P2X receptors are membrane ion channels gated by extracellular ATP1,2 that are found widely in vertebrates, but not previously in microbes. Here we identify a weakly related gene in the genome of the social amoeba Dictyostelium discoideum, and show, with the use of heterologous expression in human embryonic kidney cells, that it encodes a membrane ion channel activated by ATP (30-100 μM). Site-directed mutagenesis revealed essential conservation of structure-function relations with P2X receptors of higher organisms. The receptor was insensitive to the usual P2X antagonists3 but was blocked by nanomolar concentrations of Cu 2+ ions. In D. discoideum, the receptor was found on intracellular membranes, with prominent localization to an osmoregulatory organelle, the contractile vacuole. Targeted disruption of the gene in D. discoideum resulted in cells that were unable to regulate cell volume in hypotonic conditions. Cell swelling in these mutant cells was accompanied by a marked inhibition of contractile vacuole emptying. These findings demonstrate a new functional role for P2X receptors on intracellular organelles, in this case in osmoregulation.The D. discoideum genome contains five sequences predicted to encode proteins homologous to vertebrate P2X receptors. The protein most closely related to the seven human receptors is DDB0168616 (Supplementary Fig. 1). HEK cells expressing a humanized version of this complementary DNA responded to ATP with robust inward currents (Fig. 1). ATP evoked unitary currents (conductance 8.2 pS at -100 mV) in outsideout patches (Fig. 1a, b), establishing that this gene (D. discoideum p2xA) encodes an ATPgated ion channel (DdP2X). Whole-cell currents evoked by ATP were concentrationdependent (10-300 μM) with kinetic properties most similar to those observed for human P2X 2 or P2X 4 receptors1 (Fig. 1d). With sodium as the only cation, the current-voltage relation reversed at zero and showed moderate inward rectification (Fig. 1c). By measuring the current reversal potentials in different extracellular ions, we found that the channels were freely permeable to Ca 2+ ions but less so to larger cations (the relative permeability P X /P Na was 1.5 ± 0.11 (n = 8 cells), 0.54 ± 0.01 (5), 0.48 ± 0.21 (5), 0.38 ± 0.16 (5) and 0.21 ± 0.05 (8), where X represents Ca 2+ , choline, Tris, tetraethylammonium and N-methyl-D-glucamine, respectively; Fig. 1c). These values are in the range of vertebrate P2X receptors1. Western blotting of the expressed receptor showed two bands at 48 and 55 kDa (Fig. 1e). The

Progress in our understanding of sociobiology has occurred with little knowledge of the genetic mechanisms that underlie social traits. However, several recent studies have described microbial genes that affect social traits, thereby bringing genetics to sociobiology. A key finding is that simple genetic changes can have marked social consequences, and mutations that affect cheating and recognition behaviors have been discovered. The study of these mutants confirms a central theoretical prediction of social evolution: that genetic relatedness promotes cooperation. Microbial genetics also provides an important new perspective: that the genome-to-phenome mapping of social organisms might be organized to constrain the evolution of social cheaters. This constraint can occur both through pleiotropic genes that link cheating to a personal cost and through the existence of phoenix genes, which rescue cooperative systems from selfish and destructive strategies. These new insights show the power of studying microorganisms to improve our understanding of the evolution of cooperation.

Cheaters disrupt cooperation by reaping the benefits without paying their fair share of associated costs. Cheater impact can be diminished if cooperators display a tag (‘greenbeard') and recognise and preferentially direct cooperation towards other tag carriers. Despite its popular appeal, the feasibility of such greenbeards has been questioned because the complex patterns of partner-specific cooperative behaviours seen in nature require greenbeards to come in different colours. Here we show that a locus (‘Tgr') of a social amoeba represents a polychromatic greenbeard. Patterns of natural Tgr locus sequence polymorphisms predict partner-specific patterns of cooperation by underlying variation in partner-specific protein–protein binding strength and recognition specificity. Finally, Tgr locus polymorphisms increase fitness because they help avoid potential costs of cooperating with incompatible partners. These results suggest that a polychromatic greenbeard can provide a key mechanism for the evolutionary maintenance of cooperation.

SummaryCooperation is ubiquitous across the tree of life, from simple microbes to the complex social systems of animals [1]. Individuals cooperate by engaging in costly behaviors that can be exploited by other individuals who benefit by avoiding these associated costs. Thus, if successful exploitation of social partners during cooperative interactions increases relative fitness, then we expect selection to lead to the emergence of a single optimal winning strategy in which individuals maximize their gain from cooperation while minimizing their associated costs [2]. Such social “cheating” appears to be widespread in nature [3], including in several microbial systems [4–11], but despite the fitness advantages favoring social cheating, populations tend to harbor significant variation in social success rather than a single optimal winning strategy. Using the social amoeba Dictyostelium discoideum, we provide a possible explanation for the coexistence of such variation. We find that genotypes typically designated as “cheaters” [12] because they produce a disproportionate number of spores in chimeric fruiting bodies do not actually gain higher fitness as a result of this apparent advantage because they produce smaller, less viable spores than putative “losers.” As a consequence of this trade-off between spore number and viability, genotypes with different spore production strategies, which give the appearance of differential social success, ultimately have similar realized fitness. These findings highlight the limitations of using single fitness proxies in evolutionary studies and suggest that interpreting social trait variation in terms of strategies like cheating or cooperating may be misleading unless these behaviors are considered in the context of the true multidimensional nature of fitness.

The evolution of cooperation is a paradox because natural selection should favor exploitative individuals that avoid paying their fair share of any costs. Such conflict between the self-interests of cooperating individuals often results in the evolution of complex, opponent-specific, social strategies and counterstrategies. However, the genetic and biological mechanisms underlying complex social strategies, and therefore the evolution of cooperative behavior, are largely unknown. To address this dearth of empirical data, we combine mathematical modeling, molecular genetic, and developmental approaches to test whether variation in the production of and response to social signals is sufficient to generate the complex partner-specific social success seen in the social amoeba Dictyostelium discoideum. Firstly, we find that the simple model of production of and response to social signals can generate the sort of apparent complex changes in social behavior seen in this system, without the need for partner recognition. Secondly, measurements of signal production and response in a mutant with a change in a single gene that leads to a shift in social behavior provide support for this model. Finally, these simple measurements of social signaling can also explain complex patterns of variation in social behavior generated by the natural genetic diversity found in isolates collected from the wild. Our studies therefore demonstrate a novel and elegantly simple underlying mechanistic basis for natural variation in complex social strategies in D. discoideum. More generally, they suggest that simple rules governing interactions between individuals can be sufficient to generate a diverse array of outcomes that appear complex and unpredictable when those rules are unknown.