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Transposable temperate phages randomly insert into bacterial genomes, providing increased supply and altered spectra of mutations available to selection, thus opening alternative evolutionary trajectories. Transposable phages accelerate bacterial adaptation to new environments, but their effect on adaptation to the social environment is unclear. Using experimental evolution of Pseudomonas aeruginosa in iron-limited and iron-rich environments, where the cost of producing cooperative iron-chelating siderophores is high and low, respectively, we show that transposable phages promote divergence into extreme siderophore production phenotypes. Iron-limited populations with transposable phages evolved siderophore overproducing clones alongside siderophore non-producing cheats. Low siderophore production was associated with parallel mutations in pvd genes, encoding pyoverdine biosynthesis, and pqs genes, encoding quinolone signalling, while high siderophore production was associated with parallel mutations in phenazine-associated gene clusters. Notably, some of these parallel mutations were caused by phage insertional inactivation. These data suggest that transposable phages, which are widespread in microbial communities, can mediate the evolutionary divergence of social strategies.

Pathogenic bacteria engage in social interactions to colonize hosts, which include quorum-sensing-mediated communication and the secretion of virulence factors that can be shared as "public goods" between individuals. While in-vitro studies demonstrated that cooperative individuals can be displaced by "cheating" mutants freeriding on social acts, we know less about social interactions in infections. Here, we developed a live imaging system to track virulence factor expression and social strain interactions in the human pathogen Pseudomonas aeruginosa colonizing the gut of Caenorhabditis elegans. We found that shareable siderophores and quorum-sensing systems are expressed during infections, affect host gut colonization, and benefit non-producers. However, non-producers were unable to successfully cheat and outcompete producers. Our results indicate that the limited success of cheats is due to a combination of the down-regulation of virulence factors over the course of the infection, the fact that each virulence factor examined contributed to but was not essential for host colonization, and the potential for negative frequency-dependent selection. Our findings shed new light on bacterial social interactions in infections and reveal potential limits of therapeutic approaches that aim to capitalize on social dynamics between strains for infection control.

Policing occurs in insect, animal and human societies, where it evolved as a mechanism maintaining cooperation. Recently, it has been suggested that policing might even be relevant in enforcing cooperation in much simpler organisms such as bacteria. Here, we used individual-based modelling to develop an evolutionary concept for policing in bacteria and identify the conditions under which it can be adaptive. We modelled interactions between cooperators, producing a beneficial public good, cheaters, exploiting the public good without contributing to it, and public good-producing policers that secrete a toxin to selectively target cheaters. We found that toxin-mediated policing is favoured when (a) toxins are potent and durable, (b) toxins are cheap to produce, (c) cell and public good diffusion is intermediate, and (d) toxins diffuse farther than the public good. Although our simulations identify the parameter space where toxin-mediated policing can evolve, we further found that policing decays when the genetic linkage between public good and toxin production breaks. This is because policing is itself a public good, offering protection to toxin-resistant mutants that still produce public goods, yet no longer invest in toxins. Our work thus highlights that not only specific environmental conditions are required for toxin-mediated policing to evolve, but also strong genetic linkage between the expression of public goods, toxins and toxin resistance is essential for this mechanism to remain evolutionarily stable in the long run., (© 2019 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2019 European Society For Evolutionary Biology.)

How unicellular organisms optimize the production of compounds is a fundamental biological question. While it is typically thought that production is optimized at the individual-cell level, secreted compounds could also allow for optimization at the group level, leading to a division of labor where a subset of cells produces and shares the compound with everyone. Using mathematical modeling, we show that the evolution of such division of labor depends on the cost function of compound production. Specifically, for any trait with saturating benefits, linear costs promote the evolution of uniform production levels across cells. Conversely, production costs that diminish with higher output levels favor the evolution of specialization-especially when compound shareability is high. When experimentally testing these predictions with pyoverdine, a secreted iron-scavenging compound produced by Pseudomonas aeruginosa, we found linear costs and, consistent with our model, detected uniform pyoverdine production levels across cells. We conclude that for shared compounds with saturating benefits, the evolution of division of labor is facilitated by a diminishing cost function. More generally, we note that shifts in the level of selection from individuals to groups do not solely require cooperation, but critically depend on mechanistic factors, including the distribution of compound synthesis costs., (© 2019 The Author(s). Evolution © 2019 The Society for the Study of Evolution.)

Bacterial opportunistic pathogens are feared for their difficult-to-treat nosocomial infections and for causing morbidity in immunocompromised patients. Here, we study how such a versatile opportunist, Pseudomonas aeruginosa, adapts to conditions inside and outside its model host Caenorhabditis elegans, and use phenotypic and genotypic screens to identify the mechanistic basis of virulence evolution. We found that virulence significantly dropped in unstructured environments both in the presence and absence of the host, but remained unchanged in spatially structured environments. Reduction of virulence was either driven by a substantial decline in the production of siderophores (in treatments without hosts) or toxins and proteases (in treatments with hosts). Whole-genome sequencing of evolved clones revealed positive selection and parallel evolution across replicates, and showed an accumulation of mutations in regulator genes controlling virulence factor expression. Our study identifies the spatial structure of the non-host environment as a key driver of virulence evolution in an opportunistic pathogen.

