Dictyostelium, the Social

Joan E. Strassmann1, Sandra L. Baldauf2 1Washington University in St. Louis MO USA 2Uppsala University, Uppsala Sweden [email protected] [email protected]

Glossary entries: : A behavior that is costly to the performer’s fitness, but beneficial to others.

Greenbeard gene: A gene that affects copies of itself via three effects: production of trait, recognition of the trait in others, and differential treatment based on that trait. Sometimes not considered as part of kin selection because benefits go not to relatives but to actual bearers of the gene.

Mutualism: An interaction that benefits both parties. Can be used for interactions within and between species.

Social amoeba: A in the Dictyostelia, a in the .

Social evolution: Evolution of traits of organisms that have fitness consequences for others of the same species, in particular those traits that may benefit others at a cost to oneself. Evolution of social interactions.

Sociogenomics: Study of the genetic and genomic foundations of social behaviors.

Symbiosis: Living together in close association in ways that may be beneficial or harmful for either party.

Keywords Altruism Greenbeard gene Mutualism Social amoeba Social evolution Sociogenomics

Abstract: The Dictyostelia present a splendid opportunity for the study of mutualism, sociality and genetic conflicts of interest. These amoebae aggregate upon starvation to form cooperative multicellular structures in which some formerly independent cells die to form a stalk. This serves to lift the other cells above the substrate where their chances of dispersal are greatly enhanced, for example by sticking to passing invertebrates. Dictyostelia vary in their social organization and the cells can be cultured from soil samples from nearly all parts of the world. Furthermore, they have complex symbiotic interactions with . Together these make many kinds of studies possible. sequences are also available for increasing numbers of species; many molecular pathways are known; and experimental evolution is feasible. Who lives, who dies, and how sociality and mutualism are structured are great questions that are easily addressed in this group.

INTRODUCTION The social amoeba is an odd model system for behavior: it lacks a nervous system, is not an , and is only briefly multi-celled (Kessin, 2001, Baldauf and Strassmann, 2017). On the other hand, it is hard to imagine an organism more ideally suited to advancing our understanding of social behavior. Its social life is fascinating and tools developed by hundreds of cell and molecular biologists over the last few decades allow a gene-based approach to understanding its sociality (Strassmann and Queller, 2011, Madgwick et al., 2018). Studies of Dictyostelium provide a crucial independent test of social evolution theories, since these theories were developed with social insects and vertebrates in mind, not social amoebae. D. discoideum is a eukaryote that lives most of its life dispersed as independent amoebae primarily in the forest soil. They eat bacteria, and divide around every 4 h when food is abundant. But when they run out of food, a much more intense social stage begins (Figure 1). The amoebae aggregate by the hundreds of thousands and form a multicellular motile organism (). Ultimately, the multicellular slug organizes itself further into a fruiting body in which about 25% of the cells die to form a rigid cellulose-walled stalk while the remaining cells ascend this stalk and form a bolus of hardy at the top of the stalk (sorus). In this way, these tiny amoebae greatly increase their chances to be dispersed (Smith et al., 2014). This is essentially one-stop sociality, with a single, magnificent altruistic act whereby a subset of formerly independent cells benefits the rest. It can be compared both to a major transition to multicellularity and to the altruism of social insect workers. In some ways, the social-insect comparison is apt because, unlike most multicellular organisms, which consist of clones of cells, Dictyostelium sp. arrive at multicellularity by aggregation. Therefore, as in social insects, we might expect both altruism favored by kin selection and conflicts between the different genotypes in an aggregate. Given the genetic tools available for dicty, there is great potential for understanding the mechanisms of altruism and the control of conflict in this organism, making it a rich field for graduate students. This piece introduces the group, points out some of the most important molecular and genomic tools, summarizes what is known of its social behavior, and suggests promising future directions.

BACKGROUND

Where Is Dictyostelium on the Tree of Life? D. discoideum is the best-studied member of the Dictyostelia which is in the Amoebozoa, a superkingdom closely related to and fungi (Sheikh et al., 2017, Schaap et al., 2006). We will henceforth call D. discoideum by its vernacular name, dicty. The rest of the approximately 150 described species of Dictyostelia are much less well studied and will be referred to here by their scientific names. Dicty occupies a fascinating place on the Tree of Life, with important cellular traits shared uniquely with fungi and animals, including humans. Given the ancient age of Dictyostelia of about ~600 mya (Fiz-Palacios et al., 2013) and the small number of described species, it is highly likely that much of its diversity is undiscovered. This should change greatly with sequencing of more wild-collected clones as well as direct sequencing (culture independent sampling) of soils from around the world (Baldauf et al., 2018). The of Dictyostelia has recently been revised to match the new molecular trees (Sheikh et al., 2017). As a result the rather simple traditional morphology-based taxonomy, which recognized three genera, has now been replaced with two orders, four families and 12 genera (Fig. 2, (Sheikh et al., 2017). Dicty is found in the Dictyosteliaceae, home to most of the hardiest and easily cultured species (Dictyostelium sp. and sp.).

