Bacterial lifestyle shapes pangenomes.

Pangenomes vary across bacteria. Some species have fluid pangenomes, with a high proportion of genes varying between individual genomes. Other species have less fluid pangenomes, with different genomes tending to contain the same genes. Two main hypotheses have been suggested to explain this variation: differences in species' bacterial lifestyle and effective population size. However, previous studies have not been able to test between these hypotheses because the different features of lifestyle and effective population size are highly correlated with each other, and phylogenetically conserved, making it hard to disentangle their relative importance. We used phylogeny-based analyses, across 126 bacterial species, to tease apart the causal role of different factors. We found that pangenome fluidity was lower in i) host-associated compared with free-living species and ii) host-associated species that are obligately dependent on a host, live inside cells, and are more pathogenic and less motile. In contrast, we found no support for the competing hypothesis that larger effective population sizes lead to more fluid pangenomes. Effective population size appears to correlate with pangenome variation because it is also driven by bacterial lifestyle, rather than because of a causal relationship.

Microbial Primer: Cooperation in bacteria.

The growth and success of many bacteria appear to rely on a stunning range of cooperative behaviours. But what is cooperation and how is it studied?

The collapse of cooperation during range expansion of Pseudomonas aeruginosa.

Cooperation is commonly believed to be favourable in spatially structured environments, as these systems promote genetic relatedness that reduces the likelihood of exploitation by cheaters. Here we show that a Pseudomonas aeruginosa population that exhibited cooperative swarming was invaded by cheaters when subjected to experimental evolution through cycles of range expansion on solid media, but not in well-mixed liquid cultures. Our results suggest that cooperation is disfavoured in a more structured environment, which is the opposite of the prevailing view. We show that spatial expansion of the population prolongs cooperative swarming, which was vulnerable to cheating. Our findings reveal a mechanism by which spatial structures can suppress cooperation through modulation of the quantitative traits of cooperation, a process that leads to population divergence towards distinct colonization strategies.

Genes for cooperation are not more likely to be carried by plasmids.

Cooperation is prevalent across bacteria, but risks being exploited by non-cooperative cheats. Horizontal gene transfer, particularly via plasmids, has been suggested as a mechanism to stabilize cooperation. A key prediction of this hypothesis is that genes which are more likely to be transferred, such as those on plasmids, should be more likely to code for cooperative traits. Testing this prediction requires identifying all genes for cooperation in bacterial genomes. However, previous studies used a method which likely misses some of these genes for cooperation. To solve this, we used a new genomics tool, SOCfinder, which uses three distinct modules to identify all kinds of genes for cooperation. We compared where these genes were located across 4648 genomes from 146 bacterial species. In contrast to the prediction of the hypothesis, we found no evidence that plasmid genes are more likely to code for cooperative traits. Instead, we found the opposite-that genes for cooperation were more likely to be carried on chromosomes. Overall, the vast majority of genes for cooperation are not located on plasmids, suggesting that the more general mechanism of kin selection is sufficient to explain the prevalence of cooperation across bacteria.

SOCfinder: a genomic tool for identifying social genes in bacteria.

Bacteria cooperate by working collaboratively to defend their colonies, share nutrients, and resist antibiotics. Nevertheless, our understanding of these remarkable behaviours primarily comes from studying a few well-characterized species. Consequently, there is a significant gap in our understanding of microbial social traits, particularly in natural environments. To address this gap, we can use bioinformatic tools to identify genes that control cooperative or otherwise social traits. Existing tools address this challenge through two approaches. One approach is to identify genes that encode extracellular proteins, which can provide benefits to neighbouring cells. An alternative approach is to predict gene function using annotation tools. However, these tools have several limitations. Not all extracellular proteins are cooperative, and not all cooperative behaviours are controlled by extracellular proteins. Furthermore, existing functional annotation methods frequently miss known cooperative genes. We introduce SOCfinder as a new tool to find bacterial genes that control cooperative or otherwise social traits. SOCfinder combines information from several methods, considering if a gene is likely to [1] code for an extracellular protein [2], have a cooperative functional annotation, or [3] be part of the biosynthesis of a cooperative secondary metabolite. We use data on two extensively-studied species (P. aeruginosa and B. subtilis) to show that SOCfinder is better at finding known cooperative genes than existing tools. We also use theory from population genetics to identify a signature of kin selection in SOCfinder cooperative genes, which is lacking in genes identified by existing tools. SOCfinder opens up a number of exciting directions for future research, and is available to download from https://github.com/lauriebelch/SOCfinder.

