Shifts in mutation spectra enhance access to beneficial mutations.

Biased mutation spectra are pervasive, with wide variation in the magnitude of mutational biases that influence genome evolution and adaptation. How do such diverse biases evolve? Our experiments show that changing the mutation spectrum allows populations to sample previously undersampled mutational space, including beneficial mutations. The resulting shift in the distribution of fitness effects is advantageous: Beneficial mutation supply and beneficial pleiotropy both increase, while deleterious load reduces. More broadly, simulations indicate that reducing or reversing the direction of a long-term bias is always selectively favored. Such changes in mutation bias can occur easily via altered function of DNA repair genes. A phylogenetic analysis shows that these genes are repeatedly gained and lost in bacterial lineages, leading to frequent bias shifts in opposite directions. Thus, shifts in mutation spectra may evolve under selection and can directly alter the outcome of adaptive evolution by facilitating access to beneficial mutations.

Revisiting the Role of Genetic Variation in Adaptation.

AbstractEvolutionary biologists have thought about the role of genetic variation during adaptation for a very long time-before we understood the organization of the genetic code, the provenance of genetic variation, and how such variation influenced the phenotypes on which natural selection acts. Half a century after the discovery of the structure of DNA and the unraveling of the genetic code, we have a rich understanding of these problems and the means to both delve deeper and widen our perspective across organisms and natural populations. The 2022 Vice Presidential Symposium of the American Society of Naturalists highlighted examples of recent insights into the role of genetic variation in adaptive processes, which are compiled in this special section. The work was conducted in different parts of the world, included theoretical and empirical studies with diverse organisms, and addressed distinct aspects of how genetic variation influences adaptation. In our introductory article to the special section, we discuss some important recent insights about the generation and maintenance of genetic variation, its impacts on phenotype and fitness, its fate in natural populations, and its role in driving adaptation. By placing the special section articles in the broader context of recent developments, we hope that this overview will also serve as a useful introduction to the field.

Alteration of diet microbiota limits the experimentally evolved immune priming response in flour beetles, but not pathogen resistance.

Host-associated microbiota play a fundamental role in the training and induction of different forms of immunity, including inducible as well as constitutive components. However, direct experiments analysing the relative importance of microbiota on diverse forms of evolved immune functions are missing. We addressed this gap by using experimentally evolved lines of Tribolium castaneum that either produced inducible immune memory-like responses (immune priming) or constitutively expressed basal resistance (without priming), as divergent counterstrategies against Bacillus thuringiensis infection. We altered the microbial communities present in the diet (i.e. wheat flour) of these evolved lines using UV irradiation and estimated the impact on the beetle's ability to mount a priming response versus basal resistance. Populations that had evolved immune priming lost the ability to mount a priming response upon alteration of diet microbiota. Microbiota manipulation also caused a drastic reduction in their reproductive output and post-infection longevity. In contrast, in pathogen-resistant beetles, microbiota manipulation did not affect post-infection survival or reproduction. The divergent evolution of immune responses across beetle lines was thus associated with divergent reliance on the microbiome. Whether the latter is a direct outcome of differential pathogen exposure during selection or reflects evolved immune functions remains unclear. We hope that our results will motivate further experiments to understand the mechanistic basis of these complex evolutionary associations between microbiota, host immune strategies and fitness outcomes.

Global mistranslation increases cell survival under stress in Escherichia coli.

Mistranslation is typically deleterious for cells, although specific mistranslated proteins can confer a short-term benefit in a particular environment. However, given its large overall cost, the prevalence of high global mistranslation rates remains puzzling. Altering basal mistranslation levels of Escherichia coli in several ways, we show that generalized mistranslation enhances early survival under DNA damage, by rapidly activating the SOS response. Mistranslating cells maintain larger populations after exposure to DNA damage, and thus have a higher probability of sampling critical beneficial mutations. Both basal and artificially increased mistranslation increase the number of cells that are phenotypically tolerant and genetically resistant under DNA damage; they also enhance survival at high temperature. In contrast, decreasing the normal basal mistranslation rate reduces cell survival. This wide-ranging stress resistance relies on Lon protease, which is revealed as a key effector that induces the SOS response in addition to alleviating proteotoxic stress. The new links between error-prone protein synthesis, DNA damage, and generalised stress resistance indicate surprising coordination between intracellular stress responses and suggest a novel hypothesis to explain high global mistranslation rates.

