LONG-TERM EXPERIMENTAL EVOLUTION IN ESCHERICHIA COLI. III. VARIATION AMONG REPLICATE POPULATIONS IN CORRELATED RESPONSES TO NOVEL ENVIRONMENTS

Twelve populations of Escherichia coli were founded from a single clone and propagated for 2000 generations in identical glucose-limited environments. During this time, the mean fitnesses of the evolving populations relative to their common ancestor improved greatly, but their fitnesses relative to one another diverged only slightly. Although the populations showed similar fitness increases, they may have done so by different underlying adaptations, or they may have diverged in other respects by random genetic drift. Therefore, we examined the relative fitnesses of independently derived genotypes in two other sugars, maltose and lactose, to determine whether they were homogeneous or heterogeneous in these environments. The genetic variation among the derived lines in fitness on maltose and lactose was more than 100-times greater than their variation in fitness on glucose. Moreover, the glucose-adapted genotypes, on average, showed significant adaptation to lactose, but not to maltose. That pathways for use of maltose and glucose are virtually identical in E. coli, except for their distinct mechanisms of uptake, suggests that the derived genotypes have adapted primarily by improved glucose transport. From consideration of the number of generations of divergence, the mutation rate in E. coli, and the proportion of its genome required for growth on maltose (but not glucose), we hypothesize that pleiotropy involving the selected alleles, rather than random genetic drift of alleles at other loci, was the major cause of the variation among the derived genotypes in fitness on these other sugars.     

Fitness evolution and the rise of mutator alleles in experimental Escherichia coli populations

We studied the evolution of high mutation rates and the evolution of fitness in three experimental populations of Escherichia coli adapting to a glucose-limited environment. We identified the mutations responsible for the high mutation rates and show that their rate of substitution in all three populations was too rapid to be accounted for simply by genetic drift. In two of the populations, large gains in fitness relative to the ancestor occurred as the mutator alleles rose to fixation, strongly supporting the conclusion that mutator alleles fixed by hitchhiking with beneficial mutations at other loci. In one population, no significant gain in fitness relative to the ancestor occurred in the population as a whole while the mutator allele rose to fixation, but a substantial and significant gain in fitness occurred in the mutator subpopulation as the mutator neared fixation. The spread of the mutator allele from rarity to fixation took >1000 generations in each population. We show that simultaneous adaptive gains in both the mutator and wild-type subpopulations (clonal interference) retarded the mutator fixation in at least one of the populations. We found little evidence that the evolution of high mutation rates accelerated adaptation in these populations.     

Experimental evolution: Assortative mating and sexual selection,independent of local adaptation,lead to reproductive isolation in the nematode Caenorhabditis remanei

Using experimental evolution, we investigated the contributions of ecological divergence, sexual selection, and genetic drift to the evolution of reproductive isolation in Caenorhabditis remanei. The nematodes were reared on two different environments for 100 generations. They were assayed for fitness on both environments after 30, 64, and 100 generations, and hybrid fitness were analyzed after 64 and 100 generations. Mating propensity within and between populations was also analyzed. The design allowed us to determine whether local adaptation was synchronous with pre‐ and postzygotic reproductive isolation. Prezygotic isolation evolved quickly but was unconnected with adaptation to the divergent environments. Instead, prezygotic isolation was driven by mate preferences favoring individuals from the same replicate population. A bottleneck treatment, meant to enhance the opportunity for genetic drift, had no effect on prezygotic isolation. Postzygotic isolation occurred in crosses where at least one population had a large fitness advantage in its “home” environment. Taken together, our results suggest that prezygotic isolation did not depend on drift or adaptation to divergent environments, but instead resulted from differences in sexual interactions within individual replicates. Furthermore, our results suggest that postzygotic isolation can occur between populations even when only one population has greater fitness in its home environment.     