Signatures of kin selection in a natural population of the bacteria Bacillus subtilis.

Laboratory experiments have suggested that bacteria perform a range of cooperative behaviors, which are favored because they are directed toward relatives (kin selection). However, there is a lack of evidence for cooperation and kin selection in natural bacterial populations. Molecular population genetics offers a promising method to study natural populations because the theory predicts that kin selection will lead to relaxed selection, which will result in increased polymorphism and divergence at cooperative genes. Examining a natural population of Bacillus subtilis, we found consistent evidence that putatively cooperative traits have higher polymorphism and greater divergence than putatively private traits expressed at the same rate. In addition, we were able to eliminate alternative explanations for these patterns and found more deleterious mutations in genes controlling putatively cooperative traits. Overall, our results suggest that cooperation is favored by kin selection, with an average relatedness of r = .79 between interacting individuals.

Equilibria and oscillations in cheat-cooperator dynamics.

Cooperative societies can be threatened by cheats, who invest less in cooperation and exploit the contributions of others. The impact of cheats depends on the extent to which they are maintained in the population. However, different empirical studies, across organisms ranging from RNA replicators to bacteria, have shown diverse cheat-cooperator dynamics. These vary from approaching a stable equilibrium to dynamic cyclical oscillations. The reason for this variation remains unclear. Here, we develop a theoretical model to identify the factors that determine whether dynamics should tend toward stable equilibria or cyclical oscillations. Our analyses show that (1) a combination of both periodic population bottlenecks and density-dependent selection on cheating is required to produce cyclical oscillations and (2) the extent of frequency-dependent selection for cheating can influence the amplitude of these oscillations but does not lead to oscillations alone. Furthermore, we show that stochastic group formation (demographic stochasticity) can generate different forms of oscillation, over a longer time scale, across growth cycles. Our results provide experimentally testable hypotheses for the processes underlying cheat-cooperator dynamics.

Host-parasite coevolution and the stability of genetic kin recognition.

Crozier's paradox suggests that genetic kin recognition will not be evolutionarily stable. The problem is that more common tags (markers) are more likely to be recognized and helped. This causes common tags to increase in frequency, eliminating the genetic variability that is required for genetic kin recognition. Two potential solutions to this problem have been suggested: host-parasite coevolution and multiple social encounters. We show that the host-parasite coevolution hypothesis does not work as commonly assumed. Host-parasite coevolution only stabilizes kin recognition at a parasite resistance locus if parasites adapt rapidly to hosts and cause intermediate or high levels of damage (virulence). Additionally, when kin recognition is stabilized at a parasite resistance locus, this can have an additional cost of making hosts more susceptible to parasites. However, we show that if the genetic architecture is allowed to evolve, meaning natural selection can choose the recognition locus, genetic kin recognition is more likely to be stable. The reason for this is that host-parasite coevolution can maintain tag diversity at another (neutral) locus by genetic hitchhiking, allowing that other locus to be used for genetic kin recognition. These results suggest a way that host-parasite coevolution can resolve Crozier's paradox, without making hosts more susceptible to parasites. However, the opportunity for multiple social encounters may provide a more robust resolution of Crozier's paradox.

Symbioses shape feeding niches and diversification across insects.

For over 300 million years, insects have relied on symbiotic microbes for nutrition and defence. However, it is unclear whether specific ecological conditions have repeatedly favoured the evolution of symbioses, and how this has influenced insect diversification. Here, using data on 1,850 microbe-insect symbioses across 402 insect families, we found that symbionts have allowed insects to specialize on a range of nutrient-imbalanced diets, including phloem, blood and wood. Across diets, the only limiting nutrient consistently associated with the evolution of obligate symbiosis was B vitamins. The shift to new diets, facilitated by symbionts, had mixed consequences for insect diversification. In some cases, such as herbivory, it resulted in spectacular species proliferation. In other niches, such as strict blood feeding, diversification has been severely constrained. Symbioses therefore appear to solve widespread nutrient deficiencies for insects, but the consequences for insect diversification depend on the feeding niche that is invaded.

Is cooperation favored by horizontal gene transfer?

