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9
Evolution of Cooperation and Control
of Cheating in a Social Microbe
JOAN E. STRASSMANN*† AND DAVID C. QUELLER*
Much of what we know about the evolution of altruism comes from ani-
mals. Here, we show that studying a microbe has yielded unique insights, par-
ticularly in understanding how social cheaters are controlled. The social stage
of Dictylostelium discoideum occurs when the amoebae run out of their bacterial
prey and aggregate into a multicellular, motile slug. This slug forms a fruit-
ing body in which about a fifth of cells die to form a stalk that supports the
remaining cells as they form hardy dispersal-ready spores. Because this social
stage forms from aggregation, it is analogous to a social group, or a chimeric
multicellular organism, and is vulnerable to internal conflict. Advances in cell
labeling, microscopy, single-gene knockouts, and genomics, as well as the results
of decades of study of D. discoideum as a model for development, allow us to
explore the genetic basis of social contests and control of cheaters in unprec-
edented detail. Cheaters are limited from exploiting other clones by high
relatedness, kin discrimination, pleiotropy, noble resistance, and lottery-like
role assignment. The active nature of these limits is reflected in the elevated
rates of change in social genes compared with nonsocial genes. Despite control
of cheaters, some conflict is still expressed in chimeras, with slower movement
of slugs, slightly decreased investment in stalk compared with spore cells,
and differential contributions to stalk and spores. D. discoideum is rapidly
becoming a model system of choice for molecular studies of social evolution.
*Department of Biology, Washington University, One Brookings Dr., Campus Box 1137,
St. Louis, MO 63130. †To whom correspondence should be addressed. E-mail: strassmann@
wustl.edu.
191
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N
atural selection favors cooperation when genes underlying it
increase in frequency compared with their noncooperative coun-
terparts (Hamilton, 1964a; Frank, 1998; West et al., 2007b). Evo -
lutionary studies of cooperative interactions have focused on the selec -
tive advantages of cooperating, how cooperation is organized, whether
cheating a cooperative system can occur, and how cheaters are controlled
(Ratnieks, 1990; West et al., 2002c; Beekman and Ratnieks, 2003; Griffin
et al., 2004; Sachs et al., 2004; Travisano and Velicer, 2004; Ratnieks et al.,
2006; Wenseleers and Ratnieks, 2006b). These studies generally, but not
always, focus on within-species interactions and have been behaviorally
oriented. Social insects have been a major focus (Bourke and Franks,
1995; Robinson, 2002; Strassmann and Queller, 2007), with cooperative
birds and mammals also getting considerable attention (Cockburn, 1998;
Clutton-Brock et al., 2001; Cornwallis et al., 2010). The past few decades
have seen phenomenal progress in understanding cooperation in these
organisms by applying the powerful logic of kin selection (Queller, 1992a;
Frank, 1998; West et al., 2007b).
Our advances in understanding the evolution of social behavior
through kin selection have been very satisfying, but they have been
isolated in some respects. This is because most organisms have not been
s een to be particularly cooperative. They may come together briefly
for mating but otherwise go about the business of securing nutrients,
avoiding disease and predation, and producing progeny largely on
their own.
COOPERATION IS WIDESPREAD
Behavioral ecologists have begun to study a wider selection of organ -
isms and are finding cooperative interactions to be much more perva-
sive than previously appreciated. This is particularly true for microbes,
wherein the structured environments necessary for cooperation have been
discovered to be pervasive (Kerr et al., 2002; Griffin et al., 2004; Vos and
Velicer, 2009). Microbes are particularly affected by the actions of their
neighbors, because many functions that are internal in multicellular
organisms are external in single-celled organisms. Secreted compounds
involved in processes like iron sequestration or food digestion are vul-
nerable to exploitation by neighboring individuals (Travisano and Velicer,
2004; Buckling et al., 2007; West et al., 2007a). Microorganisms evaluate
their numbers with quorum sensing, kill nonclonemates with bacteriocins,
hunt in groups, and cooperatively swarm through their environment, to
name just a few examples of their social attributes (Crespi, 2001; Riley and
Wertz, 2002; Diggle et al., 2007a; West et al., 2007a). Sociality in nontra -
ditional study organisms is only beginning to be understood, however.
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Evolution of Cooperation and Control of Cheating in a Social Microbe / 193
COOPERATION, ORGANISMALITY, AND
MAJOR TRANSITIONS IN EVOLUTION
The second reason for expanded interest in cooperation is a growing
a ppreciation that it is important for how organisms came to be. Coop-
erative major transitions in life alter the raw material for natural selec -
tion in fundamental ways (Buss, 1987; Maynard Smith and Szathmáry,
1995). One of the earliest transitions brought molecules together into
cells in which the fates of all were intertwined in a cooperative network.
Eukaryotes themselves represent a major transition resulting from the
capture of a bacterium that becomes the mitochondrion (Margulis, 1970).
The level of cooperation between these partners is profound but not
complete. Mitochondria are maternally inherited and do not go through
meiosis, and thus will favor daughter production and have no interest
in son production.
Another major transition resulted in multicellularity (Queller, 1997,
2000; Grosberg and Strathmann, 1998; Herron and Michod, 2008). Mul-
ticellularity has evolved multiple times in both bacterial and eukaryote
lineages. Animals and plants have elaborated multicellularity into a
p lethora of diverse types. There are also a number of comparatively
simple multicellular forms, like some single-species biofilms, the algal
group Volvocales, or Dictyostelium (Herron and Michod, 2008; Strassmann
and Queller, 2010). The transition to multicellularity is different from the
transition to eukaryotes because the former involves an aggregate of
like entities, whereas the latter binds different elements. The major
transitions can thus be categorized as fraternal, with like cooperating with
like, or egalitarian, where the cooperating units bring different things to
the collaboration (Queller, 1997). Either kind of collaborative organism
will usually retain conflicts, but these conflicts must be controlled if the
partnership is to survive. How these controls operate is a major research
topic under this view of life.
