Determining the Effects of a Spatially Heterogeneous Selection Pressure on Bacterial Population Structure at the Sub-millimetre Scale

A key interest of microbial ecology is to understand the role of environmental heterogeneity in shaping bacterial diversity and fitness. However, quantifying relevant selection pressures and their effects is challenging due to the number of parameters that must be considered and the multiple scales over which they act. In the current study, a model system was employed to investigate the effects of a spatially heterogeneous mercuric ion (Hg²⁺) selection pressure on a population comprising Hg-sensitive and Hg-resistant pseudomonads. The Hg-sensitive bacteria were Pseudomonas fluorescens SBW25::rfp and Hg-resistant bacteria were P. fluorescens SBW25 carrying a gfp-labelled, Hg resistance plasmid. In the absence of Hg, the plasmid confers a considerable fitness cost on the host, with µ_max for plasmid-carrying cells relative to plasmid-free cells of only 0.66. Two image analysis techniques were developed to investigate the structure that developed in biofilms about foci of Hg (cellulose fibres imbued with HgCl₂). Both techniques indicated selection for the resistant phenotype occurred only in small areas of approximately 178–353 μm (manually defined contour region analysis) or 275–350 μm (daime analysis) from foci. Hg also elicited toxic effects that reduced the growth of both Hg-sensitive and Hg-resistant bacteria up to 250 μm from foci. Selection for the Hg resistance phenotype was therefore highly localised when Hg was spatially heterogeneous. As such, for this model system, we define here the spatial scale over which selection operates. The ability to quantify changes in the strength of selection for particular phenotypes over sub-millimetre scales is useful for understanding the scale over which environmental variables affect bacterial populations.     

Evolution of unconditional dispersal in periodic environments

Organisms modulate their fitness in heterogeneous environments by dispersing. Prior work shows that there is selection against 'unconditional' dispersal in spatially heterogeneous environments. 'Unconditional' means individuals disperse at a rate independent of their location. We prove that if within-patch fitness varies spatially and between two values temporally, then there is selection for unconditional dispersal: any evolutionarily stable strategy (ESS) or evolutionarily stable coalition (ESC) includes a dispersive phenotype. Moreover, at this ESS or ESC, there is at least one sink patch (i.e. geometric mean of fitness less than one) and no sources patches (i.e. geometric mean of fitness greater than one). These results coupled with simulations suggest that spatial-temporal heterogeneity is due to abiotic forcing result in either an ESS with a dispersive phenotype or an ESC with sedentary and dispersive phenotypes. In contrast, the spatial-temporal heterogeneity due to biotic interactions can select for higher dispersal rates that ultimately spatially synchronize population dynamics.     

Antagonistic coevolution across productivity gradients: an experimental test of the effects of dispersal

Coevolution commonly occurs in spatially heterogeneous environments, resulting in variable selection pressures acting on coevolving species. Dispersal across such environments is predicted to have a major impact on local coevolutionary dynamics. Here, we address how co‐dispersal of coevolving populations of host and parasite across an environmental productivity gradient affected coevolution in experimental populations of bacteria and their parasitic viruses (phages). The rate of coevolution between bacteria and phages was greater in high‐productivity environments. High‐productivity immigrants (～2% of the recipient population) caused coevolutionary dynamics (rates of coevolution and degree of generalist evolution) in low‐productivity environments to be largely indistinguishable from high‐productivity environments, whereas immigration from low‐productivity environments (～0.5% of the population) had no discernable impact. These results could not be explained by demography alone, but rather high‐productivity immigrants had a selective advantage in low‐productivity environments, but not vice versa. Coevolutionary interactions in high‐productivity environments are therefore likely to have a disproportionate impact on coevolution across the landscape as a whole.     

Emergence of long‐term balanced polymorphism under cyclic selection of spatially variable magnitude

A fundamental question in evolutionary biology is what promotes genetic variation at nonneutral loci, a major precursor to adaptation in changing environments. In particular, balanced polymorphism under realistic evolutionary models of temporally varying environments in finite natural populations remains to be demonstrated. Here, we propose a novel mechanism of balancing selection under temporally varying fitnesses. Using forward‐in‐time computer simulations and mathematical analysis, we show that cyclic selection that spatially varies in magnitude, such as along an environmental gradient, can lead to elevated levels of nonneutral genetic polymorphism in finite populations. Balanced polymorphism is more likely with an increase in gene flow, magnitude and period of fitness oscillations, and spatial heterogeneity. This polymorphism‐promoting effect is robust to small systematic fitness differences between competing alleles or to random environmental perturbation. Furthermore, we demonstrate analytically that protected polymorphism arises as spatially heterogeneous cyclic fitness oscillations generate a type of storage effect that leads to negative frequency dependent selection. Our findings imply that spatially variable cyclic environments can promote elevated levels of nonneutral genetic variation in natural populations.     