FUNCTIONAL GENETIC DIVERGENCE IN HIGH CO2 ADAPTED EMILIANIA HUXLEYI POPULATIONS

Predicting the impacts of environmental change on marine organisms, food webs, and biogeochemical cycles presently relies almost exclusively on short‐term physiological studies, while the possibility of adaptive evolution is often ignored. Here, we assess adaptive evolution in the coccolithophore Emiliania huxleyi, a well‐established model species in biological oceanography, in response to ocean acidification. We previously demonstrated that this globally important marine phytoplankton species adapts within 500 generations to elevated CO₂. After 750 and 1000 generations, no further fitness increase occurred, and we observed phenotypic convergence between replicate populations. We then exposed adapted populations to two novel environments to investigate whether or not the underlying basis for high CO₂‐adaptation involves functional genetic divergence, assuming that different novel mutations become apparent via divergent pleiotropic effects. The novel environment “high light” did not reveal such genetic divergence whereas growth in a low‐salinity environment revealed strong pleiotropic effects in high CO₂ adapted populations, indicating divergent genetic bases for adaptation to high CO₂. This suggests that pleiotropy plays an important role in adaptation of natural E. huxleyi populations to ocean acidification. Our study highlights the potential mutual benefits for oceanography and evolutionary biology of using ecologically important marine phytoplankton for microbial evolution experiments.     

Genetic Divergence under Uniform Selection. II. Different Responses to Selection for Knockdown Resistance to Ethanol among DROSOPHILA MELANOGASTER Populations and Their Replicate Lines

We have tested the hypothesis that genetic differences among conspecific populations may result in diverse responses to selection, using natural populations of Drosophila melanogaster. Selection for ethanol tolerance in a tube measuring knockdown resistance was imposed on five West Coast populations. In 24 generations the selected lines increased their mean knockdown times, on average, by a factor of 2.7. An initially weak latitudinal cline was steepened by selection. The two southernmost populations showed the same increases in the selected character, but differed consistently in their correlated responses in characters related to ethanol tolerance. This result indicates that the populations responded to selection by different genetic changes. Selection decreased female body weight and increased resistance to acetone, suggesting components of the response unrelated to ethanol metabolism. The Adhs allele was favored by selection in all populations at the onset, but increased in frequency only in the selected lines of the southernmost population. There was a correlation between latitude and Adh frequency changes, suggesting that fitnesses of the Adh alleles were dependent on the genetic background. Genetic background also had a large effect on the loss of fitness due to selection. Genetic drift between replicate lines caused more variation in selection response than initial genetic differences between populations. This result demonstrates the importance of genetic drift in divergence among natural populations undergoing uniform selection, since the effective population sizes approached those of small natural populations. Drift caused greater divergence between selected replicates than control replicates. Implications of this result for the genetic model of selection response are discussed.     

Evolution of pleiotropic costs in experimental populations

The fitness of populations adapting to new environments is expected to decline in different environments, but empirical studies often do not lend support for such adaptation costs. We test the idea that the initial fitness of the selected populations in the environment where the cost is estimated is key for interpreting tests of ecological trade‐offs. We isolated single clones of the yeast Saccharomyces cerevisiae every ~250 generations from replicate experimental lineages that had been selected during 5000 generations in a glucose‐limited environment. We then selected these clones in a galactose‐limited environment for ~120 generations. Finally, we estimated single‐clone fitness in both environments, before and after selection on galactose. The pleiotropic effects on glucose of selection on galactose evolved from positive to negative as fitness in glucose increased, providing strong support for the importance of initial fitness for determining the sign and magnitude of pleiotropic effects. This demonstrates that the sign of pleiotropic effects for fitness following adaptation to a new environment can change during long‐term adaptation to an original environment. We also found no relationship between the size of the fitness changes in galactose and glucose, such that pleiotropic effects in glucose became relatively smaller as the sizes of direct effects on galactose increased.     

The yield of experimental yeast populations declines during selection

The trade-off between growth rate and yield can limit population productivity. Here we tested for this life-history trade-off in replicate haploid and diploid populations of Saccharomyces cerevisiae propagated in glucose-limited medium in batch cultures for 5000 generations. The yield of single clones isolated from the haploid lineages, measured as both optical and population density at the end of a growth cycle, declined during selection and was negatively correlated with growth rate. Initially, diploid populations did not pay this cost of adaptation but haploidized after about 1000–3000 generations of selection, and this ploidy transition was associated with a decline in yield caused by reduced cell size. These results demonstrate the experimental evolution of a trade-off between growth rate and yield, caused by antagonistic pleiotropy, during adaptation in haploids and after an adaptive transition from diploidy to haploidy.     

