Cheating and resistance to cheating in natural populations of the bacterium Pseudomonas fluorescens

Bacteria perform cooperative behaviors that are exploitable by noncooperative cheats, and cheats frequently arise and coexist with cooperators in laboratory microcosms. However, evidence of competitive dynamics between cooperators and cheats in nature remains limited. Using the production of pyoverdine, an iron‐scavenging molecule, and natural soil populations of Pseudomonas fluorescens, we found that (1) nonproducers are present in the population; (2) they co‐occur (<1cm³) with pyoverdine producers; (3) they retain functional pyoverdine receptors; and (4) they can use the pyoverdine of on average 52% of producers. This suggests nonproducers can potentially act as social cheats in soil: utilizing the pyoverdine of others while producing little or none themselves. However, we found considerable variation in the extent to which nonproducers can exploit producers, as some isolates appear to produce exclusive forms of pyoverdine or kill nonproducers with toxins. We examined the consequences of this variation using theoretical modeling. We found variance in exploitability leads to some cheats gaining increased fitness benefits and others decreased benefits. However, the absolute gain in fitness from high exploitation is lower than the drop in fitness from low exploitation, decreasing the mean fitness of cheats and subsequently lowering the proportion of cheats maintained in the population. Our results suggest that although cooperator‐cheat dynamics can occur in soil, a range of mechanisms can prevent nonproducers from exploiting producers.     

Co‐evolutionary dynamics between public good producers and cheats in the bacterium Pseudomonas aeruginosa

The production of beneficial public goods is common in the microbial world, and so is cheating – the exploitation of public goods by nonproducing mutants. Here, we examine co‐evolutionary dynamics between cooperators and cheats and ask whether cooperators can evolve strategies to reduce the burden of exploitation, and whether cheats in turn can improve their exploitation abilities. We evolved cooperators of the bacterium Pseudomonas aeruginosa, producing the shareable iron‐scavenging siderophore pyoverdine, together with cheats, defective in pyoverdine production but proficient in uptake. We found that cooperators managed to co‐exist with cheats in 56% of all replicates over approximately 150 generations of experimental evolution. Growth and competition assays revealed that co‐existence was fostered by a combination of general adaptions to the media and specific adaptions to the co‐evolving opponent. Phenotypic screening and whole‐genome resequencing of evolved clones confirmed this pattern, and suggest that cooperators became less exploitable by cheats because they significantly reduced their pyoverdine investment. Cheats, meanwhile, improved exploitation efficiency through mutations blocking the costly pyoverdine‐signalling pathway. Moreover, cooperators and cheats evolved reduced motility, a pattern that likely represents adaptation to laboratory conditions, but at the same time also affects social interactions by reducing strain mixing and pyoverdine sharing. Overall, we observed parallel evolution, where co‐existence of cooperators and cheats was enabled by a combination of adaptations to the abiotic and social environment and their interactions.     

Repression of competition favours cooperation: experimental evidence from bacteria

Repression of competition (RC) within social groups has been suggested as a key mechanism driving the evolution of cooperation, because it aligns the individual’s proximate interest with the interest of the group. Despite its enormous potential for explaining cooperation across all levels of biological organization, ranging from fair meiosis, to policing in insect societies, to sanctions in mutualistic interactions between species, there has been no direct experimental test of whether RC favours the spread of cooperators in a well‐mixed population with cheats. To address this, we carried out an experimental evolution study to test the effect of RC upon a cooperative trait – the production of iron‐scavenging siderophore molecules – in the bacterium Pseudomonas aeruginosa. We found that cooperation was favoured when competition between siderophore producers and nonsiderophore‐producing cheats was repressed, but not in a treatment where competition between the two strains was permitted. We further show that RC altered the cost of cooperation, but did not affect the relatedness among interacting individuals. This confirms that RC per se, as opposed to increased relatedness, has driven the observed increase in bacterial cooperation.     

Evolutionary dynamics of interlinked public goods traits: an experimental study of siderophore production in Pseudomonas aeruginosa

