More than one-third of the fruiting bodies we examined were chimeric and 95% of the experiments contained at least one chimeric fruiting body. This shows that, at least in a lab setting, D. discoideum and D. purpureum cells can interact, aggregate, and form chimeric fruiting bodies, although the average percentage of one clone in any given fruiting body is 90%, which indicates that the two species prefer to segregate but do so imperfectly. Both species had an equivalent proportion of the other species in the chimeric fruiting bodies, but because chimeras were more frequent in D. discoideum, there were more foreign spores found in fruiting bodies with the D. discoideum morphology. We measured relatedness to determine how much mixing and sorting is happening between the two species. The higher the relatedness, the less intermixing is occurring between the two species. An r-value of 0.5 means that the cells are randomly mixing, while an r-value of 1 means that the cells are completely sorting. Despite the presence of chimeric fruiting bodies, relatedness remained high within fruiting bodies of each phenotype (0.89 for D. discoideum, 0.94 for D. purpureum) on plates that began with an equal number of cells of each species.
It is remarkable, for several reasons, that we found such a high incidence of chimerism First, D. discoideum and D. purpureum are not particularly closely related. In the current phylogeny , the node separating the two species has 4 other species in the branch including D. discoideum and 17 others in the branch including D. purpureum. This phylogenetic distance is manifest in several developmental differences between the two species that may impact the level of sorting. D. purpureum forms a stalk as the slug migrates, while D. discoideum forms its stalk after the slug finishes migrating . Additionally, D. purpureum develops faster than D. discoideum. As a result, cells of D. purpureum may differentiate first leading to an increase in sorting if the genes responsible for cell-type partitioning and development up-regulate at different times. We found evidence of this pattern when we used time-lapse microscopy. Cells of both species aggregate together for a short time, but then D. purpureum slugs break off and migrate away from the initial mound, leaving mostly D. discoideum cells. A short time later, D. discoideum slugs begin to migrate and then form fruiting bodies. Interestingly, slugs contained cells from both species, indicating only partial disassociation.
Our chimerism result is also surprising because prior research failed to show chimerism, despite having mixed different species in a variety of ways. Raper and Thom  first mixed spores of D. discoideum and D. purpureum and reported the absence of intermediate phenotypes, which was in accordance with our results, but does not preclude chimerism. They then mixed D. discoideum spores with spores of D. mucoroides, a species that is as equally distant phylogentically as D. purpureum . They used the bacterium S. marcescens as a food source. S. marcescens contains a red pigment that D. discoideum is unable to digest, resulting in dyed cells [13, 25] while D. mucoroides digests the pigment and remains white. They found that the red cells initially aggregated together with the white cells but separated into red and white fruiting bodies. This shows that most cells segregated, but it is not clear if some individual cells of the wrong type might have been present.
Raper and Thom  also tried making grafts between different portions of the slugs of D. discoideum and D. purpureum, but were unsuccessful in getting the segments to permanently coalesce and form chimeric fruiting bodies. Using these data, they concluded that Dictyostelium species did not form chimeras. In one final experiment, they were able to obtain fruiting bodies with intermediate phenotypes by allowing cells of each species to form slugs and then crushing those slugs and mixing them . These fruiting bodies contained spores from both species. However, those fruiting bodies that retained the phenotype of only one parent only produced fruiting bodies of that same phenotype, seemingly indicating that those fruiting bodies consisted of one species. Bonner and Adams  also failed to find chimeras after they completed a series of experiments where they attempted to make intermediate fruiting bodies by grafting different species together during the aggregation stage. Neither group reported the density of spores that they used.
Perhaps we were able to find chimeras while the others did not because we plated out individual spores from fruiting bodies carefully at a very low density so we could detect low levels of mixing. Overall, there was mostly sorting, but there was some mixing, which may have been missed if not looked for carefully. We also used multiple clones, and had we used only one pair, an unlucky choice (for example mix 10 between clones QS75 and QSPu13 in Figure 2A–B) could have led us to the false conclusion that there was little mixing.
Finally, the finding of chimerism between species is surprising because both species apparently avoid chimerism even with other clones of their own species. Gilbert et al.  found that the relatedness for naturally occurring fruiting bodies collected in the wild that contained multiple clones of D. discoideum was 0.68, which was much lower than the overall relatedness of 0.98, because there were many clonal fruiting bodies. This result could be due either to sorting or to patchy distribution of clones. However, clear sorting was shown in fruiting bodies of D. purpureum when pairs of clones were mixed in 50:50 ratios; the result was an overall relatedness of 0.81 . Recently, somewhat weaker sorting has also been demonstrated between D. discoideum clones (Ostrowski et al. submitted). Our relatedness values for the two species mixed 50:50, were 0.89 for D. discoideum and 0.94 for D. purpureum. The higher values indicate greater clonal sorting than within-species mixes.
