The directed mutation controversy and neo-Darwinism.

According to neo-Darwinian theory, random mutation produces genetic differences among organisms whereas natural selection tends to increase the frequency of advantageous alleles. However, several recent papers claim that certain mutations in bacteria and yeast occur at much higher rates specifically when the mutant phenotypes are advantageous. Various molecular models have been proposed that might explain these directed mutations, but the models have not been confirmed. Critics contend that studies purporting to demonstrate directed mutation lack certain controls and fail to account adequately for population dynamics. Further experiments that address these criticisms do not support the existence of directed mutations.

Punctuated evolution caused by selection of rare beneficial mutations.

For more than two decades there has been intense debate over the hypothesis that most morphological evolution occurs during relatively brief episodes of rapid change that punctuate much longer periods of stasis. A clear and unambiguous case of punctuated evolution is presented for cell size in a population of Escherichia coli evolving for 3000 generations in a constant environment. The punctuation is caused by natural selection as rare, beneficial mutations sweep successively through the population. This experiment shows that the most elementary processes in population genetics can give rise to punctuated evolution dynamics.

Experimental tests of the roles of adaptation, chance, and history in evolution.

The contributions of adaptation, chance, and history to the evolution of fitness and cell size were measured in two separate experiments using bacteria. In both experiments, populations propagated in identical environments achieved similar fitnesses, regardless of prior history or subsequent chance events. In contrast, the evolution of cell size, a trait weakly correlated with fitness, was more strongly influenced by history and chance.

Diminishing returns from mutation supply rate in asexual populations.

Mutator genotypes with increased mutation rates may be especially important in microbial evolution if genetic adaptation is generally limited by the supply of mutations. In experimental populations of the bacterium Escherichia coli, the rate of evolutionary adaptation was proportional to the mutation supply rate only in particular circumstances of small or initially well-adapted populations. These experiments also demonstrate a "speed limit" on adaptive evolution in asexual populations, one that is independent of the mutation supply rate.

Historical and contingent factors affect re-evolution of a complex feature lost during mass extinction in communities of digital organisms.

Re-evolution of complex biological features following the extinction of taxa bearing them remains one of evolution's most interesting phenomena, but is not amenable to study in fossil taxa. We used communities of digital organisms (computer programs that self-replicate, mutate and evolve), subjected to periods of low resource availability, to study the evolution, loss and re-evolution of a complex computational trait, the function EQU (bit-wise logical equals). We focused our analysis on cases where the pre-extinction EQU clade had surviving descendents at the end of the extinction episode. To see if these clades retained the capacity to re-evolve EQU, we seeded one set of multiple subreplicate 'replay' populations using the most abundant survivor of the pre-extinction EQU clade, and another set with the actual end-extinction ancestor of the organism in which EQU re-evolved following the extinction episode. Our results demonstrate that stochastic, historical, genomic and ecological factors can lead to constraints on further adaptation, and facilitate or hinder re-evolution of a complex feature.

Evolutionary genetics. Directed mutations slip-sliding away?

Adaptive frameshift mutations in the lacZ gene of Escherichia coli are, unusually, nearly all short deletions, perhaps caused by slipped-strand mispairings in mononucleotide runs. But are they directed?

Adaptive evolution of highly mutable loci in pathogenic bacteria.

Bacteria have specific loci that are highly mutable. We argue that the coexistence within bacterial genomes of such 'contingency' genes with high mutation rates, and 'housekeeping' genes with low mutation rates, is the result of adaptive evolution, and facilitates the efficient exploration of phenotypic solutions to unpredictable aspects of the host environment while minimizing deleterious effects on fitness.

Two-step resistance by Escherichia coli B to bacteriophage T2.