Public goods cooperation is common in microbes, and there is much interest in understanding how such traits evolve. Research in recent years has identified several important factors that shape the evolutionary dynamics of such systems, yet few studies have investigated scenarios involving interactions between multiple public goods. Here, we offer general predictions about the evolutionary trajectories of two public goods traits having positive, negative or neutral regulatory influence on one another's expression, and we report on a test of some of our predictions in the context of Pseudomonas aeruginosa's production of two interlinked iron‐scavenging siderophores. First, we confirmed that both pyoverdine and pyochelin siderophores do operate as public goods under appropriate environmental conditions. We then tracked their production in lines experimentally evolved under different iron‐limitation regimes known to favour different siderophore expression profiles. Under strong iron limitation, where pyoverdine represses pyochelin, we saw a decline in pyoverdine and a concomitant increase in pyochelin – consistent with expansion of pyoverdine‐defective cheats derepressed for pyochelin. Under moderate iron limitation, pyochelin declined – again consistent with an expected cheat invasion scenario – but there was no concomitant shift in pyoverdine because cross‐suppression between the traits is unidirectional only. Alternating exposure to strong and moderate iron limitation caused qualitatively similar though lesser shifts compared to the constant‐environment regimes. Our results confirm that the regulatory interconnections between public goods traits can significantly modulate the course of evolution, yet also suggest how we can start to predict the impacts such complexities will have on phenotypic divergence and community stability.     

No effect of intraspecific relatedness on public goods cooperation in a complex community

Many organisms—notably microbes—are embedded within complex communities where cooperative behaviors in the form of excreted public goods can benefit other species. Under such circumstances, intraspecific interactions are likely to be less important in driving the evolution of cooperation. We first illustrate this idea with a simple theoretical model, showing that relatedness—the extent to which individuals with the same cooperative alleles interact with each other—has a reduced impact on the evolution of cooperation when public goods are shared between species. We test this empirically using strain of Pseudomonas aeruginosa that vary in their production of metal‐chelating siderophores in copper contaminated compost (an interspecific public good). We show that nonsiderophore producers grow poorly relative to producers under high relatedness, but this cost can be alleviated by the presence of the isogenic producer (low relatedness) and/or the compost microbial community. Hence, relatedness can become unimportant when public goods provide interspecific benefits.     

Evolution of altruistic cooperation among nascent multicellular organisms

Cooperation is a classic solution to hostile environments that limit individual survival. In extreme cases this may lead to the evolution of new types of biological individuals (e.g., eusocial super‐organisms). We examined the potential for interindividual cooperation to evolve via experimental evolution, challenging nascent multicellular “snowflake yeast” with an environment in which solitary multicellular clusters experienced low survival. In response, snowflake yeast evolved to form cooperative groups composed of thousands of multicellular clusters that typically survive selection. Group formation occurred through the creation of protein aggregates, only arising in strains with high (>2%) rates of cell death. Nonetheless, it was adaptive and repeatable, although ultimately evolutionarily unstable. Extracellular protein aggregates act as a common good, as they can be exploited by cheats that do not contribute to aggregate production. These results highlight the importance of group formation as a mechanism for surviving environmental stress, and underscore the remarkable ease with which even simple multicellular entities may evolve—and lose—novel social traits.     

Phenotypic plasticity of a cooperative behaviour in bacteria

There is strong evidence that natural selection can favour phenotypic plasticity as a mechanism to maximize fitness in animals. Here, we aim to investigate phenotypic plasticity of a cooperative trait in bacteria – the production of an iron‐scavenging molecule (pyoverdin) by Pseudomonas aeruginosa. Pyoverdin production is metabolically costly to the individual cell, but provides a benefit to the local group and can potentially be exploited by nonpyoverdin‐producing cheats. Here, we subject bacteria to changes in the social environment in media with different iron availabilities and test whether cells can adjust pyoverdin production in response to these changes. We found that pyoverdin production per cell significantly decreased at higher cell densities and increased in the presence of cheats. This phenotypic plasticity significantly influenced the costs and benefits of cooperation. Specifically, the investment of resources into pyoverdin production was reduced in iron‐rich environments and at high cell densities, but increased under iron limitation, and when pyoverdin was exploited by cheats. Our study demonstrates that phenotypic plasticity in a cooperative trait as a response to changes in the environment occurs in even the simplest of organisms, a bacterium.     

Hypermutability impedes cooperation in pathogenic bacteria

When the supply of beneficial mutations limits adaptation, bacterial mutator alleles can reach high frequencies by hitchhiking with advantageous mutations. However, when populations are well adapted to their environments, the increased rate of deleterious mutations makes hypermutability selectively disadvantageous. Here, we consider a further cost of hypermutability: its potential to break down cooperation (group-beneficial behavior that is costly to the individual). This probably occurs for three reasons. First, an increased rate at which 'cheating' genotypes are generated; second, an increased probability of producing efficient cheats; and third, a decrease in relatedness (not addressed in the present study). We used Pseudomonas aeruginosa's production of extracellular iron-scavenging molecules, siderophores, to determine if cheating evolved more readily in mutator populations. Siderophore production is costly to individual bacteria but benefits all nearby cells. Siderophore-deficient cheats therefore have a selective advantage within populations. We observed the de novo evolution and subsequent increase in frequency of siderophore cheats within both wild-type and mutator populations for 200 generations. Cheats appeared and increased in frequency more rapidly in mutator populations. The presence of cheats was costly to the group, as shown by a negative correlation between cheat frequency and population density.     