Many bacteria rely on the secretion of siderophores to scavenge iron from the environment. Laboratory studies revealed that abiotic and biotic factors together determine how much siderophores bacteria make, and whether siderophores can be exploited by non-producing cheaters or be deployed by producers to inhibit competitors. Here, we explore whether these insights apply to natural communities, by comparing the production of the siderophore pyoverdine among 930 Pseudomonas strains from 48 soil and pond communities. We found that pH, iron content, carbon concentration and community diversity determine pyoverdine production levels, and the extent to which strains are either stimulated or inhibited by heterologous (non-self) pyoverdines. While pyoverdine non-producers occurred in both habitats, their prevalence was higher in soils. Environmental and genetic analyses suggest that non-producers can evolve as cheaters, exploiting heterologous pyoverdine, but also due to pyoverdine disuse in environments with increased iron availability. Overall, we found that environmental factors explained between-strain variation in pyoverdine production much better in soils than in ponds, presumably because high strain mixing in ponds impedes local adaption. Our study sheds light on the complexity of natural bacterial communities, and provides first insights into the multivariate nature of siderophore-based iron acquisition and competition among environmental pseudomonads., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Lay Summary: We probed the evolutionary robustness of two antivirulence drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine in the opportunistic pathogen Pseudomonas aeruginosa . Using an experimental evolution approach in human serum, we showed that antivirulence treatments are not evolutionarily robust per se, but vary in their propensity to select for resistance., Background and Objectives: Treatments that inhibit the expression or functioning of bacterial virulence factors hold great promise to be both effective and exert weaker selection for resistance than conventional antibiotics. However, the evolutionary robustness argument, based on the idea that antivirulence treatments disarm rather than kill pathogens, is controversial. Here, we probe the evolutionary robustness of two repurposed drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine of the opportunistic human pathogen Pseudomonas aeruginosa ., Methodology: We subjected replicated cultures of bacteria to two concentrations of each drug for 20 consecutive days in human serum as an ex vivo infection model. We screened evolved populations and clones for resistance phenotypes, including the restoration of growth and pyoverdine production, and the evolution of iron uptake by-passing mechanisms. We whole-genome sequenced evolved clones to identify the genetic basis of resistance., Results: We found that mutants resistant against antivirulence treatments readily arose, but their selective spreading varied between treatments. Flucytosine resistance quickly spread in all populations due to disruptive mutations in upp , a gene encoding an enzyme required for flucytosine activation. Conversely, resistance against gallium arose only sporadically, and was based on mutations in transcriptional regulators, upregulating pyocyanin production, a redox-active molecule promoting siderophore-independent iron acquisition. The spread of gallium resistance was presumably hampered because pyocyanin-mediated iron delivery benefits resistant and susceptible cells alike., Conclusions and Implications: Our work highlights that antivirulence treatments are not evolutionarily robust per se . Instead, evolutionary robustness is a relative measure, with specific treatments occupying different positions on a continuous scale.

Organisms as simple as bacteria can engage in complex collective actions, such as group motility and fruiting body formation. Some of these actions involve a division of labor, where phenotypically specialized clonal subpopulations or genetically distinct lineages cooperate with each other by performing complementary tasks. Here, we combine experimental and computational approaches to investigate potential benefits arising from division of labor during biofilm matrix production. We show that both phenotypic and genetic strategies for a division of labor can promote collective biofilm formation in the soil bacterium Bacillus subtilis. In this species, biofilm matrix consists of two major components, exopolysaccharides (EPSs) and TasA. We observed that clonal groups of B. subtilis phenotypically segregate into three subpopulations composed of matrix non-producers, EPS producers, and generalists, which produce both EPSs and TasA. This incomplete phenotypic specialization was outperformed by a genetic division of labor, where two mutants, engineered as specialists, complemented each other by exchanging EPSs and TasA. The relative fitness of the two mutants displayed a negative frequency dependence both in vitro and on plant roots, with strain frequency reaching a stable equilibrium at 30% TasA producers, corresponding exactly to the population composition where group productivity is maximized. Using individual-based modeling, we show that asymmetries in strain ratio can arise due to differences in the relative benefits that matrix compounds generate for the collective and that genetic division of labor can be favored when it breaks metabolic constraints associated with the simultaneous production of two matrix components., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Phenotypic plasticity in response to competition is a well-described phenomenon in higher organisms. Here, we show that also bacteria have the ability to sense the presence of competitors and mount fine-tuned responses to match prevailing levels of competition. In our experiments, we studied interspecific competition for iron between the bacterium Pseudomonas aeruginosa (PA) and its competitor Burkholderia cenocepacia (BC). We focused on the ability of PA to phenotypically adjust the production of pyoverdine, an iron-scavenging siderophore. We found that PA upregulates pyoverdine production early on during competition under condition of low iron availability. This plastic upregulation was fine-tuned in response to the level of competition imposed by BC, and seems to confer a relative fitness benefit to PA in the form of an earlier initiation of growth. At later time points, however, PA showed reduced growth in mixed compared to monoculture, suggesting that competitive responses are costly. Altogether, our results demonstrate that phenotypic plasticity in siderophore production plays an important role in interspecific competition for iron. Upregulating siderophore production may be a powerful strategy to lock iron away from competing species, and to reserve this nutrient for strain members possessing the compatible receptor for uptake., (© 2018 The Author(s). Evolution © 2018 The Society for the Study of Evolution.)