Numerous authors have noted the difficulty in obtaining mutants of E. coli B that are resistant to bacteriophage T2 using standard procedures of plating large numbers of cells in the presence of excess phage. Yet, T2-resistant mutants appear in continuous culture at rates in consistent with this difficulty. This paradoxical result derives from the fact that resistance to T2 usually arises as a consequence of two nonindependent mutations. Mutant bacteria resistant to phage T4 are very common and increase rapidly in continuous culture with phage T2 owing to an approximate halving of the rate at which T2 adsorbs to and kills these partially resistant mutants. The rate at which these partially resistant mutants then give rise to fully resistant mutants is approximately two orders of magnitude higher than the rate obtained by direct selection. These results are consistent with biochemical evidence that T2 adsorption to E. coli B involves both the bacterial lipopolysaccharide (to which phage T4 adsorbs) and a bacterial surface protein. However, this genetic evidence suggests that T2 can adsorb to either receptor type alone, whereas the biochemical evidence suggests that T2 requires a complex of the two receptors for adsorption to E. coli B. These results also indicate that the effects of genetic background can influence not only the selective advantage associated with particular mutations but also the rate at which certain selectively defined characteristics arise via mutation.

Long-term experimental evolution in Escherichia coli. IV. Targets of selection and the specificity of adaptation.

This study investigates the physiological manifestation of adaptive evolutionary change in 12 replicate populations of Escherichia coli that were propagated for 2000 generations in a glucose-limited environment. Representative genotypes from each population were assayed for fitness relative to their common ancestor in the experimental glucose environment and in 11 novel single-nutrient environments. After 2000 generations, the 12 derived genotypes had diverged into at least six distinct phenotypic classes. The nutrients were classified into four groups based upon their uptake physiology. All 12 derived genotypes improved in fitness by similar amounts in the glucose environment, and this pattern of parallel fitness gains was also seen in those novel environments where the limiting nutrient shared uptake mechanisms with glucose. Fitness showed little or no consistent improvement, but much greater genetic variation, in novel environments where the limiting nutrient differed from glucose in its uptake mechanisms. This pattern of fitness variation in the novel nutrient environments suggests that the independently derived genotypes adapted to the glucose environment by similar, but not identical, changes in the physiological mechanisms for moving glucose across both the inner and outer membranes.

Long-term experimental evolution in Escherichia coli. IX. Characterization of insertion sequence-mediated mutations and rearrangements.

As part of a long-term evolution experiment, two populations of Escherichia coli B adapted to a glucose minimal medium for 10,000 generations. In both populations, multiple IS-associated mutations arose that then went to fixation. We identify the affected genetic loci and characterize the molecular events that produced nine of these mutations. All nine were IS-mediated events, including simple insertions as well as recombination between homologous elements that generated inversions and deletions. Sequencing DNA adjacent to the insertions indicates that the affected genes are involved in central metabolism (knockouts of pykF and nadR), cell wall synthesis (adjacent to the promoter of pbpA-rodA), and ill-defined functions (knockouts of hokB-sokB and yfcU). These genes are candidates for manipulation and competition experiments to determine whether the mutations were beneficial or merely hitchhiked to fixation.

Epistasis between new mutations and genetic background and a test of genetic canalization.

The importance for fitness of epistatic interactions among mutations is poorly known, yet epistasis can exert important effects on the dynamics of evolving populations. We showed previously that epistatic interactions are common between pairs of random insertion mutations in the bacterium Escherichia coli. In this paper, we examine interactions between these mutations and other mutations by transducing each of twelve insertion mutations into two genetic backgrounds, one ancestral and the other having evolved in, and adapted to, a defined laboratory environment for 10,000 generations. To assess the effect of the mutation on fitness, we allowed each mutant to compete against its unmutated counterpart in that same environment. Overall, there was a strong positive correlation between the mutational effects on the two genetic backgrounds. Nonetheless, three of the twelve mutations had significantly different effects on the two backgrounds, indicating epistasis. There was no significant tendency for the mutations to be less harmful on the derived background. Thus, there is no evidence supporting the hypothesis that the derived bacteria had adapted, in part, by becoming buffered against the harmful effects of mutations.