LONG-TERM EXPERIMENTAL EVOLUTION IN ESCHERICHIA COLI. VII. MECHANISMS MAINTAINING GENETIC VARIABILITY WITHIN POPULATIONS

Six replicate populations of the bacterium Escherichia coli were propagated for more than 10,000 generations in a defined environment. We sought to quantify the variation among clones within these populations with respect to their relative fitness, and to evaluate the roles of three distinct population genetic processes in maintaining this variation. On average, a pair of clones from the same population differed from one another in their relative fitness by approximately 4%. This within-population variation was small compared with the average fitness gain relative to the common ancestor, but it was statistically significant. According to one hypothesis, the variation in fitness is transient and reflects the ongoing substitution of beneficial alleles. We used Fisher's fundamental theorem to compare the observed rate of each population's change in mean fitness with the extent of variation for fitness within that population, but we failed to discern any correspondence between these quantities. A second hypothesis supposes that the variation in fitness is maintained by recurrent deleterious mutations that give rise to a mutation-selection balance. To test this hypothesis, we made use of the fact that two of the six replicate populations had evolved mutator phenotypes, which gave them a genomic mutation rate approximately 100-fold higher than that of the other populations. There was a marginally significant correlation between a population's mutation rate and the extent of its within-population variance for fitness, but this correlation was driven by only one population (whereas two of the populations had elevated mutation rates). Under a third hypothesis, this variation is maintained by frequency-dependent selection, whereby genotypes have an advantage when they are rare relative to when they are common. In all six populations, clones were more fit, on average, when they were rare than when they were common, although the magnitude of the advantage when rare was usually small (~1% in five populations and ~5% in the other). These three hypotheses are not mutually exclusive, but frequency-dependent selection appears to be the primary force maintaining the fitness variation within these experimental populations.     

Long-term experimental evolution in Escherichia coli. V. Effects of recombination with immigrant genotypes on the rate of bacterial evolution

This study builds upon an earlier experiment that examined the dynamics of mean fitness in evolving populations of Escherichia coli in which mutations were the sole source of genetic variation. During thousands of generations in a constant environment, the rate of improvement in mean fitness of these asexual populations slowed considerably from an initially rapid pace. In this study, we sought to determine whether sexual recombination with novel genotypes would reaccelerate the rate of adaption in these populations. To that end, treatment populations were propagated for an additional 1000 generations in the same environment as their ancestors, but they were periodically allowed to mate with an immigrant pool of genetically distinct Hfr (high frequency recombination) donors. These donors could transfer genes to the resident populations by conjugation, but the donors themselves could not grow in the experimental environment. Control populations were propagated under identical conditions, but in the absence of sexual recombination with the donors. All twelve control populations retained the ancestral alleles at every locus that was scored. In contrast, the sexual recombination treatment yielded dramatic increases in genetic variation. Thus, there was a profound effect of recombination on the rate of genetic change. However, the increased genetic variation in the treatment populations had no significant effect on the rate of adaptive evolution, as measured by changes in mean fitness relative to a common competitor. We then considered three hypotheses that might reconcile these two outcomes: recombination pressure, hitchhiking of recombinant genotypes in association with beneficial mutations, and complex selection dynamics whereby certain genotypes may have a selective advantage only within a particular milieu of competitors. The estimated recombination rate was too low to explain the observed rate of genetic change, either alone or in combination with hitchhiking effects. However, we documented comple x ecological interactions among some recombinant genotypes, suggesting that our method for estimating fitness relative to a common competitor might have underestimated the rate of adaptive evolution in the treatment populations.     

Constraints on adaptation of Escherichia coli to mixed‐resource environments increase over time

Can a population evolved in two resources reach the same fitness in both as specialist populations evolved in each of the individual resources? This question is central to theories of ecological specialization, the maintenance of genetic variation, and sympatric speciation, yet relatively few experiments have examined costs of generalism over long‐term adaptation. We tested whether selection in environments containing two resources limits a population's ability to adapt to the individual resources by comparing the fitness of replicate Escherichia coli populations evolved for 6000 generations in the presence of glucose or lactose alone (specialists), or in varying presentations of glucose and lactose together (generalists). We found that all populations had significant fitness increases in both resources, though the magnitude and rate of these increases differed. For the first 4000 generations, most generalist populations increased in fitness as quickly in the individual resources as the corresponding specialist populations. From 5000 generations, however, a widespread cost of adaptation affected all generalists, indicating a growing constraint on their abilities to adapt to two resources simultaneously. Our results indicate that costs of generalism are prevalent, but may influence evolutionary trajectories only after a period of cost‐free adaptation.     