The selective factors that favored a past transition are not easy to
study because they have already completed their work. There are living
systems that could be considered to be more representative of transi-
tional stages, however. These, we believe, may be the most productive
for investigation into the advantages of cooperation and how conflict
is controlled. We have argued elsewhere that organisms themselves can
be defined as adapted bundles of cooperative elements, wherein actual
conflict is at a minimum (Queller and Strassmann, 2009; Strassmann and
Queller, 2010). In a 2D space, with one axis being cooperation and the
other being conflict, organisms are those collaborative living units at the
high end of cooperation and the low end of conflict. There is variation
in the level of organismality, however, and those lacking complete coop-
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eration and retaining conflict represent the best choices for studying the
origins of cooperation.
LABORATORY-FRIENDLY, SOCIAL MODEL ORGANISMS
Kin selection has been very successful for generating predictions on
t he impact of queen number, mate number, and caste on sociality in
social insects (Bourke and Franks, 1995; Bourke, 2011). Nevertheless, one
would have to say that social insects fall short as an ideal model for
studies of social evolution. They are long-lived, often do poorly in the
laboratory (except ants), are not amenable to genetic experimentation,
and have mostly already crossed the threshold to obligate sociality.
Thus, social evolution research has not found its Drosophila here.
Another problem with the organisms currently favored for studies
of cooperation is that the actual genes underlying cooperative behavior
are elusive. This is particularly true for long-lived social insects and ver-
tebrates, although the advances of genomics are slowly mitigating this
(Robinson, 2002; Honeybee Genome Sequencing Consortium, 2006). Still,
the twin powers of experimental evolution and single-gene knockouts are
beyond the reach of most currently studied social organisms.
A social evolution Drosophila would need to address these issues;
thus, it would probably be single-celled. In addition to being amenable
for experimental evolution and single-gene knockouts, it should have
full altruism, with some individuals dying to help others. This makes it
easier to interpret the actions of different partners. Other attributes of
the ideal social Drosophila include feasibility of study in a fairly natural
environment, placement in a rich phylogeny with related species that
vary in social traits, a sequenced set of genomes, and a collegial com -
munity of fellow investigators. Here, we make the case that the ideal
m odel organism for social evolution has been found and is the social
amoeba Dictyostelium discoideum. This choice is supported by the enor-
mous progress in understanding social evolution that has been made
w ith this organism in the past decade. In addition to D. discoideum, Vol-
vox and its relatives are great for studying the origins of multicellularity
in a clonal organism (Herron and Michod, 2008). M yxococcus xanthus
o ffers all the advantages of a bacterial system (Velicer and Stredwick,
2002). There are also others, but we focus here on D. discoideum (Fig. 9.1).
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Evolution of Cooperation and Control of Cheating in a Social Microbe / 195
FIGURE 9.1 D. discoideum fruiting bodies on an agar plate.
DICTYOSTELIUM DISCOIDEUM AS A MODEL
SYSTEM FOR COOPERATION
What Is a Social Amoeba?
Social amoebae are in the eukaryote kingdom Amoebozoa, sister to
the Opisthokonts, or animals plus fungi (Baldauf et al., 2000). This king -
dom is composed of solitary amoebae-like Entamoeba and Acathamoeba,
the acellular slime molds such as Physarum, and the Dictyostelidae. There
are over 100 species of Dictyostelium, divided into four major taxonomic
groups (Raper, 1984; Schaap et al., 2006). D. discoideum is in group four
and is the focal species here.
Individual amoebae of D. discoideum live in the upper layers of soil
and leaf litter in the eastern Northern Hemisphere and in eastern Asia.
The most intensely studied clone, NC4, and its derivatives such as Ax4,
come from a temperate forest near Mount Mitchell in western North
Carolina (Raper, 1984). D. discoideum amoebae are solitary predators on
bacteria, which they consume by engulfment (Bonner, 1967). Although
this is usually viewed as a solitary stage, they are always able to sense
the density of nearby amoebae with a molecule called prestarvation
f actor (Kessin, 2001). Response to this factor is inhibited when bacteria
are present (Kessin, 2001). When bacteria get scarce, and amoeba density
is sufficient, they enter one of two stages, a sexual one, discussed later, or
a social one (Fig. 9.2).
Social Cycle
The social stage, often called the developmental stage, occurs when D.
discoideum amoebae begin to starve (Fig. 9.2). Amoebae have a quorum-
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FIGURE 9.2 Colony cycles of D. discoideum. This study focuses on the social cycle,
but the sexual cycle is a promising area for future study.
sensing mechanism; if there are enough other amoebae in the area,
they begin to release cAMP and to make receptors to it, products of the
CAR genes (Kessin, 2001; Alvarez-Curto et al., 2005). A signal relay system
causes the amoebae to move up the cAMP gradient and form a mound
of hundreds of thousands of cells. Differentiation begins in the mound
stage, wherein some cells sort out toward the tip and express prestalk
g enes. The tip becomes the anterior of the slug and organizes forward
movement. During movement, cells are lost from the slug posterior. At
least some of these are capable of dedifferentiating and consuming any
bacteria encountered (Kuzdzal-Fick et al., 2007). The slug itself will not
fall apart on encountering bacteria. Some shed cells are former sentinel
cells, full of toxins, and bacteria mopped up as they traverse through the
slug (G. Chen et al., 2007).
