Genomic factors shaping codon usage across the Saccharomycotina subphylum.

Codon usage bias, or the unequal use of synonymous codons, is observed across genes, genomes, and between species. The biased use of synonymous codons has been implicated in many cellular functions, such as translation dynamics and transcript stability, but can also be shaped by neutral forces. The Saccharomycotina, the fungal subphylum containing the yeasts Saccharomyces cerevisiae and Candida albicans , has been a model system for studying codon usage. We characterized codon usage across 1,154 strains from 1,051 species to gain insight into the biases, molecular mechanisms, evolution, and genomic features contributing to codon usage patterns across the subphylum. We found evidence of a general preference for A/T-ending codons and correlations between codon usage bias, GC content, and tRNA-ome size. Codon usage bias is also distinct between the 12 orders within the subphylum to such a degree that yeasts can be classified into orders with an accuracy greater than 90% using a machine learning algorithm trained on codon usage. We also characterized the degree to which codon usage bias is impacted by translational selection. Interestingly, the degree of translational selection was influenced by a combination of genome features and assembly metrics that included the number of coding sequences, BUSCO count, and genome length. Our analysis also revealed an extreme bias in codon usage in the Saccharomycodales associated with a lack of predicted arginine tRNAs. The order contains 24 species, and 23 are computationally predicted to lack tRNAs that decode CGN codons, leaving only the AGN codons to encode arginine. Analysis of Saccharomycodales gene expression, tRNA sequences, and codon evolution suggests that extreme avoidance of the CGN codons is associated with a decline in arginine tRNA function. Codon usage bias within the Saccharomycotina is generally consistent with previous investigations in fungi, which show a role for both genomic features and GC bias in shaping codon usage. However, we find cases of extreme codon usage preference and avoidance along yeast lineages, suggesting additional forces may be shaping the evolution of specific codons.

Ploidy evolution in a wild yeast is linked to an interaction between cell type and metabolism.

Ploidy is an evolutionarily labile trait, and its variation across the tree of life has profound impacts on evolutionary trajectories and life histories. The immediate consequences and molecular causes of ploidy variation on organismal fitness are frequently less clear, although extreme mating type skews in some fungi hint at links between cell type and adaptive traits. Here, we report an unusual recurrent ploidy reduction in replicate populations of the budding yeast Saccharomyces eubayanus experimentally evolved for improvement of a key metabolic trait, the ability to use maltose as a carbon source. We find that haploids have a substantial, but conditional, fitness advantage in the absence of other genetic variation. Using engineered genotypes that decouple the effects of ploidy and cell type, we show that increased fitness is primarily due to the distinct transcriptional program deployed by haploid-like cell types, with a significant but smaller contribution from absolute ploidy. The link between cell-type specification and the carbon metabolism adaptation can be traced to the noncanonical regulation of a maltose transporter by a haploid-specific gene. This study provides novel mechanistic insight into the molecular basis of an environment-cell type fitness interaction and illustrates how selection on traits unexpectedly linked to ploidy states or cell types can drive karyotypic evolution in fungi.

Codon Optimization Improves the Prediction of Xylose Metabolism from Gene Content in Budding Yeasts.

Xylose is the second most abundant monomeric sugar in plant biomass. Consequently, xylose catabolism is an ecologically important trait for saprotrophic organisms, as well as a fundamentally important trait for industries that hope to convert plant mass to renewable fuels and other bioproducts using microbial metabolism. Although common across fungi, xylose catabolism is rare within Saccharomycotina, the subphylum that contains most industrially relevant fermentative yeast species. The genomes of several yeasts unable to consume xylose have been previously reported to contain the full set of genes in the XYL pathway, suggesting the absence of a gene-trait correlation for xylose metabolism. Here, we measured growth on xylose and systematically identified XYL pathway orthologs across the genomes of 332 budding yeast species. Although the XYL pathway coevolved with xylose metabolism, we found that pathway presence only predicted xylose catabolism about half of the time, demonstrating that a complete XYL pathway is necessary, but not sufficient, for xylose catabolism. We also found that XYL1 copy number was positively correlated, after phylogenetic correction, with xylose utilization. We then quantified codon usage bias of XYL genes and found that XYL3 codon optimization was significantly higher, after phylogenetic correction, in species able to consume xylose. Finally, we showed that codon optimization of XYL2 was positively correlated, after phylogenetic correction, with growth rates in xylose medium. We conclude that gene content alone is a weak predictor of xylose metabolism and that using codon optimization enhances the prediction of xylose metabolism from yeast genome sequence data.

