Replicate Populations Research Articles

Spatial replication can best advance our understanding of population responses to climate

Understanding the responses of plant populations dynamics to climatic variability is frustrated by the need for long‐term datasets. Here, we advocate for new studies that estimate the effects of climate by sampling replicate populations in locations with similar climate. We first use data analysis on spatial locations in the conterminous USA to assess how far apart spatial replicates should be from each other to minimize temporal correlations in climate. We find that on average spatial locations separated by 316 km (SD = 149 km) have moderate (0.5) correlations in annual precipitation. Second, we use simulations to demonstrate that spatial replication can lead to substantial gains in the range of climates sampled during a given set of years so long as the climate correlations between the populations are at low to moderate levels. Third, we use simulations to quantify how many spatial replicates would be necessary to achieve the same statistical power of a single‐population, long‐term data set under different strengths and directions of spatial correlations in climate between spatial replicates. Our results indicate that spatial replication is an untapped opportunity to study the effects of climate on demography and to rapidly fill important knowledge gaps in the field of population ecology.

Selection on plastic adherence leads to hyper-multicellular strains and incidental virulence in the budding yeast.

Many disease-causing microbes are not obligate pathogens; rather, they are environmental microbes taking advantage of an ecological opportunity. The existence of microbes whose life cycle does not require a host and are not normally pathogenic, yet are well-suited to host exploitation, is an evolutionary puzzle. One hypothesis posits that selection in the environment may favor traits that incidentally lead to pathogenicity and virulence, or serve as pre-adaptations for survival in a host. An example of such a trait is surface adherence. To experimentally test the idea of 'accidental virulence', replicate populations of Saccharomyces cerevisiae were evolved to attach to a plastic bead for hundreds of generations. Along with plastic adherence, two multicellular phenotypes- biofilm formation and flor formation- increased; another phenotype, pseudohyphal growth, responded to the nutrient limitation. Thus, experimental selection led to the evolution of highly-adherent, hyper-multicellular strains. Wax moth larvae injected with evolved hyper-multicellular strains were significantly more likely to die than those injected with evolved non-multicellular strains. Hence, selection on plastic adherence incidentally led to the evolution of enhanced multicellularity and increased virulence. Our results support the idea that selection for a trait beneficial in the open environment can inadvertently generate opportunistic, 'accidental' pathogens.

The interaction and mediation effects between the host genetic factors and Epstein-Barr virus VCA-IgA in the risk of nasopharyngeal carcinoma.

Previous studies have demonstrated strong associations between host genetic factors and Epstein-Barr virus (EBV) VCA-IgA with the risk of nasopharyngeal carcinoma (NPC). However, the specific interplay between host genetics and EBV VCA-IgA on NPC risk is not well understood. In this two-stage case-control study (N = 4804), we utilized interaction and mediation analysis to investigate the interplay between host genetics (genome-wide association study-derived polygenic risk score [PRS]) and EBV VCA-IgA antibody level in the NPC risk. We employed a four-way decomposition analysis to assess the extent to which the genetic effect on NPC risk is mediated by or interacts with EBV VCA-IgA. We consistently found a significant interaction between the PRS and EBV VCA-IgA on NPC risk (discovery population: synergy index [SI] = 2.39, 95% confidence interval [CI] = 1.85-3.10; replication population: SI = 3.10, 95% CI = 2.17-4.44; all pinteraction < 0.001). Moreover, the genetic variants included in the PRS demonstrated similar interactions with EBV VCA-IgA antibody. We also observed an obvious dose-response relationship between the PRS and EBV VCA-IgA antibody on NPC risk (all ptrend < 0.001). Furthermore, our decomposition analysis revealed that a substantial proportion (approximately 90%) of the genetic effects on NPC risk could be attributed to host genetic-EBV interaction, while the risk effects mediated by EBV VCA-IgA antibody were weak and statistically insignificant. Our study provides compelling evidence for an interaction between host genetics and EBV VCA-IgA antibody in the development of NPC. These findings emphasize the importance of implementing measures to control EBV infection as a crucial strategy for effectively preventing NPC, particularly in individuals at high genetic risk.

Evolutionary consequences of nascent multicellular life cycles.