The multicellular slug moves toward heat and light and away from
ammonia (Kessin, 2001; Bonner, 2006). The cells at the tip then migrate
down through the center of the aggregate and initiate stalk formation in
a process called culmination. The stalk cells vacuolate and die, forming
sturdy cellulose walls in the process that give them the strength to hold
up the spherical ball of spores. The final fruiting body consists of about
20% stalk cells and 80% spore cells. Thus, the social stage is triggered by
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Evolution of Cooperation and Control of Cheating in a Social Microbe / 197
starvation and involves altruism, because the stalk cells die to support
the spore cells (Kessin, 2001).
D. discoideum arrives at multicellularity not through development
from a single cell but through aggregation of dispersed cells. Therefore,
the social stage of D. discoideum is vulnerable to cheaters. This makes
it fundamentally different from a metazoan that has gone through a
s ingle-cell bottleneck and had the interests of all cells in the organism
reset to complete cooperation every generation (Maynard Smith, 1989b).
This conflict, its control, and the resulting cooperation are what make D.
discoideum such a great model for social evolution.
Why Have a Social Stage?
During the social process, three things happen, and we predict that
all three are adaptive. First, spores are made. The adaptive value of a
hardy spore is clear and has been demonstrated; it is not easily digested
by predators and can withstand long periods of cold, heat, or drought
(Raper, 1984). Second, the spores are only made atop a relatively long stalk
composed of dead cells. These stalks can be anywhere from 1 to about
4 mm long, and their construction is the most vital part of the altruism
story of D. discoideum. Why are spores made only atop stalks? It could be
that cells are vulnerable during the transformation to spore, and doing
so atop a stalk protects them from hazards in the soil. Another possibility
is that dispersal is facilitated when the spores are lifted above the soil
and that this is the main purpose of the stalk. In D. discoideum, spores
are likely to be actively transported on small invertebrates, although
the guts of vertebrates and stalks could increase the chance that they
are contacted. The third advantage to grouping is slug movement;
slugs move farther than amoebae, which could position them into
a better place for dispersal. The complex orchestration of fruiting body
formation could only have arisen through natural selection, but more
work on the actual advantages is needed. In this review, we focus on the
interactions of genetically different clones in this social process and not
on the reasons why it is adaptive.
Chimerism and Cheating the Social Contract
Mixing of two or more genetically distinct clones is likely for social
groups that form by aggregation. To see if this actually occurs, we col -
lected tiny soil samples of 0.2 g at Mountain Lake Biological Station
(Fortunato et al., 2003b). We reasoned that this was a reasonable scale
over which social aggregation might occur. We found that our 0.2-g
samples contained zero to five clones and that relatedness within the
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samples was about 0.52. These data support the view that chimerism
is possible, at least in this population.
Later, we were able to find and genotype individual wild-fruiting
bodies collected from the very rich resource of deer dung and nearby
soil. This approach gave much higher relatednesses, between 0.86 and
0.98 depending on the sample and technique (Gilbert et al., 2007). Thus,
relatedness is clearly high enough for kin selection under reasonable
values of costs and benefits, and chimerism is common enough for
social competition to be favored evolutionarily. Nevertheless, for coop -
eration to occur, there must be control of cheating. Here, we discuss
what cheating is and then move on to evidence for it and its control in
D. discoideum.
Complications with Defining Cheating
Cheating can only happen when one organism takes advantage of
another; however, it is more than that. We would not say the lion cheated
the gazelle out of its life with the lion’s pounce and suffocating bite. This
is because there is no expectation that the lion would behave in any
other way. So, for an exploitative behavior to be considered cheating,
there must be some expectation of cooperation that is not met. Cheating,
therefore, is a fundamentally social action that takes place in the context
of ordinarily cooperative acts, which the cheater somehow violates.
In D. discoideum, we talk of cheating in the context of cell allocation
to the somatic, dead stalk and the living spores. The expected social
contract is that the frequency of each clone among the spores will be the
s ame as it was in the original mixture of aggregated cells. The same
should be true in the stalk tissue. If this is not the case, we can say that
the dominant clone cheated the minority clone by getting more than its
fair share into spores, and cooperation can be put at risk when cheaters
gain an advantage.
In many kinds of interactions, the starting and ending frequency may
be viewed as enough information to determine if one partner is cheating
the other. The formation of a fruiting body from an initial population of
spores is a process that could vary for reasons other than social competi -
tion, however. Some clones may make longer or more robust stalks than
others when they are entirely on their own. Some clones may migrate
farther than others, losing cells in the process. Some clones may lose more
cells from the slug than others even if they migrate the same distance.
Variation is particularly expected in the highly variable environment of
the soil. For example, a loose-grained soil may favor longer stalks
f or a given number of cells than a tighter-grained soil if the adapted
trait is to rise above the surface. Selection on these traits can occur inde -
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Evolution of Cooperation and Control of Cheating in a Social Microbe / 199
FIGURE 9.3 In the social stage, clones
may take advantage of their partner in
three different ways. They may allocate
cells to spore and stalk in the same pro -
portions as alone but allocate less to
stalk than their partner, fixed cheating.