TEMPORAL VARIATION FAVORS THE EVOLUTION OF GENERALISTS IN EXPERIMENTAL POPULATIONS OF DROSOPHILA MELANOGASTER

In variable environments, selection should favor generalists that maintain fitness across a range of conditions. However, costs of adaptation may generate fitness trade‐offs and lead to some compromise between specialization and generalization that maximizes fitness. Here, we evaluate the evolution of specialization and generalization in 20 populations of Drosophila melanogaster experimentally evolved in constant and variable thermal environments for 3 years. We developed genotypes from each population at two temperatures after which we measured fecundity across eight temperatures. We predicted that constant environments would select for thermal specialists and that variable environments would select for thermal generalists. Contrary to our predictions, specialists and generalists did not evolve in constant and spatially variable environments, respectively. However, temporal variation produced a type of generalist that has rarely been considered by theoretical models of developmental plasticity. Specifically, genotypes from the temporally variable selective environment were more fecund across all temperatures than were genotypes from other environments. These patterns suggest certain allelic effects and should inspire new directions for modeling adaptation to fluctuating environments.     

Variation in natural selection for growth and phlorotannins in the brown alga Fucus vesiculosus

Directional selection for plant traits associated with resistance to herbivory tends to eliminate genetic variation in such traits. On the other hand, balancing selection arising from trade-offs between resistance and growth or spatially variable selection acts against the elimination of genetic variation. We explore both the amount of genetic variation and variability of natural selection for growth and concentration of phenolic secondary compounds, phlorotannins, in the brown alga Fucus vesiculosus. We measured variation in selection at two growing depths and two levels of nutrient availability in algae that had faced two kinds of past growing environments. Genetic variation was low for growth but high for phlorotannins. The form and strength of selection for both focal traits depended on the past growing environment of the algae. We found strong directional selection for growth rate in algae previously subjected to higher ultraviolet radiation, but not in algae previously subjected to higher nutrient availability. Stabilizing selection for growth occurred especially in the deep growing environment. Selection for phlorotannins was generally weak, but in some past-environment-current-environment combinations we detected either directional selection against phlorotannins or stabilizing selection. Thus, phlorotannins are not selectively neutral but affect the fitness of F. vesiculosus. In particular, there may be a fitness cost of producing phlorotannins, but the realization of such a cost varies from one environment to another. Genetic correlations between selective environments were high for growth but nonexistent for phlorotannins, emphasizing the high phenotypic plasticity of phlorotannin production. The highly heterogeneous selection, including directional, stabilizing, and spatially variable selection as well as temporal change in selection due to responses to past environmental conditions, probably maintains a high amount of genetic variation in phlorotannins. Such variation provides the potential for rapid evolutionary response of phlorotannins under directional selection.     

Legacy of prior host and soil selection on rhizobial fitness in planta

Measuring selection acting on microbial populations in natural or even seminatural environments is challenging because many microbial populations experience variable selection. The majority of rhizobial bacteria are found in the soil. However, they also live symbiotically inside nodules of legume hosts and each nodule can release thousands of daughter cells back into the soil. We tested how past selection (i.e., legacies) by two plant genotypes and by the soil alone affected selection and genetic diversity within a population of 101 strains of Ensifer meliloti. We also identified allelic variants most strongly associated with soil‐ and host‐dependent fitness. In addition to imposing direct selection on rhizobia populations, soil and host environments had lasting effects across host generations. Host presence and genotype during the legacy period explained 22% and 12% of the variance in the strain composition of nodule communities in the second cohort, respectively. Although strains with high host fitness in the legacy cohort tended to be enriched in the second cohort, the diversity of the strain community was greater when the second cohort was preceded by host rather than soil legacies. Our results indicate the potential importance of soil selection driving the evolution of these plant‐associated microbes.     