A key step in the evolutionary transition to multicellularity is the origin of multicellular groups as biological individuals capable of adaptation. Comparative work, supported by theory, suggests clonal development should facilitate this transition, although this hypothesis has never been tested in a single model system. We evolved 20 replicate populations of otherwise isogenic clonally reproducing 'snowflake' yeast (Δace2/∆ace2) and aggregative 'floc' yeast (GAL1p::FLO1 /GAL1p::FLO1) with daily selection for rapid growth in liquid media, which favors faster cell division, followed by selection for rapid sedimentation, which favors larger multicellular groups. While both genotypes adapted to this regime, growing faster and having higher survival during the group-selection phase, there was a stark difference in evolutionary dynamics. Aggregative floc yeast obtained nearly all their increased fitness from faster growth, not improved group survival; indicating that selection acted primarily at the level of cells. In contrast, clonal snowflake yeast mainly benefited from higher group-dependent fitness, indicating a shift in the level of Darwinian individuality from cells to groups. Through genome sequencing and mathematical modeling, we show that the genetic bottlenecks in a clonal life cycle also drive much higher rates of genetic drift-a result with complex implications for this evolutionary transition. Our results highlight the central role that early multicellular life cycles play in the process of multicellular adaptation.

Trade-offs constrain adaptive pathways to type VI secretion system survival

The Type VI Secretion System (T6SS) is a nano-harpoon used by many bacteria to inject toxins into neighboring cells. While much is understood about mechanisms of T6SS-mediated toxicity, less is known about the ways that competitors can defend themselves against this attack, especially in the absence of their own T6SS. Here we subjected eight replicate populations of Escherichia coli to T6SS attack by Vibrio cholerae. Over ∼500 generations of competition, isolates of the E.coli populations evolved to survive T6SS attack an average of 27-fold better, through two convergently evolved pathways: apaH was mutated in six of the eight replicate populations, while the other two populations each had mutations in both yejM and yjeP. However, the mutations we identified are pleiotropic, reducing cellular growth rates, and increasing susceptibility to antibiotics and elevated pH. These trade-offs help us understand how the T6SS shapes the evolution of bacterial interactions.

Rapidly evolving zooplankton in a salinizing world: To what extent does microevolutionary adaptation to one salt increase tolerance to another one?

AbstractSalinization is a worldwide emerging threat to freshwater systems. It can be caused by various salts with different toxicity to freshwater organisms. Populations of freshwater organisms have been shown to rapidly evolve adaptations to salinity, yet it remains unclear whether adaptation to one salt increases tolerance or, conversely, susceptibility to other salts. Using an experimental evolution approach, we subjected replicate populations of a planktonic monogonont rotifer species (Brachionus calyciflorus Pallas) to non‐saline and NaCl‐enriched environments. A subsequent multigenerational common garden (CG) transplant experiment demonstrated rapid microevolutionary adaptation to salinity. A second CG experiment provided evidence that adaptation to NaCl increases tolerance mainly to CaCl2 but to a much lesser extent to Na2SO4. Increased tolerance to CaCl2 was associated with increased tolerance to Ca2+ ion toxicity. In contrast, acquired tolerance to Na2SO4 could not be shown to involve an improved ability to cope with ion toxicity. Our results highlight that the evolution of cross‐tolerance may expand the temporal and spatial dimensions under which the ecological consequences of salt adaptation apply.

Selection and the direction of phenotypic evolution.

Predicting adaptive phenotypic evolution depends on invariable selection gradients and on the stability of the genetic covariances between the component traits of the multivariate phenotype. We describe the evolution of six traits of locomotion behavior and body size in the nematode Caenorhabditis elegans for 50 generations of adaptation to a novel environment. We show that the direction of adaptive multivariate phenotypic evolution can be predicted from the ancestral selection differentials, particularly when the traits were measured in the new environment. Interestingly, the evolution of individual traits does not always occur in the direction of selection, nor are trait responses to selection always homogeneous among replicate populations. These observations are explained because the phenotypic dimension with most of the ancestral standing genetic variation only partially aligns with the phenotypic dimension under directional selection. These findings validate selection theory and suggest that the direction of multivariate adaptive phenotypic evolution is predictable for tens of generations.

