Tim F. Cooper

Parallel changes in gene expression after 20,000 generations of evolution in Escherichia coli

Twelve populations of Escherichia coli, derived from a common ancestor, evolved in a glucose-limited medium for 20,000 generations. Here we use DNA expression arrays to examine whether gene-expression profiles in two populations evolved in parallel, which would indicate adaptation, and to gain insight into the mechanisms underlying their adaptation. We compared the expression profile of the ancestor to that of clones sampled from both populations after 20,000 generations. The expression of 59 genes had changed significantly in both populations. Remarkably, all 59 were changed in the same direction relative to the ancestor. Many of these genes were members of the cAMP-cAMP receptor protein (CRP) and guanosine tetraphosphate (ppGpp) regulons. Sequencing of several genes controlling the effectors of these regulons found a nonsynonymous mutation in spoT in one population. Moving this mutation into the ancestral background showed that it increased fitness and produced many of the expression changes manifest after 20,000 generations. The same mutation had no effect on fitness when introduced into the other evolved population, indicating that a mutation of similar effect was present already. Our study demonstrates the utility of expression arrays for addressing evolutionary issues including the quantitative measurement of parallel evolution in independent lineages and the identification of beneficial mutations.

Negative epistasis between beneficial mutations in an evolving bacterial population

Interactions between genes reduce the benefits of a mutation and decrease the rate of fitness gain during adaptation. Epistatic interactions between mutations play a prominent role in evolutionary theories. Many studies have found that epistasis is widespread, but they have rarely considered beneficial mutations. We analyzed the effects of epistasis on fitness for the first five mutations to fix in an experimental population of Escherichia coli. Epistasis depended on the effects of the combined mutations—the larger the expected benefit, the more negative the epistatic effect. Epistasis thus tended to produce diminishing returns with genotype fitness, although interactions involving one particular mutation had the opposite effect. These data support models in which negative epistasis contributes to declining rates of adaptation over time. Sign epistasis was rare in this genome-wide study, in contrast to its prevalence in an earlier study of mutations in a single gene.

Second-order selection for evolvability in a large Escherichia coli population

Descendants of bacterial lineages that retained adaptation potential outcompeted competitors of higher fitness. In theory, competition between asexual lineages can lead to second-order selection for greater evolutionary potential. To test this hypothesis, we revived a frozen population of Escherichia coli from a long-term evolution experiment and compared the fitness and ultimate fates of four genetically distinct clones. Surprisingly, two clones with beneficial mutations that would eventually take over the population had significantly lower competitive fitness than two clones with mutations that later went extinct. By replaying evolution many times from these clones, we showed that the eventual winners likely prevailed because they had greater potential for further adaptation. Genetic interactions that reduce the benefit of certain regulatory mutations in the eventual losers appear to explain, at least in part, why they were outcompeted.

Recombination speeds adaptation by reducing competition between beneficial mutations in populations of Escherichia coli.

Identification of the selective forces contributing to the origin and maintenance of sex is a fundamental problem in biology. The Fisher–Muller model proposes that sex is advantageous because it allows beneficial mutations that arise in different lineages to recombine, thereby reducing clonal interference and speeding adaptation. I used the F plasmid to mediate recombination in the bacterium Escherichia coli and measured its effect on adaptation at high and low mutation rates. Recombination increased the rate of adaptation ∼3-fold more in the high mutation rate treatment, where beneficial mutations had to compete for fixation. Sequencing of candidate loci revealed the presence of a beneficial mutation in six high mutation rate lines. In the absence of recombination, this mutation took longer to fix and, over the course of its substitution, conferred a reduced competitive advantage, indicating interference between competing beneficial mutations. Together, these results provide experimental support for the Fisher–Muller model and demonstrate that plasmid-mediated gene transfer can accelerate bacterial adaptation.