Comparative chemical genomic profiling across plant-based hydrolysate toxins reveals widespread antagonism in fitness contributions.

The budding yeast Saccharomyces cerevisiae has been used extensively in fermentative industrial processes, including biofuel production from sustainable plant-based hydrolysates. Myriad toxins and stressors found in hydrolysates inhibit microbial metabolism and product formation. Overcoming these stresses requires mitigation strategies that include strain engineering. To identify shared and divergent mechanisms of toxicity and to implicate gene targets for genetic engineering, we used a chemical genomic approach to study fitness effects across a library of S. cerevisiae deletion mutants cultured anaerobically in dozens of individual compounds found in different types of hydrolysates. Relationships in chemical genomic profiles identified classes of toxins that provoked similar cellular responses, spanning inhibitor relationships that were not expected from chemical classification. Our results also revealed widespread antagonistic effects across inhibitors, such that the same gene deletions were beneficial for surviving some toxins but detrimental for others. This work presents a rich dataset relating gene function to chemical compounds, which both expands our understanding of plant-based hydrolysates and provides a useful resource to identify engineering targets.

Yeasts from temperate forests.

Yeasts are ubiquitous in temperate forests. While this broad habitat is well-defined, the yeasts inhabiting it and their life cycles, niches, and contributions to ecosystem functioning are less understood. Yeasts are present on nearly all sampled substrates in temperate forests worldwide. They associate with soils, macroorganisms, and other habitats and no doubt contribute to broader ecosystem-wide processes. Researchers have gathered information leading to hypotheses about yeasts' niches and their life cycles based on physiological observations in the laboratory as well as genomic analyses, but the challenge remains to test these hypotheses in the forests themselves. Here, we summarize the habitat and global patterns of yeast diversity, give some information on a handful of well-studied temperate forest yeast genera, discuss the various strategies to isolate forest yeasts, and explain temperate forest yeasts' contributions to biotechnology. We close with a summary of the many future directions and outstanding questions facing researchers in temperate forest yeast ecology. Yeasts present an exciting opportunity to better understand the hidden world of microbial ecology in this threatened and global habitat.

Substrate, temperature, and geographical patterns among nearly 2000 natural yeast isolates.

Yeasts have broad importance as industrially and clinically relevant microbes and as powerful models for fundamental research, but we are only beginning to understand the roles yeasts play in natural ecosystems. Yeast ecology is often more difficult to study compared to other, more abundant microbes, but growing collections of natural yeast isolates are beginning to shed light on fundamental ecological questions. Here, we used environmental sampling and isolation to assemble a dataset of 1962 isolates collected from throughout the contiguous United States of America (USA) and Alaska, which were then used to uncover geographic patterns, along with substrate and temperature associations among yeast taxa. We found some taxa, including the common yeasts Torulaspora delbrueckii and Saccharomyces paradoxus, to be repeatedly isolated from multiple sampled regions of the USA, and we classify these as broadly distributed cosmopolitan yeasts. A number of yeast taxon-substrate associations were identified, some of which were novel and some of which support previously reported associations. Further, we found a strong effect of isolation temperature on the phyla of yeasts recovered, as well as for many species. We speculate that substrate and isolation temperature associations reflect the ecological diversity of and niche partitioning by yeast taxa.

Overdominant Mutations Restrict Adaptive Loss of Heterozygosity at Linked Loci.

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.

Synthetic hybrids of six yeast species.