Warning symptoms associated with imminent sudden cardiac arrest: a population-based case-control study with external validation

Sudden cardiac arrest is a global public health problem with a mortality rate of more than 90%. Prearrest warning symptoms could be harnessed using digital technology to potentially improve survival outcomes. We aimed to estimate the strength of association between symptoms and imminent sudden cardiac arrest. We conducted a case-control study of individuals with sudden cardiac arrest and participants without sudden cardiac arrest who had similar symptoms identified from two US community-based studies of patients with sudden cardiac arrest in California state, USA (discovery population; the Ventura Prediction of Sudden Death in Multi-Ethnic Communities [PRESTO] study), and Oregon state, USA (replication population; the Oregon Sudden Unexpected Death Study [SUDS]). Participant data were obtained from emergency medical services reports for people aged 18-85 years with witnessed sudden cardiac arrest (between Feb 1, 2015, and Jan 31, 2021) and an inclusion symptom. Data were also obtained from corresponding control populations without sudden cardiac arrest who were attended by emergency medical services for similar symptoms (between Jan 1 and Dec 31, 2019). We evaluated the association of symptoms with sudden cardiac arrest in the discovery population and validated our results in the replication population by use of logistic regression models. We identified 1672 individuals with sudden cardiac arrest from the PRESTO study, of whom 411 patients (mean age 65·7 [SD 12·4] years; 125 women and 286 men) were included in the analysis for the discovery population. From a total of 76 734 calls to emergency medical services, 1171 patients (mean age 61·8 [SD 17·3] years; 643 women, 514 men, and 14 participants without data for sex) were included in the control group. Patients with sudden cardiac arrest were more likely to have dyspnoea (168 [41%] of 411 vs 262 [22%] of 1171; p<0·0001), chest pain (136 [33%] vs 296 [25%]; p=0·0022), diaphoresis (50 [12%] vs 90 [8%]; p=0·0059), and seizure-like activity (43 [11%] vs 77 [7%], p=0·011). Symptom frequencies and patterns differed significantly by sex. Among men, chest pain (odds ratio [OR] 2·2, 95% CI 1·6-3·0), dyspnoea (2·2, 1·6-3·0), and diaphoresis (1·7, 1·1-2·7) were significantly associated with sudden cardiac arrest, whereas among women, only dyspnoea was significantly associated with sudden cardiac arrest (2·9, 1·9-4·3). 427 patients with sudden cardiac arrest (mean age 62·2 [SD 13·5]; 122 women and 305 men) were included in the analysis for the replication population and 1238 patients (mean age 59·3 [16·5] years; 689 women, 548 men, and one participant missing data for sex) were included in the control group. Findings were mostly consistent in the replication population; however, notable differences included that, among men, diaphoresis was not associated with sudden cardiac arrest and chest pain was associated with sudden cardiac arrest only in the sex-stratified multivariable analysis. The prevalence of warning symptoms was sex-specific and differed significantly between patients with sudden cardiac arrest and controls. Warning symptoms hold promise for prediction of imminent sudden cardiac arrest but might need to be augmented with additional features to maximise predictive power. US National Heart Lung and Blood Institute.

Population‐level food availability affects postcopulatory sexual selection dynamics in the guppy

Abstract Postcopulatory sexual selection (PCSS), namely sperm competition and cryptic female choice, is typically investigated in benign environments, with a fixed number of partners, which mate at the same time intervals; all conditions that are rarely met in natural populations. Although there is increasing evidence that environmental fluctuations affect sexual selection before mating, whether and to which extent they influence postcopulatory trajectories is still little explored. PCSS was investigated in replicate populations of guppies (Poecilia reticulata) in which males and females mated after maintained for 2 weeks on either restricted (RE) or ad libitum (AL) diet and the paternity of the offspring produced by multiply mated females was assigned using microsatellite markers. Compared to AL fish, RE females (i) had fewer mating partners, but the time interval between the first and the last mating was not affected; (ii) produced broods with a lower variance in male fertilization success (a measure of the opportunity for PCSS); and (iii) produced broods with a paternity bias towards the first mate (reversing the last sperm precedence observed in AL populations), and associated more towards males with higher courtship rate. Our results demonstrate that short‐term limitation in food availability significantly influence PCSS by modifying both fertilization success variance and sperm precedence pattern. Environmental variation should therefore become part of the research paradigm to improve our understanding of postcopulatory evolutionary dynamics. Read the free Plain Language Summary for this article on the Journal blog.

A genetically supported drug repurposing pipeline for diabetes treatment using electronic health records.

