The Red Queen hypothesis proposes that coevolution of interacting species (such as hosts and parasites) should drive molecular evolution through continual natural selection for adaptation and counter-adaptation 1-3 . Although the divergence observed at some host-resistance 4-6 and parasiteinfectivity 7-9 genes is consistent with this, the long time periods typically required to study coevolution have so far prevented any direct empirical test. Here we show, using experimental populations of the bacterium Pseudomonas fluorescens SBW25 and its viral parasite, phage Φ2 (refs 10, 11), that the rate of molecular evolution in the phage was far higher when both bacterium and phage coevolved with each other than when phage evolved against a constant host genotype. Coevolution also resulted in far greater genetic divergence between replicate populations, which was correlated with the range of hosts that coevolved phage were able to infect. Consistent with this, the most rapidly evolving phage genes under coevolution were those involved in host infection. These results demonstrate, at both the genomic and phenotypic level, that antagonistic coevolution is a cause of rapid and divergent evolution, and is likely to be a major driver of evolutionary change within species.

The evolution of antibiotic resistance carries a fitness cost, expressed in terms of reduced competitive ability in the absence of antibiotics. This cost plays a key role in the dynamics of resistance by generating selection against resistance when bacteria encounter an antibiotic-free environment. Previous work has shown that the cost of resistance is highly variable, but the underlying causes remain poorly understood. Here, we use a meta-analysis of the published resistance literature to determine how the genetic basis of resistance influences its cost. We find that on average chromosomal resistance mutations carry a larger cost than acquiring resistance via a plasmid. This may explain why resistance often evolves by plasmid acquisition. Second, we find that the cost of plasmid acquisition increases with the breadth of its resistance range. This suggests a potentially important limit on the evolution of extensive multidrug resistance via plasmids. We also find that epistasis can significantly alter the cost of mutational resistance. Overall, our study shows that the cost of antimicrobial resistance can be partially explained by its genetic basis. It also highlights both the danger associated with plasmidborne resistance and the need to understand why resistance plasmids carry a relatively low cost.

SummaryThe widespread evolution of resistance to herbicides is a pressing issue in global agriculture. Evolutionary principles and practices are key to the management of this threat to global food security. The application of mixtures of herbicides has been advocated as an anti-resistance strategy, without substantial empirical support for its validation.We evolved experimentally populations of the unicellular green chlorophyte, Chlamydomonas reinhardtii, to minimum inhibitory concentrations (MICs) of single-herbicide modes of action and to pair-wise and three-way mixtures between different herbicides at various total combined doses.Herbicide mixtures were most effective when each component was applied at or close to its MIC. When doses were high, increasing the number of mixture components was also effective in reducing the evolution of resistance. Employing mixtures at low combined doses did not retard resistance evolution, even accelerating the evolution of resistance to some components. At low doses, increasing the number of herbicides in the mixture tended to select for more generalist resistance (cross-resistance).Our results reinforce findings from the antibiotic resistance literature and confirm that herbicide mixtures can be very effective for resistance management, but that mixtures should only be employed where the economic and environmental context permits the applications of high combined doses.

Antibiotic resistance often evolves by mutations at conserved sites in essential genes, resulting in parallel molecular evolution between divergent bacterial strains and species. Whether these resistance mutations are having parallel effects on fitness across bacterial taxa, however, is unclear. This is an important point to address, because the fitness effects of resistance mutations play a key role in the spread and maintenance of resistance in pathogen populations. We address this idea by measuring the fitness effect of a collection of rifampicin resistance mutations in the β subunit of RNA polymerase (rpoB) across eight strains that span the diversity of the genus Pseudomonas. We find that almost 50% of rpoB mutations have background-dependent fitness costs, demonstrating that epistatic interactions between rpoB and the rest of the genome are common. Moreover, epistasis is typically strong, and it is the dominant genetic determinant of the cost of resistance mutations. To investigate the functional basis of epistasis, and because rpoB plays a central role in transcription, we measured the effects of common rpoB mutations on transcriptional efficiency across three strains of Pseudomonas. Transcriptional efficiency correlates strongly to fitness across strains, and epistasis arises because individual rpoB mutations have differential effects on transcriptional efficiency in different genetic backgrounds.