Evolutionary adaptation to temperature. IX. Preadaptation to novel stressful environments of Escherichia coli adapted to high temperature.

Stressful environments may be considered as those that reduce fitness, sometimes due in part to the increased metabolic expenditure required to sustain life. Direct adaptation to a stressor is expected to increase fitness and reduce maintenance metabolism, with the latter leading to increased biomass production. In this study, we test the general hypothesis that such adaptation to one stressor can preadapt organisms to novel stressful environments. Six lines of Escherichia coli propagated for 2,000 generations at 41-42 degrees C (42 group), a stressful temperature, were compared to six control lines propagated for 2,000 generations at 37degrees C (37 group) and to the common ancestor of both groups. We assayed biovolume yield (a measure of growth efficiency) and competitive fitness in the 42 group's selective high temperature environment as well as five novel stressful environments-acid, alkali, ethanol, high osmolarity and peroxide. As previously reported, at high temperature the 42 group had both higher yield and fitness than the 37 group and ancestor. In the novel environments, the 42 group generally produced yields higher than the 37 group (and marginally higher than the ancestor), but we found no differences in competitive fitness among the 37 and 42 groups and the ancestor. We also found that the performance of lines within groups was not correlated across stressful environments for either yield or relative fitness. Because previous adaptation to one stressor did not improve our measure of Darwinian fitness in novel stressful environments, we conclude that the 42 group shows no useful pre-adaptation, or cross-tolerance, to these types of environments.

Evolutionary adaptation to temperature. VIII. Effects of temperature on growth rate in natural isolates of Escherichia coli and Salmonella enterica from different thermal environments.

Are enteric bacteria specifically adapted to the thermal environment of their hosts? In particular, do the optimal temperatures and thermal niches of the bacterial flora reflect seasonal, geographic, or phylogenetic differences in their hosts' temperatures? We examined these questions by measuring the relationship between the temperature-dependent growth rates of enteric bacteria in a free-living ectothermic host. We sampled two species of enteric bacteria (Escherichia coli and Salmonella enterica) from three natural populations of slider turtles (Trachemys scripta elegans) seasonally over two years. Despite pronounced differences in turtle body temperatures at different seasons and in different locations, we found no evidence that the thermal growth profiles of these bacteria mirrored this variation. Optimal temperatures and maximal growth rates in rich medium were nearly the same for both bacterial species (35-36 degrees C, 2.5 doublings per hour). The thermal niche (defined as the range of temperatures over which 75% of maximal growth rate occurred) was slightly higher for E. coli (28.5-41.0 degrees C) than for S. enterica (27.7-39.8 degrees C), but the niche breadth was about the same for both. We also measured the thermal dependence of growth rate in these same bacterial species isolated from mammalian hosts. Both bacterial species had temperatures of maximal growth and thermal niches that were about 2 degrees C higher than those of their respective conspecifics sampled from turtles; niche breadths were not different. These data suggest that these bacterial species are thermal generalists that do not track fine-scale changes in their thermal environments. Even major differences in body temperatures, as great as those between ectothermic and endothermic hosts, may result in the evolution of rather modest changes in thermal properties.

Evolution of thermal dependence of growth rate of Escherichia coli populations during 20,000 generations in a constant environment.

Twelve experimental populations of the bacterium Escherichia coli evolved for 20,000 generations in a defined medium at 37 degrees C. We measured their maximum growth rates across a broad range of temperatures and at several evolutionary time points to quantify the extent to which they became thermal specialists with diminished performance at other temperatures. We also sought to determine whether antagonistic pleiotropy (genetic trade-offs) or mutation accumulation (drift decay) was primarily responsible for any thermal specialization. Populations showed consistent improvement in growth rate at moderate temperatures (27-39 degrees C), but tended to have decreased growth rate at both low (20 degrees C) and high (41-42 degrees C) temperatures. Most loss occurred early in the experiment, when adaptation was most rapid. This dynamic is predicted by antagonistic pleiotropy but not by mutation accumulation. Several populations evolved high mutation rates due to defects in their DNA repair, but they did not subsequently undergo a greater decrease in growth rate at thermal extremes than populations that retained low mutation rates, contrary to the acceleration of decay predicted by mutation accumulation. Antagonistic pleiotropy therefore is more likely to be responsible for the evolution of thermal specialization observed in maximum growth rate.

