1-P344T mutation reached high frequency. We performed whole-genome sequencing on these populations to identify genes in which mutations arose specifically in the kel1-P344T background. We reconstructed mutations in the ancestral and kel1-P344T backgrounds to validate positive genetic interactions. We identify several genetic interactors with KEL1, we validate these interactions by reconstruction experiments, and we show these interactions are not recapitulated by loss-of-function mutations. Our results demonstrate the power of experimental evolution to identify genetic interactions that are positive, allele specific, and not readily detected by other methods, and sheds light on a previously under-explored region of the yeast genetic interaction network.

Loss of heterozygosity is a common mode of adaptation in asexual diploid populations. Because mitotic recombination frequently extends the full length of a chromosome arm, the selective benefit of loss of heterozygosity may be constrained by linked heterozygous mutations. In a previous laboratory evolution experiment with diploid yeast, we frequently observed homozygous mutations in the WHI2 gene on the right arm of Chromosome XV. However, when heterozygous mutations arose in the STE4 gene, another common target on Chromosome XV, loss of heterozygosity at WHI2 was not observed. Here we show that mutations at WHI2 are partially dominant and that mutations at STE4 are overdominant. We test whether beneficial heterozygous mutations at these two loci interfere with one another by measuring loss of heterozygosity at WHI2 over 1,000 generations for ∼300 populations that differed initially only at STE4 and WHI2. We show that the presence of an overdominant mutation in STE4 reduces, but does not eliminate, loss of heterozygosity at WHI2. By sequencing 40 evolved clones, we show that populations with linked overdominant and partially dominant mutations show less parallelism at the gene level, more varied evolutionary outcomes, and increased rates of aneuploidy. Our results show that the degree of dominance and the phasing of heterozygous beneficial mutations can constrain loss of heterozygosity along a chromosome arm, and that conflicts between partially dominant and overdominant mutations can affect evolutionary outcomes.SIGNIFICANCE STATEMENTIn diploid populations, it is beneficial for partially dominant beneficial mutations to lose heterozygosity, but it is deleterious for overdominant beneficial mutations to do so. Because loss-of-heterozygosity tracts often encompass entire chromosome arms, a conflict will arise when a partially dominant beneficial mutation and an overdominant beneficial mutation exist in close proximity. We demonstrate that this conflict occurs, and that it restricts loss of heterozygosity, resulting in more variable evolutionary outcomes.

Ploidy varies considerably in nature. However, our understanding of the impact of ploidy on adaptation is incomplete. Many microbial evolution experiments characterize adaptation in haploid organisms, but few focus on diploid organisms. Here, we perform a 4,000-generation evolution experiment using diploid strains of the yeast Saccharomyces cerevisiae. We show that the rate of adaptation and spectrum of beneficial mutations are influenced by ploidy. Haldane’s sieve effectively alters access to recessive beneficial mutations in diploid populations, leading to a slower rate of adaptation and a spectrum of beneficial mutations that is shifted towards dominant mutations. Genomic position also has an important role, as the prevalence of homozygous mutations is largely dependent on their proximity to a recombination hotspot. Our results demonstrate key aspects of diploid adaptation that have previously been understudied and provide support for several proposed theories. Adaptation in diploids is poorly understood. Here, the authors show a slower rate of adaptation and a shift towards adaptive dominant mutations in long-term experimental evolution of diploid strains of the yeast Saccharomyces cerevisiae.

DNA mismatch repair is a highly conserved DNA repair pathway. In humans, germline mutations in hMSH2 or hMLH1, key components of mismatch repair, have been associated with Lynch syndrome, a leading cause of inherited cancer mortality. Current estimates of the mutation rate and the mutational spectra in mismatch repair defective cells are primarily limited to a small number of individual reporter loci. Here we use the yeast Saccharomyces cerevisiae to generate a genome-wide view of the rates, spectra, and distribution of mutation in the absence of mismatch repair. We performed mutation accumulation assays and next generation sequencing on 19 strains, including 16 msh2 missense variants implicated in Lynch cancer syndrome. The mutation rate for DNA mismatch repair null strains was approximately 1 mutation per genome per generation, 225-fold greater than the wild-type rate. The mutations were distributed randomly throughout the genome, independent of replication timing. The mutation spectra included insertions/deletions at homopolymeric runs (87.7%) and at larger microsatellites (5.9%), as well as transitions (4.5%) and transversions (1.9%). Additionally, repeat regions with proximal repeats are more likely to be mutated. A bias toward deletions at homopolymers and insertions at (AT)n microsatellites suggests a different mechanism for mismatch generation at these sites. Interestingly, 5% of the single base pair substitutions might represent double-slippage events that occurred at the junction of immediately adjacent repeats, resulting in a shift in the repeat boundary. These data suggest a closer scrutiny of tumor suppressors with homopolymeric runs with proximal repeats as the potential drivers of oncogenesis in mismatch repair defective cells.

