Embracing Complexity: Yeast Evolution Experiments Featuring Standing Genetic Variation.

The yeast Saccharomyces cerevisiae has a long and esteemed history as a model system for laboratory selection experiments. The majority of yeast evolution experiments begin with an isogenic ancestor, impose selection as cells divide asexually, and track mutations that arise and accumulate over time. Within the last decade, the popularity of S. cerevisiae as a model system for exploring the evolution of standing genetic variation has grown considerably. As a facultatively sexual microbe, it is possible to initiate experiments with populations that harbor diversity and also to maintain that diversity by promoting sexual recombination as the experiment progresses. These experimental choices expand the scope of evolutionary hypotheses that can be tested with yeast. And, in this review, I argue that yeast is one of the best model systems for testing such hypotheses relevant to eukaryotic species. Here, I compile a list of yeast evolution experiments that involve standing genetic variation, initially and/or by implementing protocols that induce sexual recombination in evolving populations. I also provide an overview of experimental methods required to set up such an experiment and discuss the unique challenges that arise in this type of research. Throughout the article, I emphasize the best practices emerging from this small but growing niche of the literature.

My Wellness Coach: evaluation of a mobile app designed to promote integrative health among underserved populations.

Underserved populations, including those from racial and ethnic groups and with low socioeconomic status, often lack access to mobile apps aimed at reducing health risk factors. This study evaluated the feasibility, acceptability, and preliminary effectiveness of the mobile app, My Wellness Coach (MWC), designed to promote behavior change in seven core areas of integrative health among underserved populations. Patients and staff were recruited from clinic and other settings. Some participants used MWC in a weekly group setting (n = 5); others on their own with support from a coordinator (n = 36). Health outcomes were assessed at baseline and 3 months. Mobile app ratings were collected at 5 weeks and 3 months. Goal setting data were analyzed at 3 months. Most participants (76%) set at least one goal, 71% created action steps for goals, and 29% completed a goal. Patients in the group setting had the highest rate of goal completion (60%) compared to patients (20%) and staff (27%) using the app on their own. Significant (p < .05) changes in pre- and post-test scores were documented for overall wellbeing, global physical health, BMI, vigorous physical activity, and eHealth literacy. Most participants (75%-91%) gave MWC high ratings for impact on behavior change, help seeking, intent to change, attitudes, knowledge, and awareness. This study documented preliminary evidence of the potential benefits of MWC among underserved communities. Future evaluations of Spanish and Android versions and comparisons between group and individual administration will inform implementation strategies for scaling MWC-based interventions to reach underserved communities nationally.

Many underserved populations, including those from diverse racial and ethnic groups and with low income, do not have access to mobile apps to improve health. This study examined whether using the My Wellness Coach (MWC) app was feasible, acceptable, and effective. MWC was designed to promote behavior change in integrative health (Movement, Nutrition, Spirituality, Resilience, Relationships, Sleep, and Environment) among underserved populations. Five participants used MWC in a group setting and 36 participants used MWC on their own with assistance from a coordinator. Participants completed surveys at the beginning of the study and 3 months later. Most participants (76%) set at least one health goal, 71% created action steps for goals, and 29% completed a goal. Participants who used MWC with the weekly group had the highest rate of goal completion (60%). Participants reported significant changes in wellbeing, physical health, body mass index, physical activity, and ability to find and understand electronic health information. Most participants (75%­91%) gave MWC positive ratings. This study provided evidence of the potential benefits of MWC among underserved communities. Future studies with Spanish and Android versions and comparisons between group and individual administration will inform strategies for expanding the reach of MWC-based interventions to underserved communities.

From microbes to mammals: The experimental evolution of aging and longevity across species.

