Experimental Evolution System Research Articles

Interactions between chaperone and energy storage networks during the evolution of Legionella pneumophila under heat shock.

Waterborne transmission of the bacterium Legionella pneumophila has emerged as a major cause of severe nosocomial infections of major public health impact. The major route of transmission involves the uptake of aerosolized bacteria, often from the contaminated hot water systems of large buildings. Public health regulations aimed at controlling the mesophilic pathogen are generally concerned with acute pasteurization and maintaining high temperatures at the heating systems and throughout the plumbing of hot water systems, but L. pneumophila is often able to survive these treatments due to both bacterium-intrinsic and environmental factors. Previous work has established an experimental evolution system to model the observations of increased heat resistance in repeatedly but unsuccessfully pasteurized L. pneumophila populations. Here, we show rapid fixation of novel alleles in lineages selected for resistance to heat shock and shifts in mutational profile related to increases in the temperature of selection. Gene-level and nucleotide-level parallelisms between independently-evolving lineages show the centrality of the DnaJ/DnaK chaperone system in the heat resistance of L. pneumophila. Inference of epistatic interactions through reverse genetics shows an unexpected interaction between DnaJ/DnaK and the polyhydroxybutyrate-accumulation energy storage mechanism used by the species to survive long-term starvation in low-nutrient environments.

Insecticide resistance reduces the profitability of insect-resistant rice cultivars

IntroductionPreventing crop yield loss caused by pests is critical for global agricultural production. Agricultural pest control has largely relied on chemical pesticides. The interaction between insecticide resistance and the adaptation of herbivorous pests to host plants may represent an emerging threat to future food security. ObjectivesThis study aims to unveil genetic evidence for the reduction in the profitability of resistant cultivars derived from insecticide resistance in target pest insects. MethodsAn experimental evolution system encompassing resistant rice and its major monophagous pest, the brown planthopper Nilaparvata lugens, was constructed. Whole genome resequencing and selective sweep analysis were utilized to identify the candidate gene loci related to the adaptation. RNA interference and induced expression assay were conducted to validate the function of the candidate loci. ResultsWe found that the imidacloprid-resistant population of N. lugens rapidly adapted to resistant rice IR36. Gene loci related to imidacloprid resistance may contribute to this phenomenon. Multiple alleles in the nicotinic acetylcholine receptor (nAChR)-7-like and P450 CYP4C61 were significantly correlated with changes in virulence to IR36 rice and insecticide resistance of N. lugens. One avirulent/susceptible genotype and two virulent/resistant genotypes could be inferred from the corresponding alleles. Importantly, we found that the virulent/resistant genotypes already exist in the wild in China, exhibiting increasing frequencies along with insecticide usage. We validated the relevance of these genotypes and the virulence to three more resistant rice cultivars. Knockdown of the above two genes in N. lugens significantly decreased both the resistance to imidacloprid and the virulence towards resistant rice. ConclusionOur findings provide direct genetic evidence to the eco-evolutionary consequence of insecticide resistance, and suggest an urgent need for the implementation of predictably sustainable pest management.

Experimental evolution of dispersal: Unifying theory, experiments and natural systems.

Dispersal is a central life history trait that affects the ecological and evolutionary dynamics of populations and communities. The recent use of experimental evolution for the study of dispersal is a promising avenue for demonstrating valuable proofs of concept, bringing insight into alternative dispersal strategies and trade-offs, and testing the repeatability of evolutionary outcomes. Practical constraints restrict experimental evolution studies of dispersal to a set of typically small, short-lived organisms reared in artificial laboratory conditions. Here, we argue that despite these restrictions, inferences from these studies can reinforce links between theoretical predictions and empirical observations and advance our understanding of the eco-evolutionary consequences of dispersal. We illustrate how applying an integrative framework of theory, experimental evolution and natural systems can improve our understanding of dispersal evolution under more complex and realistic biological scenarios, such as the role of biotic interactions and complex dispersal syndromes.

An analysis of direct and indirect effects in Drosophila melanogaster undergoing a few cycles of experimental evolution for stress-related traits.

