BACKGROUND
the evolution of altruism is difficult to explain because altruistic acts benefit other individuals while reducing the fitness of the actor.
according to hamilton's rule, cooperative and altruistic interactions are favored by genetic similarity   <cit> .
this is consistent with high levels of relatedness observed in cooperative organisms, such as social insects  <cit>  and spores in a fruiting body of the social amoeba dictyostelium discoideum  <cit> .
hamilton proposed that genetic similarity among interactors can be increased if individuals can recognize genetically similar individuals or, more passively, if limited dispersal increases the likelihood that neighbors are genetically similar.
this suggests that in spatially structured populations where interactions are often limited to neighbors, interactions are also more likely to take place among genetically similar individuals.
theoretical models suggest that spatial structure can select for the evolution of cooperation but can also increase competition among relatives  <cit> .
however, if there is a dispersal stage followed by a high relatedness stage, as is likely to be the case in d. discoideum, competition is less likely to counter cooperation.
the impact of spatial structure and relatedness on the evolution of social interactions can be studied in microbial experimental systems  <cit> .
the social amoeba dictyostelium discoideum is particularly well suited to test the effect of spatial structure and relatedness on the evolution of cooperation, because it has a complex asexual life cycle composed of a unicellular stage and a multicellular stage with clear altruism   <cit> .
during the unicellular, vegetative stage, spores germinate into single-celled amoebae that grow and divide by binary fission.
these amoebae live in soil and feed on microorganisms.
upon starvation, the multicellular stage of the life cycle begins.
single cells aggregate and form a motile slug that eventually develops into a fruiting body that consists of a stalk made of dead cells and a sorus containing the fertile spores.
during fruiting body formation, about 20% of the cells die to form the stalk.
in contrast to other multicellular organisms, the multicellular stage of d. discoideum does not go through a single cell bottleneck, but rather is the result of aggregation by thousands of cells.
these aggregating amoebae can be genetically distinct from their co-aggregants, which can lead to conflict over spore and stalk allocation  <cit>  such that one strain produces proportionately more spores and does not contribute its fair share of dead cells to the stalk.
accordingly, we define a cheater as a strain that increases in frequency during the multicellular stage when aggregating with cells of a genetically distinct strain.
despite the potential for conflict among aggregating cells, there is a clear benefit gained from the social interaction during the multicellular stage, because d. discoideum cells are unable to form spores without the formation of a fruiting body  <cit> .
the development of spores is advantageous, because only spores are able to survive harsh, nutrient deprived and desiccating environments.
the stalk of the fruiting body raises the sorus above the ground and increases the likelihood that spores will be dispersed to a nutrient rich patch either through falling a little farther from the stalk or through passive dispersal by animals.
the two stages of the life cycle are dominated by different interactions and selection pressures.
during the feeding stage, access to food and protection from predators are likely to be important.
since d. discoideum feeds by engulfing bacteria in an essentially solitary process and is not known to mount any group defenses, the impact of social interactions is not expected to be as great as during the social stage when cells cease to grow and 20% of amoebae sacrifice their lives for the benefit of others.
exactly how they compete with each other for food or other resources during the solitary growth stage is not known.
since growth and replication only occur during the solitary stage and cells in nature are likely go through several to hundreds of vegetative generations between social generations, differences during solitary growth may result in strong selection.
to test the effects of relatedness on the evolution of altruism, we created genetically diverse populations by mixing equal numbers of spores of eight genetically distinct wild isolates.
these populations were maintained for ten rounds of their life cycle under conditions that promoted social interactions at high or low relatedness.
since the amoebae undergo one multicellular social stage per cycle by definition, we will henceforth refer to one completion of the asexual life cycle as one social generation.
we manipulated spatial structure and hence relatedness of aggregating cells by plating the populations at the beginning of each social generation at either high or low density.
at high plating density, single cells encounter more non-relatives than at low plating density, which is reflected in lower relatedness among spores in single fruiting bodies at high density.
we manipulated relatedness by manipulating density, so we should consider what effects density alone might have, though nothing specific is documented in the literature on d. discoideum densities.
in our experimental conditions, the low-density populations will go through a few more vegetative generations per cycle than the high-density populations, giving greater selective opportunity to any clone that grows faster.