Senescence, the functional deterioration of cells or organisms associated with increased age, is pervasive across the tree of life. Yet our understanding of the genetic and physiological basis underlying age-related declines in health and reproduction remains limited. Experimental evolution allows empirical examination of the question of why aging occurs; imposing selection for age-specific fitness traits shifts patterns of aging in experimental populations, enabling investigations of the variation underlying senescence and the mechanisms governing it. Whole-genome sequencing of experimentally evolved populations may reveal candidate genomic variants underlying particular aging patterns; unfortunately, most study systems suffer from limitations that weaken associations between genotypes and phenotypes. In this review, we provide a survey of experimental evolution studies that have altered population-level patterns of reproductive timing and senescence in a variety of species. We discuss the specific selection conditions that have increased longevity, the phenotypic responses and trade-offs that accompany these increases, and examine genomic data collected from these experiments. Additionally, we consider how selected field studies complement laboratory experiments on life-history evolution. Finally, we address the strengths and weaknesses of existing study systems, and evaluate which model organisms appear most promising for future genomic investigations of the evolutionary biology of aging.

Crossing design shapes patterns of genetic variation in synthetic recombinant populations of Saccharomyces cerevisiae.

"Synthetic recombinant" populations have emerged as a useful tool for dissecting the genetics of complex traits. They can be used to derive inbred lines for fine QTL mapping, or the populations themselves can be sampled for experimental evolution. In the latter application, investigators generally value maximizing genetic variation in constructed populations. This is because in evolution experiments initiated from such populations, adaptation is primarily fueled by standing genetic variation. Despite this reality, little has been done to systematically evaluate how different methods of constructing synthetic populations shape initial patterns of variation. Here we seek to address this issue by comparing outcomes in synthetic recombinant Saccharomyces cerevisiae populations created using one of two strategies: pairwise crossing of isogenic strains or simple mixing of strains in equal proportion. We also explore the impact of the varying the number of parental strains. We find that more genetic variation is initially present and maintained when population construction includes a round of pairwise crossing. As perhaps expected, we also observe that increasing the number of parental strains typically increases genetic diversity. In summary, we suggest that when constructing populations for use in evolution experiments, simply mixing founder strains in equal proportion may limit the adaptive potential.

Corrigendum: Heat Shock Improves Random Spore Analysis in Diverse Strains of Saccharomyces cerevisiae.

[This corrects the article DOI: 10.3389/fgene.2020.597482.].

Can laboratory evolution experiments teach us about natural populations?

The ability to predict how natural populations will evolve and adapt to major changes in environmental conditions has long been of interest to evolutionary biologists and ecologists alike. The reality of global climate change has also created a pressing need for advancement in this particular area of research, as species are increasingly faced with rapid shifts in abiotic and biotic conditions. Evolutionary genomics has the potential to be incredibly useful as we move forward in addressing this need and in particular, evolve and resequence (E&R) studies-where researchers combine experimental evolution with whole-genome sequencing-have an important role to play. However, while E&R studies have shown a great deal of promise in tackling fundamental questions regarding the genetics of adaptation (Long et al., 2015; Schlötterer et al., 2014), it is unclear whether results from laboratory experiments can be directly translated to natural populations. In a From the Cover article in this issue of Molecular Ecology, Hsu et al. (Mol Ecol, 29, 2020) explicitly contend with this issue by examining the overlap between genes implicated in thermal adaptation in a Drosophila melanogaster E&R study and genes identified by comparing natural populations from different latitudinal clines. They report significant correlations between the two sets of temperature-adaptive genes and ultimately conclude that E&R studies can indeed generate insights applicable to populations inhabiting complex natural environments. While more work is needed to assess the generality of these conclusions, Hsu and Belmouaden (Mol Ecol, 29, 2020) contribute an important precedent.

Heat Shock Improves Random Spore Analysis in Diverse Strains of Saccharomyces cerevisiae.

Random spore analysis (RSA) is a classic method in yeast genetics that allows high-throughput purification of recombinant haploid spores following specific crosses. RSA typically involves a number of steps to induce sporulation, purge vegetative cells that fail to sporulate, and disrupt the ascus walls of sporulated cells to release haploid spores. These steps generally require expensive chemicals and/or enzymes that kill diploid cells but have few effects on spores. In the fission yeast Schizosaccharomcyes pombe, heat shock has been reported as an effective addition to RSA protocols, but to our knowledge heat shock has not been used for this purpose in the budding yeast Saccharomyces cerevisiae. Here, we evaluate the effects of heat shock on vegetative and sporulated cultures of four diverse yeast strains: a European wine strain (DBVPG6765), a Japanese sake strain (Y12), a West African palm wine strain (DBVPG6044) and a North American strain isolated from the soil beneath an oak tree (YPS128). We characterize this phenotype under multiple combinations of temperature and incubation time, and find specific conditions that lead to the exclusion of vegetative cells and an enrichment in spores, which differ by strain. We also collected genome sequence data from a recombinant population that experienced multiple rounds of RSA, including one round with a heat shock treatment. These data suggest that when incorporated into an RSA protocol, heat shock leads to increased genetic diversity among the cells that survive and mate. Ultimately, our work provides evidence that short heat treatments can improve existing RSA protocols, though in a strain-specific manner. This result informs applications of high-throughput RSA protocols, such as QTL mapping and experimental evolution research.