They may modify their behavior in chi -
mera to take advantage of their part -
ner, facultative cheating. Third, a social
parasite can only make fruiting bodies
in chimera with a victim.
pendently of cheating but then have consequences in chimeras. If one
clone in isolation allocates more to spore and continues to do so in the
chimera with another clone that allocates less to spore, the first clone may
then be viewed as a cheater, although it has behaved no differently in
the chimera.
We will argue that even this case should be called cheating, because
one clone does take advantage of the other. It might even have evolved
for that purpose: Selection in chimeras could have favored variants that
do suboptimal things on their own. We call this type of cheating “fixed,”
following Buttery et al. (2009). Cheating that results from behavior differ-
ent from what they would do when clonal, in recognition that there is a
partner to cheat, we then call “facultative” (Fig. 9.3). If the only informa -
tion we have is how they behave in a chimera compared with starting
frequencies, we cannot distinguish between these two and just call it
“cheating.”
It is probably worth pointing out that we are not implying any sort
of conscious awareness to cheating in D. discoideum. In humans, cheating
is value-based and assumes a certain awareness of the moral grounds
of an act. This, of course, is impossible in an organism lacking a nervous
system.
EVIDENCE FOR CHEATING IN D. DISCOIDEUM
Do Wild Clones Cheat?
When wild clones are mixed together, one clone often prevails over
the other (Strassmann et al., 2000). Furthermore, there is a transitive
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hierarchy of cheaters (Fortunato et al., 2003a; Buttery et al., 2009). In all
these cases, the clones are perfectly able to produce fruiting bodies with
normal, although variable, spore/stalk ratios as pure clones. Buttery et
al. (2009) found both fixed and facultative cheating among the clones.
Other evidence for social conflict among wild clones in the social
stage comes from comparing chimeras with pure clones in their ability to
migrate as slugs and to form tall fruiting bodies (Fig. 9.4). Chimeric slugs
move less far than clonal slugs when cell number is controlled (Foster et
al., 2002). This may be the result of increased competition to stay out of
the front control region that becomes the sterile stalk. The other effect
i s that there are more spore cells in chimeric mixtures, presumably
because there is less selective benefit to becoming a stalk to lift nonrela -
tives (Buttery et al., 2009).
Cheating by Single-Gene Knockouts
Nearly all the research by cell, developmental, and molecular biolo-
gists on D. discoideum has used a single clone, or descendants of that
clone. This means that these studies could not reveal cheating even
if it were common. The exception is that they could reveal circum -
stances under which a clone with a single gene that was knocked out
cheated its immediate ancestor. Kessin and colleagues (Ennis et al., 2000)
did just such a study. They made a large random collection of clones
that each had a single gene disrupted by restriction enzyme-mediated
integration (REMI), a process that randomly inserts a known sequence
containing both restriction cut sites, and an antibiotic resistance gene
(Kuspa and Loomis, 1992). Kessin and colleagues (Ennis et al., 2000) put
a pool of REMI knockouts through 20 generations of selection in a well-
mixed (low-relatedness) environment. At each round, they harvested
the spores and began the next round from them; thus, any clone that
FIGURE 9.4 Conflict is manifested in
c himeras in the form of shorter stalk
lengths, shorter migration distances,
and unequal spore/stalk ratios.
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Evolution of Cooperation and Control of Cheating in a Social Microbe / 201
cheated the others increased in frequency over these rounds. They then
characterized one mutant, fbxA. The fbxA knockout cheats its ancestor
but cannot make spores on its own.
Pools of REMI mutants can also be screened to obtain cheat-
ers that are able to make normal fruiting bodies on their own but cheat
their ancestor in a chimera. A large study of this type used pools of
REMI mutants and required that every mutant be able to fruit on its
own (Santorelli et al., 2008). This approach identified over 100 different
k nockout mutants that cheated their ancestor. If knockout cheaters
are so easy to generate and cheating is advantageous, one has to ask why
these genes have not lost function in the wild. We discuss the answer to
this question below in the section on the control of cheating.
CONTROL OF CHEATING
When wild clones come together in the social stage, cheating occurs
between pairs of co-occurring wild clones. This could be the result of
genetic or environmental factors. The work on single-gene knockouts
suggests that at least some of the differences are genetic. Why are genes
underlying victim status not eliminated from the population? We think
t he answer lies in the ways cheating is controlled. It can be controlled
by high relatedness within social groups, which could result from kin
discrimination. It can be controlled by positive pleiotropy, wherein a
cooperation gene also has another essential function. Cheating can also
be controlled if spore vs. stalk fate is the result of environmental rather
than genetic factors. For example, spore fate could be the result of posi -
tion in the mitotic cell cycle or it could be dependent on who starved
first. Here, we take up these issues (Fig. 9.5).
Control of Cheating by High Relatedness
Cheaters can be controlled if relatedness within social groups is high
enough. This is because the benefits of the sacrifice that stalk cells make
will mostly go to relatives, and thus could be favored under kin selec -
tion. The importance of high relatedness can be seen in an experiment
that used the knockout cheater fbxA (Gilbert et al., 2007). In this study, we
showed that at low relatedness, the fbxA cheater knockout wins within
groups at all mixture frequencies. This means that it should increase in
frequency in the population. There is a tradeoff, however. The higher
the frequency of the cheater in a group, the lower the spore production
becomes, hurting the fbxA knockout and WT alike within that group.
This means that the cheater knockout can only flourish at low related -
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FIGURE 9.5 Cheating can be controlled
in the social stage if fruiting bodies are
clonal, as might happen if they arise
from different patches. They may mix
but then sort into nearly clonal fruit -
ing bodies through kin discrimination.
