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Abstract Multicellular organisms that form by aggregation of cells arguably do not achieve high levels of complexity. Conflict among the cells is a widely accepted explanation for this, but an alternative hypothesis is that mixing cells of different genotypes leads to failures of coordination, which we call the “coordination hypothesis.” We empirically tested the coordination hypothesis in the social amoeba Dictyostelium discoideum. We mixed D. discoideum clones that had evolved in isolation for generations and acquired mutations that have not been tested against each other by selection. To quantify the effect of incompatibilities, we measured performance in terms of the developmental traits of slug migration and spore production. Importantly, we mixed lines evolved from the same ancestor under conditions that would not select for the evolution of de novo kin recognition. Our results show no evidence of incompatibilities in four traits related to the coordinated movement of slugs toward light in the social amoeba. Spore production was higher than expected in mixtures, in apparent contradiction to the coordination hypothesis. However, we found support for coordination incompatibilities in an interaction between migration and spore production: in mixtures, fewer cells succeeded at both migrating and becoming spores. 

Abstract Bacterial endosymbionts can provide benefits for their eukaryotic hosts, but it is often unclear if endosymbionts benefit from these relationships. The social amoeba Dictyostelium discoideum associates with three species of Paraburkholderia endosymbionts, including P. agricolaris and P. hayleyella. These endosymbionts can be costly to the host but are beneficial in certain contexts because they allow D. discoideum to carry prey bacteria through the dispersal stage. In experiments where no other species are present, P. hayleyella benefits from D. discoideum while P. agricolaris does not. However, the presence of other species may influence this symbiosis. We tested if P. agricolaris and P. hayleyella benefit from D. discoideum in the context of resource competition with Klebsiella pneumoniae, the typical laboratory prey of D. discoideum. Without D. discoideum, K. pneumoniae depressed the growth of both Paraburkholderia symbionts, consistent with competition. P. hayleyella was more harmed by interspecific competition than P. agricolaris. We found that P. hayleyella was rescued from competition by D. discoideum, while P. agricolaris was not. This may be because P. hayleyella is more specialized as an endosymbiont; it has a highly reduced genome compared to P. agricolaris and may have lost genes relevant for resource competition outside of its host. 

Chlamydiae represent a diverse group of obligate intracellular bacteria with elusive hosts in environmental settings. This study used one of the largest collections of wild amoebae (Dictyostelium discoideum and D. giganteum, 106 clones) collected over the past two decades to screen for novel environmental chlamydiae. We found that novel environmental chlamydiae are prevalent in two wild Dictyostelium species and assembled 42 novel chlamydiae metagenome-assembled genomes (MAGs). The MAGs represent three chlamydiae species previously only reported using 16S sequencing. Their genomes are divergent enough from other species to warrant placing them in two new genera (tentatively called Ca. Dictychlamydia sp. LF1, Ca. Dictychlamydia sp. LF2, and Ca. Feichlamydia sp. LF3). In addition, these chlamydiae species show strong host specificity with two Dictyostelium amoeba hosts, except one amoeba sample. Ca. Dictychlamydia sp. LF1 and Ca. Feichlamydia sp. LF3 was exclusively observed in D. discoideum, while Ca. Dictychlamydia sp. LF2 was found only in D. giganteum. Phylogenetic and comparative genomic analyses suggest that all three chlamydiae are close to arthropod-associated chlamydiae and likely have some intermediate characteristics between previously reported amoeba-associated and vertebrate-associated chlamydiae. This study significantly broadens our understanding of the chlamydial host range and underscores the role of amoebae as vital hosts for environmental chlamydiae. 

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Symbiotic interactions may change depending on third parties like predators or prey. Third-party interactions with prey bacteria are central to the symbiosis betweenDictyostelium discoideumsocial amoeba hosts andParaburkholderiabacterial symbionts. Symbiosis with inedibleParaburkholderiaallows hostD. discoideumto carry prey bacteria through the dispersal stage where hosts aggregate and develop into fruiting bodies that disperse spores. Carrying prey bacteria benefits hosts when prey are scarce but harms hosts when prey bacteria are plentiful, possibly because hosts leave some prey bacteria behind while carrying. Thus, understanding benefits and costs in this symbiosis requires measuring how many prey bacteria are eaten, carried and left behind by infected hosts. We found thatParaburkholderiainfection makes hosts leave behind both symbionts and prey bacteria. However, the number of prey bacteria left uneaten was too small to explain why infected hosts produced fewer spores than uninfected hosts. Turning to carried bacteria, we found that hosts carry prey bacteria more often after developing in prey-poor environments than in prey-rich ones. This suggests that carriage is actively modified to ensure hosts have prey in the harshest conditions. Our results show that multi-faceted interactions with third parties shape the evolution of symbioses in complex ways. 