Parallel evolution is the independent evolution of the same phenotype or genotype in response to the same selection pressure. There are examples of parallel molecular evolution across divergent genetic backgrounds, suggesting that genetic background may not play an important role in determining the outcome of adaptation. Here, we measure the influence of genetic background on phenotypic and molecular adaptation by combining experimental evolution with comparative analysis. We selected for resistance to the antibiotic rifampicin in eight strains of bacteria from the genus Pseudomonas using a short term selection experiment. Adaptation occurred by 47 mutations at conserved sites in rpoB, the target of rifampicin, and due to the high diversity of possible mutations the probability of within-strain parallel evolution was low. The probability of between-strain parallel evolution was only marginally lower, because different strains substituted similar rpoB mutations. In contrast, we found that more than 30% of the phenotypic variation in the growth rate of evolved clones was attributable to among-strain differences. Parallel molecular evolution across strains resulted in divergent phenotypic evolution because rpoB mutations had different effects on growth rate in different strains. This study shows that genetic divergence between strains constrains parallel phenotypic evolution, but had little detectable impact on the molecular basis of adaptation in this system.

Spatial synchrony is widespread in natural populations but the mechanisms that underpin it are not yet fully understood. Two key biotic drivers of spatial synchrony have been identified: dispersal and trophic interactions (e.g. natural enemies). We used spatially structured, patchy bacterial populations to show that although increased dispersal always enhanced spatial synchrony of fluctuations in bacterial abundance, this effect was far stronger in the presence of a bacteriophage parasite. Bacteriophages drove strong within patch fluctuations in bacterial abundance that became phase locked through dispersal. Furthermore, the way in which stability, measured as constancy, responded to increasing dispersal was qualitatively different depending on whether parasites were present or not. Patch-level constancy decreased with dispersal in the presence of parasites, whereas dispersal increased patch-level constancy in the absence of parasites. Population-level constancy also decreased with dispersal in the presence of parasites, but was unaffected by dispersal in the absence of parasites. These contrasting patterns were likely due to the different role played by dispersal in the presence and absence of parasites, synchronizing dynamics in the former case and averaging stochastic fluctuations in the latter. Taken together, our findings suggest that dispersal and natural enemies can interact to drive spatially synchronous population fluctuations that decrease stability at both the patch and population level.

Introductory paragraphThere is an urgent need to develop novel approaches for predicting and preventing the evolution of antibiotic resistance. Here we show that the ability to evolve de novo resistance to a clinically important β-lactam antibiotic, ceftazidime, varies drastically across the genus Pseudomonas. This variation arises because strains possessing the ampR global transcriptional regulator evolve resistance at a high rate. This does not arise because of mutations in ampR. Instead, this regulator potentiates evolution by allowing mutations in conserved peptidoglycan biosynthesis genes to induce high levels of β-lactamase expression. Crucially, blocking this evolutionary pathway by co-administering ceftazidime with the β-lactamase inhibitor avibactam can be used to eliminate pathogenic P. aeruginosa populations before they can evolve resistance. In summary, our study shows that identifying potentiator genes that act as evolutionary catalysts can be used to both predict and prevent the evolution of antibiotic resistance.

There is growing evidence that parallel molecular evolution is common, but its causes remain poorly understood. Demographic parameters such as population bottlenecks are predicted to be major determinants of parallelism. Here, we test the hypothesis that bottleneck intensity shapes parallel evolution by elucidating the genomic basis of adaptation to antibiotic-supplemented media in hundreds of populations of the bacterium Pseudomonas fluorescens Pf0-1. As expected, bottlenecking decreased the rate of phenotypic and molecular adaptation. Surprisingly, bottlenecking had no impact on the likelihood of parallel adaptive molecular evolution at a genome-wide scale. However, bottlenecking had a profound impact on the genes involved in antibiotic resistance. Specifically, under either intense or weak bottlenecking, resistance predominantly evolved by strongly beneficial mutations which provide high levels of antibiotic resistance. In contrast with intermediate bottlenecking regimes, resistance evolved by a greater diversity of genetic mechanisms, significantly reducing the observed levels of parallel genetic evolution. Our results demonstrate that population bottlenecking can be a major predictor of parallel evolution, but precisely how may be more complex than many simple theoretical predictions.