Evolthon: A community endeavor to evolve lab evolution.

In experimental evolution, scientists evolve organisms in the lab, typically by challenging them to new environmental conditions. How best to evolve a desired trait? Should the challenge be applied abruptly, gradually, periodically, sporadically? Should one apply chemical mutagenesis, and do strains with high innate mutation rate evolve faster? What are ideal population sizes of evolving populations? There are endless strategies, beyond those that can be exposed by individual labs. We therefore arranged a community challenge, Evolthon, in which students and scientists from different labs were asked to evolve Escherichia coli or Saccharomyces cerevisiae for an abiotic stress-low temperature. About 30 participants from around the world explored diverse environmental and genetic regimes of evolution. After a period of evolution in each lab, all strains of each species were competed with one another. In yeast, the most successful strategies were those that used mating, underscoring the importance of sex in evolution. In bacteria, the fittest strain used a strategy based on exploration of different mutation rates. Different strategies displayed variable levels of performance and stability across additional challenges and conditions. This study therefore uncovers principles of effective experimental evolutionary regimens and might prove useful also for biotechnological developments of new strains and for understanding natural strategies in evolutionary arms races between species. Evolthon constitutes a model for community-based scientific exploration that encourages creativity and cooperation.

The road not taken: Could stress-specific mutations lead to different evolutionary paths?

Organisms often encounter stressful conditions, some of which damage their DNA. In response, some organisms show a high expression of error-prone DNA repair machinery, causing a temporary increase in the genome-wide mutation rate. Although we now have a detailed map of the molecular mechanisms underlying such stress-induced mutagenesis (SIM), it has been hotly debated whether SIM alters evolutionary dynamics. Key to this controversy is our poor understanding about which stresses increase mutagenesis and their long-term consequences for adaptation. In a new study with Escherichia coli, Maharjan and Ferenci show that while only some nutritional stresses (phosphorous and carbon limitation) increase total mutation rates, each stress generates a unique spectrum of mutations. Their results suggest the potential for specific stresses to shape evolutionary dynamics and highlight the necessity for explicit tests of the long-term evolutionary impacts of SIM.

Wobbling Forth and Drifting Back: The Evolutionary History and Impact of Bacterial tRNA Modifications.

Along with tRNAs, enzymes that modify anticodon bases are a key aspect of translation across the tree of life. tRNA modifications extend wobble pairing, allowing specific ("target") tRNAs to recognize multiple codons and cover for other ("nontarget") tRNAs, often improving translation efficiency and accuracy. However, the detailed evolutionary history and impact of tRNA modifying enzymes has not been analyzed. Using ancestral reconstruction of five tRNA modifications across 1093 bacteria, we show that most modifications were ancestral to eubacteria, but were repeatedly lost in many lineages. Most modification losses coincided with evolutionary shifts in nontarget tRNAs, often driven by increased bias in genomic GC and associated codon use, or by genome reduction. In turn, the loss of tRNA modifications stabilized otherwise highly dynamic tRNA gene repertoires. Our work thus traces the complex history of bacterial tRNA modifications, providing the first clear evidence for their role in the evolution of bacterial translation.

Disrupting butterfly caterpillar microbiomes does not impact their survival and development.

Associations with gut microbes are believed to play crucial roles in the physiology, immune function, development and behaviour of insects. However, microbiome sequencing has recently suggested that butterflies are an anomaly, because their microbiomes do not show strong host- and developmental stage-specific associations. We experimentally manipulated butterfly larval gut microbiota and found that disrupting gut microbes had little influence on larval survival and development. Larvae of the butterflies Danaus chrysippus and Ariadne merione that fed on chemically sterilized or antibiotic-treated host plant leaves had significantly reduced bacterial loads, and their gut bacterial communities were disrupted substantially. However, neither host species treated this way suffered a significant fitness cost: across multiple experimental blocks, treated and control larvae had similar survival, growth and development. Furthermore, re-introducing microbes from the excreta of control larvae did not improve larval growth and survival. Thus, these butterfly larvae did not appear to rely on specialized gut bacteria for digestion, detoxification, biomass accumulation and metamorphosis. Our experiments thus show that dependence on gut bacteria for growth and survival is not a universal phenomenon across insects. Our findings also caution that strategies which target gut microbiomes may not always succeed in controlling Lepidopteran pests.

