Magnitude and sign epistasis among deleterious mutations in a positive-sense plant RNA virus

How epistatic interactions between mutations determine the genetic architecture of fitness is of central importance in evolution. The study of epistasis is particularly interesting for RNA viruses because of their genomic compactness, lack of genetic redundancy, and apparent low complexity. Moreover, interactions between mutations in viral genomes determine traits such as resistance to antiviral drugs, virulence and host range. In this study we generated 53 Tobacco etch potyvirus genotypes carrying pairs of single-nucleotide substitutions and measured their separated and combined deleterious fitness effects. We found that up to 38% of pairs had significant epistasis for fitness, including both positive and negative deviations from the null hypothesis of multiplicative effects. Interestingly, the sign of epistasis was correlated with viral protein-protein interactions in a model network, being predominantly positive between linked pairs of proteins and negative between unlinked ones. Furthermore, 55% of significant interactions were cases of reciprocal sign epistasis (RSE), indicating that adaptive landscapes for RNA viruses maybe highly rugged. Finally, we found that the magnitude of epistasis correlated negatively with the average effect of mutations. Overall, our results are in good agreement to those previously reported for other viruses and further consolidate the view that positive epistasis is the norm for small and compact genomes that lack genetic robustness.     

Perspective: Sign epistasis and genetic constraint on evolutionary trajectories

Epistasis for fitness means that the selective effect of a mutation is conditional on the genetic background in which it appears. Although epistasis is widely observed in nature, our understanding of its consequences for evolution by natural selection remains incomplete. In particular, much attention focuses only on its influence on the instantaneous rate of changes in frequency of selected alleles via epistatic contribution to the additive genetic variance for fitness. Thus, in this framework epistasis only has evolutionary importance if the interacting loci are simultaneously segregating in the population. However, the selective accessibility of mutational trajectories to high fitness genotypes may depend on the genetic background in which novel mutations appear, and this effect is independent of population polymorphism at other loci. Here we explore this second influence of epistasis on evolution by natural selection. We show that it is the consequence of a particular form of epistasis, which we designate sign epistasis. Sign epistasis means that the sign of the fitness effect of a mutation is under epistatic control; thus, such a mutation is beneficial on some genetic backgrounds and deleterious on others. Recent experimental innovations in microbial systems now permit assessment of the fitness effects of individual mutations on multiple genetic backgrounds. We review this literature and identify many examples of sign epistasis, and we suggest that the implications of these results may generalize to other organisms. These theoretical and empirical considerations imply that strong genetic constraint on the selective accessibility of trajectories to high fitness genotypes may exist and suggest specific areas of investigation for future research.     

Reciprocal sign epistasis between frequently experimentally evolved adaptive mutations causes a rugged fitness landscape

The fitness landscape captures the relationship between genotype and evolutionary fitness and is a pervasive metaphor used to describe the possible evolutionary trajectories of adaptation. However, little is known about the actual shape of fitness landscapes, including whether valleys of low fitness create local fitness optima, acting as barriers to adaptive change. Here we provide evidence of a rugged molecular fitness landscape arising during an evolution experiment in an asexual population of Saccharomyces cerevisiae. We identify the mutations that arose during the evolution using whole-genome sequencing and use competitive fitness assays to describe the mutations individually responsible for adaptation. In addition, we find that a fitness valley between two adaptive mutations in the genes MTH1 and HXT6/HXT7 is caused by reciprocal sign epistasis, where the fitness cost of the double mutant prohibits the two mutations from being selected in the same genetic background. The constraint enforced by reciprocal sign epistasis causes the mutations to remain mutually exclusive during the experiment, even though adaptive mutations in these two genes occur several times in independent lineages during the experiment. Our results show that epistasis plays a key role during adaptation and that inter-genic interactions can act as barriers between adaptive solutions. These results also provide a new interpretation on the classic Dobzhansky-Muller model of reproductive isolation and display some surprising parallels with mutations in genes often associated with tumors.     

The impact of high‐order epistasis in the within‐host fitness of a positive‐sense plant RNA virus

RNA viruses are the main source of emerging infectious diseases because of the evolutionary potential bestowed by their fast replication, large population sizes and high mutation and recombination rates. However, an equally important property, which is usually neglected, is the topography of the fitness landscape. How many fitness maxima exist and how well they are connected is especially interesting, as this determines the number of accessible evolutionary pathways. To address this question, we have reconstructed a region of the fitness landscape of tobacco etch potyvirus constituted by mutations observed during the experimental adaptation of the virus to the novel host Arabidopsis thaliana. Fitness was measured for many genotypes and showed the existence of multiple peaks and holes in the landscape. We found prevailing epistatic effects between mutations, with cases of reciprocal sign epistasis being common among pairs of mutations. We also found that high‐order epistasis was as important as pairwise epistasis in their contribution to fitness. Therefore, results suggest that the landscape was rugged due to the existence of holes caused by lethal genotypes, that a very limited number of potential neutral paths exist and that it contained a single adaptive peak.     