Polysphondylium sp. are some of the most striking . Their fruiting bodies are decorated with delicate whorls of side branches that are evenly spaced along the stalk and each topped by a deep purple sorus. (Fig 2). Like dicty, the polysphondylids tend to have large, robust, easily-cultured fruiting bodies built from hundreds of thousands of formerly independent amoebae. Another notable group of Dictyostelids are the heterostelids (Heterostelium sp.). Many of these also form ornate polysphondylid-type sorocarps, but these are pale in color and tiny, thus requiring the cooperation of far fewer amoebae to build the sorocarps. Heterostelium is quite a distant relative of Polysphondylium, meaning that this very distinctive morphology has evolved at least twice . At the other extreme are the sp.. These also have a social stage, but one that does not require the sacrifice of any cells to build the sorocarp. Thus, no cells die in the formation of an acytostelid sorocarp. Instead, it forms tiny stalks made entirely from cellular secretions, and all the aggregating amoebae survive to form spores. Although this behavior was once considered primitive (for dictyostelids), the molecular trees show instead that acytostelid simplicity is derived, that is, it evolved from an ancestor with altruistic development. This also means that altruism was probably lost and regained at least once. All this social variation in the Dictyostelia greatly enhances their value as a model social group.

Where Dicty Lives Dictyostelia live in the upper layers of soil where they are predatory on bacteria, eating them by engulfment (Raper, 1984). Dicty is particularly common in autumn when leaf litter is abundant. Some species are more widespread than others, with D. mucoroides and Polysphondylium violaceum among the most ubiquitous. Dicty was first described by Kenneth Raper from a site just off the Blue Ridge Parkway near Mount Mitchell, NC, USA. It is abundant in forest soils of the Appalachians above about 1000 m elevation, but it also occurs generally in the eastern United States, with collections made from Houston, TX, to northern Minnesota and Massachusetts. Other samples assigned to this species have been collected as far South as Costa Rica. It has also been found along the eastern coast of Asia, including China, Japan and India, but not in Europe or Africa. However, other Dictyostelia can be found throughout the world, including clones detected in Antarctic lake sediments and New Zealand forest canopy soils (Baldauf et al. 2018). Life Cycle There are three important cycles in the life of dicty, asexual division, sexual aggregation and , and the social cycle (Figure 1). During the feeding stage of their life, dicty exists as independent amoebae that move through the soil by advancing pseudopods and engulfing any bacteria they encounter (Bonner, 1967). The amoebae divide about every 4 h when bacteria are plentiful (Figure 1, steps 2 and 3). At this stage in their life, their existence is essentially solitary since they do not depend on others to eat, move, or divide. However, it is clear that communication among amoebae is maintained through small signaling proteins like CMF and PSF, which function as quorum sensing molecules and more (Kessin, 2001). This communication is important because starvation may be near. When an amoeba has stopped finding enough bacteria for food and senses that there are sufficient other amoebae nearby, a dramatic change transpires. When starvation occurs in dark, moist, warm conditions lacking in phosphorus, with calcium present, the sexual stage is initiated if different mating types are in close proximity (Figure 1, steps A and B). There are three mating types (Bloomfield et al., 2010). Two cells of opposite mating types fuse, forming a diploid zygote. During the amoeba stage, the cells are haploid, so meiosis does not occur before fusion. The zygote is attractive to the thousands of nearby amoebae, which are engulfed and eaten by the zygote, which grows to an enormous size (for a dicty), forming a (Figure 3(d)). The macrocyst then undergoes meiosis and then to form thousands of recombinant cells. Unfortunately for students of the system, laboratory conditions for hatching these recombinant cells have not been well worked out, but, in a major advance, the genetics of mating types is now known (Bloomfield et al., 2010). The other form of aggregation which leads to a multicellular slug and ultimately a fruiting body, has little cell division and no recombination (Figure 1, steps 4–7). The starving amoebae begin to signal to each other with cAMP released to the environment. They not only release cAMP, but also move toward it. The cells elongate as they move, and a cAMP gradient is produced that radiates outward so that o all the cells move toward a single point. As more and more cells starve and being to migrate, they form great streams, flowing toward a center. Altogether this process is called aggregation (Figure 3(a)). After a few hours, migration ceases and the center concentrates into a mound. This mound then elongates upward until it falls over and begins to crawl around like a small slug, with the same end (head) oriented toward light and heat and away from ammonia (Figure 3(b)). This translucent slug looks like a tiny worm, but differs from it in some important ways. As it crawls through a sheath largely made up of cellulose, it drops cells at the rear, and these cells can feed on any bacteria they discover, effectively recovering the solitary stage (Kuzdzal-Fick et al., 2007). A critical point for dicty is that the slug moves more quickly and farther than any individual amoeba could move. Though the slug lacks a nervous system, there are differences among the constituent cells. Those at the front direct movement and ultimately become the stalk. There is another recently discovered class of slug cells called sentinel cells. These sweep through the slug from front to back picking up toxins and bacteria, functioning simultaneously as liver, kidney, and , before they are shed at the rear of the slug (Chen et al., 2007). move farther and for a longer time when the environment lacks electrolytes, when it is very moist, and when there is either directional light or no light. When they cease moving, the cells of the slug concentrate into a tight aggregate. Here, in a process called culmination, the cells that were at the front of the slug begin to form cellulose walls and to rise up out of the mass as a very slender but rigid stalk (Figure 3). These cells die. The remaining three-quarters or so of the cells flow up this stalk, and at the top they form hardy spores in the sorus. At this point, the sorus, stalk, and basal disk comprise an erect structure called a fruiting body (sorocarp, Figure 3(c)). Thus, some of the dicty cells sacrifice their lives so that the others may rise up and sporulate a millimeter or so above the soil surface, or into a gap between soil particles. Other cells sacrifice themselves as sentinel cells picking up toxins and bacteria as they made their way through the slug. Still others were shed from the rear of the slug during their normal movement. If these do not encounter bacteria for food, or enough other shed cells to form a new, smaller fruiting body, then they also perish.