Background: A common form of cooperation in bacteria is based on the secretion of beneficial metabolites, shareable as public good among cells within a group. Because cooperation can be exploited by "cheating" mutants, which contribute less or nothing to the public good, there has been great interest in understanding the conditions required for cooperation to remain evolutionarily stable. In contrast, much less is known about whether cheats, once fixed in the population, are able to revert back to cooperation when conditions change. Here, we tackle this question by subjecting experimentally evolved cheats of Pseudomonas aeruginosa, partly deficient for the production of the iron-scavenging public good pyoverdine, to conditions previously shown to favor cooperation., Results: Following approximately 200 generations of experimental evolution, we screened 720 evolved clones for changes in their pyoverdine production levels. We found no evidence for the re-evolution of full cooperation, even in environments with increased spatial structure, and reduced costs of public good production - two conditions that have previously been shown to maintain cooperation. In contrast, we observed selection for complete abolishment of pyoverdine production. The patterns of complete trait degradation were likely driven by "cheating on cheats" in unstructured, iron-limited environments where pyoverdine is important for growth, and selection against a maladaptive trait in iron-rich environments where pyoverdine is superfluous., Conclusions: Our study shows that the path to re-evolve public-goods cooperation can be constrained. While a limitation of the number of mutational targets potentially leading to reversion might be one reason for the observed pattern, an alternative explanation is that the selective conditions required for revertants to spread from rarity are much more stringent than those needed to maintain cooperation.

All social organisms experience dilemmas between cooperators performing group-beneficial actions and cheats selfishly exploiting these actions. Although bacteria have become model organisms to study social dilemmas in laboratory systems, we know little about their relevance in natural communities. Here, we show that social interactions mediated by a single shareable compound necessary for growth (the iron-scavenging pyoverdine) have important consequences for competitive dynamics in soil and pond communities of Pseudomonas bacteria. We find that pyoverdine non- and low-producers co-occur in many natural communities. While non-producers have genes coding for multiple pyoverdine receptors and are able to exploit compatible heterologous pyoverdines from other community members, producers differ in the pyoverdine types they secrete, offering protection against exploitation from non-producers with incompatible receptors. Our findings indicate that there is both selection for cheating and cheating resistance, which could drive antagonistic co-evolution and diversification in natural bacterial communities.Lab strains of Pseudomonas are model systems for the evolution of cooperation over public goods (iron-scavenging siderophores). Here, Butaitė et al. add ecological and evolutionary insight into this system by showing that cheating and resistance to cheating both shape competition for iron in natural Pseudomonas communities.

Bacteria secrete a variety of compounds important for nutrient scavenging, competition mediation and infection establishment. While there is a general consensus that secreted compounds can be shared and therefore have social consequences for the bacterial collective, we know little about the physical limits of such bacterial social interactions. Here, we address this issue by studying the sharing of iron-scavenging siderophores between surface-attached microcolonies of the bacterium Pseudomonas aeruginosa Using single-cell fluorescence microscopy, we show that siderophores, secreted by producers, quickly reach non-producers within a range of 100 µm, and significantly boost their fitness. Producers in turn respond to variation in sharing efficiency by adjusting their pyoverdine investment levels. These social effects wane with larger cell-to-cell distances and on hard surfaces. Thus, our findings reveal the boundaries of compound sharing, and show that sharing is particularly relevant between nearby yet physically separated bacteria on soft surfaces, matching realistic natural conditions such as those encountered in soft tissue infections., (© 2017 The Author(s).)

Explaining the enormous biodiversity observed in bacterial communities is challenging because ecological theory predicts that competition between species occupying the same niche should lead to the exclusion of less competitive community members. Competitive exclusion should be particularly strong when species compete for a single limiting resource or live in unstructured habitats that offer no refuge for weaker competitors. Here, we describe the 'cheating effect', a form of intra-specific competition that can counterbalance between-species competition, thereby fostering biodiversity in unstructured habitats. Using experimental communities consisting of the strong competitor Pseudomonas aeruginosa (PA) and its weaker counterpart Burkholderia cenocepacia (BC), we show that co-existence is impossible when the two species compete for a single limiting resource, iron. However, when introducing a PA cheating mutant, which specifically exploits the iron-scavenging siderophores produced by the PA wild type, we found that biodiversity was preserved under well-mixed conditions where PA cheats could outcompete the PA wild type. Cheating fosters biodiversity in our system because it creates strong intra-specific competition, which equalizes fitness differences between PA and BC. Our study identifies cheating - typically considered a destructive element - as a constructive force in shaping biodiversity.