Increased time sampling in an evolve-and-resequence experiment with outcrossing Saccharomyces cerevisiae reveals multiple paths of adaptive change.

"Evolve and resequence" (E&R) studies combine experimental evolution and whole-genome sequencing to interrogate the genetics underlying adaptation. Due to ease of handling, E&R work with asexual organisms such as bacteria can employ optimized experimental design, with large experiments and many generations of selection. By contrast, E&R experiments with sexually reproducing organisms are more difficult to implement, and design parameters vary dramatically among studies. Thus, efforts have been made to assess how these differences, such as number of independent replicates, or size of experimental populations, impact inference. We add to this work by investigating the role of time sampling-the number of discrete time points sequence data are collected from evolving populations. Using data from an E&R experiment with outcrossing Saccharomyces cerevisiae in which populations were sequenced 17 times over ~540 generations, we address the following questions: (a) Do more time points improve the ability to identify candidate regions underlying selection? And (b) does high-resolution sampling provide unique insight into evolutionary processes driving adaptation? We find that while time sampling does not improve the ability to identify candidate regions, high-resolution sampling does provide valuable opportunities to characterize evolutionary dynamics. Increased time sampling reveals three distinct trajectories for adaptive alleles: one consistent with classic population genetic theory (i.e., models assuming constant selection coefficients), and two where trajectories suggest more context-dependent responses (i.e., models involving dynamic selection coefficients). We conclude that while time sampling has limited impact on candidate region identification, sampling eight or more time points has clear benefits for studying complex evolutionary dynamics.

Consequences of Cryopreservation in Diverse Natural Isolates of Saccharomyces cerevisiae.

Experimental evolution allows the observation of change over time as laboratory populations evolve in response to novel, controlled environments. Microbial evolution experiments take advantage of cryopreservation to archive experimental populations in glycerol media, creating a frozen, living "fossil" record. Prior research with Escherichia coli has shown that cryopreservation conditions can affect cell viability and that allele frequencies across the genome can change in response to a freeze-thaw event. We expand on these observations by characterizing fitness and genomic consequences of multiple freeze-thaw cycles in diploid yeast populations. Our study system is a highly recombinant Saccharomyces cerevisiae population (SGRP-4X) that harbors standing genetic variation that cryopreservation may threaten. We also investigate the four parental isogenic strains crossed to create the SGRP-4X. We measure cell viability over five consecutive freeze-thaw cycles; whereas we find that viability increases over time in the evolved recombinant populations, we observe no such viability improvements in the parental strains. We also collect genome-wide sequence data from experimental populations initially, after one freeze-thaw, and after five freeze-thaw cycles. In the recombinant evolved populations, we find a region of significant allele frequency change on chromosome 15 containing the ALR1 gene. In the parental strains, we find little evidence for new mutations. We conclude that cryopreserving yeast populations with standing genetic variation may have both phenotypic and genomic consequences, though the same cryopreservation practices may have only small impacts on populations with little or no initial variation.

Genome-Wide Analysis of Starvation-Selected Drosophila melanogaster-A Genetic Model of Obesity.