The physiological mechanisms underpinning adaptations to starvation and cold stresses have been extensively studied in Drosophila, yet the understanding of correlated changes in stress-related and life-history traits, as well as the energetics of stress tolerance, still remains elusive. To answer the questions empirically in this context, we allowed D. melanogaster to evolve for either increased starvation or cold tolerance (24-generations / regime) in an experimental evolution system, and examined whether selection of either trait affects un-selected stress trait, as well as the impacts potential changes in life-history and mating success-related traits. Our results revealed remarkable changes in starvation/cold tolerance (up to 1.5-fold) as a direct effect of selection, while cold tolerance had been dramatically reduced (1.26-fold) in the starvation tolerant (ST) lines compared to control counterparts, although no such changes were evident in cold-tolerant (CT) lines. ST lines exhibited a higher level of body lipids and a reduced level of trehalose content, while CT lines accumulated a greater levels of body lipid and trehalose contents. Noticeably, we found that selection for starvation or cold tolerance positively correlates with larval development time, longevity, and copulation duration, indicating that these traits are among the most common targets of selection trajectories shaping stress tolerance. Altogether, this study highlights the complexity of mechanisms evolved in ST lines that contribute to enhanced starvation tolerance, but also negatively impact cold tolerance. Nevertheless, mechanisms foraging enhanced cold tolerance in CT lines appear not to target starvation tolerance. Moreover, the parallel changes in life history/mating success traits across stress regimes could indicate some generic pathways evolved in stressful environments, targeting life-history and mating success characteristics to optimize fitness.

Experimental evolution links post-transcriptional regulation to Leishmania fitness gain.

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network associated with parasite fitness gain, with genome instability causing highly reproducible, gene dosage-independent and -dependent changes. Reduction of flagellar transcripts and increase in coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation were not correlated to dosage changes of the corresponding genes, revealing a gene dosage-independent, post-transcriptional mechanism of regulation. In contrast, abundance of gene products implicated in post-transcriptional regulation itself correlated to corresponding gene dosage changes. Thus, RNA abundance during parasite adaptation is controled by direct and indirect gene dosage changes. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of modified ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.

Functional variability in adhesion and flocculation of yeast megasatellite genes.

Megasatellites are large tandem repeats found in all fungal genomes but especially abundant in the opportunistic pathogen Candida glabrata. They are encoded in genes involved in cell-cell interactions, either between yeasts or between yeast and human cells. In the present work, we have been using an iterative genetic system to delete several Candida glabrata megasatellite-containing genes and found that 2 of them were positively involved in adhesion to epithelial cells, whereas 3 genes negatively controlled adhesion. Two of the latter, CAGL0B05061g or CAGL0A04851g, were also negative regulators of yeast-to-yeast adhesion, making them central players in controlling Candida glabrata adherence properties. Using a series of synthetic Saccharomyces cerevisiae strains in which the FLO1 megasatellite was replaced by other tandem repeats of similar length but different sequences, we showed that the capacity of a strain to flocculate in liquid culture was unrelated to its capacity to adhere to epithelial cells or to invade agar. Finally, to understand how megasatellites were initially created and subsequently expanded, an experimental evolution system was set up, in which modified yeast strains containing different megasatellite seeds were grown in bioreactors for more than 200 generations and selected for their ability to sediment at the bottom of the culture tube. Several flocculation-positive mutants were isolated. Functionally relevant mutations included general transcription factors as well as a 230-kbp segmental duplication.

Application of a Bacterial Experimental Evolution System to Visualize and Teach Evolution in Action: A Course-Based Undergraduate Research Experience