Pathogen susceptibility and fitness costs explain variation in immune priming across natural populations of flour beetles.

In many insects, individuals primed with low doses of pathogens early in life have higher survival after exposure to the same pathogen later in life. Yet, our understanding of the evolutionary and ecological history of priming of immune response in natural insect populations is limited. Previous work demonstrated population-, sex- and stage-specific variation in the survival benefit of priming response in flour beetles (Tribolium castaneum) infected with their natural pathogen Bacillus thuringiensis. However, the evolutionary forces responsible for this natural variation remained unclear. In the present work, we tested whether the strength of the priming response (measured as the survival benefit after priming and subsequent infection, relative to unprimed controls) was associated with multiple fitness parameters and immune components across 10 flour beetle populations collected from different locations in India. Our results suggest two major selective pressures that may explain the observed inter-population variation in priming: (a) Basal pathogen susceptibility - populations that were more susceptible to infection produced a stronger priming response, and (b) Short-term early reproductive success - populations where primed females produced more offspring early in life (measured over 2 days) had lower survival benefit (measured over 120 days), suggesting a potential trade-off between early reproduction and priming response. However, the negative association between survival and reproduction is limited to priming and infection in adults, but not in larvae. While other components of beetle fitness (starvation resistance and larval development) and immune function (haemolymph antibacterial activity and antimicrobial quinone secretion) also varied widely across populations, none of them was correlated with the variation in priming responses across populations. Our work is the first systematic empirical demonstration of multiple selective pressures that may govern the evolution of immune priming in the wild. We hope that this motivates further experiments to establish the role of pathogen-imposed selection and fitness costs in the evolution of priming in natural insect populations.

Large-Effect Beneficial Synonymous Mutations Mediate Rapid and Parallel Adaptation in a Bacterium.

Contrary to previous understanding, recent evidence indicates that synonymous codon changes may sometimes face strong selection. However, it remains difficult to generalize the nature, strength, and mechanism(s) of such selection. Previously, we showed that synonymous variants of a key enzyme-coding gene (fae) of Methylobacterium extorquens AM1 decreased enzyme production and reduced fitness dramatically. We now show that during laboratory evolution, these variants rapidly regained fitness via parallel yet variant-specific, highly beneficial point mutations in the N-terminal region of fae These mutations (including four synonymous mutations) had weak but consistently positive impacts on transcript levels, enzyme production, or enzyme activity. However, none of the proposed mechanisms (including internal ribosome pause sites or mRNA structure) predicted the fitness impact of evolved or additional, engineered point mutations. This study shows that synonymous mutations can be fixed through strong positive selection, but the mechanism for their benefit varies depending on the local sequence context.

Early-life inflammation, immune response and ageing.

Age-related diseases are often attributed to immunopathology, which results in self-damage caused by an inappropriate inflammatory response. Immunopathology associated with early-life inflammation also appears to cause faster ageing, although we lack direct experimental evidence for this association. To understand the interactions between ageing, inflammation and immunopathology, we used the mealworm beetle Tenebrio molitor as a study organism. We hypothesized that phenoloxidase, an important immune effector in insect defence, may impose substantial immunopathological costs by causing tissue damage to Malpighian tubules (MTs; functionally equivalent to the human kidney), in turn accelerating ageing. In support of this hypothesis, we found that RNAi knockdown of phenoloxidase (PO) transcripts in young adults possibly reduced inflammation-induced autoreactive tissue damage to MTs, and increased adult lifespan. Our work thus suggests a causative link between immunopathological costs of early-life inflammation and faster ageing. We also reasoned that if natural selection weakens with age, older individuals should display increased immunopathological costs associated with an immune response. Indeed, we found that while old infected individuals cleared infection faster than young individuals, possibly they also displayed exacerbated immunopathological costs (larger decline in MT function) and higher post-infection mortality. RNAi-mediated knockdown of PO response partially rescued MTs function in older beetles and resulted in increased lifespan after infection. Taken together, our data are consistent with a direct role of immunopathological consequences of immune response during ageing in insects. Our work is also the first report that highlights the pervasive role of tissue damage under diverse contexts of ageing and immune response.