Pleiotropic effects may prevent cheat-
ing genes from spreading. Caste fate
may be determined through a lottery,
with cells in the M or S stage of the cell
cycle becoming stalk and those in the G2
stage becoming spore. D. discoideum ap-
parently has no G1 stage, although this
is controversial.
ness because at high relatedness, it is selected against by its own com -
promised spore production.
We expect social parasites like this one to fail in nature because of
the high relatedness within fruiting bodies found in the wild. If this is true,
we should not find any clones within wild fruiting bodies that are unable
to form fruiting bodies on their own. We tested this by plating cells from
wild fruiting bodies clonally. Of 3,316 clonal isolates from 95 wild fruit -
ing bodies, all were able to make completely normal fruiting bodies on
their own. There was not a single social parasite like fbxA. Clearly, high
relatedness within fruiting bodies is a powerful evolutionary deterrent
to cheating. This does not mean cheater mutants that are competent on
their own are equally controlled, however (Santorelli et al., 2008).
Control of Cheating by Kin Discrimination
One way of achieving high relatedness is to exclude nonkin from the
group. This behavior could explain the difference in relatedness between
small soil samples and fruiting bodies. Different clones might aggregate
together to cAMP and then sort into genetically homogeneous slugs.
Even different species coaggregate to cAMP and then separate (Jack et
al., 2008); thus, it is not unreasonable to postulate a similar process
within species.
Studies of chimerism between two clones of D. discoideum have gener-
ally found fairly homogeneous mixing, however (Strassmann et al., 2000;
Fortunato et al., 2003a; Buttery et al., 2009). A couple of studies found
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Evolution of Cooperation and Control of Cheating in a Social Microbe / 203
some evidence for sorting, particularly between clones collected far
apart (Ostrowski et al., 2008) or, in another study, particularly between
c lones found close together (Flowers et al., 2010). Neither approached
the levels of sorting found in another species, Dictylostelium purpureum
(Mehdiabadi et al., 2006).
At this point, we have a puzzle. Tiny soil samples have multiple clones
of D. discoideum, but fruiting bodies are nearly clonal. Kin discrimination
is weak as far as we can tell in laboratory mixtures of equal numbers of
cells from two clones. The finding of an apparently selected molecular
mechanism for sorting deepens the puzzle. Our supposition that sort -
ing will occur in the aggregation stage or later means that cells are likely
to discriminate when they are in direct contact with each other. This
suggests that adhesion genes are likely candidates for recognition. To
function as recognition genes, adhesion genes would have to be highly
variable. The variability would provide an opportunity for discrimina -
tion that favors others carrying the same adhesion protein variant over
others carrying different forms of the molecule. They should recognize
self, with a homophilic binding site, or they should recognize a highly
variable receptor.
There is excellent evidence that two cell adhesion genes, initially
called lagC and lagB but now called tgrC and tgrB, are the kin discrimina-
tion genes in D. discoideum (Benabentos et al., 2009). These two genes
are extremely variable and are part of a large gene family of generally
much less variable genes. The protein produced by tgrC is hypothesized
to adhere to the protein produced by tgrB. If one is knocked out, it
causes development to fail at the aggregation stage. In that case, the
amoebae aggregate begins to make a mound but then falls apart, as if a
crucial component of recognition necessary for the subsequent altruistic
steps were missing. The temporal coexpression, knockout behavior,
high variability, and impact on sorting make these likely kin recogni -
tion genes. More work is clearly needed on this system to see if there
are consequences of recognition other than sorting. It could be that it
is advantageous to remain in the group but that the chimeric nature is
recognized and responded to, causing reduced migration distances and
shorter stalks, for example.
Control of Cheating by Pleiotropy
Pleiotropy means that a single gene has an impact on multiple phe-
notypic traits. It is therefore usually viewed as something that impedes
selection on a specific trait, because any changes in the underlying genes
will affect other traits as well. This conservative force in pleiotropic genes
can have interesting consequences for social genes. If an altruistic trait
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is piggy-backed on an essential gene, a mutation that causes selfish
behavior is unlikely to proliferate, because the essential function would
also be lost.
Exactly how important this might be in social traits is unknown,
because we know the genetic underpinnings for comparatively few traits.
There are a couple of genes having an impact on altruism in D. discoideum
that could be maintained through pleiotropy, however. They are from
very different parts of the genetic landscape underlying altruism in D.
discoideum. One is a cell adhesion gene, and the other is involved in the
differentiation-inducing factor (DIF-1) signaling system.
Cell adhesion is an essential part of the social process because it is
how the multicellular group stays together (Kessin, 2001). Variation in
adhesion can have an impact on cell fate, because the cells at the front
of the slug become stalk and the cells in the back three-quarters or so
become spore (Bracco et al., 2000). One way of increasing the likelihood of
becoming spore could therefore be to have reduced adhesion to the other
cells and to slip back in the slug (Ponte et al., 1998; Queller et al., 2003).
The knockout of the cell adhesion gene csaA has just this effect. When
csaA is knocked out, adhesion is reduced. On agar, this has the impact
of increasing the knockout’s frequency in the spores, presumably because
reduced adhesion allows it to slip out of the stalk-forming tip (Ponte et
al., 1998; Queller et al., 2003). On the more natural substrate of soil, how -
ever, csaA knockouts apparently do not hold together enough to get into
aggregations. It is therefore no surprise that the csaA gene continues to
be expressed normally and that cheater knockouts have not prospered.