Experimental evolution affords the opportunity to investigate adaptation to stressful environments. Studies combining experimental evolution with whole-genome resequencing have provided insight into the dynamics of adaptation and a new tool to uncover genes associated with polygenic traits. Here, we selected for starvation resistance in populations of Drosophila melanogaster for over 80 generations. In response, the starvation-selected lines developed an obese condition, storing nearly twice the level of total lipids than their unselected controls. Although these fats provide a ∼3-fold increase in starvation resistance, the imbalance in lipid homeostasis incurs evolutionary cost. Some of these tradeoffs resemble obesity-associated pathologies in mammals including metabolic depression, low activity levels, dilated cardiomyopathy, and disrupted sleeping patterns. To determine the genetic basis of these traits, we resequenced genomic DNA from the selected lines and their controls. We found 1,046,373 polymorphic sites, many of which diverged between selection treatments. In addition, we found a wide range of genetic heterogeneity between the replicates of the selected lines, suggesting multiple mechanisms of adaptation. Genome-wide heterozygosity was low in the selected populations, with many large blocks of SNPs nearing fixation. We found candidate loci under selection by using an algorithm to control for the effects of genetic drift. These loci were mapped to a set of 382 genes, which associated with many processes including nutrient response, catabolic metabolism, and lipid droplet function. The results of our study speak to the evolutionary origins of obesity and provide new targets to understand the polygenic nature of obesity in a unique model system.

Experimental Evolution and Heart Function in Drosophila.

Drosophila melanogaster is a good model species for the study of heart function. However, most previous work on D. melanogaster heart function has focused on the effects of large-effect genetic variants. We compare heart function among 18 D. melanogaster populations that have been selected for altered development time, aging, or stress resistance. We find that populations with faster development and faster aging have increased heart dysfunction, measured as percentage heart failure after electrical pacing. Experimental evolution of different triglyceride levels, by contrast, has little effect on heart function. Evolved differences in heart function correlate with allele frequency changes at many loci of small effect. Genomic analysis of these populations produces a list of candidate loci that might affect cardiac function at the intersection of development, aging, and metabolic control mechanisms.

Genome-wide analysis of long-term evolutionary domestication in Drosophila melanogaster.

Experimental evolutionary genomics now allows biologists to test fundamental theories concerning the genetic basis of adaptation. We have conducted one of the longest laboratory evolution experiments with any sexually-reproducing metazoan, Drosophila melanogaster. We used next-generation resequencing data from this experiment to examine genome-wide patterns of genetic variation over an evolutionary time-scale that approaches 1,000 generations. We also compared measures of variation within and differentiation between our populations to simulations based on a variety of evolutionary scenarios. Our analysis yielded no clear evidence of hard selective sweeps, whereby natural selection acts to increase the frequency of a newly-arising mutation in a population until it becomes fixed. We do find evidence for selection acting on standing genetic variation, as independent replicate populations exhibit similar population-genetic dynamics, without obvious fixation of candidate alleles under selection. A hidden-Markov model test for selection also found widespread evidence for selection. We found more genetic variation genome-wide, and less differentiation between replicate populations genome-wide, than arose in any of our simulated evolutionary scenarios.

Rapid divergence and convergence of life-history in experimentally evolved Drosophila melanogaster.

Laboratory selection experiments are alluring in their simplicity, power, and ability to inform us about how evolution works. A longstanding challenge facing evolution experiments with metazoans is that significant generational turnover takes a long time. In this work, we present data from a unique system of experimentally evolved laboratory populations of Drosophila melanogaster that have experienced three distinct life-history selection regimes. The goal of our study was to determine how quickly populations of a certain selection regime diverge phenotypically from their ancestors, and how quickly they converge with independently derived populations that share a selection regime. Our results indicate that phenotypic divergence from an ancestral population occurs rapidly, within dozens of generations, regardless of that population's evolutionary history. Similarly, populations sharing a selection treatment converge on common phenotypes in this same time frame, regardless of selection pressures those populations may have experienced in the past. These patterns of convergence and divergence emerged much faster than expected, suggesting that intermediate evolutionary history has transient effects in this system. The results we draw from this system are applicable to other experimental evolution projects, and suggest that many relevant questions can be sufficiently tested on shorter timescales than previously thought.

Standing genetic variation drives repeatable experimental evolution in outcrossing populations of Saccharomyces cerevisiae.