Bacterial traits that contribute to disease are termed "virulence factors" and there is much interest in therapeutic approaches that disrupt such traits. What remains less clear is whether a virulence factor identified as such in a particular context is also important in infections involving different host and pathogen types. Here, we address this question using a meta-analytic approach. We statistically analyzed the infection outcomes of 81 experiments associated with one well-studied virulence factor-pyoverdine, an iron-scavenging compound secreted by the opportunistic pathogen Pseudomonas aeruginosa . We found that this factor is consistently involved with virulence across different infection contexts. However, the magnitude of the effect of pyoverdine on virulence varied considerably. Moreover, its effect on virulence was relatively minor in many cases, suggesting that pyoverdine is not indispensable in infections. Our works supports theoretical models from ecology predicting that disease severity is multifactorial and context dependent, a fact that might complicate our efforts to identify the most important virulence factors. More generally, our study highlights how comparative approaches can be used to quantify the magnitude and general importance of virulence factors, key knowledge informing future anti-virulence treatment strategies.

Given the rise of bacterial resistance against antibiotics, we urgently need alternative strategies to fight infections. Some propose we should disarm rather than kill bacteria, through targeted disruption of their virulence factors. It is assumed that this approach (i) induces weak selection for resistance because it should only minimally impact bacterial fitness, and (ii) is specific, only interfering with the virulence factor in question. Given that pathogenicity emerges from complex interactions between pathogens, hosts and their environment, such assumptions may be unrealistic. To address this issue in a test case, we conducted experiments with the opportunistic human pathogen Pseudomonas aeruginosa , where we manipulated the availability of a virulence factor, the iron-scavenging pyoverdine, within the insect host Galleria mellonella . We observed that pyoverdine availability was not stringently predictive of virulence and affected bacterial fitness in nonlinear ways. We show that this complexity could partly arise because pyoverdine availability affects host responses and alters the expression of regulatorily linked virulence factors. Our results reveal that virulence factor manipulation feeds back on pathogen and host behaviour, which in turn affects virulence. Our findings highlight that realizing effective and evolutionarily robust antivirulence therapies will ultimately require deeper engagement with the intrinsic complexity of host-pathogen systems.

The antibiotic pipeline is running dry and infectious disease remains a major threat to public health. An efficient strategy to stay ahead of rapidly adapting pathogens should include approaches that replace, complement or enhance the effect of both current and novel antimicrobial compounds. In recent years, a number of innovative approaches to manage disease without the aid of traditional antibiotics and without eliminating the pathogens directly have emerged. These include disabling pathogen virulence-factors, increasing host tissue damage control or altering the microbiota to provide colonization resistance, immune resistance or disease tolerance against pathogens. We discuss the therapeutic potential of these approaches and examine their possible consequences for pathogen evolution. To guarantee a longer half-life of these alternatives to directly killing pathogens, and to gain a full understanding of their population-level consequences, we encourage future work to incorporate evolutionary perspectives into the development of these treatments., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Foundation for Evolution, Medicine, and Public Health.)

The term 'quorum sensing' (QS) is generally used to describe the phenomenon that bacteria release and perceive signal molecules to coordinate cooperative behaviour in response to their population size. QS-based communication has therefore been considered a social trait. Here we show that QS signals (N-acyl-homoserine lactones, AHLs) are stochastically produced in young biofilms of Pseudomonas putida and act mainly as self-regulatory signals rather than inducing neighbouring cells. We demonstrate that QS induces the expression of putisolvin biosurfactants that are not public goods, thereby triggering asocial motility of induced cells out of microcolonies. Phenotypic heterogeneity is most prominent in the early stages of biofilm development, whereas at later stages behaviour patterns across cells become more synchronized. Our findings broaden our perspective on QS by showing that AHLs can control the expression of asocial (self-directed) traits, and that heterogeneity in QS can serve as a mechanism to drive phenotypic heterogeneity in self-directed behaviour.

Bacteria typically rely on secreted metabolites, potentially shareable at the community level, to scavenge resources from the environment. The evolution of diffusible, shareable metabolites is, however, difficult to explain because molecules can get lost, or be exploited by cheating mutants. A key question is whether natural selection can act on molecule structure to control loss and shareability. We tested this possibility by collating information on diffusivity properties of 189 secreted iron-scavenging siderophores and the natural habitats occupied by the siderophore-producing species. In line with evolutionary theory, we found that highly diffusible siderophores have preferentially evolved in species living in structured habitats, such as soil and hosts, because structuring can keep producers and their shareable goods together. Poorly diffusible siderophores, meanwhile, have preferentially evolved in species living in unstructured habitats, such as seawater, indicating that these metabolites are less shareable and more likely provide direct benefits to the producers., (© 2014 John Wiley & Sons Ltd/CNRS.)