Another gene that could be a cheater were it not for pleiotropic effects
is dimA (Foster et al., 2004). This gene was isolated in a screen of REMI
mutants that are unresponsive to DIF-1, a small molecule that forces
some cells to become stalk (more on this later). In chimeras with WT,
dimA knockouts predominate in the prespore zone, presumably because
they are insensitive to DIF (Thompson et al., 2004). Ultimately, however,
they are in a minority in the actual spores. This could be true if they trans -
differentiate from prespore cells to prestalk cells later in development,
and this was shown to be the case (Foster et al., 2004). We interpreted
t his to be the result of another unknown function of dimA, an essential
function that made the knockouts worse spore cells. This is another case
in which pleiotropy inhibits the spread of a cheater.
Control of Cheating by Lottery
When two or more individuals take unequal roles in a social interac-
tion, with one being the recipient and the other being the beneficiary,
conflict can result. One way of controlling this conflict is if the partners
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Evolution of Cooperation and Control of Cheating in a Social Microbe / 205
do not know which role they will assume on entering the interaction. A
human equivalent is called the veil of ignorance (Rawls, 1971), and it calls
for resource allocation between partners by someone who does not know
which lot he or she will get. A familiar example is the common family
situation of dividing up a cake. If the child cutting the pieces does not
get to decide which piece he or she gets, under the veil of ignorance
model, he or she will be more likely to make the pieces equally sized.
Cheating could be controlled in D. discoideum if there were a lottery to
become spore based on the cell cycle.
The D. discoideum cell cycle has a very short G1 phase; thus, imme-
diately after the mitosis (M) phase, cells enter the synthesis (S) phase and
cytokinesis occurs during the S phase (Weijer et al., 1984). Therefore, in a
population, the cells in S and early growth after synthesis (G2) phases tend
to be the smallest cells with the fewest nutrient reserves. An experiment
on a thin layer of cells not touching other cells, followed with videog -
raphy, indicated that stalk cells were most likely to arise from cells that
happened to be in the S or early G2 phase of the cell cycle at the time of
starvation, whereas cells that happened to be in the late G2 phase became
prespore (Gomer and Firtel, 1987). A variety of other experiments have
also shown this (Araki et al., 1994; Azhar et al., 2001). If weaker cells are
more likely to become stalk, this makes sense, because recently divided
cells would have fewer nutrients. This cell cycle lottery system fits the
veil of ignorance model. As cells encounter less and less food, however,
it could be that those dividing earlier than others are selected against
because these cells will be in the stage that sends them to stalk.
Another interesting result of this paper (Gomer and Firtel, 1987) sug -
gests that delaying cell division may not be necessary for a cell to avoid
becoming a stalk cell. This result was derived from careful observation
of the fate of sister cells through videography. Every time a cell divided,
one sister cell became prestalk or prespore according to the above musi -
cal chairs lottery mechanism, whereas the sister cell became a null cell, a
third cell type that stained with neither prespore nor prestalk markers
(Gomer and Firtel, 1987). The fate of these null cells is unclear. These null
cells could become pstO, because that region of the slug also did not stain
with prespore or prestalk markers. This region can be viewed as the most
flexible area, with cells in that region remaining pstO on exposure to DIF,
and perhaps becoming prespore otherwise. These interesting results
remain controversial, however, and should be followed up on carefully
(Shaulskyand Loomis, 1993; Jang and Gomer, 2011).
If a recently divided cell becomes stalk because it is smaller and
weaker, cell division could be disfavored as starvation approached for
this social reason. Under normal circumstances, however, amoebae
will be selected to eat and proliferate as rapidly as possible. These two
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counterforces might achieve a compromise that could support altruism
under a wide variety of conditions in D. discoideum, if one of two recently
divided cells becomes stalk and the other becomes spore. This scenario
i s consistent with the data.
CONTROL OF CONFLICT BY POWER
We began the section on control of cheating with a discussion of
social contracts and defined cheating as the violation of those contracts.
In this case, we mean evolved contracts that favor the evolution of coop -
eration. One form of contract may be that the stronger individuals take
the best roles. Here, we explore the evidence for this idea in D. discoideum.
First-Strike Power
One of the most common determinants of whether an individual in a
social interaction becomes the altruist or the beneficiary is that individual’s
relative strength, or ability to prevail in a contest. Such contests under
social and cooperative circumstances may look very similar to contests
between nonsocial organisms for scarce resources such as good territo -
ries. The difference is that if the contest is between relatives, or mutually
dependent individuals, after the contest is decided, the loser may acqui -
esce and go to work for the winner. Such contests can be valuable for
all concerned, particularly if weaker individuals that lose contests are
more effective in taking on the helping role than they would be with the
winning, reproductive role.
How do we evaluate power in D. discoideum interactions? In some
ways, all predictors of fate also involve power. The lottery system has
a power element, because cells that recently divided may be weaker and
go to stalk. If becoming a spore cell is competitive, the first amoebae to
depart from growth and binary fission and enter the social stage may
get a head start on preparing their weapons. Under this hypothesis, the
first to starve would become spore. That this is the case has been very
nicely demonstrated in both an experiment that manipulates timing
o f starvation in genetically identical cells and an experiment that uses
an aggregation-initiation knockout. In the first experiment, cells were put
into nutrient-free medium 4 hours apart. Those with the 4-hour head
start in the social stage preferentially became spores (Kuzdzal-Fick et
al., 2010). The other experiment used a knockout that was incapable of
initiating aggregation but was capable of responding to the initial signal
from others and relaying it (Huang et al., 1997). In this case, the single
cell initiating aggregation became a spore.
