Antimicrobial resistance in the wild: Insights from epigenetics.

Spreading of bacterial and fungal strains that are resistant to antimicrobials poses a serious threat to the well-being of humans, animals, and plants. Antimicrobial resistance has been mainly investigated in clinical settings. However, throughout their evolutionary history microorganisms in the wild have encountered antimicrobial substances, forcing them to evolve strategies to combat antimicrobial action. It is well known that many of these strategies are based on genetic mechanisms, but these do not fully explain important aspects of the antimicrobial response such as the rapid development of resistance, reversible phenotypes, and hetero-resistance. Consequently, attention has turned toward epigenetic pathways that may offer additional insights into antimicrobial mechanisms. The aim of this review is to explore the epigenetic mechanisms that confer antimicrobial resistance, focusing on those that might be relevant for resistance in the wild. First, we examine the presence of antimicrobials in natural settings. Then we describe the documented epigenetic mechanisms in bacteria and fungi associated with antimicrobial resistance and discuss innovative epigenetic editing techniques to establish causality in this context. Finally, we discuss the relevance of these epigenetic mechanisms on the evolutionary dynamics of antimicrobial resistance in the wild, emphasizing the critical role of priming in the adaptation process. We underscore the necessity of incorporating non-genetic mechanisms into our understanding of antimicrobial resistance evolution. These mechanisms offer invaluable insights into the dynamics of antimicrobial adaptation within natural ecosystems.

Parental effects in a filamentous fungus: Phenotype, fitness and mechanism.

In nature, organisms have to cope with constantly changing environments. In certain conditions, it may be advantageous for the parents to pass on information about the environment, or resources to their offspring. Such transfers are known as parental effects, and they are well documented in plants and animals, but not in other eukaryotes, such as fungi. Many fungi disperse through spores, and fungal spores can potentially carry information or resources to the next generation. Understanding parental effects and their evolutionary consequences in fungi is of vital importance as they perform crucial ecosystem functions. In this study, we investigated whether parental effects are present in the filamentous fungus Neurospora crassa, how long do they last, whether the effects are adaptive, and what is their mechanism. We performed a fully factorial match/mismatch experiment for a good and a poor quality environment, in which we measured the initial growth of strains that experienced either a matched or mismatched environment in their previous generation. We found a strong silver-spoon effect in initial mycelium growth, which lasted for one generation, and increased fitness during competition experiments. By using deletion mutants that lacked key genes in epigenetic processes, we show that epigenetic mechanisms are not involved in this effect. Instead, we show that spore glycogen content, glucose availability and a radical transcription shift in spores are the main mechanisms behind this parental effect.

Experimental evolution of evolutionary potential in fluctuating environments.

Variation is the raw material for evolution. Evolutionary potential is determined by the amount of genetic variation, but evolution can also alter the visibility of genetic variation to natural selection. Fluctuating environments are suggested to maintain genetic variation but they can also affect environmental variance, and thus, the visibility of genetic variation to natural selection. However, experimental studies testing these ideas are relatively scarce. In order to determine differences in evolutionary potential we quantified variance attributable to population, genotype and environment for populations of the bacterium Serratia marcescens. These populations had been experimentally evolved in constant and two fluctuating environments. We found that strains that evolved in fluctuating environments exhibited larger environmental variation suggesting that adaptation to fluctuations has decreased the visibility of genetic variation to selection.

Chromatin structure influences rate and spectrum of spontaneous mutations in Neurospora crassa.

Although mutation rates have been extensively studied, variation in mutation rates throughout the genome is poorly understood. To understand patterns of genetic variation, it is important to understand how mutation rates vary. Chromatin modifications may be an important factor in determining variation in mutation rates in eukaryotic genomes. To study variation in mutation rates, we performed a mutation accumulation (MA) experiment in the filamentous fungus Neurospora crassa and sequenced the genomes of the 40 MA lines that had been propagated asexually for approximately 1015 [Formula: see text] mitoses. We detected 1322 mutations in total and observed that the mutation rate was higher in regions of low GC, in domains of H3K9 trimethylation, in centromeric regions, and in domains of H3K27 trimethylation. The rate of single-nucleotide mutations in euchromatin was [Formula: see text] In contrast, the mutation rate in H3K9me3 domains was 10-fold higher: 2.43 [Formula: see text] We also observed that the spectrum of single-nucleotide mutations was different between H3K9me3 and euchromatic domains. Our statistical model of mutation rate variation predicted a moderate amount of extant genetic variation, suggesting that the mutation rate is an important factor in determining levels of natural genetic variation. Furthermore, we characterized mutation rates of structural variants, complex mutations, and the effect of local sequence context on the mutation rate. Our study highlights that chromatin modifications are associated with mutation rates, and accurate evolutionary inferences should take variation in mutation rates across the genome into account.