Dicty Bacteria Interaction: Dinner, Symbiont, Macrophage, and Trojan Horse Dicty eats by engulfing bacteria and then digesting them in the resulting (Cosson and Soldati, 2008). This is similar to the way macrophages in animals engulf bacteria for destruction. However, recent work on dicty has discovered that some bacteria are not digested but instead form a long-lasting symbiotic association. Several new species of , a gram negative betaproteobacterium reside indefinitely inside dicty (Brock et al., 2011, DiSalvo et al., 2015). Burkholderia sp. do not normally serve as food but facilitate uptake and carriage of other bacteria that are food. Dicty that carry bacteria and release them into the environment where they can proliferate and be consumed later can be called farmers (Brock et al., 2011). Besides facilitating bacterial carriage, Burkholderia have a number of other impacts on dicty. The carriers of Burkholderia have fewer sentinel cells. They move less far in the slug stage. Dicty that were collected from the wild harboring Burkholderia are less damaged by them indicating an evolutionary tolerance of the symbiosis (Brock et al., 2015). And importantly, carriers of Burkholderia secrete small molecules that are harmless to themselves and toxic to non-farmers (Brock et al., 2013). Investigations into bacteria-dicty interactions are in their infancy and are sure to reveal much about this symbiosis and its importance as a refuge for harmful bacteria (Gerstenmaier et al., 2015).

How Dictyostelids Are Obtained, Collected, and Cultured Many studies can be performed using previously collected clones obtained from the stock center for a modest price. This stock center is accessed through www.dictybase.org and preserves thousands of clones. Most of these are genetically modified versions of the type clone, NC4. Early modifications allowed for growth in a bacteria-free shaking medium; these axenically grown clones are referred to as Ax2, Ax4, and related names. There are many single-gene knock-out clones that are of interest to students of social genes. Chris Thompson’s group has taken a methodical approach to knocking out all possible genes and has a Genome Wide Dictyostelium Insertion (GWDI) library of 21,529 strains with 12,247 different insertions in 5,705 of the around 12,000 genes in dicty. These mutants can be seen at remi-seq.org and ordered from the stock center mentioned above. The stock center also has hundreds of unmodified clones collected from the wild. Many are dicty, but there are also quite a few other species represented in this stock center. These clones are preserved in liquid nitrogen tanks and shipped out on request to researchers for a modest fee. It is a lot of fun to culture your own dicty or other Dictyostelids from the wild. This process involves placing a few drops of a dilute soil sample on a low nutrient agar plate (e.g. hay-infusion agar), optionally inoculated with a bacterial strain to provide food for the Dictyostelids. The isolation process is basically a race to visualize and isolate Dictyostelids from other competing organisms in the soil, especially fungi and bacteria. Detailed instructions for collecting soil samples and culturing and isolating Dictyostelids are easy to follow (Douglas et al., 2013). Clones of Dictyostelium and Polysphondylium are more easily isolated than the smaller, more delicate, and slower developing Raperostelium, Speleostelium, Heterostelium, Acytostelium and Cavenderia.

WHAT CAN SOCIAL EVOLUTION THEORY TELL US ABOUT DICTY?