The population biology of bacterial plasmids: a hidden Markov model approach

Horizontal plasmid transfer plays a key role in bacterial adaptation. In harsh environments, bacterial populations adapt by sampling genetic material from a horizontal gene pool through self-transmissible plasmids, and that allows persistence of these mobile genetic elements. In the absence of selection for plasmid-encoded traits it is not well understood if and how plasmids persist in bacterial communities. Here we present three models of the dynamics of plasmid persistence in the absence of selection. The models consider plasmid loss (segregation), plasmid cost, conjugative plasmid transfer, and observation error. Also, we present a stochastic model in which the relative fitness of the plasmid-free cells was modeled as a random variable affected by an environmental process using a hidden Markov model (HMM). Extensive simulations showed that the estimates from the proposed model are nearly unbiased. Likelihood-ratio tests showed that the dynamics of plasmid persistence are strongly dependent on the host type. Accounting for stochasticity was necessary to explain four of seven time-series data sets, thus confirming that plasmid persistence needs to be understood as a stochastic process. This work can be viewed as a conceptual starting point under which new plasmid persistence hypotheses can be tested.     

Evolution in alternating environments with tunable interlandscape correlations

Natural populations are often exposed to temporally varying environments. Evolutionary dynamics in varying environments have been extensively studied, although understanding the effects of varying selection pressures remains challenging. Here, we investigate how cycling between a pair of statistically related fitness landscapes affects the evolved fitness of an asexually reproducing population. We construct pairs of fitness landscapes that share global fitness features but are correlated with one another in a tunable way, resulting in landscape pairs with specific correlations. We find that switching between these landscape pairs, depending on the ruggedness of the landscape and the interlandscape correlation, can either increase or decrease steady‐state fitness relative to evolution in single environments. In addition, we show that switching between rugged landscapes often selects for increased fitness in both landscapes, even in situations where the landscapes themselves are anticorrelated. We demonstrate that positively correlated landscapes often possess a shared maximum in both landscapes that allows the population to step through sub‐optimal local fitness maxima that often trap single landscape evolution trajectories. Finally, we demonstrate that switching between anticorrelated paired landscapes leads to ergodic‐like dynamics where each genotype is populated with nonzero probability, dramatically lowering the steady‐state fitness in comparison to single landscape evolution.     

Genotype‐by‐environment interactions due to antibiotic resistance and adaptation in Escherichia coli

Mutations that are beneficial in one environment can have different fitness effects in other environments. In the context of antibiotic resistance, the resulting genotype‐by‐environment interactions potentially make selection on resistance unpredictable in heterogeneous environments. Furthermore, resistant bacteria frequently fix additional mutations during evolution in the absence of antibiotics. How do these two types of mutations interact to determine the bacterial phenotype across different environments? To address this, I used Escherichia coli as a model system, measuring the effects of nine different rifampicin resistance mutations on bacterial growth in 31 antibiotic‐free environments. I did this both before and after approximately 200 generations of experimental evolution in antibiotic‐free conditions (LB medium), and did the same for the antibiotic‐sensitive wild type after adaptation to the same environment. The following results were observed: (i) bacteria with and without costly resistance mutations adapted to experimental conditions and reached similar levels of competitive fitness; (ii) rifampicin resistance mutations and adaptation to LB both indirectly altered growth in other environments; and (iii) resistant‐evolved genotypes were more phenotypically different from the ancestor and from each other than resistant‐nonevolved and sensitive‐evolved genotypes. This suggests genotype‐by‐environment interactions generated by antibiotic resistance mutations, observed previously in short‐term experiments, are more pronounced after adaptation to other types of environmental variation, making it difficult to predict long‐term selection on resistance mutations from fitness effects in a single environment.     

ON THE EVOLUTION OF PHENOTYPIC PLASTICITY IN A SPATIALLY HETEROGENEOUS ENVIRONMENT

A genetic model for the dynamics of a quantitative trait is analyzed in terms of gene frequencies, linkage disequilibria, and environmental effects on the trait. In a randomly mating population, at each generation progeny move to niches where they are subject to weak Gaussian selection on the trait, with different fitness levels in the different niches. Initially, the variability of the trait is due to additive loci with heterozygous homeostasis. The evolution of plasticity is then described in terms of the invasion of the population by genetic modifiers that may epistatically affect the trait, its optimum in each niche, the strengths of selection, and other parameters characteristic of the niches. We show that the evolution of trait means within niches depends on the overall evolution in the whole system, and in general, optimum phenotypic values are not attained. The reaction norm and genotype-environment interaction may evolve even if the only effects of the modifier are on individual rates of dispersal, or on fitness effects resulting from the different environments in the different niches; this evolution does not require that the modifier affect parameters that influence the values of the trait. It is conjectured that in the least frequently reached niches with low fitness levels, the deviations from the trait optima should be larger than those in more commonly experienced and less stringent niches. Our analysis makes explicit the different contribution of between- and within-niche effects on the evolutionary dynamics of phenotypic plasticity in heterogeneous environments.     

