Presence of a loner strain maintains cooperation and diversity in well-mixed bacterial communities.

Cooperation and diversity abound in nature despite cooperators risking exploitation from defectors and superior competitors displacing weaker ones. Understanding the persistence of cooperation and diversity is therefore a major problem for evolutionary ecology, especially in the context of well-mixed populations, where the potential for exploitation and displacement is greatest. Here, we demonstrate that a 'loner effect', described by economic game theorists, can maintain cooperation and diversity in real-world biological settings. We use mathematical models of public-good-producing bacteria to show that the presence of a loner strain, which produces an independent but relatively inefficient good, can lead to rock-paper-scissor dynamics, whereby cooperators outcompete loners, defectors outcompete cooperators and loners outcompete defectors. These model predictions are supported by our observations of evolutionary dynamics in well-mixed experimental communities of the bacterium Pseudomonas aeruginosa. We find that the coexistence of cooperators and defectors that produce and exploit, respectively, the iron-scavenging siderophore pyoverdine, is stabilized by the presence of loners with an independent iron-uptake mechanism. Our results establish the loner effect as a simple and general driver of cooperation and diversity in environments that would otherwise favour defection and the erosion of diversity.

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.

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.

Interaction effects of cell diffusion, cell density and public goods properties on the evolution of cooperation in digital microbes.

Microbial cooperation typically consists in the sharing of secreted metabolites (referred to as public goods) within the community. Although public goods generally promote population growth, they are also vulnerable to exploitation by cheating mutants, which no longer contribute, but still benefit from the public goods produced by others. Although previous studies have identified a number of key factors that prevent the spreading of cheaters, little is known about how these factors interact and jointly shape the evolution of microbial cooperation. Here, we address this issue by investigating the interaction effects of cell diffusion, cell density, public good diffusion and durability (factors known to individually influence costs and benefits of public goods production) on selection for cooperation. To be able to quantify these effects across a wide parameter space, we developed an individual-based simulation platform, consisting of digital cooperator and cheater bacteria inhabiting a finite two-dimensional continuous toroidal surface. Our simulations, which closely mimic microbial microcolony growth, revealed that: (i) either reduced cell diffusion (which keeps cooperators together) or reduced public good diffusion (which keeps the public goods closer to the producer) is not only essential but also sufficient for cooperation to be promoted; (ii) the sign of selection for or against cooperation can change as a function of cell density and in interaction with diffusion parameters; and (iii) increased public goods durability has opposing effects on the evolution of cooperation depending on the level of cell and public good diffusion. Our work highlights that interactions between key parameters of public goods cooperation give rise to complex fitness landscapes, a finding that calls for multifactorial approaches when studying microbial cooperation in natural systems.

Cost of cooperation rules selection for cheats in bacterial metapopulations.

Bacteria secrete a large variety of beneficial metabolites into the environment, which can be shared as public goods among producing bacteria, but also be exploited by nonproducing cheats. Here, we focus on cooperative production of iron-chelating molecules (siderophores) in the bacterium Pseudomonas aeruginosa to study how relevant ecological factors influence selection for cheating. We designed patch-structured metapopulations that allowed us introducing among-patch ecological variation. We found that cheating readily evolved in uniform iron-limited environments. This finding is explained by severe iron limitation demanding high siderophore-production efforts, which results in high metabolic costs accruing to cooperators, and thereby facilitates the spread of cheats. In contrast, we observed a significant reduction or even negation of selection for cheating in metapopulations where we introduced patches with increased iron availability and/or opportunities to recycle siderophores. These findings are compatible with the view that cheats are less likely to invade in environments that allow bacteria to reduce siderophore-production efforts, as this lowers the overall metabolic costs accruing to cooperators. Because we increased iron availability and siderophore recycling opportunities moderately, and only in some patches, our findings demonstrate that already-small local variations in ecological conditions as occurring in nature can significantly affect selection for public-goods secretion in microbes. In addition, we found that most (84.6%) of the evolved cheats were partially deficient for siderophore production and not loss-of-function mutants. Genetic considerations indicate that mutations leading to partial deficiency occur more frequent than mutations leading to loss of function, but also suggest that partially deficient mutants might often be the more competitive cheats.

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.

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.