it is possible that the clones' relative growth rates change with density.
to be able to account for possible effects of different plating densities, we measured vegetative growth rates and germination rates of each strain individually.
in addition, the low plating densities of spores of the high relatedness populations could be more susceptible to drift.
to be able to distinguish the effects of drift and selection on these populations, we performed the experiment with twelve replicated populations at both high and low relatedness.
in most organisms, competition for limited resources and social interactions are highly intertwined, which makes it difficult to disentangle the effects of social traits from other life history traits  <cit> .
though selection can act on different traits in our experimental system, the temporal separation of the unicellular stage and the social, multicellular stage allows us to test the effects of social traits and other life history traits separately.
in addition, by forcing a global dispersal stage between social generations we can avoid complications that arise with limited dispersal, which can increase both relatedness and local competition  <cit> .
to assess the success of the different strains in our selection experiment, we measured the frequencies of each strain in the population and estimated genetic diversity by calculating shannon's index of diversity , which has its highest value when all strains are present in equal frequencies and declines with the loss of strains or unequal frequencies of the strains.
over the course of ten social generations, we expected that genetic diversity within populations would decline at both high and low relatedness as a result of competitive exclusion in our simple homogeneous environment.
we expected that this decline would be more pronounced at high relatedness, where the success of a strain is less affected by interactions with other clones and the fittest strain will be able to out-compete other strains.
further, we expected to see higher genetic diversity in the low relatedness populations, where the added complexity of the social environment provides a way for other clones to succeed.
specifically, we expected that social cheaters would be better able to take advantage of other cooperators at low relatedness.
this expectation assumes that there is some trade-off between the two ways of achieving higher fitness.
RESULTS
the social environment affects clone survivorship with high repeatability
relatedness within fruiting bodies was significantly higher under high relatedness conditions  as compared to low relatedness conditions  with no significant block effect, as determined by a fixed factor anova .
after one social generation, all strains were present in all twelve replicate populations, both at high and at low relatedness .
after ten social generations, however, a majority of the strains had decreased greatly in frequency and some had been completely eliminated.
at high relatedness, one strain  overwhelmingly predominated in all twelve populations.
two strains  were no longer present in any population.
one strain  was present in only one population and another strain  in three populations.
at low relatedness, three strains  predominated in every one of the twelve populations.
on average, the frequencies of the three persisting strains increased significantly above their starting frequency of 1/ <dig> .
two strains  were both present in three populations at very low frequencies.
three strains  could not be detected in any of the twelve low relatedness populations.
relatedness significantly affected strain composition and genetic diversity in the populations , with a significant decrease between social generations  <dig> and  <dig>  as determined by a full factor ancova with relatedness and generation as fixed factors.
more importantly, the decrease in genetic diversity was significantly stronger at high than at low relatedness, as indicated by a significant interaction term between relatedness and social generation .
a test for normality of the residuals indicated that residuals were not normally distributed , which seemed to be driven mostly by one of the replicated populations .
to test whether this population could affect the significance of the result, we repeated the analysis with hr <dig> excluded.
this resulted in normally distributed residuals , without qualitatively changing the results of the ancova.
strains that increased in frequency produced more spores and formed fruiting bodies more quickly than the other strains when grown in isolation
to assess the effects of life history traits, we grew each of the eight strains in the absence of the other strains and measured vegetative doubling rates, germination rates, spore production, and duration of the unicellular stage.
vegetative doubling rates during exponential growth did not differ significantly among the eight strains as indicated by a non-significant interaction term of strain and time  in a full factor ancova with strain, time and block as fixed factors .
nor did any of the clones show any change in doubling rate with increasing density .
germination rates varied significantly among the eight strains .
however, the three winning strains did not germinate significantly differently from the five losing strains , which suggests that the differences in germination rate cannot explain the persistence of the three winning clones.
the eight strains differed significantly in spore production , with the three strains that increased in frequency over the course of the selection experiment producing significantly more spores from a constant starting number of spores than did the strains that decreased .
however, the strain predominating in the high relatedness populations, qs <dig> , did not produce significantly more spores than the rest of the strains .