Over the past decade, there has been enormous interest in understanding the great diversity of microbial cooperative behaviors, including communication, group-based swarming, fruiting-body formation, and the secretion of group-beneficial enzymes and food-scavenging molecules. Zhang and Rainey, henceforth Z&R, recently contended that sociomicrobiologists have been overzealous in their casting of microbes as inherently social organisms, and too hasty in interpreting microbial behaviors in a social evolutionary framework. This challenge accompanied a set of experiments in which they revisited one of the best-studied social behaviors in bacteria-the production of diffusible, sharable iron-scavenging siderophore molecules. Z&R posit that their findings challenge the view that siderophore production is a cooperative trait. Here, we demonstrate that their arguments are flawed, and stem from both technical mistakes and misunderstandings of social evolution theory., (© 2013 The Author(s). Evolution © 2014 The Society for the Study of Evolution.)

Microbes are intensely social organisms that routinely cooperate and coordinate their activities to express elaborate population level phenotypes. Such coordination requires a process of collective decision-making, in which individuals detect and collate information not only from their physical environment, but also from their social environment, in order to arrive at an appropriately calibrated response. Here, we present a conceptual overview of collective decision-making as it applies to all group-living organisms; we introduce key concepts and principles developed in the context of animal and human group decisions; and we discuss, with appropriate examples, the applicability of each of these concepts in microbial contexts. In particular, we discuss the roles of information pooling, control skew, speed vs. accuracy trade-offs, local feedbacks, quorum thresholds, conflicts of interest, and the reliability of social information. We conclude that collective decision-making in microbes shares many features with collective decision-making in higher taxa, and we call for greater integration between this fledgling field and other allied areas of research, including in the humanities and the physical sciences.

Background and Objectives: Conventional antibiotics select strongly for resistance and are consequently losing efficacy worldwide. Extracellular quenching of shared virulence factors could represent a more promising strategy because (i) it reduces the available routes to resistance (as extracellular action precludes any mutations blocking a drug's entry into cells or hastening its exit) and (ii) it weakens selection for resistance, as fitness benefits to emergent mutants are diluted across all cells in a cooperative collective. Here, we tested this hypothesis empirically., Methodology: We used gallium to quench the iron-scavenging siderophores secreted and shared among pathogenic Pseudomonas aeruginosa bacteria, and quantitatively monitored its effects on growth in vitro. We assayed virulence in acute infections of caterpillar hosts (Galleria mellonella), and tracked resistance emergence over time using experimental evolution., Results: Gallium strongly inhibited bacterial growth in vitro, primarily via its siderophore quenching activity. Moreover, bacterial siderophore production peaked at intermediate gallium concentrations, indicating additional metabolic costs in this range. In vivo, gallium attenuated virulence and growth-even more so than in infections with siderophore-deficient strains. Crucially, while resistance soon evolved against conventional antibiotic treatments, gallium treatments retained their efficacy over time., Conclusions: Extracellular quenching of bacterial public goods could offer an effective and evolutionarily robust control strategy.

Adaptations by hosts in response to parasitism are generally believed to reduce the susceptibility of the adapted individual. However, recent work on Escherichia coli showed that bacteria can fight deadly phage attacks by committing altruistic suicide upon infection, in order to prevent parasite transmission to nearby relatives. Here, we compare the efficiency of suicidal host defense with individual-based resistance. We show that in unstructured environments suicidal host defense is futile since suicide cannot preferentially protect relatives, whereas individual-based resistance is highly efficient in defying phages. In contrast, we found that in structured environments suicidal host defense and individual-based resistance were both efficient in withstanding phages, with the latter type performing slightly better. We propose that the putative lower efficiency of suicidal host defense might be compensated by the fact that suicidal systems usually do not bear pleiotropic costs of resistance, as it is usually the case for individual-based resistance mechanisms.

Bacteria often possess multiple siderophore-based iron uptake systems for scavenging this vital resource from their environment. However, some siderophores seem redundant, because they have limited iron-binding efficiency and are seldom expressed under iron limitation. Here, we investigate the conundrum of why selection does not eliminate this apparent redundancy. We focus on Pseudomonas aeruginosa, a bacterium that can produce two siderophores-the highly efficient but metabolically expensive pyoverdine, and the inefficient but metabolically cheap pyochelin. We found that the bacteria possess molecular mechanisms to phenotypically switch from mainly producing pyoverdine under severe iron limitation to mainly producing pyochelin when iron is only moderately limited. We further show that strains exclusively producing pyochelin grew significantly better than strains exclusively producing pyoverdine under moderate iron limitation, whereas the inverse was seen under severe iron limitation. This suggests that pyochelin is not redundant, but that switching between siderophore strategies might be beneficial to trade off efficiencies versus costs of siderophores. Indeed, simulations parameterized from our data confirmed that strains retaining the capacity to switch between siderophores significantly outcompeted strains defective for one or the other siderophore under fluctuating iron availabilities. Finally, we discuss how siderophore switching can be viewed as a form of collective decision-making, whereby a coordinated shift in behaviour at the group level emerges as a result of positive and negative feedback loops operating among individuals at the local scale.