The causes of epistasis

Since Batesons discovery that genes can suppress the phenotypic effects of other genes, gene interactions—called epistasis—have been the topic of a vast research effort. Systems and developmental biologists study epistasis to understand the genotype–phenotype map, whereas evolutionary biologists recognize the fundamental importance of epistasis for evolution. Depending on its form, epistasis may lead to divergence and speciation, provide evolutionary benefits to sex and affect the robustness and evolvability of organisms. That epistasis can itself be shaped by evolution has only recently been realized. Here, we review the empirical pattern of epistasis, and some of the factors that may affect the form and extent of epistasis. Based on their divergent consequences, we distinguish between interactions with or without mean effect, and those affecting the magnitude of fitness effects or their sign. Empirical work has begun to quantify epistasis in multiple dimensions in the context of metabolic and fitness landscape models. We discuss possible proximate causes (such as protein function and metabolic networks) and ultimate factors (including mutation, recombination, and the importance of natural selection and genetic drift). We conclude that, in general, pleiotropy is an important prerequisite for epistasis, and that epistasis may evolve as an adaptive or intrinsic consequence of changes in genetic robustness and evolvability.

Growth of Western Australian corals in the anthropocene.

Heat or Acid? The question of how tropical coral reefs will respond to increasing atmospheric greenhouse gas concentrations and concomitant climate change is widely debated. Model predictions and laboratory experiments suggest that decreasing carbonate saturation and decreasing pH may reduce calcification in carbonate-depositing organisms, including corals, yet field data are sparse, and recent declines in coral growth rates have been variously attributed to thermal stress or ocean acidification. Cooper et al. (p. 593) demonstrate that there has been no large-scale decline in calcification rates of massive Porites on coral reefs along the Indian Ocean coast of Western Australia. Instead, coral growth has increased significantly in the past 110 years, particularly at high latitudes. Thus, coral calcification appears to increase as ocean waters warm, but—at excessive temperatures—coral bleaching and reduced ocean carbonate saturation may lead to growth declines as observed on the Great Barrier Reef. Cores taken from massive corals indicate that temperature rather than ocean acidification has governed reef growth. Anthropogenic increases of atmospheric carbon dioxide lead to warmer sea surface temperatures and altered ocean chemistry. Experimental evidence suggests that coral calcification decreases as aragonite saturation drops but increases as temperatures rise toward thresholds optimal for coral growth. In situ studies have documented alarming recent declines in calcification rates on several tropical coral reef ecosystems. We show there is no widespread pattern of consistent decline in calcification rates of massive Porites during the 20th century on reefs spanning an 11° latitudinal range in the southeast Indian Ocean off Western Australia. Increasing calcification rates on the high-latitude reefs contrast with the downward trajectory reported for corals on Australia’s Great Barrier Reef and provide additional evidence that recent changes in coral calcification are responses to temperature rather than ocean acidification.

Experimental evolution with E. coli in diverse resource environments. I. Fluctuating environments promote divergence of replicate populations

BackgroundEnvironmental conditions affect the topology of the adaptive landscape and thus the trajectories followed by evolving populations. For example, a heterogeneous environment might lead to a more rugged adaptive landscape, making it more likely that replicate populations would evolve toward distinct adaptive peaks, relative to a uniform environment. To date, the influence of environmental variability on evolutionary dynamics has received relatively little experimental study.ResultsWe report findings from an experiment designed to test the effects of environmental variability on the adaptation and divergence of replicate populations of E. coli. A total of 42 populations evolved for 2000 generations in 7 environmental regimes that differed in the number, identity, and presentation of the limiting resource. Regimes were organized in two sets, having the sugars glucose and maltose singly and in combination, or glucose and lactose singly and in combination. Combinations of sugars were presented either simultaneously or as temporally fluctuating resource regimes. This design allowed us to compare the effects of resource identity and presentation on the evolutionary trajectories followed by replicate populations. After 2000 generations, the fitness of all populations had increased relative to the common ancestor, but to different extents. Populations evolved in glucose improved the least, whereas populations evolving in maltose or lactose increased the most in their respective sets. Among-population divergence also differed across regimes, with variation higher in those groups that evolved in fluctuating environments than in those that faced constant resource regimens. This divergence under the fluctuating conditions increased between 1000 and 2000 generations, consistent with replicate populations evolving toward distinct adaptive peaks.ConclusionsThese results support the hypothesis that environmental heterogeneity can give rise to more rugged adaptive landscapes, which in turn promote evolutionary diversification. These results also demonstrate that this effect depends on the form of environmental heterogeneity, with greater divergence when the pairs of resources fluctuated temporally rather than being presented simultaneously.