The effect of a temperature-sensitive prophage on the evolution of virulence in an opportunistic bacterial pathogen.

Viruses are key actors of ecosystems and have major impacts on global biogeochemical cycles. Prophages deserve particular attention as they are ubiquitous in bacterial genomes and can enter a lytic cycle when triggered by environmental conditions. We explored how temperature affects the interactions between prophages and other biological levels using an opportunistic pathogen, the bacterium Serratia marcescens, which harbours several prophages and that had undergone an evolution experiment under several temperature regimes. We found that the release of one of the prophages was temperature-sensitive and malleable to evolutionary changes. We further discovered that the virulence of the bacterium in an insect model also evolved and was positively correlated with phage release rates. We determined through analysis of genetic and epigenetic data that changes in the bacterial outer cell wall structure possibly explain this phenomenon. We hypothezise that the temperature-dependent phage release rate acted as a selection pressure on S. marcescens and that it resulted in modified bacterial virulence in the insect host. Our study system illustrates how viruses can mediate the influence of abiotic environmental changes to other biological levels and thus be involved in ecosystem feedback loops.

Evolution of anticipatory effects mediated by epigenetic changes.

Anticipatory effects mediated by epigenetic changes occur when parents modify the phenotype of their offspring by making epigenetic changes in their gametes, guided by information from an environmental cue. To investigate when do anticipatory effects mediated by epigenetic changes evolve in a fluctuating environment, I use an individual-based simulation model with explicit genetic architecture. The model allows for the population to respond to environmental changes by evolving plasticity, bet hedging, or by tracking the environment with genetic adaptation, in addition to the evolution of anticipatory effects. The results show that anticipatory effects evolve when the environmental cue provides reliable information about the environment and the environment changes at intermediate rates, provided that fitness costs of anticipatory effects are rather low. Moreover, evolution of anticipatory effects is quite robust to different genetic architectures when reliability of the environmental cue is high. Anticipatory effects always give smaller fitness benefits than within-generation plasticity, suggesting a possible reason for generally small observed anticipatory effects in empirical studies.

Roles of adenine methylation and genetic mutations in adaptation to different temperatures in Serratia marcescens.

Epigenetic modifications can contribute to adaptation, but the relative contributions of genetic and epigenetic variation are unknown. Previous studies on the role of epigenetic changes in adaptation in eukaryotes have nearly exclusively focused on cytosine methylation (m5C), while prokaryotes exhibit a richer system of methyltransferases targetting adenines (m6A) or cytosines (m4C, m5C). DNA methylation in prokaryotes has many roles, but its potential role in adaptation still needs further investigation. We collected phenotypic, genetic, and epigenetic data using single molecule real-time sequencing of clones of the bacterium Serratia marcescens that had undergone experimental evolution in contrasting temperatures to investigate the relationship between environment and genetic, epigenetic, and phenotypic changes. The genomic distribution of GATC motifs, which were the main target for m6A methylation, and of variable m6A epiloci pointed to a potential link between m6A methylation and regulation of gene expression in S. marcescens. Evolved strains, while genetically homogeneous, exhibited many polymorphic m6A epiloci. There was no strong support for a genetic control of methylation changes in our experiment, and no clear evidence of parallel environmentally induced or environmentally selected methylation changes at specific epiloci was found. Both genetic and epigenetic variants were associated with some phenotypic traits. Overall, our results suggest that both genetic and adenine methylation changes have the potential to contribute to phenotypic adaptation in S. marcescens, but that any environmentally induced epigenetic change occurring in our experiment would probably have been quite labile.

Crowdsourced analysis of fungal growth and branching on microfluidic platforms.