Fitness landscapes emerging from pharmacodynamic functions in the evolution of multidrug resistance

Adaptive evolution often involves beneficial mutations at more than one locus. In this case, the trajectory and rate of adaptation is determined by the underlying fitness landscape, that is, the fitness values and mutational connectivity of all genotypes under consideration. Drug resistance, especially resistance to multiple drugs simultaneously, is also often conferred by mutations at several loci so that the concept of fitness landscapes becomes important. However, fitness landscapes underlying drug resistance are not static but dependent on drug concentrations, which means they are influenced by the pharmacodynamics of the drugs administered. Here, I present a mathematical framework for fitness landscapes of multidrug resistance based on Hill functions describing how drug concentrations affect fitness. I demonstrate that these ‘pharmacodynamic fitness landscapes’ are characterized by pervasive epistasis that arises through (i) fitness costs of resistance (even when these costs are additive), (ii) nonspecificity of resistance mutations to drugs, in particular cross‐resistance, and (iii) drug interactions (both synergistic and antagonistic). In the latter case, reciprocal drug suppression may even lead to reciprocal sign epistasis, so that the doubly resistant genotype occupies a local fitness peak that may be difficult to access by evolution. Simulations exploring the evolutionary dynamics on some pharmacodynamic fitness landscapes with both constant and changing drug concentrations confirm the crucial role of epistasis in determining the rate of multidrug resistance evolution.     

The rank ordering of genotypic fitness values predicts genetic constraint on natural selection on landscapes lacking sign epistasis

Sewall Wright's genotypic fitness landscape makes explicit one mechanism by which epistasis for fitness can constrain evolution by natural selection. Wright distinguished between landscapes possessing multiple fitness peaks and those with only a single peak and emphasized that the former class imposes substantially greater constraint on natural selection. Here I present novel formalism that more finely partitions the universe of possible fitness landscapes on the basis of the rank ordering of their genotypic fitness values. In this report I focus on fitness landscapes lacking sign epistasis (i.e., landscapes that lack mutations the sign of whose fitness effect varies epistatically), which constitute a subset of Wright's single peaked landscapes. More than one fitness rank ordering lacking sign epistasis exists for L > 2 (where L is the number of interacting loci), and I find that a highly statistically significant effect exists between landscape membership in fitness rank-ordering partition and two different proxies for genetic constraint, even within this subset of landscapes. This statistical association is robust to population size, permitting general inferences about some of the characteristics of fitness rank orderings responsible for genetic constraint on natural selection.     

Rapid evolutionary escape by large populations from local fitness peaks is likely in nature

Fitness interactions between loci in the genome, or epistasis, can result in mutations that are individually deleterious but jointly beneficial. Such epistasis gives rise to multiple peaks on the genotypic fitness landscape. The problem of evolutionary escape from such local peaks has been a central problem of evolutionary genetics for at least 75 years. Much attention has focused on models of small populations, in which the sequential fixation of valley genotypes carrying individually deleterious mutations operates most quickly owing to genetic drift. However, valley genotypes can also be subject to mutation while transiently segregating, giving rise to copies of the high fitness escape genotype carrying the jointly beneficial mutations. In the absence of genetic recombination, these mutations may then fix simultaneously. The time for this process declines sharply with increasing population size, and it eventually comes to dominate evolutionary behavior. Here we develop an analytic expression for N(crit), the critical population size that defines the boundary between these regimes, which shows that both are likely to operate in nature. Frequent recombination may disrupt high-fitness escape genotypes produced in populations larger than N(crit) before they reach fixation, defining a third regime whose rate again slows with increasing population size. We develop a novel expression for this critical recombination rate, which shows that in large populations the simultaneous fixation of mutations that are beneficial only jointly is unlikely to be disrupted by genetic recombination if their map distance is on the order of the size of single genes. Thus, counterintuitively, mass selection alone offers a biologically realistic resolution to the problem of evolutionary escape from local fitness peaks in natural populations.     