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Evolution of Cooperation and Control of Cheating in a Social Microbe / 207
Glucose Feeding, Condition, and Power
Power based on condition has also been studied directly by making
chimeras of cells that were well fed with cells that were poorly fed. This
was done by varying the amount of glucose in the medium of axenically
grown cells. The cells fed with glucose were more likely to become spore
than the glucose-starved cells (Leach et al., 1973). This effect holds with
other metabolizable sugars and is absent with other sugars (Takeuchi et
al., 1986). This is strong support for the hypothesis, but there could be
something special about sugars; thus, we repeated this experiment with
a g lucose treatment and added another treatment to separate cells. In
this treatment, we stressed the cells by growing them in a more acid pH
t han usual (Castillo et al., 2011). We affirmed the weakening effects of
both treatments by documenting that they increased doubling times in
the solitary stage. As expected, both acid-stressed and glucose-starved
cells ended up preferentially in the stalk. Both treatments also made
fewer spores when grown alone, however; thus, the chimera results are
not attributable to competition alone (Castillo et al., 2011).
DIF-1 and Power
One of the delights in working with a microbial system is the acces -
sibility of mechanisms. Whether a cell becomes spore or stalk is medi -
ated by DIF-1, a small, secreted, chlorinated alkyl phenone (Kay, 1998).
Stronger cells that are immune to its effects at biological levels produce
DIF-1. Weaker cells can break it down but become stalk cells from its
impact, mostly ending up in the lower cup or the basal disk, both of
which are dead parts of the stalk (Thompson and Kay, 2000a,b). DIF-1
is unlikely simply to be a signal rather than a mediator of competition
for several reasons. Signals are unlikely to include chlorine, something
that is common for poisons. Levels of DIF-1 in the slug are about 62 nm
(Kay, 1998), which is high, given that it can be lethal at concentrations as
low as 200 nm (Masento et al., 1988). Signals have receptors and poisons
do not, and no receptor has ever been found for DIF-1. Its small, toxic
nature is just what might be expected of a poison (Atzmony et al., 1997).
Unlike most morphogens, it is distributed evenly through the social
stage and varies on its cell-specific impact (Kay and Thompson, 2009;
Chattwood and Thompson, 2011; Parkinson et al., 2011). In some respects,
it is a tame poison, incorporated into social life to mediate condition in
a homogeneous mixture into different cell fates.
The condition variants resulting from position in the cell cycle or glu-
cose feeding are tied to DIF-1 levels with weaker, more recently divided
cells more vulnerable to DIF-1. There are single-gene knockouts with an
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impact on cell cycle and nutritional responses that further support the
involvement of DIF-1, in a story nicely summarized by Chattwood and
Thompson (2011). Cells that have rtoA knocked out lose the specificity
toward stalk of the M and/or S cell cycle phase, producing fruiting bod-
ies that are mostly stalk with tiny spore heads (Wood et al., 1996). This
has been shown to be the result of high intracellular calcium, which has
independently been shown to bias cell fate toward stalk (Baskar et al.,
2000; Azhar et al., 2001; Chattwood and Thompson, 2011). Cells with
high intracellular calcium are far more sensitive to DIF-1 (Schaap et al.,
1996; Baskar et al., 2000). A similar story can be told with a gene that
links nutritional status to cell fate, a D. discoideum homolog of the human
retinoblastoma gene, rblA (MacWilliams et al., 2006; Chattwood and
Thompson, 2011). Knockouts of rblA are hypersensitive to DIF-1 and
preferentially become stalk.
Other work by Thompson and colleagues (Parkinson et al., 2011) has
shown that the patterns linking DIF-1, or more generally stalk-inducing
factors (StIFs), are also important in spore-stalk hierarchies of natural
clones. These hierarchies are based on whether clones become spore
or stalk when mixed pairwise with other clones (Fortunato et al., 2003a;
Buttery et al., 2009). They separately evaluated response to and produc -
tion of StIFs and found a threefold difference in production and a 15-fold
difference in response; the latter was most powerful in explaining the
hierarchy observed in natural clones (Parkinson et al., 2011). Thus, we
know a satisfying amount about how power affects cell fate through
DIF-1. There is more to learn, however, particularly because cheating
can result from knocking out so many different genes (Santorelli et al.,
2008). This led to another general approach to identifying resistance
genes.
Genetic Control of Cheating by Noble Resistors
The evolution of resistance to cheater genes may limit their spread.
To test this idea, we selected for resistors of cheater genes. We took one
cheater, chtC, and exposed a pool of REMI mutants to it over successive
rounds (Khare et al., 2009). We allowed selection of the REMI pool but
not of the chtC knockout. We did this by removing the G418 resistance
from the chtC clone so that we could kill it at each round, leaving the
mutants we were selecting intact. We then simply added back in the
naive chtC clone for the next round. This process resulted in a number of
mutants that were resistant to chtC knockouts and could not be cheated by
it. Interestingly, they were not cheaters of their ancestor; thus, we called
them noble resistors (Khare et al., 2009).
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Evolution of Cooperation and Control of Cheating in a Social Microbe / 209
SOCIAL GENES, ARMS RACES, AND THE RED QUEEN
Cheating and countering cheating are social processes that we
predict will result in rapid evolution in the underlying genes. Our test
of this hypothesis used the newly sequenced species D. purpureum and
compared it with D. discoideum (Sucgang et al., 2011). Unfortunately, this
is not an ideal pair of species because their proteins are as diverged as
t hose of humans and fish. This means that silent amino acid changes
(ds) are saturated, and thus are not useful in comparisons. Instead, we
compared homologs; rates of amino acid change; and conservation scores,
a measure of similarity that includes indels. We used two sets of social
genes for comparisons. The first set was the 100 or so REMI mutants
that cheated their ancestors when mixed equally with them (Santorelli
et al., 2008). These genes did not show more rapid evolution, and thus
failed to support our hypothesis that social genes evolve more rapidly.
