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.

Adaptation to a limiting element involves mitigation of multiple elemental imbalances.

About 20 elements underlie biology and thus constrain biomass production. Recent systems-level observations indicate that altered supply of one element impacts the processing of most elements encompassing an organism (i.e. ionome). Little is known about the evolutionary tendencies of ionomes as populations adapt to distinct biogeochemical environments. We evolved the bacterium Serratia marcescens under five conditions (i.e. low carbon, nitrogen, phosphorus, iron or manganese) that limited the yield of the ancestor compared with replete medium, and measured the concentrations and use efficiency of these five, and five other elements. Both physiological responses of the ancestor, as well as evolutionary responses of descendants to experimental environments involved changes in the content and use efficiencies of the limiting element, and several others. Differences in coefficients of variation in elemental contents based on biological functions were evident, with those involved in biochemical building (C, N, P, S) varying least, followed by biochemical balance (Ca, K, Mg, Na), and biochemical catalysis (Fe, Mn). Finally, descendants evolved to mitigate elemental imbalances evident in the ancestor in response to limiting conditions. Understanding the tendencies of such ionomic responses will be useful to better forecast biological responses to geochemical changes.

Heterozygote advantage and pleiotropy contribute to intraspecific color trait variability.

The persistence of intrapopulation phenotypic variation typically requires some form of balancing selection because drift and directional selection eventually erode genetic variation. Heterozygote advantage remains a classic explanation for the maintenance of genetic variation in the face of selection. However, examples of heterozygote advantage, other than those associated with disease resistance, are rather uncommon. Across most of its distribution, males of the aposematic moth Arctia plantaginis have two hindwing phenotypes determined by a heritable one locus-two allele polymorphism (genotypes: WW/Wy = white morph, yy = yellow morph). Using genotyped moths, we show that the presence of one or two copies of the yellow allele affects several life-history traits. Reproductive output of both males and females and female mating success are negatively affected by two copies of the yellow allele. Females carrying one yellow allele (i.e., Wy) have higher fertility, hatching success, and offspring survival than either homozygote, thus leading to strong heterozygote advantage. Our results indicate strong female contribution especially at the postcopulatory stage in maintaining the color polymorphism. The interplay between heterozygote advantage, yellow allele pleiotropic effect, and morph-specific predation pressure may exert balancing selection on the color locus, suggesting that color polymorphism may be maintained through complex interactions between natural and sexual selection.

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.

Rich resource environment of fish farms facilitates phenotypic variation and virulence in an opportunistic fish pathogen.

Phenotypic variation is suggested to facilitate the persistence of environmentally growing pathogens under environmental change. Here, we hypothesized that the intensive farming environment induces higher phenotypic variation in microbial pathogens than natural environment, because of high stochasticity for growth and stronger survival selection compared to the natural environment. We tested the hypothesis with an opportunistic fish pathogen Flavobacterium columnare isolated either from fish farms or from natural waters. We measured growth parameters of two morphotypes from all isolates in different resource concentrations and two temperatures relevant for the occurrence of disease epidemics at farms and tested their virulence using a zebrafish (Danio rerio) infection model. According to our hypothesis, isolates originating from the fish farms had higher phenotypic variation in growth between the morphotypes than the isolates from natural waters. The difference was more pronounced in higher resource concentrations and the higher temperature, suggesting that phenotypic variation is driven by the exploitation of increased outside-host resources at farms. Phenotypic variation of virulence was not observed based on isolate origin but only based on morphotype. However, when in contact with the larger fish, the less virulent morphotype of some of the isolates also had high virulence. As the less virulent morphotype also had higher growth rate in outside-host resources, the results suggest that both morphotypes can contribute to F. columnare epidemics at fish farms, especially with current prospects of warming temperatures. Our results suggest that higher phenotypic variation per se does not lead to higher virulence, but that environmental conditions at fish farms could select isolates with high phenotypic variation in bacterial population and hence affect evolution in F. columnare at fish farms. Our results highlight the multifaceted effects of human-induced environmental alterations in shaping epidemiology and evolution in microbial pathogens.

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.

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.

Town population size and structuring into villages and households drive infectious disease risks in pre-healthcare Finland.