It has been hypothesized that horizontal gene transfer on plasmids can facilitate the evolution of cooperation, by allowing genes to jump between bacteria, and hence increase genetic relatedness at the cooperative loci. However, we show theoretically that horizontal gene transfer only appreciably increases relatedness when plasmids are rare, where there are many plasmid-free cells available to infect (many opportunities for horizontal gene transfer). In contrast, when plasmids are common, there are few opportunities for horizontal gene transfer, meaning relatedness is not appreciably increased, and so cooperation is not favored. Plasmids, therefore, evolve to be rare and cooperative, or common and noncooperative, meaning plasmid frequency and cooperativeness are never simultaneously high. The overall level of plasmid-mediated cooperation, given by the product of plasmid frequency and cooperativeness, is therefore consistently negligible or low.

Gene transferability and sociality do not correlate with gene connectivity.

The connectivity of a gene, defined as the number of interactions a gene's product has with other genes' products, is a key characteristic of a gene. In prokaryotes, the complexity hypothesis predicts that genes which undergo more frequent horizontal transfer will be less connected than genes which are only very rarely transferred. We tested the role of horizontal gene transfer, and other potentially important factors, by examining the connectivity of chromosomal and plasmid genes, across 134 diverse prokaryotic species. We found that (i) genes on plasmids were less connected than genes on chromosomes; (ii) connectivity of plasmid genes was not correlated with plasmid mobility; and (iii) the sociality of genes (cooperative or private) was not correlated with gene connectivity.

Multiple social encounters can eliminate Crozier's paradox and stabilise genetic kin recognition.

Crozier's paradox suggests that genetic kin recognition will not be evolutionarily stable. The problem is that more common tags (markers) are more likely to be recognised and helped. This causes common tags to increase in frequency, and hence eliminates the genetic variability that is required for genetic kin recognition. It has therefore been assumed that genetic kin recognition can only be stable if there is some other factor maintaining tag diversity, such as the advantage of rare alleles in host-parasite interactions. We show that allowing for multiple social encounters before each social interaction can eliminate Crozier's paradox, because it allows individuals with rare tags to find others with the same tag. We also show that rare tags are better indicators of relatedness, and hence better at helping individuals avoid interactions with non-cooperative cheats. Consequently, genetic kin recognition provides an advantage to rare tags that maintains tag diversity, and stabilises itself.

Kin selection for cooperation in natural bacterial populations.

Bacteria produce a range of molecules that are secreted from the cell and can provide a benefit to the local population of cells. Laboratory experiments have suggested that these "public goods" molecules represent a form of cooperation, favored because they benefit closely related cells (kin selection). However, there is a relative lack of data demonstrating kin selection for cooperation in natural populations of bacteria. We used molecular population genetics to test for signatures of kin selection at the genomic level in natural populations of the opportunistic pathogen Pseudomonas aeruginosa We found consistent evidence from multiple traits that genes controlling putatively cooperative traits have higher polymorphism and greater divergence and are more likely to harbor deleterious mutations relative to genes controlling putatively private traits, which are expressed at similar rates. These patterns suggest that cooperative traits are controlled by kin selection, and we estimate that the relatedness for social interactions in P. aeruginosa is r = 0.84. More generally, our results demonstrate how molecular population genetics can be used to study the evolution of cooperation in natural populations.

The Black Box as a Control for Payoff-Based Learning in Economic Games.

The black box method was developed as an "asocial control" to allow for payoff-based learning while eliminating social responses in repeated public goods games. Players are told they must decide how many virtual coins they want to input into a virtual black box that will provide uncertain returns. However, in truth, they are playing with each other in a repeated social game. By "black boxing" the game's social aspects and payoff structure, the method creates a population of self-interested but ignorant or confused individuals that must learn the game's payoffs. This low-information environment, stripped of social concerns, provides an alternative, empirically derived null hypothesis for testing social behaviours, as opposed to the theoretical predictions of rational self-interested agents (Homo economicus). However, a potential problem is that participants can unwittingly affect the learning of other participants. Here, we test a solution to this problem in a range of public goods games by making participants interact, unknowingly, with simulated players ("computerised black box"). We find no significant differences in rates of learning between the original and the computerised black box, therefore either method can be used to investigate learning in games. These results, along with the fact that simulated agents can be programmed to behave in different ways, mean that the computerised black box has great potential for complementing studies of how individuals and groups learn under different environments in social dilemmas.

The evolution of cheating in viruses.

