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.

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.

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.

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.

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.

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.

Impact of Phyllosphere Methylobacterium on Host Rice Landraces.

The genus Methylobacterium includes widespread plant-associated bacteria that are abundant in the plant phyllosphere (leaf surfaces), consume plant-secreted methanol, and can produce plant growth-promoting metabolites. However, despite the potential to increase agricultural productivity, their impact on host fitness in the natural environment is relatively poorly understood. Here, we conducted field experiments with three traditionally cultivated rice landraces from northeastern India. We inoculated seedlings with native versus nonnative phyllosphere Methylobacterium strains and found significant impacts on plant growth and grain yield. However, these effects were variable. Whereas some Methylobacterium isolates were beneficial for their host, others had no impact or were no more beneficial than the bacterial growth medium on its own. Host plant benefits were not consistently associated with Methylobacterium colonization and did not have altered phyllosphere microbiome composition, changes in the early expression of plant stress response pathways, or bacterial auxin production. We provide the first demonstration of the benefits of phyllosphere Methylobacterium for rice yield under field conditions and highlight the need for further analysis to understand the mechanisms underlying these benefits. Given that the host landrace-Methylobacterium relationship was not generalizable, future agricultural applications will require careful testing to identify coevolved host-bacterium pairs that may enhance the productivity of high-value rice varieties. IMPORTANCE Plants are associated with diverse microbes in nature. Do the microbes increase host plant health, and can they be used for agricultural applications? This is an important question that must be answered in the field rather than in the laboratory or greenhouse. We tested the effects of native, leaf-inhabiting bacteria (genus Methylobacterium) on traditionally cultivated rice varieties in a crop field. We found that inoculation with some bacteria increased rice grain production substantially while a nonnative bacterium reduced plant health. Overall, the effect of bacterial inoculation varied across pairs of rice varieties and their native bacteria. Thus, knowledge of evolved associations between specific bacteria hosted by specific rice varieties is necessary to develop ways to increase the yield of traditional rice landraces and preserve these important sources of cultural and genetic diversity.

Evolutionary jumps in bacterial GC content.

Genomic GC (Guanine-Cytosine) content is a fundamental molecular trait linked with many key genomic features such as codon and amino acid use. Across bacteria, GC content is surprisingly diverse and has been studied for many decades; yet its evolution remains incompletely understood. Since it is difficult to observe GC content evolve on laboratory time scales, phylogenetic comparative approaches are instrumental; but this dimension is rarely studied systematically in the case of bacterial GC content. We applied phylogenetic comparative models to analyze GC content evolution in multiple bacterial groups across 2 major bacterial phyla. We find that GC content diversifies via a combination of gradual evolution and evolutionary "jumps." Surprisingly, unlike prior reports that solely focused on reductions in GC, we found a comparable number of jumps with both increased and decreased GC content. Overall, many of the identified jumps occur in lineages beyond the well-studied peculiar examples of endosymbiotic and AT-rich marine bacteria and do not support the predicted role of oxygen dependence. Our analysis of rapid and large shifts in GC content thus identifies new clades and novel contexts to further understand the ecological and evolutionary drivers of this important genomic trait.

Selection bias in mutation accumulation.

Mutation accumulation (MA) experiments, in which de novo mutations are sampled and subsequently characterized, are an essential tool in understanding the processes underlying evolution. In microbial populations, MA protocols typically involve a period of population growth between severe bottlenecks, such that a single individual can form a visible colony. While it has long been appreciated that the action of positive selection during this growth phase cannot be eliminated, it is typically assumed to be negligible. Here, we quantify the effect of both positive and negative selection in MA studies, demonstrating that selective effects can substantially bias the distribution of fitness effects (DFE) and mutation rates estimated from typical MA protocols in microbes. We then present a simple correction for this bias that applies to both beneficial and deleterious mutations, and can be used to correct the observed DFE in multiple environments. We use simulated MA experiments to illustrate the extent to which the MA-inferred DFE differs from the underlying true DFE, and demonstrate that the proposed correction accurately reconstructs the true DFE over a wide range of scenarios; we also provide an example of these corrections applied to experimental data. These results highlight that positive selection during microbial MA experiments is in fact not negligible, but can be corrected to gain a more accurate understanding of fundamental evolutionary parameters.