Expression Profiles Reveal Parallel Evolution of Epistatic Interactions Involving the CRP Regulon in Escherichia coli

The extent and nature of epistatic interactions between mutations are issues of fundamental importance in evolutionary biology. However, they are difficult to study and their influence on adaptation remains poorly understood. Here, we use a systems-level approach to examine epistatic interactions that arose during the evolution of Escherichia coli in a defined environment. We used expression arrays to compare the effect on global patterns of gene expression of deleting a central regulatory gene, crp. Effects were measured in two lineages that had independently evolved for 20,000 generations and in their common ancestor. We found that deleting crp had a much more dramatic effect on the expression profile of the two evolved lines than on the ancestor. Because the sequence of the crp gene was unchanged during evolution, these differences indicate epistatic interactions between crp and mutations at other loci that accumulated during evolution. Moreover, a striking degree of parallelism was observed between the two independently evolved lines; 115 genes that were not crp-dependent in the ancestor became dependent on crp in both evolved lines. An analysis of changes in crp dependence of well-characterized regulons identified a number of regulatory genes as candidates for harboring beneficial mutations that could account for these parallel expression changes. Mutations within three of these genes have previously been found and shown to contribute to fitness. Overall, these findings indicate that epistasis has been important in the adaptive evolution of these lines, and they provide new insight into the types of genetic changes through which epistasis can evolve. More generally, we demonstrate that expression profiles can be profitably used to investigate epistatic interactions.

The Environment Affects Epistatic Interactions to Alter the Topology of an Empirical Fitness Landscape

The fitness effect of mutations can be influenced by their interactions with the environment, other mutations, or both. Previously, we constructed 32 ( = 25) genotypes that comprise all possible combinations of the first five beneficial mutations to fix in a laboratory-evolved population of Escherichia coli. We found that (i) all five mutations were beneficial for the background on which they occurred; (ii) interactions between mutations drove a diminishing returns type epistasis, whereby epistasis became increasingly antagonistic as the expected fitness of a genotype increased; and (iii) the adaptive landscape revealed by the mutation combinations was smooth, having a single global fitness peak. Here we examine how the environment influences epistasis by determining the interactions between the same mutations in two alternative environments, selected from among 1,920 screened environments, that produced the largest increase or decrease in fitness of the most derived genotype. Some general features of the interactions were consistent: mutations tended to remain beneficial and the overall pattern of epistasis was of diminishing returns. Other features depended on the environment; in particular, several mutations were deleterious when added to specific genotypes, indicating the presence of antagonistic interactions that were absent in the original selection environment. Antagonism was not caused by consistent pleiotropic effects of individual mutations but rather by changing interactions between mutations. Our results demonstrate that understanding adaptation in changing environments will require consideration of the combined effect of epistasis and pleiotropy across environments.

Parasites and mutational load : an experimental test of a pluralistic theory for the evolution of sex

Ecological and mutational explanations for the evolution of sexual reproduction have usually been considered independently. Although many of these explanations have yielded promising theoretical results, experimental support for their ability to overcome a twofold cost of sex has been limited. For this reason, it has recently been argued that a pluralistic approach, combining effects from multiple models, may be necessary to explain the apparent advantage of sex. One such pluralistic model proposes that parasite load and synergistic epistasis between deleterious mutations might interact to create an advantage for recombination. Here, we test this proposal by comparing the fitness functions of parasitized and parasite–free genotypes of Escherichia coli bearing known numbers of transposon–insertion mutations. In both classes, we failed to detect any evidence for synergistic epistasis. However, the average effect of deleterious mutations was greater in parasitized than parasite–free genotypes. This effect might broaden the conditions under which another proposed model combining parasite–host coevolutionary dynamics and mutation accumulation can explain the maintenance of sex. These results suggest that, on average, deleterious mutations act multiplicatively with each other but in synergy with infection in determining fitness.