Fungal hyphal growth and branching are essential traits that allow fungi to spread and proliferate in many environments. This sustained growth is essential for a myriad of applications in health, agriculture, and industry. However, comparisons between different fungi are difficult in the absence of standardized metrics. Here, we used a microfluidic device featuring four different maze patterns to compare the growth velocity and branching frequency of fourteen filamentous fungi. These measurements result from the collective work of several labs in the form of a competition named the "Fungus Olympics." The competing fungi included five ascomycete species (ten strains total), two basidiomycete species, and two zygomycete species. We found that growth velocity within a straight channel varied from 1 to 4 µm/min. We also found that the time to complete mazes when fungal hyphae branched or turned at various angles did not correlate with linear growth velocity. We discovered that fungi in our study used one of two distinct strategies to traverse mazes: high-frequency branching in which all possible paths were explored, and low-frequency branching in which only one or two paths were explored. While the high-frequency branching helped fungi escape mazes with sharp turns faster, the low-frequency turning had a significant advantage in mazes with shallower turns. Future work will more systematically examine these trends.

Evolutionary rescue at different rates of environmental change is affected by trade-offs between short-term performance and long-term survival.

As climate change accelerates and habitats free from anthropogenic impacts diminish, populations are forced to migrate or to adapt quickly. Evolutionary rescue (ER) is a phenomenon, in which a population is able to avoid extinction through adaptation. ER is considered to be more likely at slower rates of environmental change. However, the effects of correlated characters on evolutionary rescue are seldom explored yet correlated characters could play a major role in ER. We tested how evolutionary background in different fluctuating environments and the rate of environmental change affect the probability of ER by exposing populations of the bacteria Serratia marcescens to two different rates of steady temperature increase. As suggested by theory, slower environmental change allowed populations to grow more effectively even at extreme temperatures, but at the expense of long-term survival at extreme conditions due to correlated selection. Our results indicate important gap of knowledge on the effects of correlated selection during the environmental change and on evolutionary rescue at differently changing environments.

Quantitative genetics of temperature performance curves of Neurospora crassa.

Earth's temperature is increasing due to anthropogenic CO 2 emissions; and organisms need either to adapt to higher temperatures, migrate into colder areas, or face extinction. Temperature affects nearly all aspects of an organism's physiology via its influence on metabolic rate and protein structure, therefore genetic adaptation to increased temperature may be much harder to achieve compared to other abiotic stresses. There is still much to be learned about the evolutionary potential for adaptation to higher temperatures, therefore we studied the quantitative genetics of growth rates in different temperatures that make up the thermal performance curve of the fungal model system Neurospora crassa. We studied the amount of genetic variation for thermal performance curves and examined possible genetic constraints by estimating the G-matrix. We observed a substantial amount of genetic variation for growth in different temperatures, and most genetic variation was for performance curve elevation. Contrary to common theoretical assumptions, we did not find strong evidence for genetic trade-offs for growth between hotter and colder temperatures. We also simulated short-term evolution of thermal performance curves of N. crassa, and suggest that they can have versatile responses to selection.

Marked Neurospora crassa Strains for Competition Experiments and Bayesian Methods for Fitness Estimates.

The filamentous fungus Neurospora crassa, a model microbial eukaryote, has a life cycle with many features that make it suitable for studying experimental evolution. However, it has lacked a general tool for estimating relative fitness of different strains in competition experiments. To remedy this need, we constructed N. crassa strains that contain a modified csr-1 locus and developed an assay for detecting the proportion of the marked strain using a post PCR high resolution melting assay. DNA extraction from spore samples can be performed on 96-well plates, followed by a PCR step, which allows many samples to be processed with ease. Furthermore, we suggest a Bayesian approach for estimating relative fitness from competition experiments that takes into account the uncertainty in measured strain proportions. We show that there is a fitness effect of the mating type locus, as mating type mat a has a higher competitive fitness than mat A The csr-1* marker also has a small fitness effect, but is still a suitable marker for competition experiments. As a proof of concept, we estimate the fitness effect of the qde-2 mutation, a gene in the RNA interference pathway, and show that its competitive fitness is lower than what would be expected from its mycelial growth rate alone.

Adaptation to environmental stress at different timescales.