High relatedness among interacting individuals has generally been considered a precondition for the evolution of altruism. However, kin-selection theory also predicts the evolution of altruism when relatedness is low, as long as the cost of the altruistic act is minor compared with its benefit. Here, we demonstrate evidence for a low-cost altruistic act in bacteria. We investigated Escherichia coli responding to the attack of an obligately lytic phage by committing suicide in order to prevent parasite transmission to nearby relatives. We found that bacterial suicide provides large benefits to survivors at marginal costs to committers. The cost of suicide was low, because infected cells are moribund, rapidly dying upon phage infection, such that no more opportunity for reproduction remains. As a consequence of its marginal cost, host suicide was selectively favoured even when relatedness between committers and survivors approached zero. Altogether, our findings demonstrate that low-cost suicide can evolve with ease, represents an effective host-defence strategy, and seems to be widespread among microbes. Moreover, low-cost suicide might also occur in higher organisms as exemplified by infected social insect workers leaving the colony to die in isolation.

While adaptive adjustment of sex ratio in the function of colony kin structure and food availability commonly occurs in social Hymenoptera, long-term studies have revealed substantial unexplained between-year variation in sex ratio at the population level. In order to identify factors that contribute to increased between-year variation in population sex ratio, we conducted a comparative analysis across 47 Hymenoptera species differing in their breeding system. We found that between-year variation in population sex ratio steadily increased as one moved from solitary species, to primitively eusocial species, to single-queen eusocial species, to multiple-queen eusocial species. Specifically, between-year variation in population sex ratio was low (6.6% of total possible variation) in solitary species, which is consistent with the view that in solitary species, sex ratio can vary only in response to fluctuations in ecological factors such as food availability. In contrast, we found significantly higher (19.5%) between-year variation in population sex ratio in multiple-queen eusocial species, which supports the view that in these species, sex ratio can also fluctuate in response to temporal changes in social factors such as queen number and queen-worker control over sex ratio, as well as factors influencing caste determination. The simultaneous adjustment of sex ratio in response to temporal fluctuations in ecological and social factors seems to preclude the existence of a single sex ratio optimum. The absence of such an optimum may reflect an additional cost associated with the evolution of complex breeding systems in Hymenoptera societies.

In social groups where relatedness among interacting individuals is low, cooperation can often only be maintained through mechanisms that repress competition among group members. Repression-of-competition mechanisms, such as policing and punishment, seem to be of particular importance in human societies, where cooperative interactions often occur among unrelated individuals. In line with this view, economic games have shown that the ability to punish defectors enforces cooperation among humans. Here, I examine a real-world example of a repression-of-competition system, the police institutions common to modern human societies. Specifically, I test evolutionary policing theory by comparing data on policing effort, per capita crime rate, and similarity (used as a proxy for genetic relatedness) among citizens across the 26 cantons of Switzerland. This comparison revealed full support for all three predictions of evolutionary policing theory. First, when controlling for policing efforts, crime rate correlated negatively with the similarity among citizens. This is in line with the prediction that high similarity results in higher levels of cooperative self-restraint (i.e. lower crime rates) because it aligns the interests of individuals. Second, policing effort correlated negatively with the similarity among citizens, supporting the prediction that more policing is required to enforce cooperation in low-similarity societies, where individuals' interests diverge most. Third, increased policing efforts were associated with reductions in crime rates, indicating that policing indeed enforces cooperation. These analyses strongly indicate that humans respond to cues of their social environment and adjust cheating and policing behaviour as predicted by evolutionary policing theory.

Public goods cooperation abounds in nature, occurring in organisms ranging from bacteria to humans. Although previous research focused on the behavioral and ecological conditions favoring cooperation, the question of whether the molecular and regulatory properties of the public good itself can influence selection for cooperation has received little attention. Using a metapopulation model, we show that extended molecular durability of a public good--allowing multiple reuse across generations--greatly reduces selection for cheating if (and only if) the production of the public good is facultatively regulated. To test the apparent synergy between public goods durability and facultative regulation, we examined the production of iron-scavenging pyoverdin molecules by the bacterium Pseudomonas aeruginosa, a cooperative behavior that is facultatively regulated in response to iron availability. We show that pyoverdin is a very durable public good and that extended durability significantly enhances fitness. Consistent with our model, we found that nonsiderophore-producing mutants (cheats) had a relative fitness advantage over siderophore producers (cooperators) when pyoverdin durability was low but not when durability was high. This was because cooperators facultatively reduced their investment in pyoverdin production when enough pyoverdin had accumulated in the media-a cost-saving strategy that minimized the ability of cheats to invade. These findings show how molecular properties of cooperative acts can shape the costs and benefits of cooperation.