Consumers range from specialists that feed on few resources to generalists that feed on many. Generalism has the clear advantage of having more resources to exploit, but the costs that limit generalism are less clear. We explore two understudied costs of generalism in a generalist amoeba predator,Dictyostelium discoideum, feeding on naturally co-occurring bacterial prey. Both involve costs of combining prey that are suitable on their own. First, amoebas exhibit a reduction in growth rate when they switched to one species of prey bacteria from another compared to controls that experience only the second prey. The effect was consistent across all six tested species of bacteria. These switching costs typically disappear within a day, indicating adjustment to new prey bacteria. This suggests that these costs are physiological. Second, amoebas usually grow more slowly on mixtures of prey bacteria compared to the expectation based on their growth on single prey. There were clear mixing costs in three of the six tested prey mixtures, and none showed significant mixing benefits. These results support the idea that, although amoebas can consume a variety of prey, they must use partially different methods and thus must pay costs to handle multiple prey, either sequentially or simultaneously. 

Cooperation is widespread across life, but its existence can be threatened by exploitation. The rise of obligate social cheaters that are incapable of contributing to a necessary cooperative function can lead to the loss of that function. In the social amoebaDictyostelium discoideum, obligate social cheaters cannot form dead stalk cells and in chimeras instead form living spore cells. This gives them a competitive advantage within chimeras. However, obligate cheaters of this kind have thus far not been found in nature, probably because they are often enough in clonal populations that they need to retain the ability to produce stalks. In this study we discovered an additional cost to obligate cheaters. Even when there are wild-type cells to parasitize, the chimeric fruiting bodies that result have shorter stalks and these are disadvantaged in spore dispersal. The inability of obligate cheaters to form fruiting bodies when they are on their own combined with the lower functionality of fruiting bodies when they are not represent limits on obligate social cheating as a strategy. 

The evolution of symbiotic interactions may be affected by unpredictable conditions. However, a link between prevalence of these conditions and symbiosis has not been widely demonstrated. We test for these associations usingDictyostelium discoideumsocial amoebae and their bacterial endosymbionts.D. discoideumcommonly hosts endosymbiotic bacteria from three taxa:Paraburkholderia, Amoebophilusand Chlamydiae. Three species of facultativeParaburkholderiaendosymbionts are the best studied and give hosts the ability to carry prey bacteria through the dispersal stage to new environments.Amoebophilusand Chlamydiae are obligate endosymbiont lineages with no measurable impact on host fitness. We tested whether the frequency of both single infections and coinfections of these symbionts were associated with the unpredictability of their soil environments by using symbiont presence-absence data fromD. discoideumisolates from 21 locations across the eastern United States. We found that symbiosis across all infection types, symbiosis withAmoebophilusand Chlamydiae obligate endosymbionts, and symbiosis involving coinfections were not associated with any of our measures. However, unpredictable precipitation was associated with symbiosis in two species ofParaburkholderia, suggesting a link between unpredictable conditions and symbiosis. 

Abstract Aggregative multicellularity relies on cooperation among formerly independent cells to form a multicellular body. Previous work with Dictyostelium discoideum showed that experimental evolution under low relatedness profoundly decreased cooperation, as indicated by the loss of fruiting body formation in many clones and an increase of cheaters that contribute proportionally more to spores than to the dead stalk. Using whole-genome sequencing and variant analysis of these lines, we identified 38 single nucleotide polymorphisms in 29 genes. Each gene had 1 variant except for grlG (encoding a G protein-coupled receptor), which had 10 unique SNPs and 5 structural variants. Variants in the 5′ half of grlG—the region encoding the signal peptide and the extracellular binding domain—were significantly associated with the loss of fruiting body formation; the association was not significant in the 3′ half of the gene. These results suggest that the loss of grlG was adaptive under low relatedness and that at least the 5′ half of the gene is important for cooperation and multicellular development. This is surprising given some previous evidence that grlG encodes a folate receptor involved in predation, which occurs only during the single-celled stage. However, non-fruiting mutants showed little increase in a parallel evolution experiment where the multicellular stage was prevented from happening. This shows that non-fruiting mutants are not generally selected by any predation advantage but rather by something—likely cheating—during the multicellular stage. 

Some endosymbionts living within a host must modulate their hosts’ immune systems in order to infect and persist. We studied the effect of a bacterial endosymbiont on a facultatively multicellular social amoeba host. Aggregates of the amoeba Dictyostelium discoideum contain a subpopulation of sentinel cells that function akin to the immune systems of more conventional multicellular organisms. Sentinel cells sequester and discard toxins from D. discoideum aggregates and may play a central role in defence against pathogens. We measured the number and functionality of sentinel cells in aggregates of D. discoideum infected by bacterial endosymbionts in the genus Paraburkholderia. Infected D. discoideum produced fewer and less functional sentinel cells, suggesting that Paraburkholderia may interfere with its host’s immune system. Despite impaired sentinel cells, however, infected D. discoideum were less sensitive to ethidium bromide toxicity, suggesting that Paraburkholderia may also have a protective effect on its host. By contrast, D. discoideum infected by Paraburkholderia did not show differences in their sensitivity to two non-symbiotic pathogens. Our results expand previous work on yet another aspect of the complicated relationship between D. discoideum and Paraburkholderia, which has considerable potential as a model for the study of symbiosis.