Environments are changing rapidly, and to cope with these changes, organisms have to adapt. Adaptation can take many shapes and occur at different speeds, depending on the type of response, the trait, the population, and the environmental conditions. The biodiversity crisis that we are currently facing illustrates that numerous species and populations are not capable of adapting with sufficient speed to ongoing environmental changes. Here, we discuss current knowledge on the ability of animals and plants to adapt to environmental stress on different timescales, mainly focusing on thermal stress and ectotherms. We discuss within-generation responses that can be fast and induced within minutes or hours, evolutionary adaptations that are often slow and take several generations, and mechanisms that lay somewhere in between and that include epigenetic transgenerational effects. To understand and predict the impacts of environmental change and stress on biodiversity, we suggest that future studies should (1) have an increased focus on understanding the type and speed of responses to fast environmental changes; (2) focus on the importance of environmental fluctuations and the predictability of environmental conditions on adaptive capabilities, preferably in field studies encompassing several fitness components; and (3) look at ecosystem responses to environmental stress and their resilience when disturbed.

Effects of acclimation time and epigenetic mechanisms on growth of Neurospora in fluctuating environments.

Reaction norms or tolerance curves have often been used to predict how organisms deal with fluctuating environments. A potential drawback is that reaction norms measured in different constant environments may not capture all aspects of organismal responses to fluctuating environments. We examined growth of the filamentous fungus Neurospora crassa in fluctuating temperatures and tested if growth in fluctuating temperatures can be explained simply by the growth in different constant temperatures or if more complex models are needed. In addition, as previous studies on fluctuating environments have revealed that past temperatures that organisms have experienced can affect their response to current temperature, we tested the roles of different epigenetic mechanisms in response to fluctuating environments using different mutants. We found that growth of Neurospora can be predicted in fluctuating temperatures to some extent if acclimation times are taken into account in the model. Interestingly, while fluctuating environments have been linked with epigenetic responses, we found only some evidence of involvement of epigenetic mechanisms on tolerating fluctuating temperatures. Mutants which lacked H3K4 or H3K36 methylation had slightly impaired response to temperature fluctuations, in addition the H3K4 methylation mutant and a mutant in the RNA interference pathway had altered acclimation times.

Ecological plant epigenetics: Evidence from model and non-model species, and the way forward.

Growing evidence shows that epigenetic mechanisms contribute to complex traits, with implications across many fields of biology. In plant ecology, recent studies have attempted to merge ecological experiments with epigenetic analyses to elucidate the contribution of epigenetics to plant phenotypes, stress responses, adaptation to habitat, and range distributions. While there has been some progress in revealing the role of epigenetics in ecological processes, studies with non-model species have so far been limited to describing broad patterns based on anonymous markers of DNA methylation. In contrast, studies with model species have benefited from powerful genomic resources, which contribute to a more mechanistic understanding but have limited ecological realism. Understanding the significance of epigenetics for plant ecology requires increased transfer of knowledge and methods from model species research to genomes of evolutionarily divergent species, and examination of responses to complex natural environments at a more mechanistic level. This requires transforming genomics tools specifically for studying non-model species, which is challenging given the large and often polyploid genomes of plants. Collaboration among molecular geneticists, ecologists and bioinformaticians promises to enhance our understanding of the mutual links between genome function and ecological processes.

Epigenetic and Genetic Contributions to Adaptation in Chlamydomonas.

Epigenetic modifications, such as DNA methylation or histone modifications, can be transmitted between cellular or organismal generations. However, there are no experiments measuring their role in adaptation, so here we use experimental evolution to investigate how epigenetic variation can contribute to adaptation. We manipulated DNA methylation and histone acetylation in the unicellular green alga Chlamydomonas reinhardtii both genetically and chemically to change the amount of epigenetic variation generated or transmitted in adapting populations in three different environments (salt stress, phosphate starvation, and high CO2) for two hundred asexual generations. We find that reducing the amount of epigenetic variation available to populations can reduce adaptation in environments where it otherwise happens. From genomic and epigenomic sequences from a subset of the populations, we see changes in methylation patterns between the evolved populations over-represented in some functional categories of genes, which is consistent with some of these differences being adaptive. Based on whole genome sequencing of evolved clones, the majority of DNA methylation changes do not appear to be linked to cis-acting genetic mutations. Our results show that transgenerational epigenetic effects play a role in adaptive evolution, and suggest that the relationship between changes in methylation patterns and differences in evolutionary outcomes, at least for quantitative traits such as cell division rates, is complex.

Epigenetic Control of Phenotypic Plasticity in the Filamentous Fungus Neurospora crassa.