this suggests that spore production affected the persistence of the three strains at low relatedness, but is not sufficient to explain the observed differences between high and low relatedness.
the eight strains differed considerably in their speed of fruiting body formation.
when we plated equal numbers of spores in association with their bacterial food source on plates and scored the plates after three days of incubation, three strains  had developed farthest, to the stage called mexican hat .
four strains  were in the tight aggregate stage, and one strain  was still in the loose aggregate stage.
we used the speed of fruiting body formation as a proxy to estimate the length of the unicellular stage.
given that the strains did not differ in doubling rates, a shorter unicellular stage most likely arose from different threshold densities for aggregation.
the three persisting strains all either produced more spores and/or formed fruiting bodies sooner than the other strains .
this suggests that a combination of high spore production and short length of the unicellular stage affected the strains' ability to persist over ten social generations and their ability to out-compete other strains.
differences in these traits however are not sufficient to explain the observed differences between high and low relatedness.
cheating in the social stage did not explain which strains persisted
we tested whether cheating, the overrepresentation of one clone in the spores relative to its initial frequency in the cells could account for the increase in frequency of the three strains at low relatedness by mixing starved cells of each of these three strains  with a mixture of starved cells of the five strains that decreased at low relatedness over the course of the selection experiment.
all three of these strains showed an insignificant trend to decrease in frequency over the course of fruiting body formation .
since we tested the directional hypothesis that the three strains would increase, we tested whether the change was significantly greater than zero.
this effect was not significant for any of the strains individually  or combined .
the small sample sizes could potentially prevent detection of significance, but since all results went in the opposite direction from that expected for cheating, we think it is unlikely that lack of power was a problem.
this suggests that cheating did not lead to the observed increase in frequency of the three strains at low relatedness over the course of the selection experiment.
higher relatedness is expected to select for altruism over a greater range of benefits and costs  <cit> .
as a result, we expected cheaters to be more likely to persist at low relatedness where they can gain an advantage when interacting with cooperators.
to test whether some of the three strains persisting at low relatedness were able to cheat each other, we performed pair-wise cheating assays by mixing starved cells of two strains in equal proportions and counting the proportion of spores produced by each genotype .
we observed cheating among the three low relatedness strains, but found no evidence that the two clones that persisted only at low relatedness  did so by cheating the one that was present at both high and low relatedness .
qs <dig>  was cheated by both qs <dig>  and qs <dig> .
qs <dig> decreased in frequency when mixed with qs <dig>  but not quite significantly so .
we did not observe a significant change in frequency in the control mixes , nor did we observe a significant block effect in either experimental or control mix.
discussion
higher relatedness promotes the evolution of altruism all else being equal  <cit> .
spatial structure can limit interactions to neighbors and hence increase the likelihood that interactions are taking place among genetically similar individuals.
to test the effect of relatedness on the evolution of altruism, we evolved genetically diverse populations for ten social generations at low and high density, which resulted in high and low relatedness, respectively.
over the course of the selection experiment, we observed a highly repeatable pattern: the same three strains increased in frequency at low relatedness and one of these three strains predominated in all populations at high relatedness.
the remarkably high degree of repeatability among the twelve replicated, independently evolved populations indicates that drift is not playing much of a role, even in the high-relatedness populations that started each round with only  <dig> spores.
it is highly unlikely that the same strain would dominate in all twelve independently evolved populations solely due to chance  in twelve populations  out of twelve populations ).
although low relatedness populations showed higher diversity, as we expected, social advantages during the fruiting body stage did not seem to have a strong effect.
the three persisting strains did not cheat the other five strains as a group when mixed head to head.
we expected that cheaters would be able to persist at low relatedness where they could exploit other strains and increase in frequency during the social stage.
not all of the three clones that persisted at low relatedness were equally successful in social competition with each other.
however, one of the strains cheating during fruiting body formation was the most successful clone at high relatedness, indicating that it did not face a trade-off between the ability to cheat other strains and the ability to develop by itself.
differences in spore production and length of the unicellular stage best explained which three strains persisted.
germination rate differed only slightly among the strains and could not account for the persistence of the three strains.
vegetative doubling rates did not differ measurably among the eight strains.