The success of many viruses depends upon cooperative interactions between viral genomes. However, whenever cooperation occurs, there is the potential for 'cheats' to exploit that cooperation. We suggest that: (1) the biology of viruses makes viral cooperation particularly susceptible to cheating; (2) cheats are common across a wide range of viruses, including viral entities that are already well studied, such as defective interfering genomes, and satellite viruses. Consequently, the evolutionary theory of cheating could help us understand and manipulate viral dynamics, while viruses also offer new opportunities to study the evolution of cheating.

Plasmids do not consistently stabilize cooperation across bacteria but may promote broad pathogen host-range.

Horizontal gene transfer via plasmids could favour cooperation in bacteria, because transfer of a cooperative gene turns non-cooperative cheats into cooperators. This hypothesis has received support from theoretical, genomic and experimental analyses. By contrast, we show here, with a comparative analysis across 51 diverse species, that genes for extracellular proteins, which are likely to act as cooperative 'public goods', were not more likely to be carried on either: (1) plasmids compared to chromosomes; or (2) plasmids that transfer at higher rates. Our results were supported by theoretical modelling which showed that, while horizontal gene transfer can help cooperative genes initially invade a population, it has less influence on the longer-term maintenance of cooperation. Instead, we found that genes for extracellular proteins were more likely to be on plasmids when they coded for pathogenic virulence traits, in pathogenic bacteria with a broad host-range.

Cooperative interactions among females can lead to even more extraordinary sex ratios.

Hamilton's local mate competition theory provided an explanation for extraordinary female-biased sex ratios in a range of organisms. When mating takes place locally, in structured populations, a female-biased sex ratio is favored to reduce competition between related males, and to provide more mates for males. However, there are a number of wasp species in which the sex ratios appear to more female biased than predicted by Hamilton's theory. It has been hypothesized that the additional female bias in these wasp species results from cooperative interactions between females. We investigated theoretically the extent to which cooperation between related females can interact with local mate competition to favor even more female-biased sex ratios. We found that (i) cooperation between females can lead to sex ratios that are more female biased than predicted by local competition theory alone, and (ii) sex ratios can be more female biased when the cooperation occurs from offspring to mothers before dispersal, rather than cooperation between siblings after dispersal. Our models formally confirm the verbal predictions made in previous experimental studies, which could be applied to a range of organisms. Specifically, cooperation can help explain sex ratio biases in Sclerodermus and Melittobia wasps, although quantitative comparisons between predictions and data suggest that some additional factors may be operating.

A solution to a sex ratio puzzle in Melittobia wasps.

The puzzling sex ratio behavior of Melittobia wasps has long posed one of the greatest questions in the field of sex allocation. Laboratory experiments have found that, in contrast to the predictions of theory and the behavior of numerous other organisms, Melittobia females do not produce fewer female-biased offspring sex ratios when more females lay eggs on a patch. We solve this puzzle by showing that, in nature, females of Melittobia australica have a sophisticated sex ratio behavior, in which their strategy also depends on whether they have dispersed from the patch where they emerged. When females have not dispersed, they lay eggs with close relatives, which keeps local mate competition high even with multiple females, and therefore, they are selected to produce consistently female-biased sex ratios. Laboratory experiments mimic these conditions. In contrast, when females disperse, they interact with nonrelatives, and thus adjust their sex ratio depending on the number of females laying eggs. Consequently, females appear to use dispersal status as an indirect cue of relatedness and whether they should adjust their sex ratio in response to the number of females laying eggs on the patch.

Payoff-based learning best explains the rate of decline in cooperation across 237 public-goods games.

What motivates human behaviour in social dilemmas? The results of public goods games are commonly interpreted as showing that humans are altruistically motivated to benefit others. However, there is a competing 'confused learners' hypothesis: that individuals start the game either uncertain or mistaken (confused) and then learn from experience how to improve their payoff (payoff-based learning). Here we (1) show that these competing hypotheses can be differentiated by how they predict contributions should decline over time; and (2) use metadata from 237 published public goods games to test between these competing hypotheses. We found, as predicted by the confused learners hypothesis, that contributions declined faster when individuals had more influence over their own payoffs. This predicted relationship arises because more influence leads to a greater correlation between contributions and payoffs, facilitating learning. Our results suggest that humans, in general, are not altruistically motivated to benefit others but instead learn to help themselves.