Dynamic social behaviour in a bacterium: Pseudomonas aeruginosa partially compensates for siderophore loss to cheats

Cooperation underlies diverse phenomena including the origins of multicellular life, human behaviour in economic markets and the mechanisms by which pathogenic bacteria cause disease. Experiments with microorganisms have advanced our understanding of how, when and why cooperation evolves, but the extent to which microbial cooperation can recapitulate aspects of animal behaviour is debated. For instance, understanding the evolution of behavioural response rules (how should one individual respond to another's decision to cooperate or defect?) is a key part of social evolution theory, but the possible existence of such rules in social microbes has not been explored. In one specific context (biparental care in animals), cooperation is maintained if individuals respond to a partner's defection by increasing their own investment into cooperation, but not so much that this fully compensates for the defector's lack of investment. This is termed ‘partial compensation’. Here, I show that partial compensation for the presence of noncooperating ‘cheats’ is also observed in a microbial social behaviour: the cooperative production of iron‐scavenging siderophores by the bacterium Pseudomonas aeruginosa. A period of evolution in the presence of cheats maintains this response, whereas evolution in the absence of cheats leads to a loss of compensatory behaviour. These results demonstrate (i) the remarkable flexibility of bacterial social behaviour, (ii) the potential generality of partial compensation as a social response rule and (iii) the need for mathematical models to explore the evolution of response rules in multi‐player social interactions.     

The social evolution of dispersal with public goods cooperation

Selection can favour the evolution of individually costly dispersal if this alleviates competition between relatives. However, conditions that favour altruistic dispersal also mediate selection for other social behaviours, such as public goods cooperation, which in turn is likely to mediate dispersal evolution. Here, we investigate – both experimentally (using bacteria) and theoretically – how social habitat heterogeneity (i.e. the distribution of public goods cooperators and cheats) affects the evolution of dispersal. In addition to recovering the well‐known theoretical result that the optimal level of dispersal increases with genetic relatedness of patch mates, we find both mathematically and experimentally that dispersal is always favoured when average patch occupancy is low, but when average patch occupancy is high, the presence of public goods cheats greatly alters selection for dispersal. Specifically, when public goods cheats are localized to the home patch, higher dispersal rates are favoured, but when cheats are present throughout available patches, lower dispersal rates are favoured. These results highlight the importance of other social traits in driving dispersal evolution.     

An experimental test of whether cheating is context dependent

Microbial cells rely on cooperative behaviours that can breakdown as a result of exploitation by cheats. Recent work on cheating in microbes, however, has produced examples of populations benefiting from the presence of cheats and/or cooperative behaviours being maintained despite the presence of cheats. These observations have been presented as evidence for selection favouring cheating at the population level. This apparent contradiction arises when cheating is defined simply by the reduced expression of a cooperative trait and not in terms of the social costs and benefits of the trait under investigation. Here, we use two social traits, quorum sensing and iron‐scavenging siderophore production in Pseudomonas aeruginosa, to illustrate the importance of defining cheating by the social costs and benefits. We show that whether a strain is a cheat depends on the costs and benefits associated with the social and abiotic environment and not the absolute expression of a cooperative trait.     

Pyoverdin cheats fail to invade bacterial populations in stationary phase

Microbes engage in cooperative behaviours by producing and secreting public goods, the benefits of which are shared among cells, and are therefore susceptible to exploitation by nonproducing cheats. In nature, bacteria are not typically colonizing sterile, rich environments in contrast to laboratory experiments, which involve inoculating sterile culture with few bacterial cells that then race to fill the available niche. Here, we study the potential implications of this difference, using the production of pyoverdin, an iron‐scavenging siderophore that acts as a public good in the bacteria Pseudomonas aeruginosa. We show that (1) nonproducers are able to invade cultures of producers when added at the start of growth or during early exponential growth phase, but not during late exponential or stationary phase; (2) the producer strain does not produce pyoverdin in the late exponential and stationary phases and so is not paying the cost of cooperating during those phases. These results suggest that whether a nonproducing mutant can invade will depend upon when the mutation arises, as well as the population structure, and raise a potential difficulty with the use of antimicrobial treatment strategies that propose to exploit the invasive abilities of cheats.     

