ARTICLE Received 8 Apr 2013 | Accepted 31 Jul 2013 | Published 13 Sep 2013 DOI: 10.1038/ncomms3385 Social amoeba farmers carry defensive symbionts to protect and privatize their crops Debra A. Brock1, Silven Read2, Alona Bozhchenko2, David C. Queller1 & Joan E. Strassmann1 Agricultural crops are investments that can be exploited by others. Farmer clones of the social amoeba Dictyostelium discoideum carry bacteria to seed out new food populations but they also carry other non-food bacteria such as Burkholderia spp. Here we demonstrate that these farmer-carried Burkholderia inhibit the growth of non-farmer D. discoideum clones that could exploit the farmers’ crops. Using supernatants, we show that inhibition is due to molecules secreted by Burkholderia. When farmer and non-farmer amoebae are mixed together at various frequencies and allowed to complete the social stage, the ability of non-farmers to produce spores falls off rapidly with an increase in the percentage of farmers and their defensive symbionts. Conversely, farmer spore production is unaffected by the frequency of non-farmers. Our results suggest that successful farming is a complex evolutionary adaptation because it requires additional strategies, such as recruiting third parties, to effectively defend and privatize crops. 1 Department of Biology, Washington University at St. Louis, St. Louis, Missouri 63130, USA. 2 Department of Ecology and Evolutionary Biology, Rice University, Houston, Texas 77005, USA. Correspondence and requests for materials should be addressed to D.A.B. (email: [email protected]). NATURE COMMUNICATIONS | 4:2385 | DOI: 10.1038/ncomms3385 | www.nature.com/naturecommunications 1 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3385 ocial dilemmas, in which individual good conflicts with 1 2,3 Table 1 | List of Dictyostelium discoideum clones used in each collective good , are common in nature . For example, experimental assay. SDictyostelium discoideum social amoebas divide asexually until their bacterial food source runs out and then they aggregate 4,5 Clone Type Spore Supernatant Supernatant Competition and cooperate to form a multicellular fruiting body . About 20% production assay: assay: assay of the cells die in the process of building a stalk that puts spores assay in amoebae spores (the other 80% of cells) in a position to be dispersed to better Burkholderia areas. The dilemma arises because groups form by aggregation; xenovorans hence, stalk production by one clone can be exploited by others, QS1NFXXXX sometimes called cheaters, which produce little or no stalk. In this QS6 NF X case, although cheating occurs6,7, it is greatly reduced by a variety QS8A F X of means, including kin recognition and other mechanisms that QS9NFXXXX make many groups clonal5,8–11; thus, stalk cell altruism can be QS11 F X X X X favoured by kin selection. QS14 NF X Some clones of D. discoideum face another social dilemma. QS17 NF X X X X QS18 NF X X X X Farmer clones carry potentially exploitable food bacteria through 12 QS21 F X X X X their dispersing spore stage to seed out after dispersal . This QS22 F X X X X behaviour is costly because farmers leave some uneaten bacteria QS23 F X X X X behind, reducing the farmer’s ultimate reproductive spore QS154 NF X production. However, carrying bacteria pays when the spores QS155 F X X X X are dispersed to areas without good food bacteria. The ability of QS158 NF X farmers to bring bacteria that previously allowed them to flourish QS159 F X at another site now allows them to capitalize on available QS160 NF X X X X nutrients and increase their reproductive spore production. NC63.2 F X Agriculture is very rare in non-humans13–17, perhaps because NC75.2 NF X NC105.1 F X of the social dilemma inherent in investing in crops that can be NC174 NF X exploited by others. In addition, farming by humans is the classic setting for the social dilemma called the tragedy of the commons; NF, non-farmer; F, farmer. each farmer who adds a cow to a common pasturage gains the whole benefit, while imposing most of the costs on others, so the pasture becomes overgrazed, to the detriment of all18. An even consisting of 5 non-farmers and 5 farmers that carry bacteria more basic tragedy of the commons is glossed over in this B. xenovorans (498% identity using a 481-bp PCR fragment of account. If the cows themselves become commons and anyone 16S ribosomal DNA for comparison), the most common non-food can harvest their milk or meat, no farmers would invest in them. bacterium isolated from farmers. We separately cultured spores Non-human farmers, which include some ants, termites, collected from these 10 D. discoideum clones, providing only ambrosia beetles, marine snails and damselfish13–17, must B. xenovorans as a food source. Non-farmer D. discoideum clones somehow solve this second dilemma without the benefit of were unable to grow