In "evolve-and-resequence" (E&R) experiments, whole-genome sequence data from laboratory-evolved populations can potentially uncover mechanisms of adaptive change. E&R experiments with initially isogenic, asexually reproducing microbes have repeatedly shown that beneficial de novo mutations drive adaptation, and these mutations are not shared among independently evolving replicate populations. Recent E&R experiments with higher eukaryotes that maintain genetic variation via sexual reproduction implicate largely different mechanisms; adaptation may act primarily on pre-existing genetic variation and occur in parallel among independent populations. But this is currently a debated topic, and generalizing these conclusions is problematic because E&R experiments with sexual species are difficult to implement and important elements of experimental design suffer for practical reasons. We circumvent potentially confounding limitations with a yeast model capable of shuffling genotypes via sexual recombination. Our starting population consisted of a highly intercrossed diploid Saccharomyces cerevisiae initiated from four wild haplotypes. We imposed a laboratory domestication treatment on 12 independent replicate populations for 18 weeks, where each week included 2 days as diploids in liquid culture and a forced recombination/mating event. We then sequenced pooled population samples at weeks 0, 6, 12, and 18. We show that adaptation is highly parallel among replicate populations, and can be localized to a modest number of genomic regions. We also demonstrate that despite hundreds of generations of evolution and large effective population sizes, de novo beneficial mutations do not play a large role in this adaptation. Further, we have high power to detect the signal of change in these populations but show how this power is dramatically reduced when fewer timepoints are sampled, or fewer replicate populations are analyzed. As ours is the most highly replicated and sampled E&R study in a sexual species to date, this evokes important considerations for past and future experiments.

Genome-wide association study of extreme longevity in Drosophila melanogaster.

Human genome-wide association studies (GWAS) of longevity attempt to identify alleles at different frequencies in the extremely old, relative to a younger control sample. Here, we apply a GWAS approach to "synthetic" populations of Drosophila melanogaster derived from a small number of inbred founders. We used next-generation DNA sequencing to estimate allele and haplotype frequencies in the oldest surviving individuals of an age cohort and compared these frequencies with those of randomly sampled individuals from the same cohort. We used this case-control strategy in four independent cohorts and identified eight significantly differentiated regions of the genome potentially harboring genes with relevance for longevity. By modeling the effects of local haplotypes, we have more power to detect regions enriched for longevity genes than marker-based GWAS. Most significant regions occur near chromosome ends or centromeres where recombination is infrequent, consistent with these regions harboring unconditionally deleterious alleles impacting longevity. Genes in regions of normal recombination are enriched for those relevant to immune function and a gene family involved in oxidative stress response. Genetic differentiation between our experimental cohorts is comparable to that between human populations, suggesting in turn that our results may help explain heterogeneous signals in human association studies of extreme longevity when panels have diverse ancestry.

What paths do advantageous alleles take during short-term evolutionary change?

Combining experimental evolution with whole-genome resequencing is a promising new strategy for investigating the dynamics of evolutionary change. Published studies that have resequenced laboratory-selected populations of sexual organisms have typically focused on populations sampled at the end of an evolution experiment. These studies have attempted to associate particular alleles with phenotypic change and attempted to distinguish between different theoretical models of adaptation. However, neither the population used to initiate the experiment nor multiple time points sampled during the evolutionary trajectory are generally available for examination. In this issue of Molecular Ecology, Orozco-terWengel et al. (2012) take a significant step forward by estimating genome-wide allele frequencies at the start, 15 generations into and at the end of a 37-generation Drosophila experimental evolution study. The authors identify regions of the genome that have responded to laboratory selection and describe the temporal dynamics of allele frequency change. They identify two common trajectories for putatively adaptive alleles: alleles either gradually increase in frequency throughout the entire 37 generations or alleles plateau at a new frequency by generation 15. The identification of complex trajectories of alleles under selection contributes to a growing body of literature suggesting that simple models of adaptation, whereby beneficial alleles arise and increase in frequency unimpeded until they become fixed, may not adequately describe short-term response to selection.

How does adaptation sweep through the genome? Insights from long-term selection experiments.