Fitness effects of mutations in bacteria

Mutation is the primary source of variation in any organism. Without it, natural selection cannot operate and organisms cannot adapt to novel environments. Mutation is also generally a source of defect: many mutations are not neutral but cause fitness decreases in the organisms where they arise. In bacteria, another important source of variation is horizontal gene transfer. This source of variation can also cause beneficial or deleterious effects. Determining the distribution of fitness effects of mutations in different environments and genetic backgrounds is an active research field. In bacteria, knowledge of these distributions is key for understanding important traits. For example, for determining the dynamics of microorganisms with a high genomic mutation rate (mutators), and for understanding the evolution of antibiotic resistance, and the emergence of pathogenic traits. All of these characteristics are extremely relevant for human health both at the individual and population levels. Experimental evolution has been a valuable tool to address these questions. Here, we review some of the important findings of mutation effects in bacteria revealed through laboratory experiments.     

A General Model to Account for Enzyme Variation in Natural Populations

Approximate conditions for genetic polymorphism in temporally and spatially varying environments are presented for loci which are intermediate at the level of fitness or at the level of gene function. The conditions suggest that polymorphism will be more likely in more variable environments while unlikely in constant environments. Biochemical evidence is presented to justify the assumption of heterozygote intermediacy. Observations on natural populations are cited which substantiate the claim that allozymic polymorphism is primarily due to selection acting on environmental variation in gene function.     

The role of migration in the evolution of phenotypic switching

Stochastic switching is an example of phenotypic bet hedging, where an individual can switch between different phenotypic states in a fluctuating environment. Although the evolution of stochastic switching has been studied when the environment varies temporally, there has been little theoretical work on the evolution of phenotypic switching under both spatially and temporally fluctuating selection pressures. Here, we explore the interaction of temporal and spatial change in determining the evolutionary dynamics of phenotypic switching. We find that spatial variation in selection is important; when selection pressures are similar across space, migration can decrease the rate of switching, but when selection pressures differ spatially, increasing migration between demes can facilitate the evolution of higher rates of switching. These results may help explain the diverse array of non-genetic contributions to phenotypic variability and phenotypic inheritance observed in both wild and experimental populations.     

Selection history and epistatic interactions impact dynamics of adaptation to novel environmental stresses

In rapidly changing environments, selection history may impact the dynamics of adaptation. Mutations selected in one environment may result in pleiotropic fitness trade-offs in subsequent novel environments, slowing the rates of adaptation. Epistatic interactions between mutations selected in sequential stressful environments may slow or accelerate subsequent rates of adaptation, depending on the nature of that interaction. We explored the dynamics of adaptation during sequential exposure to herbicides with different modes of action in Chlamydomonas reinhardtii. Evolution of resistance to two of the herbicides was largely independent of selection history. For carbetamide, previous adaptation to other herbicide modes of action positively impacted the likelihood of adaptation to this herbicide. Furthermore, while adaptation to all individual herbicides was associated with pleiotropic fitness costs in stress-free environments, we observed that accumulation of resistance mechanisms was accompanied by a reduction in overall fitness costs. We suggest that antagonistic epistasis may be a driving mechanism that enables populations to more readily adapt in novel environments. These findings highlight the potential for sequences of xenobiotics to facilitate the rapid evolution of multiple-drug and -pesticide resistance, as well as the potential for epistatic interactions between adaptive mutations to facilitate evolutionary rescue in rapidly changing environments.     