in these pure cultures, with mortality so anything like human institutions19 that guarantee property rights severe that spore production was zero (Fig. 1). However, farmers or a just division of goods. D. discoideum farmers produce a were able to produce some spores in this treatment, although far valuable crop that could be eaten by a non-farming competitor fewer than when they are reared on a better food bacterium. It clone who does not pay the costs of farming, so why is farming is not clear whether the farmer clones were eating B. xenovorans evolutionarily stable? Living in high-relatedness groups should or whether they were feeding on carried K. pneumoniae that help but multiple D. discoideum clones often co-occur in 0.2 gm they had been grown on previously; however, it is clear that soil samples20 and sometimes even in single fruiting bodies8; thus, B. xenovorans carried by farmers is at best a very poor food source. exploitation remains an important concern. All farmers raised in the lab with the food bacterium Klebsiella B. xenovorans harm non-farmer but not farmer clones. Nor- pneumoniae will carry it while retaining one or more of a number mally, both B. xenovorans and a food bacterium like K. pneu- of other species12. Some of these carried bacteria are not good moniae would be available to proliferating D. discoideum food sources. As bacterial species produce a wide range of amoebae. Therefore, we also tested farmers and non-farmers on chemical products including antibiotics, virulence factors and bacteria mixes with initial compositions dominated by K. pneu- bacteriocins21–23, these non-food bacteria could have pathogenic moniae but with 5 or 10% B. xenovorans mixed in. The smallest effects; however, this seems inconsistent with the observed ability initial amount of B. xenovorans (5%) significantly reduced non- of farmers to proliferate as well as non-farmers12. Alternatively, farmer spore production, yielding fewer than half as many spores these bacteria might provide a benefit other than food, for as controls, whereas spore production for farmers was statistically example, defending against non-farmer D. discoideum clones to unchanged (Fig. 1). We found comparable results in the 10% help privatize farmers’ crops. Here we show that these non-food B. xenovorans treatment, although with a slight reduction in bacteria also provide advantages to their host; they secrete farmer spores. This evidence is consistent with the hypothesis molecules that promote the growth of their host but inhibit that B. xenovorans bacteria carried by farmers selectively harm the growth of non-farming conspecifics of the host. Thus, their non-farmer D. discoideum competitors. D. discoideum farmers privatize their crops by carrying defensive symbionts directed at competitors. B. xenovorans supernatants benefit farmers and harm non- farmers. To test whether the reduction in non-farmer spore Results production is because of secreted biomolecules of B. xenovorans, Burkholderia xenovorans is inedible by D. discoideum. To test we tested each of the same five farmer and five non-farmer clones this hypothesis, we used a set of 10 D. discoideum clones (Table 1) against supernatants collected from each of the five farmers’ 2 NATURE COMMUNICATIONS | 4:2385 | DOI: 10.1038/ncomms3385 | www.nature.com/naturecommunications & 2013 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3385 ARTICLE 800 300 a Starvation buffer Farmers 700 Bx supernatant Non-farmers 250 a *** *** 600 after growth a,b 6 200 500 after 3 hr growth 10 4 × b,c 10 400 × spores 150 c c 300 amoebae 200 100 D. discoideum D. 100 Total Total 50 D. discoideum D. 0 Total Total 100% Kp 5% Bx + 10% Bx + 100% Bx 95% Kp 90% Kp 0 Farmers Non-farmers Proportion of K. pneumoniae and B. xenovorans as food sources Solitary stage Figure 1 | Burkholderia xenovorans (Bx) is a poor food for D. discoideum. In 100% Bx, non-farmers are unable to produce any spores, whereas spore 120 Starvation buffer production for farmers is greatly reduced compared with 100% Klebsiella pneumoniae (Kp) control. However, small amounts of Bx harm non-farmer Bx supernatant ** clones but not farmer clones. Non-farmers produce significantly fewer 100 * spores in 5% Bx þ 95% Kp and 10% Bx þ 90% Kp compared with 100% Kp control, whereas farmers have minimal change in spore production under 80 the same conditions (mixed model analysis of variance: farmer F1,8 ¼ 0.95, after social stage 4 P ¼ 0.3588, treatment F2,16 ¼ 78.38, Po0.0001, farmer  treatment 10 F2,16 ¼ 28.59, Po0.0001, n ¼ 10); Significant differences found between × farmers and non-farmers are indicated by different letters, which reflect 60 results of a post hoc Tukey’s honestly significantly different (HSD) test (this spores analysis excluded the 100% Bx treatment that was not normally distributed 40 and not directly relevant to the question of protecting a crop).