Social life is often considered to cost in terms of increased parasite or pathogen risk. However, evidence for this in the wild remains equivocal, possibly because populations and social groups are often structured, which affects the local transmission and extinction of diseases. We test how the structuring of towns into villages and households influenced the risk of dying from three easily diagnosable infectious diseases-smallpox, pertussis and measles-using a novel dataset covering almost all of Finland in the pre-healthcare era (1800-1850). Consistent with previous results, the risk of dying from all three diseases increased with the local population size. However, the division of towns into a larger number of villages decreased the risk of dying from smallpox and to some extent of pertussis but it slightly increased the risk for measles. Dividing towns into a larger number of households increased the length of the epidemic for all three diseases and led to the expected slower spread of the infection. However, this could be seen only when local population sizes were small. Our results indicate that the effect of population structure on epidemics, disease or parasite risk varies between pathogens and population sizes, hence lowering the ability to generalize the consequences of epidemics in spatially structured populations, and mapping the costs of social life, via parasites and diseases.

Systematic Comparison of Epidemic and Non-Epidemic Carbapenem Resistant Klebsiella pneumoniae Strains.

Over the past few decades, extensively drug resistant (XDR) resistant Klebsiella pneumoniae has become a notable burden to healthcare all over the world. Especially carbapenemase-producing strains are problematic due to their capability to withstand even last resort antibiotics. Some sequence types (STs) of K. pneumoniae are significantly more prevalent in hospital settings in comparison to other equally resistant strains. This provokes the question whether or not there are phenotypic characteristics that may render certain K. pneumoniae more suitable for epidemic dispersal between patients, hospitals, and different environments. In this study, we selected seven epidemic and non-epidemic carbapenem resistant K. pneumoniae isolates for extensive systematic characterization for phenotypic and genotypic qualities in order to identify potential factors that precede or emerge from epidemic successfulness. Studied characteristics include growth rates and densities in different conditions (media, temperature, pH, resource levels), tolerance to alcohol and drought, inhibition between strains, ability to compensate pH, as well as various genomic features. Overall, there are clear differences between isolates, yet, only drought tolerance was found to notably associate with non-epidemic K. pneumoniae strains. We further report a preliminary study on the potential to control K. pneumoniae ST11 with an antimicrobial component produced by a non-epidemic K. pneumoniae. This component initially restricts bacterial growth, but stable resistance develops rapidly in vitro.

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.

Heat hardening capacity in Drosophila melanogaster is life stage-specific and juveniles show the highest plasticity.

Variations in stress resistance and adaptive plastic responses during ontogeny have rarely been addressed, despite the possibility that differences between life stages can affect species' range margins and thermal tolerance. Here, we assessed the thermal sensitivity and hardening capacity of Drosophila melanogaster across developmental stages from larval to the adult stage. We observed strong differences between life stages in heat resistance, with adults being most heat resistant followed by puparia, pupae and larvae. The impact of heat hardening (1 h at 35°C) on heat resistance changed during ontogeny, with the highest positive effect of hardening observed in puparia and pupae and the lowest in adults. These results suggest that immobile life stages ( puparia and pupae) have evolved high plasticity in upper thermal limits whereas adults and larvae rely more on behavioural responses to heat stress allowing them to escape from extreme high temperatures. While most studies on the plasticity of heat resistance in ectotherms have focused on the adult life stage, our findings emphasize the crucial importance of juvenile life stages of arthropods in understanding the thermal biology and life stage-specific physiological responses to variable and stressful high temperatures. Failure to acknowledge this complication might lead to biased estimates of species' ability to cope with environmental changes, such as climate change.

Invasion triple trouble: environmental fluctuations, fluctuation-adapted invaders and fluctuation-mal-adapted communities all govern invasion success.

BACKGROUND: It has been suggested that climate change will lead to increased environmental fluctuations, which will undoubtedly have evolutionary consequences for all biota. For instance, fluctuations can directly increase the risk of invasions of alien species into new areas, as these species have repeatedly been proposed to benefit from disturbances. At the same time increased environmental fluctuations may also select for better invaders. However, selection by fluctuations may also influence the resistance of communities to invasions, which has rarely been tested. We tested eco-evolutionary dynamics of invasion with bacterial clones, evolved either in constant or fluctuating temperatures, and conducted experimental invasions in both conditions. RESULTS: We found clear evidence that ecological fluctuations, as well as adaptation to fluctuations by both the invader and community, all affected invasions, but played different roles at different stages of invasion. Ecological fluctuations clearly promoted invasions, especially into fluctuation mal-adapted communities. The evolutionary background of the invader played a smaller role. CONCLUSIONS: Our results indicate that climate change associated disturbances can directly increase the risk of invasions by altering ecological conditions during invasions, as well as via the evolution of both the invader and communities. Our experiment provides novel information on the complex consequences of climate change on invasions in general, and also charts risk factors associated with the spread of environmentally growing opportunistic pathogens.

Broad thermal tolerance is negatively correlated with virulence in an opportunistic bacterial pathogen.