Concepts of evolution are typically taught through examples of extremely long timescales, which do not always resonate broadly. Here, we describe a course-based undergraduate research experience tailored for junior and senior undergraduate biology majors. Students visualize and learn in real-time how evolution can operate in bacteria in response to problems associated with a high-density lifestyle. Students directly evolve mutant strains, conduct whole genome sequencing to identify the causal mutations, carry out bioinformatics analysis to predict molecular consequences of the mutations, engineer their mutants to become antibiotic-resistant, and compete them head-to-head in a class-wide round-robin tournament to infer the properties of natural selection. The presented format is designed for a full semester, but the modular structure of the course allows instructors to make simple modifications for a shorter duration. A substantial portion of this course also focuses on scientific communication. Each student prepares a lab report structured as an original research article to gain experience in writing a publication quality manuscript. Individual components of their reports are prepared throughout the semester and are followed with instructor- and peer-based draft edits. Finally, students are tasked with working as a team to deliver an oral presentation, which drives them to come to a consensus on the interpretation of their group's data. Such a comprehensive research experience is difficult for a student to acquire without securing a research position in a faculty lab, but this course allows a large group of students to directly experience and actively contribute to open-ended and hypothesis-driven research.

High-throughput analysis of adaptation using barcoded strains of Saccharomyces cerevisiae.

BackgroundExperimental evolution of microbes can be used to empirically address a wide range of questions about evolution and is increasingly employed to study complex phenomena ranging from genetic evolution to evolutionary rescue. Regardless of experimental aims, fitness assays are a central component of this type of research, and low-throughput often limits the scope and complexity of experimental evolution studies. We created an experimental evolution system in Saccharomyces cerevisiae that utilizes genetic barcoding to overcome this challenge.ResultsWe first confirm that barcode insertions do not alter fitness and that barcode sequencing can be used to efficiently detect fitness differences via pooled competition-based fitness assays. Next, we examine the effects of ploidy, chemical stress, and population bottleneck size on the evolutionary dynamics and fitness gains (adaptation) in a total of 76 experimentally evolving, asexual populations by conducting 1,216 fitness assays and analyzing 532 longitudinal-evolutionary samples collected from the evolving populations. In our analysis of these data we describe the strengths of this experimental evolution system and explore sources of error in our measurements of fitness and evolutionary dynamics.ConclusionsOur experimental treatments generated distinct fitness effects and evolutionary dynamics, respectively quantified via multiplexed fitness assays and barcode lineage tracking. These findings demonstrate the utility of this new resource for designing and improving high-throughput studies of experimental evolution. The approach described here provides a framework for future studies employing experimental designs that require high-throughput multiplexed fitness measurements.

Rapid Evolution of Plant-Bacterium Mutualism in the Rhizosphere

Plant roots are associated with a diverse microbial community that consists of antagonistic, commensal and mutualistic organisms. While beneficial plant-microbe interactions are important for plant performance, direct evidence for the evolution of rhizosphere mutualism remains elusive. Here we experimentally show that initially plant-antagonistic Pseudomonas protegens bacterium can rapidly evolve into a mutualist in the rhizosphere of Arabidopsis thaliana only within six plant growth cycles (6 months). Using an in vivo experimental evolution system, we observed that P. protegens rapidly diversified into distinct phenotypes, with some having clearly positive effects on the plant performance. Crucially, this evolution of plant facilitation was also accompanied with increased bacterial fitness indicative of a mutualistic interaction. At the genetic level, P. protegens mutualism was associated with mutations in the GacS/GacA two-component global regulator system, which is known to affect several aspects of bacterial physiology including carbon metabolism, host-microbe signalling, and downregulation of phytotoxic secondary metabolites. At the phenotypic level, the evolution of mutualism could be explained via two mechanisms: improved competition for root exudates and enhanced capacity for activating the root-specific transcription factor gene MYB72, which is essential for the production and excretion of the antimicrobial scopoletin for which the mutualists evolved enhanced tolerance. Together, these results show that plant-bacteria mutualism can rapidly evolve at an agriculturally relevant evolutionary timescale in the rhizosphere.

Experimental bacterial adaptation to the zebrafish gut reveals a primary role for immigration.