The curse of the pharaoh: the evolution of virulence in pathogens with long living propagules.

We used a mathematical model to evaluate the hypothesis that parasites and pathogens with long living propagules should evolve high levels of virulence, i.e. high rates of pathogen-induced host mortality. Our model shows that the optimal level of virulence is independent of the longevity of the propagules either: (i) if the density or the prevalence of infected hosts is in (or fluctuates around) equilibrium; or (ii) if the death rate of the infected host population is high compared with that of the propagules. The level of virulence that maximizes the parasite's fitness (Multhusian parameter) increases with increasing longevity of its propagules only if the host-parasite system has not reached equilibrium and the death rate of the propagules is high relative to that of the infected hosts. Therefore, for parasites that have recently invaded a susceptible host population, greater propagule longevity may initially favour higher virulence; but once the equilibrium is reached the optimal virulence is independent of propagule longevity.

Pervasive compensatory adaptation in Escherichia coli.

To investigate compensatory adaptation (CA), we used genotypes of Escherichia coli which were identical except for one or two deleterious mutations. We compared CA for (i) deleterious mutations with large versus small effects, (ii) genotypes carrying one versus two mutations, and (iii) pairs of deleterious mutations which interact in a multiplicative versus synergistic fashion. In all, we studied 14 different genotypes, plus a control strain which was not mutated. Most genotypes showed CA during 200 generations of experimental evolution, where we define CA as a fitness increase which is disproportionately large relative to that in evolving control lines, coupled with retention of the original deleterious mutation(s). We observed greater CA for mutations of large effect than for those of small effect, which can be explained by the greater benefit to recovery in severely handicapped genotypes given the dynamics of selection. The rates of CA were similar for double and single mutants whose initial fitnesses were approximately equal. CA was faster for synergistic than for multiplicative pairs, presumably because the marginal gain which results from CA for one of the component mutations is greater in that case. The most surprising result in our view, is that compensation should be so readily achieved in an organism which is haploid and has little genetic redundancy This finding suggests a degree of versatility in the E. coil genome which demands further study from both genetic and physiological perspectives.

Phenotypic and evolutionary adaptation of a model bacterial system to stressful thermal environments.

We studied both phenotypic and evolutionary adaptation to various thermal environments using the bacterium Escherichia coli as an experimental model system. We determined that 42 degrees C was stressful to a bacterial clone adapted to 37 degrees C, based on reductions in both absolute and competitive fitness, as well as induction of a heat stress response. This clone was also used to found replicated populations that were propagated for thousands of generations under several different thermal regimes, including 42 degrees C. Evolutionary adaptation of the populations to 42 degrees C resulted in an increase in both absolute and relative fitness at that temperature, measured respectively as an increase in the number of descendants (and their biovolume) and in competitive ability relative to the ancestral clone. The replicated experimental lineages achieved their evolutionary improvement by several distinct pathways, which produced differential preadaptation to a non-stressful nutrient environment. Adaptation to this stressful temperature entailed neither a change in the ancestral thermal niche nor any pronounced trade-offs in fitness within the thermal niche, contrary to a priori predictions. This study system was several important advantages for evaluating hypotheses concerning the effects of stress on phenotypic and evolutionary adaptation, including the ability to obtain lineages that have evolved in controlled and defined environments, to make direct measurements of fitness and to quantify the degree of stress imposed by different environments.