What Are the Benefits to Grouping in Dicty? The social stage in the dicty life cycle involves the clear cost of death for about a quarter of all cells, and so there should be a compensating advantage. This advantage cannot accrue to the dying cells, but there could be a kin-selection benefit to genetically identical clonemates that joined the same aggregation. We first discuss the advantages, then the disadvantages, to grouping with non- clonemates, and then the genetic relatedness within cooperating groups. An early stage in aggregation is the slug, which can move tens of centimeters, through a protective cellulose sheath. This movement may bring the constituent cells to a new location where bacteria are more plentiful. Cells that are shed during movement can themselves take advantage of any food sources they encounter. Clearly, movement is facilitated by the social stage compared to the movement of individual amoebae, and it occurs in the relative protection of the cellulose sheath. Slugs made up of larger numbers of amoebae move farther than those with fewer amoebae. Once the slug has finished moving, it forms a stalk of dead cells that the living cells climb. This stalk provides a benefit in lifting the spores above the substrate to sporulate and be more easily and widely dispersed. Larger groups both make longer stalks and invest a slightly smaller proportion of individuals in the stalk. Nearly all species except dicty form a stalk from the beginning of migration (instead of a free slug). This facilitates gap crossing in the three-dimensional soil matrix, but it means that cells die and are lost from the migrating group (Raper, 1984, Gilbert et al., 2012a).

What Are the Costs of Grouping with Nonrelatives in Dicty? The advantages to grouping in dicty may not accrue equally to all genotypes if multiple genotypes are represented in a single fruiting body. In particular, clones that succeed in avoiding contributing to the dead stalk cells will be more represented in the next generation. Some clones may be able to avoid stalk contribution when paired with others. When two clones are mixed, one often predominates among the spores while avoiding contribution to the stalk cells. In a round robin tournament, where every clone is paired against the others, there is a dominance hierarchy in which some clones consistently dominate in contribution (Fortunato et al., 2003a). This is interesting and puzzling, for if they are consistently dominant, we would not expect the losing forms to persist in nature, particularly in the same habitat. This puzzle can be solved if different clones dominate under different conditions, if there are tradeoffs in dominance, if the environment is changeable enough that the system is not at equilibrium, or if frequency determines strategy (Parkinson et al., 2011, Madgwick et al., 2018, Wolf et al., 2015, Buttery et al., 2009). This result, that clones compete in fruiting bodies and do not pay the costs of stalk formation equally, is interesting and important and sets the stage for future investigations. If there is conflict within an aggregation over which cells become spore and which become stalk, we expect that conflict may also be expressed earlier, as the slug migrates. Since the front of the slug is the organizing center that directs movement and later becomes stalk, cells in a chimera of two or more clones may be less willing to join this altruistic region, and this hesitancy may slow slug movement. This is exactly what happened. For a given number of cells, pure clones moved farther than chimeras (Figure 4).

What Is Relatedness Within a Dicty Fruiting Body? One of the challenges of working on a is that they are hard to see. Even though fruiting bodies of dicty measure 1–4 mm and so are visible without magnification, they are hard to find in nature. Naturally occurring fruiting bodies on deer feces were first seen near the main building at Mountain Lake Biological Station on 15 October 2000. However wild-collected fruiting bodies were not successfully genotyped until a few years later, and those on dung exhibited high genetic relatedness of close to 0.90 within fruiting bodies. This is estimated from spores, not stalk, since it has not yet been possible to genotype stalks composed of dead cells. Within 0.2 g soil samples, there can be as many as six genetically distinct dicty clones, but we do not know at what frequency they form chimeric fruiting bodies. This is an area that could use more work.

Does Dicty Recognize Kin? Kin recognition and discrimination are central topics in animal behavior, for only with such recognition can kin be preferred. Microbes also have wide-ranging kin recognition abilities (Strassmann et al., 2011). In dicty, kin and non-kin occur in spatial proximity in nature (Fortunato et al., 2003b). Yet fruiting bodies from nature are largely, although not entirely, clonal Gilbert et al 2007). This is likely to be both because of the microdistribution of clones in nature and kin discrimination genes. The genes responsible for kin sorting are two highly variable cell adhesion genes called tgrB1 and tgrC1(Benabentos et al., 2009, Hirose et al., 2011). These genes stimulate segregation into different clones early in the social stage, but later different clones can fuse as separate slugs if they encounter each other. The balance between early segregation and later fusion in nature is complex (Strassmann et al., 2000, Gilbert et al., 2012b, Ostrowski et al., 2008). Clearly, dicty has a social structure that is amenable to further study. It has a solitary and a social stage. In the social stage, it is clear who is benefiting and who is paying costs. Genetic diversity occurs at a scale where interactions are likely. Chimeric groups show costs compared to groups of pure clones, as would be expected with social conflict. The standard variables of sociobiology, costs and benefits, relatedness, and recognition, are all important in dicty and can be both manipulated and measured. An exciting frontier involves experimental evolution, since dicty goes through its social stage in only a few days. Much can be learned from dicty. In the following section, we discuss how the availability of genetic approaches makes the system even more attractive.