All animals live in intimate association with microorganisms that profoundly influence their health and development, yet the traits that allow microorganisms to establish and maintain host associations are not well understood. To date, most investigations aimed at identifying traits required for host association have focused on intrahost niches. Consequently, little is known about the relative contribution of extrahost factors such as environmental growth and survival and immigration into hosts from the external environment, as promoters of host association. To address this, we developed a tractable experimental evolution system that investigates both intra- and extrahost factors contributing to bacterial adaptation to the vertebrate gut. We passaged replicate lines of a zebrafish bacterial isolate, Aeromonas veronii, through populations of germ-free larval zebrafish (Danio rerio), each time using gut-associated Aeromonas populations to inoculate the aquatic environment of the next zebrafish population. We observed rapid increased adaptation to the host in all replicate lines. The initial adaptations present in early-evolved isolates did not increase intrahost fitness but rather enhanced both immigration from the environment and interhost transmission. Only in later-evolved isolates did we find evidence for intrahost-specific adaptations, as demonstrated by comparing their competitive fitness in the host genotype to which they evolved to that in a different genotype. Our results show how selection for bacterial transmission between hosts and their environment can shape bacterial-host association. This work illuminates the nature of selective forces present in host–microbe systems and reveals specific mechanisms of increased host association. Furthermore, our findings demonstrate that the entire host–microbe–environment system must be considered when identifying microbial traits that contribute to host adaptation.

Long-term context-dependent genetic adaptation of the viral genetic cloud.

RNA viruses generate a cloud of genetic variants within each host. This cloud contains high-frequency genotypes, and many rare variants. The dynamics of these variants is crucial to understand viral evolution and their effect on their host. We use an experimental evolution system to show that the genetic cloud surrounding the Coxsackie virus master sequence slowly, but steadily, evolves over hundreds of generations. This movement is determined by strong context-dependent mutations, where the frequency and type of mutations are affected by neighboring positions, even in silent mutations. This context-dependent mutation pattern serves as a spearhead for the viral population's movement within the adaptive landscape and affects which new dominant variants will emerge. The non-local mutation patterns affect the mutated dinucleotide distribution, and eventually lead to a non-uniform dinucleotide distribution in the main viral sequence. We tested these results on other RNA viruses with similar conclusions. Supplementary data are available at Bioinformatics online.

Adaptation of Escherichia coli to long-term batch culture in various rich media

Experimental evolution studies have characterized the genetic strategies microbes utilize to adapt to their environments, mainly focusing on how microbes adapt to constant and/or defined environments. Using a system that incubates Escherichia coli in different complex media in long-term batch culture, we have focused on how heterogeneity and environment affects adaptive landscapes. In this system, there is no passaging of cells, and therefore genetic diversity is lost only through negative selection, without the experimentally-imposed bottlenecking common in other platforms. In contrast with other experimental evolution systems, because of cycling of nutrients and waste products, this is a heterogeneous environment, where selective pressures change over time, similar to natural environments. We determined that incubation in each environment leads to different adaptations by observing the growth advantage in stationary phase (GASP) phenotype. Re-sequencing whole genomes of populations identified both mutant alleles in a conserved set of genes and differences in evolutionary trajectories between environments. Reconstructing identified mutations in the parental strain background confirmed the adaptive advantage of some alleles, but also identified a surprising number of neutral or even deleterious mutations. This result indicates that complex epistatic interactions may be under positive selection within these heterogeneous environments.

A Shift in the Thermoregulatory Curve as a Result of Selection for High Activity-Related Aerobic Metabolism.

According to the “aerobic capacity model,” endothermy in birds and mammals evolved as a result of natural selection favoring increased persistent locomotor activity, fuelled by aerobic metabolism. However, this also increased energy expenditure even during rest, with the lowest metabolic rates occurring in the thermoneutral zone (TNZ) and increasing at ambient temperatures (Ta) below and above this range, depicted by the thermoregulatory curve. In our experimental evolution system, four lines of bank voles (Myodes glareolus) have been selected for high swim-induced aerobic metabolism and four unselected lines have been maintained as a control. In addition to a 50% higher rate of oxygen consumption during swimming, the selected lines have also evolved a 7.3% higher mass-adjusted basal metabolic rate. Therefore, we asked whether voles from selected lines would also display a shift in the thermoregulatory curve and an increased body temperature (Tb) during exposure to high Ta. To test these hypotheses we measured the RMR and Tb of selected and control voles at Ta from 10 to 34°C. As expected, RMR within and around the TNZ was higher in selected lines. Further, the Tb of selected lines within the TNZ was greater than the Tb of control lines, particularly at the maximum measured Ta of 34°C, suggesting that selected voles are more prone to hyperthermia. Interestingly, our results revealed that while the slope of the thermoregulatory curve below the lower critical temperature (LCT) is significantly lower in the selected lines, the LCT (26.1°C) does not differ. Importantly, selected voles also evolved a higher maximum thermogenesis, but thermal conductance did not increase. As a consequence, the minimum tolerated temperature, calculated from an extrapolation of the thermoregulatory curve, is 8.4°C lower in selected (−28.6°C) than in control lines (−20.2°C). Thus, selection for high aerobic exercise performance, even though operating under thermally neutral conditions, has resulted in the evolution of increased cold tolerance, which, under natural conditions, could allow voles to inhabit colder environments. Further, the results of the current experiment support the assumptions of the aerobic capacity model of the evolution of endothermy.