The costs and benefits of basal infection resistance vs immune priming responses in an insect.

In insects, basal pathogen resistance and immune priming can evolve as mutually exclusive strategies, with distinct infection outcomes. However, the evolutionary drivers of such diverse immune functions remain poorly understood. Here, we addressed this key issue by systematically analyzing the differential fitness costs and benefits of priming vs resistance evolution in Tribolium beetle populations infected with Bacillus thuringiensis. Surprisingly, resistant beetles had increased post-infection reproduction and a longer lifespan under both starving as well as fed conditions, with no other measurable costs. In contrast, priming reduced offspring early survival, development rate and reproduction. Priming did improve post-infection survival of offspring, but this added trans-generational benefit of immune priming might not compensate for its pervasive costs. Resistance was thus consistently more beneficial. Overall, our work demonstrates the evolutionary change in trans-generational priming response, and provides a detailed comparison of the complex fitness consequences of evolved priming vs resistance.

The impact of mistranslation on phenotypic variability and fitness.

Phenotypic variation is widespread in natural populations, and can significantly alter population ecology and evolution. Phenotypic variation often reflects underlying genetic variation, but also manifests via non-heritable mechanisms. For instance, translation errors result in about 10% of cellular proteins carrying altered sequences. Thus, proteome diversification arising from translation errors can potentially generate phenotypic variability, in turn increasing variability in the fate of cells or of populations. However, the link between protein diversity and phenotypic variability remains unverified. We manipulated mistranslation levels in Escherichia coli, and measured phenotypic variability between single cells (individual-level variation), as well as replicate populations (population-level variation). Monitoring growth and survival, we find that mistranslation indeed increases variation across E. coli cells, but does not consistently increase variability in growth parameters across replicate populations. Interestingly, although any deviation from the wild-type (WT) level of mistranslation reduces fitness in an optimal environment, the increased variation is associated with a survival benefit under stress. Hence, we suggest that mistranslation-induced phenotypic variation can impact growth and survival and has the potential to alter evolutionary trajectories.

The red flour beetle Tribolium castaneum: A model for host-microbiome interactions.

A large body of ongoing research focuses on understanding the mechanisms and processes underlying host-microbiome interactions, and predicting their ecological and evolutionary outcomes. To draw general conclusions about such interactions and understand how they are established, we must synthesize information from a diverse set of species. We analysed the microbiome of an important insect model-the red flour beetle Tribolium castaneum-which is a widespread generalist pest of stored cereals. The beetles complete their entire life cycle in flour, which thus serves multiple functions: habitat, food, and a source of microbes. We determined key factors that shape the T. castaneum microbiome, established protocols to manipulate it, and tested its consequences for host fitness. We show that the T. castaneum microbiome is derived from flour-acquired microbes, and varies as a function of (flour) resource and beetle density. Beetles gain multiple fitness benefits from their microbiome, such as higher fecundity, egg survival, and lifespan; and reduced cannibalism. In contrast, the microbiome has a limited effect on development rate, and does not enhance pathogen resistance. Importantly, the benefits are derived only from microbes in the ancestral resource (wheat flour), and not from novel resources such as finger millet, sorghum, and corn. Notably, the microbiome is not essential for beetle survival and development under any of the tested conditions. Thus, the red flour beetle is a tractable model system to understand the ecology, evolution and mechanisms of host-microbiome interactions, while closely mimicking the host species' natural niche.

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.

Phenotypic diversity of Methylobacterium associated with rice landraces in North-East India.