WHAT CAN DICTY TELL US ABOUT SOCIAL EVOLUTION THEORY? Does Cheating Have a Genetic Basis? One of the advantages to a microbial system is that genes can be knocked out and the impact of their lack can be evaluated. In dicty, one way this is achieved is by a process known as REMI, restriction enzyme mediated integration. This involves inserting a labeled cassette conferring antibiotic resistance into the DNA at sites cut by the co-introduced restriction enzyme. Dosages are tweaked so each cell receives either no insertion or a single insertion. Then those lacking insertions are killed with an antibiotic. The pool of mutants can be selected. Another approach is to simply start with a selection of the knockouts in the Chris Thompson library. One of the most interesting selection regimes for students of social behavior involved favoring knockout mutants that increased the knockout’s ability to become spore and not die as stalk. The process to attain these knockout mutants involved beginning with a pool of knockout mutants and allowing them to form fruiting bodies repeatedly, with each round beginning with spores from the previous round. Thus, knockout mutants that preferentially attain spore status will be overrepresented. The hundred or so genes identified using this process are a rich source of future study subjects. The molecular pathways involved in cheating are diverse and worthy of further study. Nevertheless, we know something about how some specific genes influence social competitiveness.

How Is Cheating Controlled in Nature? Cheating in natural populations of dicty presents a number of problems. If cheating is common, then social cooperation itself can be threatened. This is because a clone would not benefit from sociality if it became stalk while another clone became the fertile spores. If a gene conferred an advantage to its bearer in all environments, then it might increase in frequency until it was fixed in the population. Cheating can be controlled if the amoebae that aggregate together are highly related because then the benefits of cooperation would go to relatives and cheaters would cheat other cheaters. A mutant that is a cheater but confers a cost on its bearer in terms of fruiting body success will spread only when it is the rarer partner in a fruiting body. A study of the gene fbxA-, also known as chtA-, showed that the knockout mutant was consistently a cheater, becoming overrepresented among the spores compared to its frequency in the original mixture. However, another impact is that chimeric mixtures produced fewer spores, and fbxA- by itself produced defective fruiting bodies with essentially no spores. The cost of this cheater mutant means that it can only thrive at low frequencies with respect to this locus, when it is in a minority in the fruiting body and can exploit other genotypes. In fact, the point where the advantage of cheating crosses the loss of spore production is at a frequency of only 0.25, much lower than that found in wild fruiting bodies. Thus, it is no surprise that in a search of morphologically defective mutants among wild-collected spores, none were found in a sample of 3316 spores. Pleiotropy is another way that cheating may be controlled. If a gene that favors a fair balance between spore and stalk also has some other essential function, then it could not easily be defeated. This is so because it would also lose the essential function. Such a gene is dimA. When this gene is knocked out, the bearer cannot respond to differentiation inducing factor, DIF, a hormone that is normally produced by incipient spore cells and which induces other cells to become stalk. The dimA- cells do not respond to DIF and in the slug stage appear to cheat by contributing less to the prestalk region. But by the time the fruiting body is formed, dimA- cells are actually underrepresented among the mature spores, because wild-type cells have actively transdifferentiated from prestalk to prespore. The loss of an essential function, whose exact nature is still unclear, means that dimA- cells cannot lose cooperation and become cheaters, without losing more fundamentally in other ways.

What do molecular evolution studies tell us about selection on social traits? Laboratory experiments can tell us how different clones of dicty interact, whether they differ by single genes, or are genetically diverse clones. But there is always the question, as with any laboratory model, as to whether these interactions are representative of what happens in nature. One way to address this issue is to look at the evolutionary history of genes involved in the social process. These can be those mentioned above that when knocked out cause cheating, or those genes that are differentially expressed in chimera. The former set of genes seems to support balancing selection for social genes (Ostrowski et al., 2015). On the other hand, genes differentially expressed in chimera show positive selection (Noh et al., 2018). Clearly more can be done in this area but the indications so far support the laboratory findings on social interactions.

What Is the Evidence for a Greenbeard Gene in Dicty? Hamilton realized that if a single gene encoded (1) a recognizable signal, (2) recognition of the signal in others, and (3) altruistic behavior toward bearers of the gene, then altruism could evolve with respect to this gene, no matter what its implications were for the rest of the bearer’s genome(Hamilton, 1964). Dawkins quickly picked up on this and called the trait a greenbeard gene, where the recognizable trait is a green beard, but he considered genes with such complex effects improbable(Dawkins, 1976). Haig wisely surmised that if there are greenbeard traits, a possible candidate would be an adhesion gene, since in this case the multiple functions might be inseparable. It seems that the dicty gene csaA functions in this way(Queller et al., 2003). It is a homophilic adhesion gene. When this gene was first successfully knocked out, the knockout appeared to function as well as the wildtype did. But then the investigators realized that in a chimera with its parent, it was a cheater on agar, contributing more than its fair share to the spores. This was so because the reduced adhesion caused it to slide to the back of the slug where prespore cells are found. But this reduction in adhesion had another effect. On agar, the csaA knockouts suffered no deficits in aggregation, but on soil, their reduced adhesion meant that they often failed to make it into the fruiting body. On soil, chimeric mixtures produced fewer knockout mutant spores. Thus, csaA is a greenbeard gene. The recognition and the action are the homophilic binding. The binding likewise ensures that the altruism is directed preferentially toward those that share the gene. One might wonder whether variation in the csaA gene contributes to present day recognition among clones. Apparently it does not (Queller et al., 2003). There is little variation in the gene as seen in present populations. This may be something else expected from a greenbeard gene. It has become fixed in a form such that everyone has the recognized trait, the ability to recognize, and the altruistic behavior, so discrimination, stable or unstable, based on this locus, is no longer possible. Clearly, this is only the beginning of a very interesting period of research as genes for social traits in dicty are discovered and characterized, leading to new insights into social behavior and evolution.