Nucleotide diversity inflation as a genome-wide response to experimental lifespan extension in Drosophila melanogaster

BackgroundEvolutionary theory predicts that antagonistically selected alleles, such as those with divergent pleiotropic effects in early and late life, may often reach intermediate population frequencies due to balancing selection, an elusive process when sought out empirically. Alternatively, genetic diversity may increase as a result of positive frequency-dependent selection and genetic purging in bottlenecked populations.ResultsWhile experimental evolution systems with directional phenotypic selection typically result in at least local heterozygosity loss, we report that selection for increased lifespan in Drosophila melanogaster leads to an extensive genome-wide increase of nucleotide diversity in the selected lines compared to replicate control lines, pronounced in regions with no or low recombination, such as chromosome 4 and centromere neighborhoods. These changes, particularly in coding sequences, are most consistent with the operation of balancing selection and the antagonistic pleiotropy theory of aging and life history traits that tend to be intercorrelated. Genes involved in antioxidant defenses, along with multiple lncRNAs, were among those most affected by balancing selection. Despite the overwhelming genetic diversification and the paucity of selective sweep regions, two genes with functions important for central nervous system and memory, Ptp10D and Ank2, evolved under positive selection in the longevity lines.ConclusionsOverall, the ‘evolve-and-resequence’ experimental approach proves successful in providing unique insights into the complex evolutionary dynamics of genomic regions responsible for longevity.

Design and Experimental Evolution of trans-Splicing Group I Intron Ribozymes

Group I intron ribozymes occur naturally as cis-splicing ribozymes, in the form of introns that do not require the spliceosome for their removal. Instead, they catalyze two consecutive trans-phosphorylation reactions to remove themselves from a primary transcript, and join the two flanking exons. Designed, trans-splicing variants of these ribozymes replace the 3′-portion of a substrate with the ribozyme’s 3′-exon, replace the 5′-portion with the ribozyme’s 5′-exon, or insert/remove an internal sequence of the substrate. Two of these designs have been evolved experimentally in cells, leading to variants of group I intron ribozymes that splice more efficiently, recruit a cellular protein to modify the substrate’s gene expression, or elucidate evolutionary pathways of ribozymes in cells. Some of the artificial, trans-splicing ribozymes are promising as tools in therapy, and as model systems for RNA evolution in cells. This review provides an overview of the different types of trans-splicing group I intron ribozymes that have been generated, and the experimental evolution systems that have been used to improve them.

Rapid genomic changes in Drosophila melanogaster adapting to desiccation stress in an experimental evolution system.