The results of numerous economic games suggest that humans behave more cooperatively than would be expected if they were maximizing selfish interests. It has been argued that this is because individuals gain satisfaction from the success of others, and that such prosocial preferences require a novel evolutionary explanation. However, in previous games, imperfect behavior would automatically lead to an increase in cooperation, making it impossible to decouple any form of mistake or error from prosocial cooperative decisions. Here we empirically test between these alternatives by decoupling imperfect behavior from prosocial preferences in modified versions of the public goods game, in which individuals would maximize their selfish gain by completely (100%) cooperating. We found that, although this led to higher levels of cooperation, it did not lead to full cooperation, and individuals still perceived their group mates as competitors. This is inconsistent with either selfish or prosocial preferences, suggesting that the most parsimonious explanation is imperfect behavior triggered by psychological drives that can prevent both complete defection and complete cooperation. More generally, our results illustrate the caution that must be exercised when interpreting the evolutionary implications of economic experiments, especially the absolute level of cooperation in a particular treatment.

Split sex ratio-a pattern where colonies within a population specialize in either male or queen production-is a widespread phenomenon in ants and other social Hymenoptera. It has often been attributed to variation in colony kin structure, which affects the degree of queen-worker conflict over optimal sex allocation. However, recent findings suggest that split sex ratio is a more diverse phenomenon, which can evolve for multiple reasons. Here, we provide an overview of the main conditions favouring split sex ratio. We show that each split sex-ratio type arises due to a different combination of factors determining colony kin structure, queen or worker control over sex ratio and the type of conflict between colony members.

There has been extensive theoretical debate over whether population viscosity (limited dispersal) can favour cooperation. While limited dispersal increases the probability of interactions occurring between relatives, which can favour cooperation, it can also lead to an increase in competition between relatives and this can reduce or completely negate selection for cooperation. Despite much theoretical attention, there is a lack of empirical research investigating these issues. We cultured Pseudomonas aeruginosa bacteria in medium with different degrees of viscosity and examined the fitness consequences for a cooperative trait-the production of iron-scavenging siderophore molecules. We found that increasing viscosity of the growth medium (i) significantly limited bacterial dispersal and the diffusion of siderophore molecules and (ii) increased the fitness of individuals that produced siderophores relative to mutants that did not. We propose that viscosity favours siderophore-producing individuals in this system, because the benefits of siderophore production are more likely to accrue to relatives (i.e. greater indirect benefits), and, at the same time, bacteria are more likely to gain direct fitness benefits by taking up siderophore molecules produced by themselves (i.e. the trait becomes less cooperative). Our results suggest that viscosity of the microbial growth environment is a crucial factor determining the dynamics of wild-type bacteria and siderophore-deficient mutants in natural habitats, such as the viscous mucus in cystic fibrosis lung.

Numerous theoretical studies have investigated how limited dispersal may provide an explanation for the evolution of cooperation, by leading to interactions between relatives. However, despite considerable theoretical attention, there has been a lack of empirical tests. In this article, we test how patterns of dispersal influence the evolution of cooperation, using iron-scavenging in the bacterium Pseudomonas aeruginosa as our cooperative trait. We found that relatively limited dispersal does not favor cooperation. The reason for this is that although limited dispersal increases the relatedness between interacting individuals, it also leads to increased local competition for resources between relatives. This result supports Taylor's prediction that in the simplest possible scenario, the effects of increased relatedness and local competition exactly cancel out. In contrast, we show that one way for cooperation to be favored is if individuals disperse in groups (budding dispersal), because this maintains high relatedness while reducing local competition between relatives (relatively global competition).

Phenotypic noise and social evolution in microbes have recently attracted huge interdisciplinary interest. A new study highlights the interaction between these phenomena and its implications for self-destructive cooperation.

Among papionin primates, the Barbary macaque (Macaca sylvanus) shows the most extensive interactions between infants and group members other than the mother. Two different types of interactions occur: (1) long-lasting dyadic interactions between a handler and an infant, and (2) brief triadic interactions between two handlers involving an infant. Previous investigations showed that infant handling by males is best explained as use of infants to manage relationships with other males. In contrast, no adaptive explanation for infant handling by females emerged. Here, we compared the infant-handling pattern between subadult/adult males and subadult/adult females in a free-ranging group of 46 Barbary macaques on Gibraltar to test whether the relationship management hypothesis also applies to female handlers. We further investigated the infant-handling pattern of juveniles and used microsatellite markers to estimate relatedness between infant handlers and the infant's mother. We found that males, females and juveniles all participated extensively in triadic interactions using infants of above-average related females. In contrast, only males and juveniles were highly involved in dyadic interactions with infants of related females, while females rarely handled infants other than their own. The pattern of infant handling was entirely compatible with the predictions of the relationship management hypothesis for males and mostly so for females. Moreover, our genetic analysis revealed that males and females differ in their partner choice: while females preferred to interact with related females, males had no significant preference to interact with related males. We further discuss the observed above-average relatedness values between infant handlers and the infant's mother in the light of kin-selection theory.