Initial mutations direct alternative pathways of protein evolution

Whether evolution is erratic due to random historical details, or is repeatedly directed along similar paths by certain constraints, remains unclear. Epistasis (i.e. non-additive interaction between mutations that affect fitness) is a mechanism that can contribute to both scenarios. Epistasis can constrain the type and order of selected mutations, but it can also make adaptive trajectories contingent upon the first random substitution. This effect is particularly strong under sign epistasis, when the sign of the fitness effects of a mutation depends on its genetic background. In the current study, we examine how epistatic interactions between mutations determine alternative evolutionary pathways, using in vitro evolution of the antibiotic resistance enzyme TEM-1 β-lactamase. First, we describe the diversity of adaptive pathways among replicate lines during evolution for resistance to a novel antibiotic (cefotaxime). Consistent with the prediction of epistatic constraints, most lines increased resistance by acquiring three mutations in a fixed order. However, a few lines deviated from this pattern. Next, to test whether negative interactions between alternative initial substitutions drive this divergence, alleles containing initial substitutions from the deviating lines were evolved under identical conditions. Indeed, these alternative initial substitutions consistently led to lower adaptive peaks, involving more and other substitutions than those observed in the common pathway. We found that a combination of decreased enzymatic activity and lower folding cooperativity underlies negative sign epistasis in the clash between key mutations in the common and deviating lines (Gly238Ser and Arg164Ser, respectively). Our results demonstrate that epistasis contributes to contingency in protein evolution by amplifying the selective consequences of random mutations.     

The causes of epistasis

Since Bateson's 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.     

Pervasive Pairwise Intragenic Epistasis among Sequential Mutations in TEM-1 β-Lactamase

Interactions between mutations play a central role in shaping the fitness landscape, but a clear picture of intragenic epistasis has yet to emerge. To further reveal the prevalence and patterns of intragenic epistasis, we present a survey of epistatic interactions between sequential mutations in TEM-1 β-lactamase. We measured the fitness effect of ~ 12,000 pairs of consecutive amino acid substitutions and used our previous study of the fitness effects of single amino acid substitutions to calculate epistasis for over 8000 mutation pairs. Since sequential mutations are prone to physically interact, we postulated that our study would be surveying specific epistasis instead of nonspecific epistasis. We found widespread negative epistasis, especially in beta-strands, and a high frequency of negative sign epistasis among individually beneficial mutations. Negative epistasis (52%) occurred 7.6 times as frequently as positive epistasis (6.8%). Buried residues experienced more negative epistasis that surface-exposed residues. However, TEM-1 exhibited a couple of hotspots for positive epistasis, most notably L221/ R222 at which many combinations of mutations positively interacted. This study is the first to systematically examine pairwise epistasis throughout an entire protein performing its native function in its native host.     

The causes of epistasis in genetic networks

Epistasis refers to the nonadditive interactions between genes in determining phenotypes. Considerable efforts have shown that, even for a given organism, epistasis may vary both in intensity and sign. Recent comparative studies supported that the overall sign of epistasis switches from positive to negative as the complexity of an organism increases, and it has been hypothesized that this change shall be a consequence of the underlying gene network properties. Why should this be the case? What characteristics of genetic networks determine the sign of epistasis? Here we show, by evolving genetic networks that differ in their complexity and robustness against perturbations but that perform the same tasks, that robustness increased with complexity and that epistasis was positive for small nonrobust networks but negative for large robust ones. Our results indicate that robustness and negative epistasis emerge as a consequence of the existence of redundant elements in regulatory structures of genetic networks and that the correlation between complexity and epistasis is a byproduct of such redundancy, allowing for the decoupling of epistasis from the underlying network complexity.     

A gene's eye view of epistasis,selection and speciation

In this mini‐review, I discuss the effects of gene interaction or epistasis from a `gene's eye view.' By a `gene's eye view' of epistasis, I mean that I will consider a single, bi‐allelic locus, A , whose effects on fitness result only from its interactions with alleles of another, unknown locus, X . I will show how changes in the frequencies of alleles at the background locus affect the relationship of alleles at the A ‐locus to fitness. Changing the genetic background changes the fundamental characteristics of the A ‐locus, such as the magnitude and sign of allelic effects on fitness, and, consequently, it changes the strength and pattern of selection. I consider each of the four kinds of pair–wise interactions between two loci and show that some kinds of epistasis are more sensitive than others to population genetic subdivision. Lastly, I show that some kinds of epistasis are more likely than others to affect the process of speciation and contribute to or be responsible for general genetic features of interspecific hybrids, such as Haldane's rule.     