the observed difference in genetic diversity at high and low relatedness could be the result of factors other than relatedness.
to achieve conditions of high and low relatedness, we plated the populations at low and high density, respectively.
while we cannot completely rule out effects due to density-dependence, we found no evidence that density-dependent effects account for the different outcomes of our selection experiment.
first, the eight clones do not differ measurably in their vegetative growth rates.
thus, no clone gains a significant advantage by having more cell generations in one of the treatments.
the same data show that over a broad range of densities, each of our clones grows at a constant rate.
we did not test the earliest stages, but at that time cells in both high-density and low-density treatments actually experience the same density, because they are all still in plaques started from single cells.
thus clonal vegetative fitnesses do not change with density, and different densities do not explain the differences between treatments.
the difference in population size at transfer also resulted in more vegetative doublings during the unicellular stage in the high relatedness treatments.
the average number of doublings per social generations was  <dig>  at low relatedness and  <dig>  at high relatedness, which is consistent with the 100-fold increase necessary to make up the difference in plating densities.
therefore, one could hypothesize that the higher genetic diversity at low relatedness is simply a transient state on a path that, if carried on farther, would yield the identical outcome as the high relatedness populations.
several lines of evidence suggest that this is unlikely to be the case.
we did not observe any differences in doubling rates during unicellular growth.
therefore, it is unlikely that the additional doublings at high relatedness could account for the different strain compositions and diversities at high and low relatedness.
in addition, over the course of the low-relatedness experiment qs <dig>  and qs <dig>  increased from the starting frequency of 1/ <dig>  which would not be expected if they were on their way to being out-competed by the dominant one.
more tellingly, the five losing clones were virtually eliminated from the low relatedness populations .
if these populations were intermediates on the way to the dominance of qs <dig>  seen in the high relatedness populations, we would expect to see even fewer of these loser clones at high relatedness, but in fact we see many more .
this difference is significant .
lastly, we calculated the average change in frequency of each strain per vegetative generation by dividing the difference between final frequency and initial frequency  by the number of vegetative generations .
if the difference in vegetative doublings accounted for the different outcomes at high and low relatedness, we would expect a correlation between the change in frequency at high and low relatedness after correcting for vegetative generations, which is clearly not the case, except for the one clone that did well under both conditions .
even so, we cannot definitively exclude a possible effect due to the different plating densities and the resulting differences in vegetative generations at high and low relatedness.
gilbert et al.
<cit>  assessed relatedness in natural populations and observed high levels of relatedness.
high relatedness among interacting individuals can be achieved in two ways, by kin discrimination or by limited dispersal, and it is worth considering what role these mechanisms may have played in our experiment.
d. discoideum clones are sometimes able to discriminate against other clones.
in experimental mixes with wild clones, cells from genetically distinct strains sorted more with increasing genetic distance, while genetically similar strains formed chimeric fruiting bodies  <cit> .
benabentos et al.
<cit>  recently identified high levels of polymorphism among wild isolates in members of the lag  gene family which encodes trans-membrane proteins.
a strong correlation between sequence divergence in these lag genes and sorting during fruiting body formation supports the hypothesis that these genes are important in kin discrimination.
a preference for forming fruiting bodies with kin was also observed in other dictyostelid species.
different genotypes of dictyostelium purpureum preferentially aggregated and formed fruiting bodies with their clone mates  <cit> .
despite some ability to discriminate, our low-relatedness treatment did generate low relatedness among spores within fruiting bodies, and generated complete mixing in 1: <dig> mixes during the cheating assays  indicating that the eight strains used readily form chimeric fruiting bodies.
this suggests that kin discrimination did not play an important role in our experiment as genetically different strains readily mixed and formed chimeric fruiting bodies when given the opportunity.
limited dispersal could also promote interactions among genetically similar individuals  <cit> .
over the course of the life cycle, d. discoideum has different means of dispersal.
during the unicellular stage, single cells can move short distances to explore new food sources.
this kind of movement is expected to be the same in the high and low relatedness treatments.
however in the high-density, low relatedness treatment single cells are more likely to encounter and compete with genetically different cells and it is this difference that we exploited to get high versus low relatedness treatments.