Experimental evolution of insect immune memory versus pathogen resistance.

Under strong pathogen pressure, insects often evolve resistance to infection. Many insects are also protected via immune memory (immune priming), whereby sublethal exposure to a pathogen enhances survival after secondary infection. Theory predicts that immune memory should evolve when the pathogen is highly virulent, or when pathogen exposure is relatively rare. However, there are no empirical tests of these hypotheses, and the adaptive benefits of immune memory relative to direct resistance against a pathogen are poorly understood. To determine the selective pressures and ecological conditions that shape immune evolution, we imposed strong pathogen selection on flour beetle (Tribolium castaneum) populations, infecting them with Bacillus thuringiensis (Bt) for 11 generations. Populations injected first with heat-killed and then live Bt evolved high basal resistance against multiple Bt strains. By contrast, populations injected only with a high dose of live Bt evolved a less effective but strain-specific priming response. Control populations injected with heat-killed Bt did not evolve priming; and in the ancestor, priming was effective only against a low Bt dose. Intriguingly, one replicate population first evolved priming and subsequently evolved basal resistance, suggesting the potential for dynamic evolution of different immune strategies. Our work is the first report showing that pathogens can select for rapid modulation of insect priming ability, allowing hosts to evolve divergent immune strategies (generalized resistance versus specific immune memory) with potentially distinct mechanisms.

Immunosenescence and the ability to survive bacterial infection in the red flour beetle Tribolium castaneum.

In most animals, ageing is associated with a decline in immune function (immune senescence). However, different components of the immune system seem to age differentially, and many studies do not measure the ultimate fitness consequences of immune function after infection. Previous work shows that immune function may be traded off with other fitness components such as reproduction. It is possible that age alters the nature of these trade-offs, particularly in conjunction with factors such as gender and mating that can also affect investment in immune function. We tested the impact of age, sex and mating on post-infection survivorship in Tribolium castaneum flour beetles, as well as the components of baseline constitutive innate immunity and external (secreted) immune function in uninfected individuals. We also tested whether the reproductive ability of uninfected females is traded off with immune function (baseline innate and external immunity) and post-infection survivorship across age groups. We found that age, sex and mating significantly affected immune components and infection outcome, although the magnitude and nature of the impact varied in each case. We found that older beetles were more susceptible to infection by the pathogen Bacillus thuringiensis even though major components of the constitutive innate immune defence (antibacterial and phenoloxidase activity) remained unchanged or improved with age. Thus, these aspects of innate immunity cannot explain the observed decline in post-infection survival of older beetles. We did not find trade-offs between the reproductive ability of uninfected females and their immune function. In contrast to innate immunity, external immunity showed an overall decline with age but was also affected by sex and mating. Finally, we show that bacterial infection alters external immunity via complex interactions between age, sex and mating status. Our work uncovers novel interactions between age, sex and mating that can determine the evolution and outcome of immunosenescence by affecting the time course of relative investment in different immune and fitness components.

Good codons, bad transcript: large reductions in gene expression and fitness arising from synonymous mutations in a key enzyme.