HOW DOES SOCIAL BEHAVIOR VARY ACROSS THE DICTYOSTELIA? In this article, we have focused on dicty because it is by far the best-studied species, but there are other interesting species awaiting further work. Dictyostelia is an ancient group with roughly as much molecular depth as the animal kingdom and a divergence time of around 600 million years ago (Figure 2). Compared to animals, Dictyostelia vary little in form(Schaap et al., 2006, Glöckner et al., 2016). Whether this is because of the greater levels of conflict in a social organism with physical cohesiveness like a but lacking a single cell bottleneck is an interesting question. Of course, a simpler answer would be that possibilities for morphological complexity are fewer in the Dictyostelia. All Dictyostelids produce a group of hardy spores that sit on top of a dead stalk. However, how they do this differs in whether an aggregation center forms one or many fruiting bodies, in the number of spore groups there are, and in exactly where these groups are on the stalk. Species with polysphondylild morphology have tree-like fruiting bodies with both side and terminal balls of spores (Figure 2). Other species like Coremiostelium polycephalum form clusters of sporophores that are fused over most of their length, giving the impression of a group of spore balls at the end of a single thick stalk. Dictyostelium rosarium produces spore ballsalong a single stalk giving the impression of a string of beads. Form does not differ only in the final social stage; there are differences along the way. Some species do not form a migratory slug, but culminate on the spot. Of those that do form a motile slug, most begin to form the stalk immediately, leaving a growing train of dead stalk cells behind the moving slug. Dicty is one of only three species with cells that are not terminally differentiated before fruiting. There is also variation in the chemoattractant that the amoebae use to aggregate. In dicty and probably all the other species of Dictyostelium, the chemoattractant is cAMP. In Polysphondylium and probably most, if not all Heterostelium spp., the chemoattractant is a dipeptide called glorin. In still other species, it is folate, and in most cases it is simply unknown. Dicty and its relatives in the dictyostelids have lost the ability to form spores except during the social process. However this is not true for the other Dictyostelia, where many of the species have been found to form a spore stage referred to as amicrocyst, whichdoes not seem to be as hardy as spores from the social stage. Microcysts are probably the ancestral resting stage, as it is found throughout the AmoebozoaThese microcyst-forming Dictyostelia may be an interesting option to study as they have a nonsocial option for hard times. Does this solitary option make the social contract regarding fair contributions to stalk more enforceable? A tantalizing glimpse of what else might be discovered in this novel social system comes from Speleostelium. caveatum. Only a single clone of this fascinating species was ever isolated, by Kenneth Raper from a slurry of bat guano from Blanchard Cave, Arkansas. This species seems to be a predator on any Dictyostelia it encounterss. It aggregates right along with its prey, and then delays the prey’s progression through the multicellular stages while it munches on the individual cells. The efficiency of this strategy is shown by the fact that 1% initial frequency of S. caveatum in a blend can result in nearly all S. caveatum spores. No doubt other social exploiters of novel ways lurk in the bacteria-rich corners of the planet. There are collections of these other species in the dicty stock center and in personal collections particularly of Sandie Baldauf and Pauline Schaap. Wild culturing techniques most often yield D. giganteum, various D. mucoroides, and P. violaceum all members of the particularly hardy and prolific Dictyosteliaceae. There are also at least 17 sequenced representing nearly every of Dictyostelia, including at least six members of dicty’s own genus, Dictyostelium, as well asHeterostelium, Acytostelium, Cavenderia, Coremiostelium, Rostrostelium, and Synstelium.