during the multicellular stage, d. discoideum forms slugs that move towards light and away from ammonia  <cit> .
these slugs can move longer distances than single cells.
chimeric slugs have been observed to move less far than clonal slugs of the same size  <cit>  and this effect would differ between our treatments.
during the experiment, we minimized the effect of slug dispersal by keeping the plates in the dark without a light source.
long-range dispersal of d. discoideum can occur when spores are picked up by animals  <cit>  or are washed away by water.
in our experiment we collected all the spores after one social generation and thoroughly mixed the spores before plating them onto a fresh bacterial lawn for the next social generation.
therefore uncontrolled dispersal of slugs and spores did not have any effect on the structure of the next generation or on the different outcomes at high and low relatedness.
cooperating individuals are very susceptible to exploitation by cheaters.
therefore it is difficult to explain the maintenance of cooperation in the light of cheating.
one possibility is that cheaters have reduced fitness when they are unable to exploit cooperators and have to interact among themselves.
cheating assays among the persisting strains at low relatedness confirmed the presence of cheating.
one of these cheaters  was unable to persist at high relatedness, suggesting such a cost to cheating.
however, cheating did not seem to be costly for the other cheater , which dominated all high-relatedness populations.
cheaters unable to persist at high relatedness have previously been observed in d. discoideum.
a known cheater mutant  was mixed with its wild type ancestor and while able to invade at low relatedness was unable to do so at high relatedness  <cit> .
this inability to invade when the mutant was at high levels of relatedness is a result of the high fitness cost of the cheater mutation.
when grown in isolation, this cheater mutant was not able to form fruiting bodies  <cit> .
however, not all cheater clones experienced the same costs to cheating in our experiment.
this is consistent with previous results, where we identified a large set of cheater mutants in d. discoideum  <cit> .
some of these cheater clones had a cost in terms of spore production when on their own, while others performed the same as the wild type, and still others produced even more spores than the wild type.
all of these cheaters were able to form fruiting bodies with normal appearances on their own.
our results suggest that trade-offs between cheating and the ability to form fruiting bodies alone vary among strains and that a combination of other life-history traits can partly explain the outcome of the selection experiment.
even so, interactions other than the ones we tested may contribute to shaping these populations.
for example, we only tested pair-wise interactions during the multicellular stage.
it is possible that the cheating dynamics change when all three strains can interact.
alternatively, our results may suggest that some interesting social interactions occur during the unicellular stage.
since the vegetative stage is quite long, small effects early in the vegetative stage could result in a large effect over the course of one social generation  <cit> .
CONCLUSIONS
for d. discoideum, spatial environment and the resulting differences in relatedness among interactors imposed strong selection pressures, as indicated by the highly repeatable differences in genetic diversity and strain composition between high and low relatedness.
as expected, cheaters persisted better at low relatedness than at high relatedness.
interestingly, the strain that predominated in all populations at high relatedness was one of the cheaters.
this indicates variable trade-offs between the ability to cheat other strains and the ability to form fruiting bodies alone.
the persistence of the three strains was best explained by high spore production and a short unicellular stage, both traits that are strongly affected during vegetative growth.
however, none of the traits examined could account for the difference between high and low relatedness, which suggests that other possible interactions occur during the vegetative stage.
our results suggest that the costs and benefits of the social interactions vary among strains and that social interactions in this social amoeba are complex and extend into the vegetative stage.