Biased codon usage in protein-coding genes is pervasive, whereby amino acids are largely encoded by a specific subset of possible codons. Within individual genes, codon bias is stronger at evolutionarily conserved residues, favoring codons recognized by abundant tRNAs. Although this observation suggests an overall pattern of selection for translation speed and/or accuracy, other work indicates that transcript structure or binding motifs drive codon usage. However, our understanding of codon bias evolution is constrained by limited experimental data on the fitness effects of altering codons in functional genes. To bridge this gap, we generated synonymous variants of a key enzyme-coding gene in Methylobacterium extorquens. We found that mutant gene expression, enzyme production, enzyme activity, and fitness were all significantly lower than wild-type. Surprisingly, encoding the gene using only rare codons decreased fitness by 40%, whereas an allele coded entirely by frequent codons decreased fitness by more than 90%. Increasing gene expression restored mutant fitness to varying degrees, demonstrating that the fitness disadvantage of synonymous mutants arose from a lack of beneficial protein rather than costs of protein production. Protein production was negatively correlated with the frequency of motifs with high affinity for the anti-Shine-Dalgarno sequence, suggesting ribosome pausing as the dominant cause of low mutant fitness. Together, our data support the idea that, although a particular set of codons are favored on average across a genome, in an individual gene selection can either act for or against codons depending on their local context.

The evolution of bacterial DNA base composition.

Bacterial genomes exhibit a large amount of variation in their base composition, which ranges from 13% to 75% GC. The evolution and maintenance of this variation has proved to be an enduring puzzle despite decades of theoretical and empirical work. We present an overview of various aspects of this problem, focusing on results from a diverse set of recent studies that use whole-genome sequencing in combination with bioinformatic, phylogenetic, molecular biological, and experimental evolution approaches. We propose that analysis of within-genome variance in GC content is also important to understand how genome-wide base composition has evolved. We close with a discussion of open questions and fruitful avenues of inquiry that may bring us closer to understanding the evolutionary dynamics of bacterial DNA base composition.

Mutational Signatures in Wild Type Escherichia coli Strains Reveal Predominance of DNA Polymerase Errors.

While mutational processes operating in the Escherichia coli genome have been revealed by multiple laboratory experiments, the contribution of these processes to accumulation of bacterial polymorphism and evolution in natural environments is unknown. To address this question, we reconstruct signatures of distinct mutational processes from experimental data on E. coli hypermutators, and ask how these processes contribute to differences between naturally occurring E. coli strains. We show that both mutations accumulated in the course of evolution of wild-type strains in nature and in the lab-grown nonmutator laboratory strains are explained predominantly by the low fidelity of DNA polymerases II and III. By contrast, contributions specific to disruption of DNA repair systems cannot be detected, suggesting that temporary accelerations of mutagenesis associated with such disruptions are unimportant for within-species evolution. These observations demonstrate that accumulation of diversity in bacterial strains in nature is predominantly associated with errors of DNA polymerases.

Fitness landscapes reveal context-dependent benefits of oviposition behavior.

Resource choice behavior has enormous fitness consequences and can drive niche expansion. However, individual behavioral choices are often mediated by context, determined by past experience. Do such context-dependent behaviors reflect maladaptive variation or are they locally adaptive? Using Tribolium castaneum (the red flour beetle), we demonstrate that context-dependent oviposition behavior reflects distinct, context-specific local fitness peaks. We measured offspring fitness to generate fitness landscapes as a function of all possible oviposition behaviors (i.e., combinations of fecundity and resource preference) in a habitat containing optimal and suboptimal resource patches. We did this by experimentally manipulating egg allocation across patches, which allowed us to assess behaviors not typically observed in the laboratory. We found that females from different age and competition contexts exhibit distinct behaviors which optimize different fitness components, linked in a tradeoff. With prior exposure to strong competition and increasing age, females produce few but fast-developing offspring that are advantageous under high resource competition. In contrast, young naïve females produce significantly more (but slower developing) offspring, which is beneficial under weak competition. Systematically mapping complete context-dependent fitness landscapes is thus critical to infer behavioral optimality and offers predictive power in novel contexts.

The layered costs and benefits of translational redundancy.