Phenotypic plasticity is the ability of a genotype to produce different phenotypes under different environmental or developmental conditions. Phenotypic plasticity is a ubiquitous feature of living organisms, and is typically based on variable patterns of gene expression. However, the mechanisms by which gene expression is influenced and regulated during plastic responses are poorly understood in most organisms. While modifications to DNA and histone proteins have been implicated as likely candidates for generating and regulating phenotypic plasticity, specific details of each modification and its mode of operation have remained largely unknown. In this study, we investigated how epigenetic mechanisms affect phenotypic plasticity in the filamentous fungus Neurospora crassa By measuring reaction norms of strains that are deficient in one of several key physiological processes, we show that epigenetic mechanisms play a role in homeostasis and phenotypic plasticity of the fungus across a range of controlled environments. In general, effects on plasticity are specific to an environment and mechanism, indicating that epigenetic regulation is context dependent and is not governed by general plasticity genes. Specifically, we found that, in Neurospora, histone methylation at H3K36 affected plastic response to high temperatures, H3K4 methylation affected plastic response to pH, but H3K27 methylation had no effect. Similarly, DNA methylation had only a small effect in response to sucrose. Histone deacetylation mainly decreased reaction norm elevation, as did genes involved in histone demethylation and acetylation. In contrast, the RNA interference pathway was involved in plastic responses to multiple environments.

Epigenetic mutations can both help and hinder adaptive evolution.

Epigenetic variation is being integrated into our understanding of adaptation, yet we lack models on how epigenetic mutations affect evolution that includes de novo genetic change. We model the effects of epigenetic mutations on the dynamics and endpoints of adaptive walks-a process where a series of beneficial mutations move a population towards a fitness optimum. We use an individual-based model of an asexual population, where mutational effects are drawn from Fisher's geometric model. We find cases where epigenetic mutations speed adaptation or result in populations with higher fitness. However, we also find cases where they slow adaptation or result in populations with lower fitness. The effect of epigenetic mutations on adaptive walks depends crucially on their stability and fitness effects relative to genetic mutations, with small-effect epigenetic mutations generally speeding adaptation, and epigenetic mutations with the same fitness effects as genetic mutations slowing adaptation. Our work reveals a complex relationship between epigenetic mutations and natural selection and highlights the need for empirical data.

Local evolution of seed flotation in Arabidopsis.

Arabidopsis seeds rapidly release hydrophilic polysaccharides from the seed coat on imbibition. These form a heavy mucilage layer around the seed that makes it sink in water. Fourteen natural Arabidopsis variants from central Asia and Scandinavia were identified with seeds that have modified mucilage release and float. Four of these have a novel mucilage phenotype with almost none of the released mucilage adhering to the seed and the absence of cellulose microfibrils. Mucilage release was modified in the variants by ten independent causal mutations in four different loci. Seven distinct mutations affected one locus, coding the MUM2 ß-D-galactosidase, and represent a striking example of allelic heterogeneity. The modification of mucilage release has thus evolved a number of times independently in two restricted geographical zones. All the natural mutants identified still accumulated mucilage polysaccharides in seed coat epidermal cells. Using nuclear magnetic resonance (NMR) relaxometry their production and retention was shown to reduce water mobility into internal seed tissues during imbibition, which would help to maintain seed buoyancy. Surprisingly, despite released mucilage being an excellent hydrogel it did not increase the rate of water uptake by internal seed tissues and is more likely to play a role in retaining water around the seed.

Genetic basis of adaptation in Arabidopsis thaliana: local adaptation at the seed dormancy QTL DOG1.

Local adaptation provides an opportunity to study the genetic basis of adaptation and investigate the allelic architecture of adaptive genes. We study delay of germination 1 (DOG1), a gene controlling natural variation in seed dormancy in Arabidopsis thaliana and investigate evolution of dormancy in 41 populations distributed in four regions separated by natural barriers. Using F(ST) and Q(ST) comparisons, we compare variation at DOG1 with neutral markers and quantitative variation in seed dormancy. Patterns of genetic differentiation among populations suggest that the gene DOG1 contributes to local adaptation. Although Q(ST) for seed dormancy is not different from F(ST) for neutral markers, a correlation with variation in summer precipitation supports that seed dormancy is adaptive. We characterize dormancy variation in several F(2) -populations and show that a series of functionally distinct alleles segregate at the DOG1 locus. Theoretical models have shown that the number and effect of alleles segregatin at quantitative trait loci (QTL) have important consequences for adaptation. Our results provide support to models postulating a large number of alleles at quantitative trait loci involved in adaptation.