 the evolution of altruism is difficult to explain because altruistic acts benefit other individuals while reducing the fitness of the actor. according to hamilton's rule, cooperative and altruistic interactions are favored by genetic similarity. this is consistent with high levels of relatedness observed in cooperative organisms, such as social insects and spores in a fruiting body of the social amoeba dictyostelium discoideum. hamilton proposed that genetic similarity among interactors can be increased if individuals can recognize genetically similar individuals or, more passively, if limited dispersal increases the likelihood that neighbors are genetically similar. this suggests that in spatially structured populations where interactions are often limited to neighbors, interactions are also more likely to take place among genetically similar individuals. theoretical models suggest that spatial structure can select for the evolution of cooperation but can also increase competition among relatives. however, if there is a dispersal stage followed by a high relatedness stage, as is likely to be the case in d. discoideum, competition is less likely to counter cooperation. the impact of spatial structure and relatedness on the evolution of social interactions can be studied in microbial experimental systems. the social amoeba dictyostelium discoideum is particularly well suited to test the effect of spatial structure and relatedness on the evolution of cooperation, because it has a complex asexual life cycle composed of a unicellular stage and a multicellular stage with clear altruism. during the unicellular, vegetative stage, spores germinate into single-celled amoebae that grow and divide by binary fission. these amoebae live in soil and feed on microorganisms. upon starvation, the multicellular stage of the life cycle begins. single cells aggregate and form a motile slug that eventually develops into a fruiting body that consists of a stalk made of dead cells and a sorus containing the fertile spores. during fruiting body formation, about 20% of the cells die to form the stalk. in contrast to other multicellular organisms, the multicellular stage of d. discoideum does not go through a single cell bottleneck, but rather is the result of aggregation by thousands of cells. these aggregating amoebae can be genetically distinct from their co-aggregants, which can lead to conflict over spore and stalk allocation such that one strain produces proportionately more spores and does not contribute its fair share of dead cells to the stalk. accordingly, we define a cheater as a strain that increases in frequency during the multicellular stage when aggregating with cells of a genetically distinct strain. despite the potential for conflict among aggregating cells, there is a clear benefit gained from the social interaction during the multicellular stage, because d. discoideum cells are unable to form spores without the formation of a fruiting body. the development of spores is advantageous, because only spores are able to survive harsh, nutrient deprived and desiccating environments. the stalk of the fruiting body raises the sorus above the ground and increases the likelihood that spores will be dispersed to a nutrient rich patch either through falling a little farther from the stalk or through passive dispersal by animals. the two stages of the life cycle are dominated by different interactions and selection pressures. during the feeding stage, access to food and protection from predators are likely to be important. since d. discoideum feeds by engulfing bacteria in an essentially solitary process and is not known to mount any group defenses, the impact of social interactions is not expected to be as great as during the social stage when cells cease to grow and 20% of amoebae sacrifice their lives for the benefit of others. exactly how they compete with each other for food or other resources during the solitary growth stage is not known. since growth and replication only occur during the solitary stage and cells in nature are likely go through several to hundreds of vegetative generations between social generations, differences during solitary growth may result in strong selection. to test the effects of relatedness on the evolution of altruism, we created genetically diverse populations by mixing equal numbers of spores of eight genetically distinct wild isolates. these populations were maintained for ten rounds of their life cycle under conditions that promoted social interactions at high or low relatedness. since the amoebae undergo one multicellular social stage per cycle by definition, we will henceforth refer to one completion of the asexual life cycle as one social generation. we manipulated spatial structure and hence relatedness of aggregating cells by plating the populations at the beginning of each social generation at either high or low density. at high plating density, single cells encounter more non-relatives than at low plating density, which is reflected in lower relatedness among spores in single fruiting bodies at high density. we manipulated relatedness by manipulating density, so we should consider what effects density alone might have, though nothing specific is documented in the literature on d. discoideum densities. in our experimental conditions, the low-density populations will go through a few more vegetative generations per cycle than the high-density populations, giving greater selective opportunity to any clone that grows faster. it is possible that the clones' relative growth rates change with density. to be able to account for possible effects of different plating densities, we measured vegetative growth rates and germination rates of each strain individually. in addition, the low plating densities of spores of the high relatedness populations could be more susceptible to drift. to be able to distinguish the effects of drift and selection on these populations, we performed the experiment with twelve replicated populations at both high and low relatedness. in most organisms, competition for limited resources and social interactions are highly intertwined, which makes it difficult to disentangle the effects of social traits from other life history traits. though selection can act on different traits in our experimental system, the temporal separation of the unicellular stage and the social, multicellular stage allows us to test the effects of social traits and other life history traits separately. in addition, by forcing a