BackgroundExperimental evolution studies, coupled with whole genome resequencing and advances in bioinformatics, have become a powerful tool for exploring how populations respond to selection at the genome-wide level, complementary to genome-wide association studies (GWASs) and linkage mapping experiments as strategies to connect genotype and phenotype. In this experiment, we analyzed genomes of Drosophila melanogaster from lines evolving under long-term directional selection for increased desiccation resistance in comparison with control (no-selection) lines.ResultsWe demonstrate that adaptive responses to desiccation stress have exerted extensive footprints on the genomes, manifested through a high degree of fixation of alleles in surrounding neighborhoods of eroded heterozygosity. These patterns were highly convergent across replicates, consistent with signatures of ‘soft’ selective sweeps, where multiple alleles present as standing genetic variation become beneficial and sweep through the replicate populations at the same time. Albeit much less frequent, we also observed line-unique sweep regions with zero or near-zero heterozygosity, consistent with classic, or ‘hard’, sweeps, where novel rather than pre-existing adaptive mutations may have been driven to fixation. Genes responsible for cuticle and protein deubiquitination seemed to be central to these selective sweeps. High divergence within coding sequences between selected and control lines was also reflected by significant results of the McDonald-Kreitman and Ka/Ks tests, showing that as many as 347 genes may have been under positive selection.ConclusionsDesiccation stress, a common challenge to many organisms inhabiting dry environments, proves to be a very potent selecting factor having a big impact on genome diversity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2556-y) contains supplementary material, which is available to authorized users.

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.

The Effect of Mobile Element IS10 on Experimental Regulatory Evolution in Escherichia coli

Mobile genetic elements are widespread in bacteria, where they cause several kinds of mutations. Although their effects are on the whole negative, rare beneficial mutations caused by insertion sequence elements are frequently selected in some experimental evolution systems. For example, in earlier work, we found that strains of Escherichia coli that lack the sigma factor RpoS adapt to a high-osmolarity environment by the insertion of element IS10 into the promoter of the otsBA operon, rewiring expression from RpoS dependent to RpoS independent. We wished to determine how the presence of IS10 in the genome of this strain shaped the evolutionary outcome. IS10 could influence the outcome by causing mutations that confer adaptive phenotypes that cannot be achieved by strains without the element. Alternatively, IS10 could influence evolution by increasing the rate of appearance of certain classes of beneficial mutations even if they are no better than those that could be achieved by a strain without the element. We found that populations evolved from an IS10-free strain did not upregulate otsBA. An otsBA-lacZY fusion facilitated the recovery of a number of mutations that upregulate otsB without involving IS10 and found that two caused greater fitness increases than IS10 insertion, implying that evolution could have upregulated otsBA in the IS10-free strain. Finally, we demonstrate that there is epistasis between the IS10 insertion into the otsBA promoter and the other adaptive mutations, implying that introduction of IS10 into the otsBA promoter may alter the trajectory of adaptive evolution. We conclude that IS10 exerts its effect not by creating adaptive phenotypes that could not otherwise occur but by increasing the rate of appearance of certain adaptive mutations.

THE POPULATION GENETICS OF PHENOTYPIC DETERIORATION IN EXPERIMENTAL POPULATIONS OF BACILLUS SUBTILIS

Although many examples of trait loss exist in nature, the underlying population genetic mechanism responsible for the loss is usually unknown. Selective or neutral processes can result in the deterioration of a trait, and often one of these is inferred based on indirect evidence. Furthermore, selective pressures that are unique to particular environments and the effect these might have on the population genetic cause of trait loss are not well understood. Here we describe an experimental evolution system where two different environments were used for addressing the population genetic cause of trait loss throughout evolutionary time. We found that growth in minimal medium (i.e., prototrophy) was lost in all populations regardless of the experimental environment and that the pattern of trait loss in one environment was due to selection, whereas in the other environment the cause remains inconclusive.

THE POPULATION GENETICS OF PHENOTYPIC DETERIORATION IN EXPERIMENTAL POPULATIONS OF BACILLUS SUBTILIS

Although many examples of trait loss exist in nature, the underlying population genetic mechanism responsible for the loss is usually unknown. Selective or neutral processes can result in the deterioration of a trait, and often one of these is inferred based on indirect evidence. Furthermore, selective pressures that are unique to particular environments and the effect these might have on the population genetic cause of trait loss are not well understood. Here we describe an experimental evolution system where two different environments were used for addressing the population genetic cause of trait loss throughout evolutionary time. We found that growth in minimal medium (i.e., prototrophy) was lost in all populations regardless of the experimental environment and that the pattern of trait loss in one environment was due to selection, whereas in the other environment the cause remains inconclusive.