The ecology and genetics of fitness in Chlamydomonas. IX. The rate of accumulation of variation of fitness under selection

Populations of Chlamydomonas founded by single cells were cultured in chemostats for 50 days, representing about 125 generations. The mean and variance of division rate was measured daily by withdrawing cells from the effluent and culturing them for 24 h on filtered effluent medium solidified with agar. Mean fitness did not change during the period of culture, and the behavior of neutral markers indicated that no substitutions of novel beneficial mutations occurred. However, the variance of fitness increased markedly at about the same rate in two replicate populations. The standardized rate, or mutational heritability, was Vm/VE = 4-5 x 10(-3) per generation. This is substantially greater than most other estimates for characters closely correlated with fitness. Moreover, it seems difficult to reconcile with the absence of any change in mean fitness. We investigated the possibility that frequency-dependent selection was created by spatial heterogeneity within the culture vessel by testing cell populations with different phenotypes from the top, bottom, and surface of the chemostats. However, the differentiation of these populations seemed to be attributable to phenotypic plasticity, with no evidence that their characteristics were heritable. Finally, we report an experiment in which lines were selected for about 100 generations on solid or liquid medium. These lines became specifically adapted to the medium on which they were cultured, showing that liquid and solid media, even when chemically identical, provide different conditions of growth for Chlamydomonas. The genetic variance appearing in the cultures was therefore attributed to conditionally neutral mutations that were not expressed in the chemostat. This implies that rates of accumulation of mutational variance measured in the culture environment itself (where this can be done) may greatly underestimate the variation available for a response through selection to environmental change. Moreover, it suggests that chemostat populations may be more dynamic and more diverse than is usually thought.     

The number of mutations selected during adaptation in a laboratory population of Saccharomyces cerevisiae

There is currently limited empirical and theoretical support for the prevailing view that adaptation typically results from the fixation of many mutations, each with small phenotypic effects. Recent theoretical work suggests that, on the contrary, most of the phenotypic change during an episode of adaptation can result from the selection of a few mutations with relatively large effects. I studied the genetics of adaptation by populations of budding yeast to a culture regime of daily hundredfold dilution and transfer in a glucose-limited minimal liquid medium. A single haploid genotype isolated after 2000 generations showed a 76% fitness increase over its ancestor. This evolved haploid was crossed with its ancestor, and tetrad dissections were used to isolate a complete series of six meiotic tetrads. The Castle-Wright estimator of the number of loci at which adaptive mutations had been selected, modified to account for linkage and variation among mutations in the size of their effect, is 4.4. The estimate for a second haploid genotype, isolated from a separate population and with a fitness gain of 60%, was 2.7 loci. Backcrosses to the ancestor with the first evolved genotype support the inference that adaptation resulted primarily from two to five mutations. These backcrosses also indicated that deleterious mutations had hitchhiked with adaptive mutations in this evolved genotype.     

THE GENETIC BASIS OF THE ECOLOGICAL AMPLITUDE OF SPARTINA PATENS. II. VARIANCE AND CORRELATION ANALYSIS

Reciprocal transplantations of Spartina patens genotypes from adjacent salt marsh, swale, and dune habitats provided evidence for genetic differentiation among subpopulations, due at least in part to contrasting selection regimes. Genet survival in the different habitats was related to the amount of genetic divergence. In the dune habitat, marsh ramets showed the lowest survival, swale ramets showed intermediate survival, and dune ramets showed the highest survival. This relationship was not reciprocal, however. The marsh habitat afforded an environment where survival was maximal for all genotypes. Thus, by comparison, the dune environment appeared to impose a more intense selection pressure, and the swale an intermediate selection pressure on Spartina patens. In each site resident genotypes tended to show greater relative fitness than aliens. This evidence for genetic divergence corroborates that previously reported on morphometric (Silander and Antonovics, 1979) and allozymic traits (Silander, 1984). High levels of phenotypic plasticity may permit greater adaptation to the spatially and temporally heterogeneous environment occupied by S. patens than would genetic variation alone. Dune and swale genets were more phenotypically plastic across traits examined than were marsh genotypes. The higher plasticity in these peripheral subpopulations may confer increased fitness among residents and compensate for observed declines in genetic variation. A slight decrease in genetic variability was evident from marsh to dune subpopulations. However, since the differences in genetic variation among subpopulations were small, and disparities did occur, it is unlikely that evolutionary divergence is retarded primarily by a lack of genetic variability in the characters considered. Evidence is presented to indicate that evolutionary divergence among subpopulations may be retarded by negative or unfavorable correlations among characters being selected simultaneously. These negative correlations may increase extinction probabilities in small peripheral populations, such as those represented by the dune or swale, and are likely to lower fitness. Based on these observations, I hypothesize that further microevolution may be retarded in peripheral dune and swale subpopulations, primarily by unfavorable genetic correlation structures among fitness components or characters under simultaneous selection. Contributing factors may include lowered genetic variance and higher levels of phenotypic plasticity.     