The identification of new uses for existing drug therapies has the potential to identify treatments for comorbid conditions that have the added benefit of glycemic control while also providing a rapid, low-cost approach to drug (re)discovery. We developed and tested a genetically-informed drug-repurposing pipeline for diabetes management. This approach mapped genetically-predicted gene expression signals from the largest genome-wide association study for type 2 diabetes mellitus to drug targets using publicly available databases to identify drug-gene pairs. These drug-gene pairs were then validated using a two-step approach: 1) a self-controlled case-series (SCCS) using electronic health records from a discovery and replication population, and 2) Mendelian randomization (MR). After filtering on sample size, 20 candidate drug-gene pairs were validated and various medications demonstrated evidence of glycemic regulation including two anti-hypertensive classes: angiotensin-converting enzyme inhibitors as well as calcium channel blockers (CCBs). The CCBs demonstrated the strongest evidence of glycemic reduction in both validation approaches (SCCS HbA1c and glucose reduction:-0.11%, p=0.01 and-0.85mg/dL, p=0.02, respectively; MR: OR=0.84, 95% CI=0.81, 0.87, p=5.0 x 10-25). Our results support CCBs as a strong candidate medication for blood glucose reduction in addition to cardiovascular disease reduction. Further, these results support the adaptation of this approach for use in future drug-repurposing efforts for other conditions. National Institutes of Health, Medical Research Council Integrative Epidemiology Unit at the University of Bristol, UK Medical Research Council, American Heart Association, and Department of Veterans Affairs (VA) Informatics and Computing Infrastructure and VA Cooperative Studies Program.

Genome Dynamics and Temperature Adaptation During Experimental Evolution of Obligate Intracellular Bacteria.

Evolution experiments with free-living microbes have radically improved our understanding of genome evolution and how microorganisms adapt. Yet there is a paucity of such research focusing on strictly host-associated bacteria, even though they are widespread in nature. Here, we used the Acanthamoeba symbiont Protochlamydia amoebophila, a distant relative of the human pathogen Chlamydia trachomatis and representative of a large group of protist-associated environmental chlamydiae, as a model to study how obligate intracellular symbionts evolve and adapt to elevated temperature, a prerequisite for the pivotal evolutionary leap from protist to endothermic animal hosts. We established 12 replicate populations under two temperatures (20 °C, 30 °C) for 510 bacterial generations (38 months). We then used infectivity assays and pooled whole-genome resequencing to identify any evolved phenotypes and the molecular basis of adaptation in these bacteria. We observed an overall reduction in infectivity of the symbionts evolved at 30 °C, and we identified numerous nonsynonymous mutations and small indels in these symbiont populations, with several variants persisting throughout multiple time points and reaching high frequencies. This suggests that many mutations may have been beneficial and played an adaptive role. Mutated genes within the same temperature regime were more similar than those between temperature regimes. Our results provide insights into the molecular evolution of intracellular bacteria under the constraints of strict host dependance and highly structured populations and suggest that for chlamydial symbionts of protists, temperature adaptation was facilitated through attenuation of symbiont infectivity as a tradeoff to reduce host cell burden.

Sexually-selected male weapon increases the risk of population extinction under environmental change: an experimental evidence.

Exaggerated sexually-selected traits, occurring more commonly in males, help individuals to increase reproductive success, but are costly to produce and maintain. These costs on the one hand may improve population fitness by intensifying selection against maladapted males, but on the other hand may increase the risk of extinction under environmental challenge. However, the impact of sexually selected traits on extinction risk have not been investigated experimentally. We used replicate populations of a male-dimorphic mite, Rhizoglyphus robini, to test if prevalence of a sexually-selected weapon affected the risk of extinction under temperature increase (2 0C per each of three consecutive generations). In two independent experiments that utilized either inbred lines or lines mass selected for or against the weapon to establish experimental replicate populations differing in the prevalence of the weapon, we found that populations with high weapon prevalence were more likely to go extinct. Extinctions occurred despite partial suppression of the weapon expression at increased temperature and were not explained by increased male mortality. Our results provide the first, to our knowledge, experimental evidence demonstrating dramatic effect of elaborated sexual traits on the risk of extinction under environmental challenge.

Evolution of reproductive isolation in a long-term evolution experiment with Drosophila melanogaster: 30 years of divergent life-history selection.