Why do these two species cooperate at least some of the time and is it true mutualism? Our system is unique and interesting in that it defies previous explanations of mutualism. In most cooperative interactions involving different species, each partner brings different goods or services to the association, such as between the Senita cactus and Senita moth, where the moth pollinates the cactus in exchange for a place to oviposit eggs and the larvae to subsequently eat a portion of the seeds . That is not the case with these two Dictyostelids because both species provide essentially the same services – migration and stalk formation. Mutualisms are now being recognized as lying on a continuum with parasitism. Some people also hypothesize that mutualism evolved from parasitism and that mutualism is best described as mutual exploitation [27, 28]. It may be that the two species are exploiting each other differentially, with D. discoideum benefiting most in the metric we measured. If this true, it may be that this interaction lies on the boundary between the two and is heading towards mutualism.
It is possible that the mixing is a mistake. Each species may undergo its social lifecycle where certain cells altruistically form stalk cells as it would if in a clonal population. Cells of different species may aggregate and develop together because of their close proximity to each other and similar developmental characteristics. Another possibility is that although this interaction evolved to provide beneficial cooperation within species (or even within clones), different species are able to benefit from those services, such as protection from predators, migration, spore formation and dispersal when they would otherwise not be able to because of a cell number deficiency. When both species face the possibility of being unable to aggregate on their own because they lack sufficient cell number, the two species will aggregate together and form fruiting bodies, to their mutual benefit, instead of dying out. Though we are unable to fully distinguish these hypotheses, we can provide an accounting of some of the costs and benefits that result from interspecies chimerism.
In any cooperative relationship, there are costs associated with each altruistic act. One such cost is that the altruistic act is not reciprocated, which may lead to the exploitation of one partner by the other. Cheating is the greatest concern when there is an interaction between two individuals that are not genetically identical. Earlier research shows that clones of D. discoideum may cheat each other, but prior experiments involving only D. purpureum clones show that the species maintains a high degree of kin discrimination by preferentially associating with kin without displaying a consistent pattern of cheating [16, 18]. The stronger segregation seen in D. purpureum may have evolved as a way to prevent cheating between clones, but it also might mean that this species no longer has a need to maintain mechanisms of cheating, or other defenses against cheating. When the two species are mixed together, D. discoideum's ability to cheat and D. purpureum's lack of a cheating mechanism may be the reason D. purpureum was exploited.
It is possible that this association is kept stable and that cheating is kept to a minimum because the aggregates form only when necessary and that they are kept as pure as possible, as indicated by the much higher relatedness values we calculated when compared to those found in previous studies. Additionally, both species suffered in the production of spores per fruiting body, which may be why the two species tend to segregate from each other despite some of the benefits that may be gained from the interaction.
We did not observe a clear benefit to this interaction that might explain why it has persisted. In terms of spore production, D. discoideum maintained the number of spores it produces while D. purpureum decreased the number of spores produced. However, additional possible benefits result from larger slug sizes that are not measurable using spore production, the metric we tested. One possible benefit for cells from both species is protection from predators. By aggregating together, the amoebae can initiate mechanisms to avoid soil predators such as nematodes. Kessin et al.  showed that Caenorhabditis elegans feeds on individual amoebae up through early aggregation. However, in late aggregation the cells form a polysaccharide sheath that the nematodes are unable to penetrate. This sheath protects the amoebae as they migrate as a multicellular slug. Once the fruiting body is formed, C. elegans may ingest the spores, but they are unable to digest them. Kessin et al.  found an additional benefit in D. purpureum: at high cell densities, it is able to repel nematodes. Therefore, it may be beneficial to both species to aggregate together when cell numbers are low, especially in the presence of predators.
Migration distance is another potential benefit of forming a larger slug. Foster et al.  found that larger slugs of D. discoideum traveled further than slugs containing half the number of cells. Also, they found that larger chimeric slugs traveled further than smaller clonal slugs. When slugs are traveling to a new location because the current one has run out of bacteria, larger slugs are more likely, over both smaller slugs and solitary cells, to reach a new patch of bacteria [30, 31].
A final possible benefit to co-aggregation is for spore dispersal purposes. To successfully disperse spores, they must be held aloft on a stalk of sufficient height. If there are too few cells in the aggregate, a fruiting body may not form at all. Or, even if a small fruiting body is able to form, it may be at a disadvantage relative to larger fruiting bodies, making it less likely to disperse due to contact from passing invertebrates.
Although we can only hypothesize about possible benefits to both species from cooperating, a mutualism would not evolve between these two species without gaining some type of fitness benefit. In single species fruiting bodies, some cells altruistically give up reproduction so that equally related cells become reproductive spores. In our experiments, cells still forfeit their reproductive ability so that related cells benefit. However, cells of the other species also benefit through by-product altruism, as they too are able to form reproductive spores because of the sacrifice of the other cells.