Explaining the evolution of cooperation among non-relatives is one of the major challenges for evolutionary biology. In this study, we experimentally examined human cooperation in the iterated Snowdrift game (ISD), which has received little attention so far, and compared it with human cooperation in the iterated Prisoner's Dilemma (IPD), which has become the paradigm for the evolution of cooperation. We show that iteration in the ISD leads to consistently higher levels of cooperation than in the IPD. We further demonstrate that the most successful strategies known for the IPD (generous Tit-for-Tat and Pavlov) were also successfully used in the ISD. Interestingly, we found that female players cooperated significantly more often than male players in the IPD but not in the ISD. Moreover, female players in the IPD applied Tit-for-Tat-like or Pavlovian strategies significantly more often than male players, thereby achieving significantly higher pay-offs than male players did. These data demonstrate that the willingness to cooperate does not only depend on the type of the social dilemma, but also on the class of individuals involved. Altogether, our study shows that the ISD can potentially explain high levels of cooperation among non-relatives in humans. In addition, the ISD seems to reflect the social dilemma more realistically than the IPD because individuals obtain immediate direct benefits from the cooperative acts they perform and costs of cooperation are shared between cooperators.

The theory of inclusive fitness provides a powerful explanation for reproductive altruism in social insects, whereby workers gain inclusive fitness benefit by rearing the brood of related queens. Some ant species, however, have unicolonial population structures where multiple nests, each containing numerous queens, are interconnected and individuals move freely between nests. In such cases, nestmate relatedness values may often be indistinguishable from zero, which is problematic for inclusive fitness-based explanations of reproductive altruism. We conducted a detailed population genetic study in the polygynous ant Formica exsecta, which has been suggested to form unicolonial populations in its native habitat. Analyses based on adult workers indeed confirmed a genetic structuring consistent with a unicolonial population structure. However, at the population level the genetic structuring inferred from worker pupae was not consistent with a unicolonial population structure, but rather suggested a multicolonial population structure of extended family-based nests. These contrasting patterns suggest limited queen dispersal and free adult worker dispersal. That workers indeed disperse as adults was confirmed by mark-recapture measures showing consistent worker movement between nests. Together, these findings describe a new form of social organization, which possibly also characterizes other ant species forming unicolonial populations in their native habitats. Moreover, the genetic analyses also revealed that while worker nestmate relatedness was indistinguishable from zero at a small geographical scale, it was significantly positive at the population level. This highlights the need to consider the relevant geographical scale when investigating the role of inclusive fitness as a selective force maintaining reproductive altruism.

In animal societies, cooperation for the common wealth and latent conflicts due to the selfish interests of individuals are in delicate balance. In many ant species, colonies contain multiple breeders and workers interact with nestmates of varying degrees of relatedness. Therefore, workers could increase their inclusive fitness by preferentially caring for their closest relatives, yet evidence for nepotism in insect societies remains scarce and controversial. We experimentally demonstrate that workers of the ant Formica exsecta do not discriminate between highly related and unrelated brood, but that brood viability differs between queens. We further show that differences in brood viability are sufficient to explain a relatedness pattern that has previously been interpreted as evidence for nepotism. Hence, our findings support the view that nepotism remains elusive in social insects and emphasize the need for further controlled experiments.

In polygynous (multiple queens per nest) ants, queen dispersal is often limited with young queens being recruited within the parental colony. This mode of dispersal leads to local resource competition between nestmate queens and is frequently associated with extremely male-biased sex ratios at the population level. The queen-replenishment hypothesis has been recently proposed to explain colony sex ratio investment under such conditions. It predicts that colonies containing many queens (subject to high local resource competition) should only produce males, whereas colonies hosting few queens (reduced or no local resource competition) should produce new queens in addition to males. We experimentally tested this hypothesis in the ant Formica exsecta by manipulating queen number over three consecutive years in 120 colonies of a highly polygynous population. Queens were transferred from 40 colonies into another 40 colonies while queen number was not manipulated in 40 control colonies. Genetic analyses of worker offspring revealed that our treatment significantly changed the number of reproductive queens. The sex ratio of colonies was significantly different between treatments in the third breeding season following the experiment initiation. We found that, as predicted by the queen-replenishment hypothesis, queen removal resulted in a significant increase in the proportion of colonies that produced new queens. These results provide the first experimental evidence for the queen-replenishment hypothesis, which might account for sex ratio specialization in many highly polygynous ant species.