We ask if three decades and over 1,500 generations of divergent life-history selection on age at reproduction has resulted in the evolution of reproductive isolation (RI) between laboratory populations of Drosophila melanogaster. We tested for premating, postmating-prezygotic, and postzygotic reproductive isolation between three replicate population pairs. Large, evolved differences in body size between selection treatments suggested the potential for prezygotic barriers driven by sexual selection or physical incompatibilities between the sexes. Although a simple prediction would be preference for larger size, creating directional isolation, our results from individual mate choice trials indicate that populations from both selection treatments show a marked bias towards homotypic mate choice; indicative of prezygotic RI driven by sexual selection or sexual conflict. Hybridization between the focal populations resulted in the production of viable adult flies with intermediate size and developmental traits. We observed a suggestive but statistically nonsignificant trend of fitness decline in the F2 generation of hybrids, but no significant evidence suggesting the evolution of postmating-prezygotic or postzygotic RI. Our findings are in accord with extant literature that posits that premating RI evolves before postmating forms of RI.

Evolution of Metabolome and Transcriptome Supports a Hierarchical Organization of Adaptive Traits.

Most organismal phenotypes have a polygenic basis, which enables adaptive phenotypic responses on ecological time scales. While adaptive phenotypic changes are highly parallel in replicate populations, this does not apply to the contributing loci. In particular for small populations, the same phenotypic shift can be fueled by different sets of alleles at alternative loci (genetic redundancy). Although this phenomenon is empirically well supported, the molecular basis of the genetic redundancy is not yet understood. To fill this gap, we compared the heterogeneity of the evolutionary transcriptomic and metabolomic response in ten Drosophila simulans populations which evolved parallel high-level phenotypic changes in a novel temperature environment but used different allelic combinations of alternative loci. We showed that the metabolome evolved more parallel than the transcriptome, confirming a hierarchical organization of molecular phenotypes. Different sets of genes responded in each evolved population but led to the enrichment of similar biological functions and a consistent metabolic profile. Since even the metabolomic response was still highly heterogeneous across evolved populations, we propose that selection may operate on pathways/networks.

Experimental test of the influence of light availability on the evolution of eye size and behaviour in Daphnia.

There exists extensive variation in eye size. Much work has provided a connection between light availability and differences in eye size across taxa. Experimental tests of the role of the light environment on the evolution of eye size are lacking. Here, we performed a selection experiment that examined the influence of light availability on shifts in eye size and the connection between eye size and phototactic (anti-predator) behaviour in Daphnia. We set-up replicate experimental populations of Daphnia, repeatedly evaluated phenotypic shifts in eye size during the ~50-day experiment, and performed a common garden experiment at the end of the experiment to test for evolutionary shifts in eye size and behaviour. Our phenotypic analyses showed that eye size rapidly diverged between the light treatments; relative eye size was consistently larger in the low versus high light treatments. Selection on eye size was also modified by variation in density as increases in Daphnia density favoured a larger eye. However, we did not observe differences in eye size between the light treatments following two generations of common garden rearing at the end of the experiment. We instead observed strong shifts in anti-predator behaviour. Daphnia from the low light treatment exhibited decreased phototactic responses to light. Our results show that decreased light relaxes selection on anti-predator behaviour. Such trends provide new insights into selection on eye size and behaviour.

Evolution Under Dietary Restriction Increases Reproduction at the Cost of Decreased Somatic Growth.

Dietary restriction (DR) is cited as the most reliable means of extending life span in a wide range of taxa, yet the evolutionary basis of the DR effect on life span remains unclear. The resource reallocation hypothesis proposes that the longevity-extending response of DR is adaptive and stems from the reallocation of resources from reproduction to somatic maintenance under food-limited conditions. However, if DR continues for multiple generations, such a response becomes maladaptive, and genotypes with higher fecundity should be selectively favored over genotypes with longer longevity. To test this prediction, we exposed replicate populations of the rotifer Brachionus dorcas, a model organism for aging and experimental evolution studies, to DR and ad-libitum (AL) diets for 100 days. During the selection experiment, AL-selected populations showed higher growth rates and mictic ratios than DR-selected populations. After approximately 27 asexual generations of selection, populations with a DR selection history had a higher net reproductive rate but lower body volume and ingestion rate in the absence of survival costs than populations with an AL selection history when they were assayed on an AL diet. Our results are inconsistent with the prediction that evolution on sustained DR increases reproduction and reduces life span, and show for the first time that sustained DR selects for clones with higher energy investment in reproduction but lower investment in somatic growth.