The rate and accuracy of translation hinges upon multiple components - including transfer RNA (tRNA) pools, tRNA modifying enzymes, and rRNA molecules - many of which are redundant in terms of gene copy number or function. It has been hypothesized that the redundancy evolves under selection, driven by its impacts on growth rate. However, we lack empirical measurements of the fitness costs and benefits of redundancy, and we have poor a understanding of how this redundancy is organized across components. We manipulated redundancy in multiple translation components of Escherichia coli by deleting 28 tRNA genes, 3 tRNA modifying systems, and 4 rRNA operons in various combinations. We find that redundancy in tRNA pools is beneficial when nutrients are plentiful and costly under nutrient limitation. This nutrient-dependent cost of redundant tRNA genes stems from upper limits to translation capacity and growth rate, and therefore varies as a function of the maximum growth rate attainable in a given nutrient niche. The loss of redundancy in rRNA genes and tRNA modifying enzymes had similar nutrient-dependent fitness consequences. Importantly, these effects are also contingent upon interactions across translation components, indicating a layered hierarchy from copy number of tRNA and rRNA genes to their expression and downstream processing. Overall, our results indicate both positive and negative selection on redundancy in translation components, depending on a species' evolutionary history with feasts and famines.

Translation is the process by which cellular machines called ribosomes use the information encoded in genes to make proteins . Every organism requires two types of RNA molecules to make new proteins: ribosomal RNAs (rRNAs, which form part of the ribosome) and transfer RNAs (tRNAs, which transport the amino acid molecules that form proteins to the ribosomes). These RNA molecules are coded in the genome, but different organisms have different 'copy numbers': some genomes contain just a few copies of each of these genes, while others have thousands. This apparent redundancy ­ the presence of several copies of the same gene ­ is puzzling because it is costly to make and maintain DNA and RNA. This leads to an important question: how does redundancy in these important genes (coding for tRNAs and rRNAs) evolve? The answer is key to understanding how one of the most fundamental cellular processes, the making of proteins from DNA, has evolved. A possible reason for organisms to have many copies of the genes required to make proteins is to allow rapid translation, which allows cells to divide faster, and populations of cells to grow more quickly. However, this would likely mean that, when nutrients are scarce, carrying and translating many copies of the same gene would become a burden on the cell. Raval et al. set out to test this idea by measuring the costs and benefits of seemingly redundant translation components. To do this, Raval et al. deleted some of the redundant gene copies in the bacterium Escherichia coli and asked if that changed bacterial growth. The experiments showed that when nutrients were plentiful, cells with more copies of the genes (high redundancy) were better able to use the nutrients and divide rapidly. However, when nutrients were limited, bacteria with extra gene copies divided more slowly, showing that the extra genes are indeed a big burden on the cell. Raval et al. propose that nutrients available in the environment ultimately determine whether redundancy of the translation machinery is a blessing or a curse. This suggests that the redundancy and underlying growth strategies of different organisms are forged by their experiences of feast and famine during their evolutionary past. Importantly, by testing the joint effect of many different molecules involved in translation, Raval et al. uncovered several strategies that may maximize bacterial growth and protein production. Their results could thus be useful for optimizing the synthesis of important products that use growing cells as factories ­ from beer to insulin ­ where the rate of growth is critical.

Towards evolutionary predictions: Current promises and challenges.

Evolution has traditionally been a historical and descriptive science, and predicting future evolutionary processes has long been considered impossible. However, evolutionary predictions are increasingly being developed and used in medicine, agriculture, biotechnology and conservation biology. Evolutionary predictions may be used for different purposes, such as to prepare for the future, to try and change the course of evolution or to determine how well we understand evolutionary processes. Similarly, the exact aspect of the evolved population that we want to predict may also differ. For example, we could try to predict which genotype will dominate, the fitness of the population or the extinction probability of a population. In addition, there are many uses of evolutionary predictions that may not always be recognized as such. The main goal of this review is to increase awareness of methods and data in different research fields by showing the breadth of situations in which evolutionary predictions are made. We describe how diverse evolutionary predictions share a common structure described by the predictive scope, time scale and precision. Then, by using examples ranging from SARS-CoV2 and influenza to CRISPR-based gene drives and sustainable product formation in biotechnology, we discuss the methods for predicting evolution, the factors that affect predictability and how predictions can be used to prevent evolution in undesirable directions or to promote beneficial evolution (i.e. evolutionary control). We hope that this review will stimulate collaboration between fields by establishing a common language for evolutionary predictions.