Predicting the effects of global increase in temperatures on disease virulence is challenging, especially for environmental opportunistic bacteria, because pathogen fitness may be differentially affected by temperature within and outside host environment. So far, there is very little empirical evidence on the connections between optimal temperature range and virulence in environmentally growing pathogens. Here, we explored whether the virulence of an environmentally growing opportunistic fish pathogen, Flavobacterium columnare, is malleable to evolutionary changes via correlated selection on thermal tolerance. To this end, we experimentally quantified the thermal performance curves (TPCs) for maximum biomass yield of 49 F. columnare isolates from eight different geographic locations in Finland over ten years (2003-2012). We also characterized virulence profiles of these strains in a zebra fish (Danio rerio) infection model. We show that virulence among the strains increased over the years, but thermal generalism, and in particular tolerance to higher temperatures, was negatively associated with virulence. Our data suggest that temperature has a strong effect on the pathogen genetic diversity and therefore presumably also on disease dynamics. However, the observed increase in frequency and severity of F. columnare epidemics over the last decade cannot be directly linked to bacterial evolution due to increased mean temperature, but is most likely associated with factors related to increased length of growing season, or other time-dependent change in environment. Our study demonstrates that complex interactions between the host, the pathogen and the environment influence disease virulence of an environmentally growing opportunistic pathogen.

Ecological conditions alter cooperative behaviour and its costs in a chemically defended sawfly.

The evolution of cooperation and social behaviour is often studied in isolation from the ecology of organisms. Yet, the selective environment under which individuals evolve is much more complex in nature, consisting of ecological and abiotic interactions in addition to social ones. Here, we measured the life-history costs of cooperative chemical defence in a gregarious social herbivore, Diprion pini pine sawfly larvae, and how these costs vary under different ecological conditions. We ran a rearing experiment where we manipulated diet (resin content) and attack intensity by repeatedly harassing larvae to produce a chemical defence. We show that forcing individuals to allocate more to cooperative defence (high attack intensity) incurred a clear cost by decreasing individual survival and potency of chemical defence. Cooperative behaviour and the magnitude of its costs were further shaped by host plant quality. The number of individuals participating in group defence, immune responses and female growth decreased on a high resin diet under high attack intensity. We also found some benefits of cheating: non-defending males had higher growth rates across treatments. Taken together, these results suggest that ecological interactions can shape the adaptive value of cooperative behaviour and maintain variation in the frequency of cooperation and cheating.

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.

Adaptation to fluctuations in temperature by nine species of bacteria.

Rapid environmental fluctuations are ubiquitous in the wild, yet majority of experimental studies mostly consider effects of slow fluctuations on organism. To test the evolutionary consequences of fast fluctuations, we conducted nine independent experimental evolution experiments with bacteria. Experimental conditions were same for all species, and we allowed them to evolve either in fluctuating temperature alternating rapidly between 20°C and 40°C or at constant 30°C temperature. After experimental evolution, we tested the performance of the clones in both rapid fluctuation and in constant environments (20°C, 30°C and 40°C). Results from experiments on these nine species were combined meta-analytically. We found that overall the clones evolved in the fluctuating environment had evolved better efficiency in tolerating fluctuations (i.e., they had higher yield in fluctuating conditions) than the clones evolved in the constant environment. However, we did not find any evidence that fluctuation-adapted clones would have evolved better tolerance to any measured constant environments (20°C, 30°C, and 40°C). Our results back up recent empirical findings reporting that it is hard to predict adaptations to fast fluctuations using tolerance curves.

Application of high resolution melting assay (HRM) to study temperature-dependent intraspecific competition in a pathogenic bacterium.

Studies on species' responses to climate change have focused largely on the direct effect of abiotic factors and in particular temperature, neglecting the effects of biotic interactions in determining the outcome of climate change projections. Many microbes rely on strong interference competition; hence the fitness of many pathogenic bacteria could be a function of both their growth properties and intraspecific competition. However, due to technical challenges in distinguishing and tracking individual strains, experimental evidence on intraspecific competition has been limited so far. Here, we developed a robust application of the high-resolution melting (HRM) assay to study head-to-head competition between mixed genotype co-cultures of a waterborne bacterial pathogen of fish, Flavobacterium columnare, at two different temperatures. We found that competition outcome in liquid cultures seemed to be well predicted by growth yield of isolated strains, but was mostly inconsistent with interference competition results measured in inhibition tests on solid agar, especially as no growth inhibition between strain pairs was detected at the higher temperature. These results suggest that, for a given temperature, the factors driving competition outcome differ between liquid and solid environments.

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.