THE QUANTITATIVE AND MOLECULAR GENETIC ARCHITECTURE OF A SUBDIVIDED SPECIES

In an effort to elucidate the evolutionary mechanisms that determine the genetic architecture of a species, we have analyzed 17 populations of the microcrustacean Daphnia pulex for levels of genetic variation at the level of life-history characters and molecular markers in the nuclear and mitochondrial genomes. This species is highly subdivided, with approximately 30% of the variation for nuclear molecular markers and 50% of the variation for mitochondrial markers being distributed among populations. The average level of genetic subdivision for quantitative traits is essentially the same as that for nuclear markers, which superficially suggests that the life-history characters are diverging at the neutral rate. However, the existence of a strong correlation between the levels of population subdivision and broadsense heritabilities of individual traits argues against this interpretation, suggesting instead that the among-population divergence of some quantitative traits (most notably body size) is being driven by local adaptation to different environments. The fact that the mean phenotypes of the individual populations are also strongly correlated with local levels of homozygosity indicates that variation in local inbreeding plays a role in population differentiation. Rather than being a passive consequence of local founder effects, levels of homozygosity may be selected for directly for their effects on the phenotype (adaptive inbreeding depression). There is no relationship between the levels of variation within populations for molecular markers and quantitative characters, and this is explained by the fact that the average standing genetic variation for life-history characters in this species is equivalent to only 33 generations of variation generated by mutation.     

Large Genetic Change at Small Fitness Cost in Large Populations of Drosophila Melanogaster Selected for Wind Tunnel Flight: Rethinking Fitness Surfaces

The fitness effects of extreme genetic change by selection were studied in large populations subjected to prolonged, intense selection. Two replicate populations of Drosophila melanogaster, with estimated effective sizes 500 <= N(e) <= 1000, were selected for increased performance in a wind tunnel, selecting on average the fastest 4.5% of flies. The mean apparent flying speed of both lines increased from ~2 to 170 cm/sec and continued to respond at diminishing rates, without reaching a plateau, for 100 generations. Competitive fitness tests in generations 50 and 85 showed minimal or no fitness loss in selected lines compared to controls. Sublines relaxed in generations 65 and 85 showed minimal or no regression in apparent flying speed. Hybrid lines, from a cross of selected X control lines in generation 75, responded to reselection saltationally, showing that the chromosomes of the selected lines had been assembled from alleles at many loci, from many different chromosomes in the base population. Thus, major genetic change was achieved, but without the costs usually associated with strong directional selection. Large population size has been interpreted, in opposing models, as either a brake or an accelerator in its effects on long-term change by selection. These results favor the second model, and challenge the concept of rugged fitness surfaces underlying the first model.     

Sustained fitness gains and variability in fitness trajectories in the long-term evolution experiment with Escherichia coli

Many populations live in environments subject to frequent biotic and abiotic changes. Nonetheless, it is interesting to ask whether an evolving population''s mean fitness can increase indefinitely, and potentially without any limit, even in a constant environment. A recent study showed that fitness trajectories of Escherichia coli populations over 50 000 generations were better described by a power-law model than by a hyperbolic model. According to the power-law model, the rate of fitness gain declines over time but fitness has no upper limit, whereas the hyperbolic model implies a hard limit. Here, we examine whether the previously estimated power-law model predicts the fitness trajectory for an additional 10 000 generations. To that end, we conducted more than 1100 new competitive fitness assays. Consistent with the previous study, the power-law model fits the new data better than the hyperbolic model. We also analysed the variability in fitness among populations, finding subtle, but significant, heterogeneity in mean fitness. Some, but not all, of this variation reflects differences in mutation rate that evolved over time. Taken together, our results imply that both adaptation and divergence can continue indefinitely—or at least for a long time—even in a constant environment.     