Allopolyploidy generates diversity by increasing the number of copies and sources of chromosomes. Many of the best-known evolutionary radiations, crops, and industrial organisms are ancient or recent allopolyploids. Allopolyploidy promotes differentiation and facilitates adaptation to new environments, but the tools to test its limits are lacking. Here we develop an iterative method of Hybrid Production (iHyPr) to combine the genomes of multiple budding yeast species, generating Saccharomyces allopolyploids of at least six species. When making synthetic hybrids, chromosomal instability and cell size increase dramatically as additional copies of the genome are added. The six-species hybrids initially grow slowly, but they rapidly regain fitness and adapt, even as they retain traits from multiple species. These new synthetic yeast hybrids and the iHyPr method have potential applications for the study of polyploidy, genome stability, chromosome segregation, and bioenergy.

Detecting genetic interactions using parallel evolution in experimental populations.

Eukaryotic genomes contain thousands of genes organized into complex and interconnected genetic interaction networks. Most of our understanding of how genetic variation affects these networks comes from quantitative-trait loci mapping and from the systematic analysis of double-deletion (or knockdown) mutants, primarily in the yeast Saccharomyces cerevisiae. Evolve and re-sequence experiments are an alternative approach for identifying novel functional variants and genetic interactions, particularly between non-loss-of-function mutations. These experiments leverage natural selection to obtain genotypes with functionally important variants and positive genetic interactions. However, no systematic methods for detecting genetic interactions in these data are yet available. Here, we introduce a computational method based on the idea that variants in genes that interact will co-occur in evolved genotypes more often than expected by chance. We apply this method to a previously published yeast experimental evolution dataset. We find that genetic targets of selection are distributed non-uniformly among evolved genotypes, indicating that genetic interactions had a significant effect on evolutionary trajectories. We identify individual gene pairs with a statistically significant genetic interaction score. The strongest interaction is between genes TRK1 and PHO84, genes that have not been reported to interact in previous systematic studies. Our work demonstrates that leveraging parallelism in experimental evolution is useful for identifying genetic interactions that have escaped detection by other methods. This article is part of the theme issue 'Convergent evolution in the genomics era: new insights and directions'.

Adaptive genome duplication affects patterns of molecular evolution in Saccharomyces cerevisiae.

Genome duplications are important evolutionary events that impact the rate and spectrum of beneficial mutations and thus the rate of adaptation. Laboratory evolution experiments initiated with haploid Saccharomyces cerevisiae cultures repeatedly experience whole-genome duplication (WGD). We report recurrent genome duplication in 46 haploid yeast populations evolved for 4,000 generations. We find that WGD confers a fitness advantage, and this immediate fitness gain is accompanied by a shift in genomic and phenotypic evolution. The presence of ploidy-enriched targets of selection and structural variants reveals that autodiploids utilize adaptive paths inaccessible to haploids. We find that autodiploids accumulate recessive deleterious mutations, indicating an increased susceptibility for nonadaptive evolution. Finally, we report that WGD results in a reduced adaptation rate, indicating a trade-off between immediate fitness gains and long-term adaptability.

Experimental evolution in fungi: An untapped resource.

Historically, evolutionary biology has been considered an observational science. Examining populations and inferring evolutionary histories mold evolutionary theories. In contrast, laboratory evolution experiments make use of the amenability of traditional model organisms to study fundamental processes underlying evolution in real time in simple, but well-controlled, environments. With advances in high-throughput biology and next generation sequencing, it is now possible to propagate hundreds of parallel populations over thousands of generations and to quantify precisely the frequencies of various mutations over time. Experimental evolution combines the ability to simultaneously monitor replicate populations with the power to vary individual parameters to test specific evolutionary hypotheses, something that is impractical or infeasible in natural populations. Many labs are now conducting laboratory evolution experiments in nearly all model systems including viruses, bacteria, yeast, nematodes, and fruit flies. Among these systems, fungi occupy a unique niche: with a short generation time, small compact genomes, and sexual cycles, fungi are a particularly valuable and largely untapped resource for propelling future growth in the field of experimental evolution. Here, we describe the current state of fungal experimental evolution and why fungi are uniquely positioned to answer many of the outstanding questions in the field. We also review which fungal species are most well suited for experimental evolution.