SEE ALSO Behavioral ecology and sociobiology; Cooperation and sociality; Kin Recognition and Genetics; Kin Selection and Relatedness; Microbial Behavior

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Genetic signatures of microbial altruism and cheating in social amoebas in the wild. Proceedings of the National Academy of Sciences, iin press. OSTROWSKI, E. A., KATOH, M., SHAULSKY, G., QUELLER, D. C. & STRASSMANN, J. E. 2008. Kin discrimination increases with genetic distance in a social amoeba. PLoS Biology, 6, e287. OSTROWSKI, E. A., SHEN, Y., TIAN, X., SUCGANG, R., JIANG, H., QU, J., KATOH- KURASAWA, M., BROCK, D. A., DINH, C., LARA-GARDUNO, F., LEE, S., KOVAR, C., HUYEN, D., KORCHINA, V., JACKSON, L., PATIL, S., HAN, Y., CHABOUB, L., SHAULSKY, G., MUZNY, D., WORLEY, K. C., GIBBS, R. A., RICHARDS, S., KUSPA, A., STRASSMANN, J. E. & QUELLER, D. C. 2015. Genomic signatures of cooperation and conflict in the social amoeba. Current Biology, 25, 1661-1665. PARKINSON, K., BUTTERY, N. J., WOLF, J. B. & THOMPSON, C. R. L. 2011. A simple mechanism for complex social behavior. PLoS Biology, 9, e1001039. QUELLER, D. C., PONTE, E., BOZZARO, S. & STRASSMANN, J. E. 2003. Single-gene greenbeard effects in the social amoeba, Dictyostelium discoideum. Science, 299, 105-106. RAPER, K. B. 1984. The Dictyostelids, Princeton NJ, Princeton University Press. SCHAAP, P., WINCKLER, T., NELSON, M., ALVAREZ-CURTO, E., ELGIE, B., HAGIWARA, H., CAVENDER, J., MILANO-CURTO, A., ROZEN, D. E., DINGERMANN, T., MUTZEL, R. & BALDAUF, S. L. 2006. Molecular phylogeny and evolution of morphology in the social amoebas. Science, 314, 661-663. SHEIKH, S., THULIN, M., CAVENDER, J. C., ESCALANTE, R., KAWAKAMI, S.-I., LADO, C., LANDOLT, J. C., NANJUNDIAH, V., QUELLER, D. C. & STRASSMANN, J. E. 2017. A New Classification of the Dictyostelids. Protist. SMITH, J., QUELLER, D. C. & STRASSMANN, J. E. 2014. Fruiting bodies of the social amoeba Dictyostelium discoideum increase spore transport by Drosophila. BMC evolutionary biology, 14, 105. STRASSMANN, J. E., GILBERT, O. M. & QUELLER, D. C. 2011. Kin discrimination and cooperation in microbes. Annual Review of Microbiology, 65, 349-367. STRASSMANN, J. E. & QUELLER, D. C. 2011. Evolution of cooperation and control of cheating in a social microbe. Proc. Natl. Acad. Sci. USA, 108, 10855-10862. STRASSMANN, J. E., ZHU, Y. & QUELLER, D. C. 2000. Altruism and social cheating in the social amoeba, Dictyostelium discoideum. Nature, 408, 965-967. WOLF, J. B., HOWIE, J. A., PARKINSON, K., GRUENHEIT, N., MELO, D., ROZEN, D. & THOMPSON, C. R. 2015. Fitness trade-offs result in the illusion of social success. Current Biology, 25, 1086-1090. Further Reading Bonner J.T. (1967) The Cellular Slime Molds. Princeton NJ: Princeton University Press. Bourke, A. F. G. 2011. Principles of social evolution. Oxford University Press, Oxford UK. Crespi B.J. (2001) The evolution of social behavior in . Trends in Ecology and Evolution 16: 178–183. Ennis H.L., Dao D.N., Pukatzki S.U., and Kessin R.H. (2000) Dictyostelium amoebae lacking an F- box protein form spores rather than stalk in chimeras with wild type. Proc. Natl. Acad. Sci. USA 97: 3292–3297. Angelo Fortunato, Queller David C., and Strassmann Joan E. (2003) A linear dominance hierarchy among clones in chimeras of the social amoeba, Dictyostelium discoideum. Journal of Evolutionary Biology 16: 438–445. Foster K.R., Fortunato A., Strassmann J.E., and Queller D.C. (2002) The costs and benefits of being a chimera. Proceedings of the Royal Society of London, Series B 269: 2357–2362. Gilbert O.M., Foster K.R., Mehdiabadi N.J., Strassmann J.E., and Queller D.C. (2007) High relatedness maintains multicellular cooperation in a social amoeba by controlling cheater mutants. Proceedings of the National Academy of Sciences USA 104: 8913–8917. Kessin R.H. (2001) Dictyostelium: evolution, cell biology, and the development of multicellularity. Cambridge UK: Cambridge University Press. Mehdiabadi N.J., Talley-Farnum T., Jack C., Platt T.G., Shaulsky G., Queller D.C., and Strassmann J.E. (2006) Kin preference in a social microorganism. Nature 442: 881–882. Raper K.B. (1984) The Dictyostelids. Princeton NJ: Princeton University Press. Santorelli L.A., Thompson C.R.L., Villegas E., Svetz J., Dinh C., Parikh A., Sucgang R., Kuspa A., Strassmann J.E., Queller D.C., and Shaulsky G. (2008) Facultative cheater mutants reveal the genetic complexity of cooperation in social amoebae. Nature 451: 1107–1110. Schaap P., Winckler T., Nelson M., et al. (2006) Molecular phylogeny and evolution of morphology in the social amoebas. Science 314: 661–663. Shaulsky G., and Kessin R. (2007) The cold war of the social amoebae. Current Biology 17: R684– R692. Strassmann J.E., Zhu Y., and Queller D.C. (2000) Altruism and social cheating in the social amoeba, Dictyostelium discoideum. Nature 408: 965–967. Strassmann J.E., and Queller D.C. (2007) Altruism among amoebas. Natural History 116: 24–29. Strassmann J. E., and Queller D. C. (2011) Evolution of cooperation and control of cheating in a social microbe. Proc. Nat. Acad. Sci. 108: 10855-10862. West S.A., Griffin A.S., Gardner A., and Diggle S.P. (2006) Social evolution theory for microorganisms. Nature Reviews Microbiology 4:

Change history: May 2018. Joan Strassmann updated the text, the references, and the figures with the help of new co-author Sandra Baldauf. This is an update of Joan E. Strassmann, Dictyostelium, the Social Amoeba, in Encyclopedia of Animal Behavior, edited by Michael D. Breed and Janice Moore, Elsevier, New York, 2010, Pages 513 - 519

Biographical Sketch

Joan E. Strassmann must love social amoebae because she left social wasp work in Tuscany, Italy, to take up Dictyostelium studies at home. Her work is a collaborative enterprise with her husband David Queller. She received her B. S. from the University of Michigan with an honor’s thesis under Richard Alexander, then a Ph.D. from The University of Texas at Austin under Lawrence E. Gilbert and Alan R. Templeton. She has enjoyed fieldwork on wasps and stingless bees and applying kin selection theory to these systems, and using DNA microsatellites to estimate relatedness among cooperators. She is a member of the American Academy of Arts and Sciences and the US National Academy of Sciences.

Sandra Baldauf is a world expert on molecular phylogeny and pioneered the field of phylogenomic study of . Her work uncovered fundamental evolutionary relationships such as the origins of animals, fungi, and aggregating amoebae. She received her B.S., M.S, and Ph.D. from the University of Michigan before a postdoc in Nova Scotia. She was professor of bioinformatics at the University of York before joining Uppsala University in Sweden where she heads the program in systematic biology, founded by Linneas in 1763. In 2016 she was awarded the prestigious Bjorkenska priset for research excellence.

Figure 1 The life cycle of Dictyostelium discoideum. (1) A hardy spore which can last for years in the soil germinates, releasing a motile amoeba. (2) The haploid amoeba hunts bacteria, eats them, and grows. There are several kinds of social communication among amoebae, including quorum sensing. (3) After about 4 h of eating, the amoeba divides mitotically. This individual stage can last for months. (A) When food becomes scarce, under certain conditions, a sexual stage is initiated. Amoebae aggregate, and the first two individuals of opposite mating types fuse, forming a diploid individual. Thousands of others join the aggregate, and amazingly are consumed by the zygote, which becomes a giant cell. This giant cell undergoes meiosis and then divides many times. (B) A hardy macrocyst is formed of recombined spores. (4) Another pathway can also result from food scarcity: the asexual multicellular pathway which is initiated with aggregation. (5) A motile multicellular slug visible to the human eye is formed, and this slug migrates toward heat and light. (6) At a new location, if it has migrated, the slug reorganizes in a form called a Mexican hat. (7) The cells form a sorocarp or fruiting body in which about 20% of cells die to form a stalk which the other cells flow up and become hardy spores at the top. Figure 2 Figure 2. Dictyostelia is divided into two classes and four families, the latter each represented here by two to five species. There are now 12 recognized genera, all but two of which can be placed in one of the four families (the rest remain uncertain, or incertae sedis). The group is extremely old (~600 mya) and has a molecular depth roughly equivalent to the distance from hydra to human (basically, all of animals), despite having far less morphological variation (drawings are not to scale). The species indicated here are Cavenderia parvisporum, C. aureo-stipes, Acytostelium leptosomum, Heterostelium pallidum, Speleostelium caveatum, Raperostelium minutum, Polysphondylium violaceum, and Dictyosetlium citrinum, D. discoideum, D. purpureum, and D. mucoroides.

Figure 3 Multicellular stages of Dictyostelium discoideum. (a) Aggregation of formerly independent cells into a multicellular body. (b) Motile multicellular slug moving towards light. (c) Fruiting body consisting of a basal disc, a stalk, and a sorus, or spores. The basal disc and the stalk are formed of formerly living amoebae that have died to form this supporting structure. (d) , the sexual stage of D. discoideum. (Courtesy of Owen Gilbert).

Figure 4 When equal numbers of cells are mixed, those made up of multiple clones produce slugs that travel less far toward light (Foster et al., 2002).