An experimental study of strong reciprocity in bacteria

Strong reciprocity, whereby cooperators punish non-cooperators, may help to explain the evolutionary success of cooperative behaviours. However, theory suggests that selection for strong reciprocity can depend upon tight genetic linkage between cooperation and punishment, to avoid the strategy being outcompeted by non-punishing cooperators. We tested this hypothesis using experimental populations of the bacterium Pseudomonas aeruginosa, which cooperate by producing iron-scavenging siderophores and, in this context, punish non-cooperators with toxins. Consistent with theory, we show that cooperative punishers can indeed invade cheats, but only when the traits are tightly linked. These results emphasize that punishment is only likely to be favoured when the punishment itself leads to a direct or indirect fitness benefit to the actor.     

COEVOLUTION BETWEEN COOPERATORS AND CHEATS IN A MICROBIAL SYSTEM

In many circumstances organisms invest in cooperative activities to increase their mutual fitness but are susceptible to cheats that obtain the benefits of cooperation without investment. Natural selection may favor cooperators that resist cheats, and cheats that avoid such resistance; in theory the coevolutionary interaction may be sustained and dynamic. Here, we report evidence of antagonistic coevolution between cooperators and cheats involved in biofilm formation by Pseudomonas fluorescens bacteria. Two distinct phenotypes occur in static culture tubes: one that can form a biofilm at the air–broth interface and thus obtain improved access to oxygen, and one that colonizes the broth phase but which can also invade, and weaken, the biofilm produced by the other type. Over serial passage, biofilm producers (considered here as cooperators) evolve to become better at resisting invasion, and biofilm nonproducers (cheats) evolve to be more efficient invaders. Each type has higher performance (resistance in the case of cooperators and biofilm invasion for cheats) in competition with isolates of the other type from their past compared to that from their future, indicating a dynamic coevolutionary interaction. Such coevolution may have important consequences for the maintenance of cooperation.     

Short‐Term Variation in the Level of Cooperation in the Cleaner Wrasse Labroides dimidiatus: Implications for the Role of Potential Stressors

There is a wealth of game theoretical approaches to the evolution and maintenance of cooperation between unrelated individuals and accumulating empirical tests of these models. This contrasts strongly with our lack of knowledge on proximate causes of cooperative behaviour. Marine cleaning mutualism has been used as a model system to address functional aspects of conflict resolution: client reef fish benefit from cleaning interactions through parasite removal, but cleaner fish Labroides dimidiatus prefer client mucus. Hence, feeding against their preference represents cooperative behaviour in cleaners. Cleaners regularly cheat non‐predatory clients while they rarely cheat predatory clients. Here, we asked how precisely cleaners can adjust service quality from one interaction to the next. We found that non‐predatory clients receive a better service if the previous client was a predator than if the previous client was a non‐predator. In a related laboratory experiment, a hand‐net used as a stressor resulted in cleaners feeding more against their preference in subsequent interactions. The combination of the cleaners’ behaviour in the two studies shows that the cleaners’ service quality for a given client species is not fixed, but it can be manipulated. The results suggest that short‐term stress is one factor that causes cleaners to increase their levels of cooperation, a hypothesis that is amenable to further experiments manipulating the endocrine system.     

Neoproterozoic 'snowball Earth' glaciations and the evolution of altruism

We hypothesize that a demographic and ecological effect of Neoproterozoic ‘snowball Earth’ glaciations was to increase the fitness of group‐level traits and consequently the likelihood of the evolution of macroscopic form. Extreme and repeated founder effects raised genetic relatedness – and therefore the influence of kin selection on the individuals within a group. This was permissive for the evolution of some highly costly altruistic traits, including those for macroscopic differentiation. In some eukaryotic species, the harsh and fluctuating abiotic conditions made a macroscopic physiology advantageous, perhaps necessary, for collective survival. This caused population‐wide group viability selection, whereby non‐altruist ‘cheat’ genotypes killed the groups they were in, and therefore themselves, by reaching fixation. Furthermore, dispersal between refugia would reach zero under anything near a ‘hard snowball’, which would protect altruists at high local frequency from the influx of cheats from neighbouring groups. We illustrate our hypothesis analytically and with a simple spatial model. We show how removal of between‐group dispersal, in a population with initial between‐group variation in cheat frequency, causes the relative frequency of altruists to increase while the population as a whole decreases in size, as a result of group death caused by cheat invasion. This may be of particular relevance to animal multicellularity because irreversible differentiation (highly altruistic in that it imposes a high fitness cost on the individual cell) is more prevalent than in other multicellular eukaryotes. The relevance of our hypothesis should be scaled by any future consensus on the severity of snowball Earth, but it is theoretically plausible that global‐scale glaciations had a systematic influence on the level of selection during Earth history.     

