BRIEF COMMUNICATIONdoi:10.1111/j.1558-5646.2010.00959.xNOVEL COOPERATION EXPERIMENTALLYEVOLVED BETWEEN SPECIESWilliam Harcombe1,2,31Section of Integrative Biology, The University of Texas at Austin, Austin, Texas 787122E-mail: [email protected] August 4, 2009Accepted December 18, 2009Cooperation violates the view of “nature red in tooth and claw” that prevails in our understanding of evolution, yet examples ofcooperation abound. Most work has focused on maintenance of cooperation within a single species through mechanisms such askin selection. The factors necessary for the evolutionary origin of aiding unrelated individuals such as members of another specieshave not been experimentally tested. Here, I demonstrate that cooperation between species can be evolved in the laboratory if(1) there is preexisting reciprocation or feedback for cooperation, and (2) reciprocation is preferentially received by cooperativegenotypes. I used a two species system involving Salmonella enterica ser. Typhimurium and an Escherichia coli mutant unable tosynthesize an essential amino acid. In lactose media Salmonella consumes metabolic waste from E. coli, thus creating a mechanismof reciprocation for cooperation. Growth in a spatially structured environment assured that the benefits of cooperation werepreferentially received by cooperative genotypes. Salmonella evolved to aid E. coli by excreting a costly amino acid, however thisnovel cooperation disappeared if the waste consumption or spatial structure were removed. This study builds on previous work todemonstrate an experimental origin of interspecific cooperation, and to test the factors necessary for such interactions to arise.KEY WORDS: Consortia, cross-feeding, E. coli, Salmonella, spatial structure.Cooperation is a problem that has mystified biologists since theoriginal proposal of evolution by natural selection. Natural se-lection should favor selfish acts, and yet cooperation is evidentat all levels of biological organization from genes to societies. Alarge body of theory has been generated to explain the patternsobserved in nature (Sachs et al. 2004; West et al. 2007a), and re-cently, exciting empirical tests of the theory have begun to emerge(Griffin et al. 2004; MacLean and Gudelj 2006; Ross-Gillespieet al. 2007). T hese tests largely focus on the maintenance ofcooperative traits within a species. However, we lack a clear illus-tration of the mechanisms necessary for the evolutionary originof cooperation, particularly between species.Previous work suggests that several factors are important forthe evolution of interspecies cooperation (Trivers 1971; Sachset al. 2004; West et al. 2007a). When an organism aids an individ-3Current address: Organismal and Evolutionary Biology, HarvardUniversity, Cambridge, MA 02138.ual of another species it must acquire a direct benefit in return, as itis not feasible to gain inclusive fitness simply through increasing arecipients reproductive potential. Cooperation between unrelatedindividuals likely depends on (1) reciprocation between partners,and (2) direction of reciprocation to cooperating individuals. Thisraises several intriguing questions. If it is only advantageous tocooperate if your partner also cooperates, how does the processbegin? Furthermore, how can benefits be directed to specific coop-erating individuals of another species? Finally, are the conditionsthat maintain cooperation sufficient for its origin?Excretion of waste products may provide a mechanism forthe initiation of reciprocation (Sachs et al. 2004). Excretion ofwaste is clearly not a costly process that needs evolutionary ex-planation, but waste products can often be beneficial for otherorganisms. For example, some insects benefit from the feces ofcows, and bacteria often acquire metabolites from the excretionsof other microbes (Schink 1997, 2002). These benefits could pro-vide the foundation for the evolution of cooperation. A user of1C2010 The Author(s).EvolutionBRIEF COMMUNICATIONwaste products may be selected to help its partner as a way ofincreasing the waste products received. Such selection could giverise to costly cooperation, that is, costly to the producer but whichultimately benefits the producer by increasing the reciprocationfrom the partner.A spatially structured environment may provide a mechanismthat directs benefits to cooperating individuals (Griffin et al. 2004;Sachs et al. 2004). Individuals that pay a cost to help their partnerswill only spread in a population if they get more of the benefitsfrom the partner than do individuals that do not pay the cost ofhelping. Spatial structure may facilitate the direction of benefitsby localizing interactions. In the extreme, spatial structure cancreate patches that contain just one individual of each species.Patches that contain cooperators will permit more growth thanthose patches that do not. However, perhaps surprisingly, spatialstructure can also lead to the evolution of intensified antagonisticinteractions between partners (West et al. 2001), so the effect ofspatial structure is not clear.A system of two bacterial species was used to test whetherour current understandings can be used to evolve novel coopera-tion. The system involved Salmonella enterica ser. Typhimuriumand an Escherichia coli mutant unable to synthesize methionine(met-E. coli). A preexisting mechanism that would allow for re-ciprocation was created by growing the two species in lactose.Escherichia coli metabolizes lactose and then excretes costlessmetabolic byproducts on which Salmonella can feed. A methodof directing benefits was provided by growing the community onagar plates. Although evolution could have improved the growthof each species independently, a cooperative adaptation arose.Salmonella evolved to secrete the amino acid that E. coli required.This origin of cooperation was dependent on both a preexistingmechanism of reciprocation and a method of directing benefits.Materials and MethodsSYSTEMThe study system consisted of E. coli and Salmonella.TheE.coli strains used was E. coli K12 BW25113 (rrnB3 lacZ4787hsdR514 (araBAD)568 rph-1) with a metA knockout. This linewas acquired as part of the Keio collection (JW3973) (Baba et al.2006). To reenable lactose metabolism the E. coli was mated for 40min with E. coli HfrH PO1 relA1 thi-1 spoT supQ80 nad57::Tn10.The constructed E. coli line achieves no appreciable growth inminimal media in the absence of methionine. The