Research on Economic Optimal Dispatching of Microgrid Based on an Improved Bacteria Foraging Optimization

This paper proposes an improved Bacterial Foraging Optimization for economically optimal dispatching of the microgrid. Three optimized steps are presented to solve the slow convergence, poor precision, and low efficiency of traditional Bacterial Foraging Optimization. First, the self-adaptive step size equation in the chemotaxis process is present, and the particle swarm velocity equation is used to improve the convergence speed and precision of the algorithm. Second, the crisscross algorithm is used to enrich the replication population and improve the global search performance of the algorithm in the replication process. Finally, the dynamic probability and sine-cosine algorithm are used to solve the problem of easy loss of high-quality individuals in dispersal. Quantitative analysis and experiments demonstrated the superiority of the algorithm in the benchmark function. In addition, this study built a multi-objective microgrid dynamic economic dispatch model and dealt with the uncertainty of wind and solar using the Monte Carlo method in the model. Experiments show that this model can effectively reduce the operating cost of the microgrid, improve economic benefits, and reduce environmental pollution. The economic cost is reduced by 3.79% compared to the widely used PSO, and the economic cost is reduced by 5.23% compared to the traditional BFO.

Eco-evolutionary games for harvesting self-renewing common resource: effect of growing harvester population

The tragedy of the commons (TOCs) is a ubiquitous social dilemma witnessed in interactions between a population of living entities and shared resources available to them: the individuals in the population tend to selfishly overexploit a common resource as it is arguably the rational choice, or in case of non-human beings, it may be an evolutionarily uninvadable action. How to avert the TOC is a significant problem related to the conservation of resources. It is not hard to envisage situations where the resource could be self-renewing and the size of the population may be dependent on the state of the resource through the fractions of the population employing different exploitation rates. If the self-renewal rate of the resource lies between the maximum and the minimum exploitation rates, it is not a priori obvious under what conditions the TOC can be averted. In this paper, we address this question analytically and numerically using the setup of an evolutionary game theoretical replicator equation that models the Darwinian tenet of natural selection. Through the replicator equation, while we investigate how a population of replicators exploit the shared resource, the latter’s dynamical feedback on the former is also not ignored. We also present a transparent bottom-up derivation of the game-resource feedback model to facilitate future studies on the stochastic effects on the findings presented herein.

Differential genotype response to increased resource abundance helps explain parallel evolution of Daphnia populations in the wild.

Under controlled laboratory conditions, previous studies have shown that selection can produce repeatable evolutionary trajectories. Yet, the question remains for many of these studies if, given identical starting populations, evolution in the wild proceeds in a non-random direction. In the present study, we investigated the extent to which rapid evolution in the wild is parallel by monitoring the genetic composition of replicate populations of Daphnia in field mesocosms containing two clonal genotypes. We found parallel changes across all nine mesocosms, in which the same genotype increased in frequency. To probe whether genotype-specific response to resource abundance could have led to this frequency change, we conducted a life-history assay under high-resource abundance and low-resource abundance. We found that resource exploitation differed by genotype, in that, while one genotype (the winner in the field mesocosms) was more fit than the other genotype at high resources, the other genotype performed slightly better at low resources. We suspect that levels of resource abundance found in the summer field mesocosms had values in which the genotype better with abundant resources had the advantage. These findings suggest that variation in certain traits associated with resource acquisition can drive genotype frequency change.

Identifying Targets of Selection in Laboratory Evolution Experiments.

Adaptive evolution navigates a balance between chance and determinism. The stochastic processes of mutation and drift generate phenotypic variation; however, once mutations reach an appreciable frequency in the population, their fate is governed by the deterministic action of selection, enriching for favorable genotypes and purging the less-favorable ones. The net result is that replicate populations will traverse similar-but not identical-pathways to higher fitness. This parallelism in evolutionary outcomes can be leveraged to identify the genes and pathways under selection. However, distinguishing between beneficial and neutral mutations is challenging because many beneficial mutations will be lost due to drift and clonal interference, and many neutral (and even deleterious) mutations will fix by hitchhiking. Here, we review the best practices that our laboratory uses to identify genetic targets of selection from next-generation sequencing data of evolved yeast populations. The general principles for identifying the mutations driving adaptation will apply more broadly.