Whole-genome whole-population sequencing is used to examine the dynamics of genome-sequence evolution in Saccharomyces cerevisiae populations for 1,000 generations; this reveals patterns of sequence evolution driven by pervasive genetic hitchhiking and interference, and shows that beneficial mutations that escape drift and increase in frequency typically occur in cohorts. Lateral gene transfer through genetic hitchhiking and clonal interference are known to contribute to evolutionary outcomes in laboratory evolution experiments. However, the role of these phenomena in determining the dynamics of genome sequence evolution remains to be determined. This study examines in detail the long-term dynamics of genome evolution in 40 experimental yeast populations. Results demonstrate the pervasive importance of clonal interference and hitchhiking at the genomic sequence level across many replicate populations. In addition, beneficial mutations that escape drift and increase in frequency typically travel through populations in clusters. The dynamics of adaptation determine which mutations fix in a population, and hence how reproducible evolution will be. This is central to understanding the spectra of mutations recovered in the evolution of antibiotic resistance1, the response of pathogens to immune selection2,3, and the dynamics of cancer progression4,5. In laboratory evolution experiments, demonstrably beneficial mutations are found repeatedly6,7,8, but are often accompanied by other mutations with no obvious benefit. Here we use whole-genome whole-population sequencing to examine the dynamics of genome sequence evolution at high temporal resolution in 40 replicate Saccharomyces cerevisiae populations growing in rich medium for 1,000 generations. We find pervasive genetic hitchhiking: multiple mutations arise and move synchronously through the population as mutational ‘cohorts’. Multiple clonal cohorts are often present simultaneously, competing with each other in the same population. Our results show that patterns of sequence evolution are driven by a balance between these chance effects of hitchhiking and interference, which increase stochastic variation in evolutionary outcomes, and the deterministic action of selection on individual mutations, which favours parallel evolutionary solutions in replicate populations.

Natural selection optimizes an organism's genotype within the context of its environment. Adaptations to one environment can decrease fitness in another, revealing evolutionary trade-offs. Here, we show that the cost of gene expression underlies a trade-off between growth rate and mating efficiency in the yeast Saccharomyces cerevisiae . During asexual growth, mutations that eliminate the ability to mate provide an ≈2% per-generation growth-rate advantage. Some strains, including most laboratory strains, carry an allele of GPA1 (an upstream component of the mating pathway) that increases mating efficiency by ≈30% per round of mating at the cost of an ≈1% per-generation growth-rate disadvantage. In addition to demonstrating a trade-off between growth rate and mating efficiency, our results illustrate differences in the selective pressures defining fitness in the laboratory versus the natural environment and show that selection, acting on the cost of gene expression, can optimize expression levels and promote gene loss.

A primary goal of recent work in experimental evolution is to probe the molecular basis of adaptation. This requires an understanding of the individual mutations in evolving populations: their identity, their physiological and fitness effects, and the interactions between them. The combination of high-throughput methods for laboratory evolution and next-generation sequencing methods now makes it possible to identify and quantify mutations in hundreds of replicate populations over thousands of generations, and to directly measure fitness effects and epistatic interactions. Many laboratories are now leveraging these tools to study the molecular basis of adaptation and the reproducibility of evolutionary outcomes across a variety of model systems. Genetic analyses on evolved populations are shedding light on the statistics of epistasis between evolved mutations. Here we review the current understanding of the spectrum of mutations observed across these systems, with a focus on epistatic interactions between beneficial mutations and constraints on evolutionary outcomes. We emphasize evolution in asexual microbes, where next generation sequencing methods have been widely applied.