Environment changes epistasis to alter trade‐offs along alternative evolutionary paths

The fitness effect of a mutation can depend on both its genetic background, known as epistasis, and the prevailing external environment. Many examples of these dependencies are known, but few studies consider both aspects in combination, especially as they affect mutations that have been selected together. We examine interactions between five coevolved mutations in eight diverse environments. We find that mutations are, on average, beneficial across environments, but that there is high variation in their fitness effects, including many examples of mutations conferring a cost in some, but not other, genetic background‐environment combinations. Indeed, even when global interaction trends are accounted for, specific local mutation interactions are common and differed across environments. One consequence of this dependence is that the range of trade‐offs in genotype fitness across selected and alternative environments are contingent on the particular evolutionary path followed over the mutation landscape. Finally, although specific interactions were common, there was a consistent pattern of diminishing returns epistasis whereby mutation effects were less beneficial when added to genotypes of higher fitness. Our results underline that specific mutation effects are highly dependent on the combination of genetic and external environments, and support a general relationship between a genotype's current fitness and its potential to increase in fitness.     

Positive Epistasis Drives the Acquisition of Multidrug Resistance

The evolution of multiple antibiotic resistance is an increasing global problem. Resistance mutations are known to impair fitness, and the evolution of resistance to multiple drugs depends both on their costs individually and on how they interact—epistasis. Information on the level of epistasis between antibiotic resistance mutations is of key importance to understanding epistasis amongst deleterious alleles, a key theoretical question, and to improving public health measures. Here we show that in an antibiotic-free environment the cost of multiple resistance is smaller than expected, a signature of pervasive positive epistasis among alleles that confer resistance to antibiotics. Competition assays reveal that the cost of resistance to a given antibiotic is dependent on the presence of resistance alleles for other antibiotics. Surprisingly we find that a significant fraction of resistant mutations can be beneficial in certain resistant genetic backgrounds, that some double resistances entail no measurable cost, and that some allelic combinations are hotspots for rapid compensation. These results provide additional insight as to why multi-resistant bacteria are so prevalent and reveal an extra layer of complexity on epistatic patterns previously unrecognized, since it is hidden in genome-wide studies of genetic interactions using gene knockouts.     

Mapping the Fitness Landscape of Gene Expression Uncovers the Cause of Antagonism and Sign Epistasis between Adaptive Mutations

How do adapting populations navigate the tensions between the costs of gene expression and the benefits of gene products to optimize the levels of many genes at once? Here we combined independently-arising beneficial mutations that altered enzyme levels in the central metabolism of Methylobacterium extorquens to uncover the fitness landscape defined by gene expression levels. We found strong antagonism and sign epistasis between these beneficial mutations. Mutations with the largest individual benefit interacted the most antagonistically with other mutations, a trend we also uncovered through analyses of datasets from other model systems. However, these beneficial mutations interacted multiplicatively (i.e., no epistasis) at the level of enzyme expression. By generating a model that predicts fitness from enzyme levels we could explain the observed sign epistasis as a result of overshooting the optimum defined by a balance between enzyme catalysis benefits and fitness costs. Knowledge of the phenotypic landscape also illuminated that, although the fitness peak was phenotypically far from the ancestral state, it was not genetically distant. Single beneficial mutations jumped straight toward the global optimum rather than being constrained to change the expression phenotypes in the correlated fashion expected by the genetic architecture. Given that adaptation in nature often results from optimizing gene expression, these conclusions can be widely applicable to other organisms and selective conditions. Poor interactions between individually beneficial alleles affecting gene expression may thus compromise the benefit of sex during adaptation and promote genetic differentiation.     

The genetic architecture of disease resistance in plants and the maintenance of recombination by parasites

Parasites represent strong selection on host populations because they are ubiquitous and can drastically reduce host fitness. It has been hypothesized that parasite selection could explain the widespread occurrence of recombination because it is a coevolving force that favours new genetic combinations in the host. A review of deterministic models for the maintenance of recombination reveals that for recombination to be favoured, multiple genes that interact with each other must be under selection. To evaluate whether parasite selection can explain the maintenance of recombination, we review 85 studies that investigated the genetic architecture of plant disease resistance and discuss whether they conform to the requirements that emerge from theoretical models. General characteristics of disease resistance in plants and problems in evaluating resistance experimentally are also discussed. We found strong evidence that disease resistance in plants is determined by multiple loci. Furthermore, in most cases where loci were tested for interactions, epistasis between loci that affect resistance was found. However, we found weak support for the idea that specific allelic combinations determine resistance to different host genotypes and there was little data on whether epistasis between resistance genes is negative or positive. Thus, the current data indicate that it is possible that parasite selection can favour recombination, but more studies in natural populations that specifically address the nature of the interactions between resistance genes are necessary. The data summarized here suggest that disease resistance is a complex trait and that environmental effects and fitness trade-offs should be considered in future models of the coevolutionary dynamics of host and parasites.     