The ecology and distribution of many bacteria is strongly associated with specific eukaryotic hosts. However, the impact of such host association on bacterial ecology and evolution is not well understood. Bacteria from the genus Methylobacterium consume plant-derived methanol, and are some of the most abundant and widespread plant-associated bacteria. In addition, many of these species impact plant fitness. To determine the ecology and distribution of Methylobacterium in nature, we sampled bacteria from 36 distinct rice landraces, traditionally grown in geographically isolated locations in North-East (NE) India. These landraces have been selected for diverse phenotypic traits by local communities, and we expected that the divergent selection on hosts may have also generated divergence in associated Methylobacterium strains. We determined the ability of 91 distinct rice-associated Methylobacterium isolates to use a panel of carbon sources, finding substantial variability in carbon use profiles. Consistent with our expectation, across spatial scales this phenotypic variation was largely explained by host landrace identity rather than geographical factors or bacterial taxonomy. However, variation in carbon utilisation was not correlated with sugar exudates on leaf surfaces, suggesting that bacterial carbon use profiles do not directly determine bacterial colonization across landraces. Finally, experiments showed that at least some rice landraces gain an early growth advantage from their specific phyllosphere-colonizing Methylobacterium strains. Together, our results suggest that landrace-specific host-microbial relationships may contribute to spatial structure in rice-associated Methylobacterium in a natural ecosystem. In turn, association with specific bacteria may provide new ways to preserve and understand diversity in one of the most important food crops of the world.

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.

Host dietary specialization and neutral assembly shape gut bacterial communities of wild dragonflies.

Host-associated gut microbiota can have significant impacts on host ecology and evolution and are often host-specific. Multiple factors can contribute to such host-specificity: (1) host dietary specialization passively determining microbial colonization, (2) hosts selecting for specific diet-acquired microbiota, or (3) a combination of both. The latter possibilities indicate a functional association and should produce stable microbiota. We tested these alternatives by analyzing the gut bacterial communities of six species of wild adult dragonfly populations collected across several geographic locations. The bacterial community composition was predominantly explained by sampling location, and only secondarily by host identity. To distinguish the role of host dietary specialization and host-imposed selection, we identified prey in the guts of three dragonfly species. Surprisingly, the dragonflies-considered to be generalist predators-consumed distinct prey; and the prey diversity was strongly correlated with the gut bacterial profile. Such host dietary specialization and spatial variation in bacterial communities suggested passive rather than selective underlying processes. Indeed, the abundance and distribution of 72% of bacterial taxa were consistent with neutral community assembly; and fluorescent in situ hybridization revealed that bacteria only rarely colonized the gut lining. Our results contradict the expectation that host-imposed selection shapes the gut microbiota of most insects, and highlight the importance of joint analyses of diet and gut microbiota of natural host populations.

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.

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.

Antagonistic pleiotropy for carbon use is rare in new mutations.

Pleiotropic effects of mutations underlie diverse biological phenomena such as ageing and specialization. In particular, antagonistic pleiotropy ("AP": when a mutation has opposite fitness effects in different environments) generates tradeoffs, which may constrain adaptation. Models of adaptation typically assume that AP is common - especially among large-effect mutations - and that pleiotropic effect sizes are positively correlated. Empirical tests of these assumptions have focused on de novo beneficial mutations arising under strong selection. However, most mutations are actually deleterious or neutral, and may contribute to standing genetic variation that can subsequently drive adaptation. We quantified the incidence, nature, and effect size of pleiotropy for carbon utilization across 80 single mutations in Escherichia coli that arose under mutation accumulation (i.e., weak selection). Although ∼46% of the mutations were pleiotropic, only 11% showed AP; among beneficial mutations, only ∼4% showed AP. In some environments, AP was more common in large-effect mutations; and AP effect sizes across environments were often negatively correlated. Thus, AP for carbon use is generally rare (especially among beneficial mutations); is not consistently enriched in large-effect mutations; and often involves weakly deleterious antagonistic effects. Our unbiased quantification of mutational effects therefore suggests that antagonistic pleiotropy may be unlikely to cause maladaptive tradeoffs.