The ecology of cooperative breeding behaviour

Ecology is a fundamental driving force for the evolutionary transition from solitary living to breeding cooperatively in groups. However, the fact that both benign and harsh, as well as stable and fluctuating, environments can favour the evolution of cooperative breeding behaviour constitutes a paradox of environmental quality and sociality. Here, we propose a new model – the dual benefits framework – for resolving this paradox. Our framework distinguishes between two categories of grouping benefits – resource defence benefits that derive from group‐defended critical resources and collective action benefits that result from social cooperation among group members – and uses insider–outsider conflict theory to simultaneously consider the interests of current group members (insiders) and potential joiners (outsiders) in determining optimal group size. We argue that the different grouping benefits realised from resource defence and collective action profoundly affect insider–outsider conflict resolution, resulting in predictable differences in the per capita productivity, stable group size, kin structure and stability of the social group. We also suggest that different types of environmental variation (spatial vs. temporal) select for societies that form because of the different grouping benefits, thus helping to resolve the paradox of why cooperative breeding evolves in such different types of environments.     

Resource abundance and the critical transition to cooperation

Cooperation is abundant in nature, occurring at all levels of biological complexity. Yet cooperation is continually threatened by subversion from noncooperating cheaters. Previous studies have shown that cooperation can nevertheless be maintained when the benefits that cooperation provides to relatives outweigh the associated costs. These fitness costs and benefits are not fixed properties, but can be affected by the environment in which populations reside. Here, we describe how one environmental factor, resource abundance, decisively affects the evolution of cooperative public goods production in two independent evolving systems. In the Avida digital evolution platform, populations evolved in environments with different levels of a required resource, whereas populations of Vibrio cholerae evolved in the presence of different nutrient concentrations. In both systems, cooperators and cheaters co‐existed stably in resource‐rich environments, whereas cheaters dominated in resource‐poor environments. These two outcomes were separated by a sharp transition that occurred at a critical level of resource. These results offer new insights into how the environment affects the evolution of cooperation and highlight the challenges that populations of cooperators face when they experience environmental change.     

Evolution of cooperative cross-feeding could be less challenging than originally thought

The act of cross-feeding whereby unrelated species exchange nutrients is a common feature of microbial interactions and could be considered a form of reciprocal altruism or reciprocal cooperation. Past theoretical work suggests that the evolution of cooperative cross-feeding in nature may be more challenging than for other types of cooperation. Here we re-evaluate a mathematical model used previously to study persistence of cross-feeding and conclude that the maintenance of cross-feeding interactions could be favoured for a larger parameter ranges than formerly observed. Strikingly, we also find that large populations of cross-feeders are not necessarily vulnerable to extinction from an initially small number of cheats who receive the benefit of cross-feeding but do not reciprocate in this cooperative interaction. This could explain the widespread cooperative cross-feeding observed in natural populations.     

Competitive interactions in Escherichia coli populations: the role of bacteriocins

Explaining the coexistence of competing species is a major challenge in community ecology. In bacterial systems, competition is often driven by the production of bacteriocins, which are narrow-spectrum proteinaceous toxins that serve to kill closely related species, providing the producer better access to limited resources. Bacteriocin producers have been shown to competitively exclude sensitive, nonproducing strains. However, the dynamics between bacteriocin producers, each lethal to its competitor, are largely unknown. In this study, we used in vitro, in vivo and in silico models to study competitive interactions between bacteriocin producers. Two Escherichia coli strains were generated, each carrying a DNA-degrading bacteriocin (colicins E2 and E7). Using reporter-gene assays, we showed that each DNase bacteriocin is not only lethal to its opponent but, at lower doses, can also induce the expression of its opponent''s toxin. In a well-mixed habitat, the E2 producer outcompeted its adversary; however, in structured environments (on plates or in mice colons), the two producers coexisted in a spatially ‘frozen'' pattern. Coexistence occurred when the producers were initiated with a clumped spatial distribution. This suggests that a ‘clump'' of each producer can block invasion of the other producer. Agent-based simulation of bacteriocin-mediated competition further showed that mutual exclusion in a structured environment is a relatively robust result. These models imply that colicin-mediated colicin induction enables producers to successfully compete and defend their niche against invaders. This suggests that localized interactions between producers of DNA-degrading toxins can lead to stable coexistence of heterogeneously distributed strains within the bacterial community and to the maintenance of diversity.