The second set of genes we used was based on a social index,
which was higher when a gene was more expressed in the social stage
compared with the nonsocial stage. In this analysis, the more social
genes had a lower probability of having homologs, an elevated rate of
amino acid change, and a lower conservation score, supporting our
hypothesis (Sucgang et al., 2011). The result could also be attributable
to weaker purifying selection on social genes, however, and a better
analysis would be between more closely related species.
OTHER ARENAS FOR COOPERATION: MUTUALISMS AND SEX
No review of social behavior of D. discoideum would be complete with-
out mentioning two very exciting areas for future study. The sexual cycle
is also a social cycle but has been studied very little. The other area is the
discovery of a farming mutualism between D. discoideum and bacteria.
This opens up the opportunity for studies of between-species symbioses.
Sexual Cycle Has Social Elements That Involve the Ultimate
Sacrifice
The sexual cycle is triggered by starvation in the presence of sufficient
numbers of other amoebae under wet, phosphate-poor conditions and
begins with aggregation to cAMP (Bonner, 1967; Kessin, 2001). Two cells
of different mating types fuse, forming a diploid zygote. The amoeba
stage is ordinarily haploid and divides by mitosis; thus, no reduction
division is necessary before sexual fusion. Aggregation does not cease
with the formation of a diploid zygote (Urushihara, 1992; Ishida et al.,
2005). Other amoebae continue to swarm in by the thousands, up the
cAMP gradient. The zygote proceeds to consume the other cells by
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phagocytosis. The pace of consumption is slowed to a level that allows
the waiting victim amoebae to construct an envelope around the aggrega -
tion, and this slowing is also regulated by cAMP. After a time, there is a
firm wall around the zygote and its victims, and the latter are consumed
and digested. Recombination and crossing over then happen, the zygote
undergoes meiosis, and many recombinants are formed.
In a major recent advance, the sex-determining locus was identified
and the presence of three mating types was confirmed, clearly establish -
ing the genetic basis of sex (Bloomfield et al., 2010). The sexual cycle is
somewhat of an enigma because it rarely leads to recombinant progeny
under laboratory conditions (Kessin, 2001), but estimates of recombina -
tion rates of natural clones indicate they are very high, with a population
r of 37.75 and baseline linkage disequilibrium achieved between 10 and
25 kb (Flowers et al., 2010). Getting the system to work in the laboratory
would open up many interesting social questions to investigation. For
example, we could select for social traits in sexually recombined pools
and look for quantitative trait loci associated with social traits.
D. discoideum Farms Bacteria
There is another reason why D. discoideum is particularly good for
studies of cooperation: mutualism. The standard view of the social stage
of development is that all bacteria are purged from the aggregate (Kessin,
2001). There are known mechanisms for this that function at different
stages, from mound, to slug, to final fruiting body. The sorus is consid -
ered to be sterile apart from the D. discoideum spores. Very recently, we
d iscovered that this is not the case for about one-third of all clones
(Brock et al., 2011). These clones carry bacteria with them through the
social stage like a farmer might bring a flock of sheep to a different pas -
ture. These bacteria are found within the fruiting body. When the spores
hatch after favorable growing conditions have been encountered, they
can feed on the proliferating population of the bacteria they brought.
These farmed bacteria are better food than most wild bacteria. This farm -
ing mutualism is highly amenable for study, because all partners are
microbial, advantages are clear, and the relationship is not obligate, at
least at the species level. This discovery adds between-species coopera -
tion to the things that can be studied about D. discoideum.
CONCLUSION
The ultimate advantage to an ideal model organism is what you
can learn from it. In D. discoideum, we have shown that conflict exists in
the form of shorter stalk lengths, reduced migration distances, and cheat -
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Evolution of Cooperation and Control of Cheating in a Social Microbe / 211
ing to avoid the sterile caste. We have delineated cheating into fixed,
facultative, and social parasite forms. We have shown that cheating can
be controlled by high relatedness, kin discrimination, pleiotropy, or lot -
teries. We have shown that conflict can be controlled by conventions and
power. The first cells to starve become spore, as do stronger cells. A small,
toxic molecule called DIF-1 mediates social interactions. We and others
have backed up much of this work with specific genes and knockouts.
Further whole-genome outcomes are on the horizon, as is a much more
detailed understanding of kin discrimination. Frontiers include the
farming symbiosis and exploration of the sexual cycle. Clearly, this is a
system that has yielded many important secrets about the cooperative
side of major transitions.
ACKNOWLEDGMENTS
We thank John Avise and Francisco Ayala for inviting us to help
organize the Arthur M. Sackler Colloquium on Cooperation. We thank
many colleagues for helpful discussions of these points, especially Sandie
Baldauf, Koos Boomsma, Kevin Foster, Richard Gomer, Ashleigh Griffin,
Rob Kay, Richard Kessin, Adam Kuspa, Gene Robinson, Gadi Shaulsky,
Chris Thompson, Greg Velicer, and Stuart West as well as our own
laboratory group. Our research is supported by the U.S. National Science
Foundation under Grants DEB 0816690 and DEB 0918931.
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