A major goal in evolutionary biology is to understand the origins and fates of adaptive mutations. Natural selection may act to increase the frequency of de novo beneficial mutations, or those already present in the population as standing genetic variation. These beneficial mutations may ultimately reach fixation in a population, or they may stop increasing in frequency once a particular phenotypic state has been achieved. It is not yet well understood how different features of population biology, and/or different environmental circumstances affect these adaptive processes. Experimental evolution is a promising technique for studying the dynamics of beneficial alleles, as populations evolving in the laboratory experience natural selection in a replicated, controlled manner. Whole-genome sequencing, regularly obtained over the course of sustained laboratory selection, could potentially reveal insights into the mutational dynamics that most likely occur in natural populations under similar circumstances. To date, only a few evolution experiments for which whole-genome data are available exist. This review describes results from these resequenced laboratory-selected populations, in systems with and without sexual recombination. In asexual systems, adaptation from new mutations can be studied, and results to date suggest that the complete, unimpeded fixation of these mutations is not always observed. In sexual systems, adaptation from standing genetic variation can be studied, and in the admittedly few examples we have, the complete fixation of standing variants is not always observed. To date, the relative frequency of adaptation from new mutations versus standing variation has not been tested using a single experimental system, but recent studies using Caenorhabditis elegans and Saccharomyces cerevisiae suggest that this a realistic future goal.

New experiments for an undivided genetics.

There used to be a broad split within the experimental genetics research community between those who did mechanistic research using homozygous laboratory strains and those who studied patterns of genetic variation in wild populations. The former benefited from the advantage of reproducible experiments, but faced difficulties of interpretation given possible genomic and evolutionary complexities. The latter research approach featured readily interpreted evolutionary and genomic contexts, particularly phylogeny, but was poor at determining functional significance. Such burgeoning experimental strategies as genome-wide analysis of quantitative trait loci, genotype-phenotype associations, and the products of experimental evolution are now fostering a unification of experimental genetic research that strengthens its scientific power.

Genomic Croesus: experimental evolutionary genetics of Drosophila aging.

For more than 40 years, multiple laboratories have studied Drosophila stocks that have been forced to evolve slowed rates of aging and increased average longevities. These stocks have been used to test both physiological and genetic theories of aging, yielding a number of interesting findings. A little-noticed problem is that these tests have too frequently produced positive results with respect to physiological and genetic mechanisms underlying slowed aging. A genomic interpretation of this copious success is that hundreds of genetic loci have undergone changes in allele frequency or gene expression as a result of selection for slowed aging. This implicates many genetic mechanisms in the control of aging, in general, across the diversity of aging species. As the technology for surveying genomes and transcriptomes continues to improve rapidly, the loci of aging are becoming ever easier to identify. But interpreting the detailed functional consequences of all of these loci presents a radically larger challenge. Like Croesus, experimental gerontology is faced with the problem of genomic foundations for aging which are extremely rich.

Genome-wide analysis of a long-term evolution experiment with Drosophila.

Experimental evolution systems allow the genomic study of adaptation, and so far this has been done primarily in asexual systems with small genomes, such as bacteria and yeast. Here we present whole-genome resequencing data from Drosophila melanogaster populations that have experienced over 600 generations of laboratory selection for accelerated development. Flies in these selected populations develop from egg to adult ∼20% faster than flies of ancestral control populations, and have evolved a number of other correlated phenotypes. On the basis of 688,520 intermediate-frequency, high-quality single nucleotide polymorphisms, we identify several dozen genomic regions that show strong allele frequency differentiation between a pooled sample of five replicate populations selected for accelerated development and pooled controls. On the basis of resequencing data from a single replicate population with accelerated development, as well as single nucleotide polymorphism data from individual flies from each replicate population, we infer little allele frequency differentiation between replicate populations within a selection treatment. Signatures of selection are qualitatively different than what has been observed in asexual species; in our sexual populations, adaptation is not associated with 'classic' sweeps whereby newly arising, unconditionally advantageous mutations become fixed. More parsimonious explanations include 'incomplete' sweep models, in which mutations have not had enough time to fix, and 'soft' sweep models, in which selection acts on pre-existing, common genetic variants. We conclude that, at least for life history characters such as development time, unconditionally advantageous alleles rarely arise, are associated with small net fitness gains or cannot fix because selection coefficients change over time.