An evolutionary cost of separate genders revealed by male-limited evolution

Theory predicts that intralocus sexual conflict can constrain the evolution of sexual dimorphism, preventing each sex from independently maximizing its fitness. To test this idea, we limited genome-wide gene expression to males in four replicate Drosophila melanogaster populations, removing female-specific selection. Over 25 generations, male fitness increased markedly, as sexually dimorphic traits evolved in the male direction. When male-evolved genomes were expressed in females, their fitness displayed a nearly symmetrical decrease. These results suggest that intralocus conflict strongly limits sex-specific adaptation, promoting the maintenance of genetic variation for fitness. Populations may carry a heavy genetic load as a result of selection for separate genders.     

An Experimental Test of the Relationship between Genetic Variation and Environmental Variation in Tribolium Flour Beetles

The hypothesis that a component of genetic variation for polygenic fitness traits is maintained by environmental heterogeneity was tested using an experimental system involving two species of flour beetles, Tribolium castaneum and T. confusum. Replicated populations of each species from a number of environmental treatments were analyzed for various fitness components following almost 60 generations of natural selection. Environmental differences consisted of flours of cereals commonly invaded by natural populations of these insects.—Tests for adaptation to environments were based on experiments in which populations were reared factorially on each flour, such that population treatment x flour interactions could be detected. Measurements were made of survival, growth rate, larval weight, pupal weight, developmental time, fecundity of individuals at low density and fecundity and cannibalism at high density in both fresh and conditioned media.—Flour differences were found to have significant effects on most traits. Evidence for significant genetic variation and significant genotype x environment interaction was also found. However, no evidence could be found to support the hypothesis that genetic variation was maintained by environmental heterogeneity in food resources. The absence of adaptation to the experimental treatments despite the presence of genetic variation in fitness components suggests that pleiotropy may assume an important role in determining net fitness values of polygenes.     

Adaptation of Escherichia coli to glucose promotes evolvability in lactose

The selective history of a population can influence its subsequent evolution, an effect known as historical contingency. We previously observed that five of six replicate populations that were evolved in a glucose‐limited environment for 2000 generations, then switched to lactose for 1000 generations, had higher fitness increases in lactose than populations started directly from the ancestor. To test if selection in glucose systematically increased lactose evolvability, we started 12 replay populations—six from a population subsample and six from a single randomly selected clone—from each of the six glucose‐evolved founder populations. These replay populations and 18 ancestral populations were evolved for 1000 generations in a lactose‐limited environment. We found that replay populations were initially slightly less fit in lactose than the ancestor, but were more evolvable, in that they increased in fitness at a faster rate and to higher levels. This result indicates that evolution in the glucose environment resulted in genetic changes that increased the potential of genotypes to adapt to lactose. Genome sequencing identified four genes—iclR, nadR, spoT, and rbs—that were mutated in most glucose‐evolved clones and are candidates for mediating increased evolvability. Our results demonstrate that short‐term selective costs during selection in one environment can lead to changes in evolvability that confer longer term benefits.     

The process of adaptation of flour beetles to new environments

Five populations derived from a composite, genetically heterogeneous strain of the flour beetle, Tribolium castaneum, were selected on new diets (dog food, powdered rice, brewers' yeast, wheat flour and oats) for 13–16 generations. A control population was reared on the standard medium (flour + 5% brewers' yeast). Adaptedness was measured in terms of (1) fitness parameters (survival and developmental time), (2) amylase activity levels (amylase is an essential digestive enzyme in flour beetles) and (3) food preference behavior.There were significant temporal trends in fitness components in the selected populations. Survival was initially low but increased with time. Developmental time decreased in later generations of selection. The control population showed the smallest increase in survival and no decrease in developmental time. When tested at the end of the experiment, survival to adulthood of eggs from each selected population on its selective diet was higher than that of control eggs. On the standard medium, no differences in survival among populations were detected. These results suggest that the improvement in fitness may indicate adaptation to the new environments created by the different food media.Amylase activity also increased temporally in 4 selected populations but not in the control (nor on unenriched wheat flour). Tests at the end of the experiment, however, suggest that this increase was, in large part, environmentally induced. We found no evidence that an adaptive genetic change in amylase regulation took place.There were no temporal trends in food-preference behavior during the selection process.