Dispersal: a central and independent trait in life history

The study of tradeoffs among major life history components (age at maturity, lifespan and reproduction) allowed the development of a quantitative framework to understand how environmental variation shapes patterns of biodiversity among and within species. Because every environment is inherently spatially structured, and in most cases temporally variable, individuals need to move within and among habitats to maximize fitness. Dispersal is often assumed to be tightly integrated into life histories through genetic correlations with other vital traits. This assumption is particularly strong within the context of a fast‐slow continuum of life‐history variation. Such a framework is to date used to explain many aspects of population and community dynamics. Evidence for a consistent and context‐independent integration of dispersal in life histories is, however, weak. We therefore advocate the explicit integration of dispersal into life history theory as a principal axis of variation influencing fitness, that is free to evolve, independently of other life history traits. We synthesize theoretical and empirical evidence on the central role of dispersal and its evolutionary dynamics on the spatial distribution of ecological strategies and its impact on population spread, invasions and coexistence. By applying an optimality framework we show that the inclusion of dispersal as an independent dimension of life histories might substantially change our view on evolutionary trajectories in spatially structured environments. Because changes in the spatial configuration of habitats affect the costs of movement and dispersal, adaptations to reduce these costs will increase phenotypic divergence among and within populations. We outline how this phenotypic heterogeneity is anticipated to further impact population and community dynamics.     

Age-specific fitness components and their temporal variation in the barn owl

Theory predicts that temporal variability plays an important role in the evolution of life histories, but empirical studies evaluating this prediction are rare. In constant environments, fitness can be measured by the population growth rate lambda, and the sensitivity of lambda to changes in fitness components estimates selection on these traits. In variable environments, fitness is measured by the stochastic growth rate lambda(S), and stochastic sensitivities estimate selection pressure. Here we examine age-specific schedules for reproduction and survival in a barn owl population (Tyto alba). We estimated how temporal variability affected fitness and selection, accounting for sampling variance. Despite large sample sizes of old individuals, we found no strong evidence for senescence. The most variable fitness components were associated with reproduction. Survival was less variable. Stochastic simulations showed that the observed variation decreased fitness by about 30%, but the sensitivities of lambda and lambda(S) to changes in all fitness components were almost equal, suggesting that temporal variation had negligible effects on selection. We obtained these results despite high observed variability in the fitness components and relatively short generation time of the study organism, a situation in which temporal variability should be particularly important for natural selection and early senescence is expected.     

Quantification and decomposition of environment‐selection relationships

In nature, selection varies across time in most environments, but we lack an understanding of how specific ecological changes drive this variation. Ecological factors can alter phenotypic selection coefficients through changes in trait distributions or individual mean fitness, even when the trait‐absolute fitness relationship remains constant. We apply and extend a regression‐based approach in a population of Soay sheep (Ovis aries) and suggest metrics of environment‐selection relationships that can be compared across studies. We then introduce a novel method that constructs an environmentally structured fitness function. This allows calculation of full (as in existing approaches) and partial (acting separately through the absolute fitness function slope, mean fitness, and phenotype distribution) sensitivities of selection to an ecological variable. Both approaches show positive overall effects of density on viability selection of lamb mass. However, the second approach demonstrates that this relationship is largely driven by effects of density on mean fitness, rather than on the trait‐fitness relationship slope. If such mechanisms of environmental dependence of selection are common, this could have important implications regarding the frequency of fluctuating selection, and how previous selection inferences relate to longer term evolutionary dynamics.     

Bacterial adaptation to sublethal antibiotic gradients can change the ecological properties of multitrophic microbial communities

Antibiotics leak constantly into environments due to widespread use in agriculture and human therapy. Although sublethal concentrations are well known to select for antibiotic-resistant bacteria, little is known about how bacterial evolution cascades through food webs, having indirect effect on species not directly affected by antibiotics (e.g. via population dynamics or pleiotropic effects). Here, we used an experimental evolution approach to test how temporal patterns of antibiotic stress, as well as migration within metapopulations, affect the evolution and ecology of microcosms containing one prey bacterium, one phage and two protist predators. We found that environmental variability, autocorrelation and migration had only subtle effects for population and evolutionary dynamics. However, unexpectedly, bacteria evolved greatest fitness increases to both antibiotics and enemies when the sublethal levels of antibiotics were highest, indicating positive pleiotropy. Crucially, bacterial adaptation cascaded through the food web leading to reduced predator-to-prey abundance ratio, lowered predator community diversity and increased instability of populations. Our results show that the presence of natural enemies can modify and even reverse the effects of antibiotics on bacteria, and that antibiotic selection can change the ecological properties of multitrophic microbial communities by having indirect effects on species not directly affected by antibiotics.