Red Queen Dynamics with Non-Standard Fitness Interactions

Antagonistic coevolution between hosts and parasites can involve rapid fluctuations of genotype frequencies that are known as Red Queen dynamics. Under such dynamics, recombination in the hosts may be advantageous because genetic shuffling can quickly produce disproportionately fit offspring (the Red Queen hypothesis). Previous models investigating these dynamics have assumed rather simple models of genetic interactions between hosts and parasites. Here, we assess the robustness of earlier theoretical predictions about the Red Queen with respect to the underlying host-parasite interactions. To this end, we created large numbers of random interaction matrices, analysed the resulting dynamics through simulation, and ascertained whether recombination was favoured or disfavoured. We observed Red Queen dynamics in many of our simulations provided the interaction matrices exhibited sufficient ‘antagonicity’. In agreement with previous studies, strong selection on either hosts or parasites favours selection for increased recombination. However, fast changes in the sign of linkage disequilibrium or epistasis were only infrequently observed and do not appear to be a necessary condition for the Red Queen hypothesis to work. Indeed, recombination was often favoured even though the linkage disequilibrium remained of constant sign throughout the simulations. We conclude that Red Queen-type dynamics involving persistent fluctuations in host and parasite genotype frequencies appear to not be an artefact of specific assumptions about host-parasite fitness interactions, but emerge readily with the general interactions studied here. Our results also indicate that although recombination is often favoured, some of the factors previously thought to be important in this process such as linkage disequilibrium fluctuations need to be reassessed when fitness interactions between hosts and parasites are complex.     

The role of pleiotropy vs signaller-receiver gene epistasis in life history trade-offs: dissecting the genomic architecture of organismal design in social systems

Traditional life history theory ignores trade-offs due to social interactions, yet social systems expand the set of possible trade-offs affecting a species evolution--by introducing asymmetric interactions between the sexes, age classes and invasion of alternative strategies. We outline principles for understanding gene epistasis due to signaller-receiver dynamics, gene interactions between individuals, and impacts on life history trade-offs. Signaller-receiver epistases create trade-offs among multiple correlated traits that affect fitness, and generate multiple fitness optima conditional on frequency of alternative strategies. In such cases, fitness epistasis generated by selection can maintain linkage disequilibrium, even among physically unlinked loci. In reviewing genetic methods for studying life history trade-offs, we conclude that current artificial selection or gene manipulation experiments focus on pleiotropy. Multi-trait selection experiments, multi-gene engineering methods or multiple endocrine manipulations can test for epistasis and circumvent these limitations. In nature, gene mapping in field pedigrees is required to study social gene epistases and associated trade-offs. Moreover, analyses of correlational selection and frequency-dependent selection are necessary to study epistatic social system trade-offs, which can be achieved with group-structured versions of Price's (1970) equation.     

SELECTION BIASES THE PREVALENCE AND TYPE OF EPISTASIS ALONG ADAPTIVE TRAJECTORIES

The contribution to an organism's phenotype from one genetic locus may depend upon the status of other loci. Such epistatic interactions among loci are now recognized as fundamental to shaping the process of adaptation in evolving populations. Although little is known about the structure of epistasis in most organisms, recent experiments with bacterial populations have concluded that antagonistic interactions abound and tend to deaccelerate the pace of adaptation over time. Here, we use the NK model of fitness landscapes to examine how natural selection biases the mutations that substitute during evolution based on their epistatic interactions. We find that, even when beneficial mutations are rare, these biases are strong and change substantially throughout the course of adaptation. In particular, epistasis is less prevalent than the neutral expectation early in adaptation and much more prevalent later, with a concomitant shift from predominantly antagonistic interactions early in adaptation to synergistic and sign epistasis later in adaptation. We observe the same patterns when reanalyzing data from a recent microbial evolution experiment. These results show that when the order of substitutions is not known, standard methods of analysis may suggest that epistasis retards adaptation when in fact it accelerates it.