global dispersal stage between social generations we can avoid complications that arise with limited dispersal, which can increase both relatedness and local competition. to assess the success of the different strains in our selection experiment, we measured the frequencies of each strain in the population and estimated genetic diversity by calculating shannon's index of diversity, which has its highest value when all strains are present in equal frequencies and declines with the loss of strains or unequal frequencies of the strains. over the course of ten social generations, we expected that genetic diversity within populations would decline at both high and low relatedness as a result of competitive exclusion in our simple homogeneous environment. we expected that this decline would be more pronounced at high relatedness, where the success of a strain is less affected by interactions with other clones and the fittest strain will be able to out-compete other strains. further, we expected to see higher genetic diversity in the low relatedness populations, where the added complexity of the social environment provides a way for other clones to succeed. specifically, we expected that social cheaters would be better able to take advantage of other cooperators at low relatedness. this expectation assumes that there is some trade-off between the two ways of achieving higher fitness.  the social environment affects clone survivorship with high repeatability relatedness within fruiting bodies was significantly higher under high relatedness conditions as compared to low relatedness conditions with no significant block effect, as determined by a fixed factor anova. after one social generation, all strains were present in all twelve replicate populations, both at high and at low relatedness. after ten social generations, however, a majority of the strains had decreased greatly in frequency and some had been completely eliminated. at high relatedness, one strain overwhelmingly predominated in all twelve populations. two strains were no longer present in any population. one strain was present in only one population and another strain in three populations. at low relatedness, three strains predominated in every one of the twelve populations. on average, the frequencies of the three persisting strains increased significantly above their starting frequency of 1/. two strains were both present in three populations at very low frequencies. three strains could not be detected in any of the twelve low relatedness populations. relatedness significantly affected strain composition and genetic diversity in the populations, with a significant decrease between social generations and as determined by a full factor ancova with relatedness and generation as fixed factors. more importantly, the decrease in genetic diversity was significantly stronger at high than at low relatedness, as indicated by a significant interaction term between relatedness and social generation. a test for normality of the residuals indicated that residuals were not normally distributed, which seemed to be driven mostly by one of the replicated populations. to test whether this population could affect the significance of the result, we repeated the analysis with hr excluded. this resulted in normally distributed residuals, without qualitatively changing the results of the ancova. strains that increased in frequency produced more spores and formed fruiting bodies more quickly than the other strains when grown in isolation to assess the effects of life history traits, we grew each of the eight strains in the absence of the other strains and measured vegetative doubling rates, germination rates, spore production, and duration of the unicellular stage. vegetative doubling rates during exponential growth did not differ significantly among the eight strains as indicated by a non-significant interaction term of strain and time in a full factor ancova with strain, time and block as fixed factors. nor did any of the clones show any change in doubling rate with increasing density. germination rates varied significantly among the eight strains. however, the three winning strains did not germinate significantly differently from the five losing strains, which suggests that the differences in germination rate cannot explain the persistence of the three winning clones. the eight strains differed significantly in spore production, with the three strains that increased in frequency over the course of the selection experiment producing significantly more spores from a constant starting number of spores than did the strains that decreased. however, the strain predominating in the high relatedness populations, qs, did not produce significantly more spores than the rest of the strains. this suggests that spore production affected the persistence of the three strains at low relatedness, but is not sufficient to explain the observed differences between high and low relatedness. the eight strains differed considerably in their speed of fruiting body formation. when we plated equal numbers of spores in association with their bacterial food source on plates and scored the plates after three days of incubation, three strains had developed farthest, to the stage called mexican hat. four strains were in the tight aggregate stage, and one strain was still in the loose aggregate stage. we used the speed of fruiting body formation as a proxy to estimate the length of the unicellular stage. given that the strains did not differ in doubling rates, a shorter unicellular stage most likely arose from different threshold densities for aggregation. the three persisting strains all either produced more spores and/or formed fruiting bodies sooner than the other strains. this suggests that a combination of high spore production and short length of the unicellular stage affected the strains' ability to persist over ten social generations and their ability to out-compete other strains. differences in these traits however are not sufficient to explain the observed differences between high and low relatedness. cheating in the social stage did not explain which strains persisted we tested whether cheating, the overrepresentation of one clone in the spores relative to its initial frequency in the cells could account for the increase in frequency of the three strains at low relatedness by mixing starved cells of each of these three strains with a mixture of starved cells of the five strains that decreased at low relatedness over the course of the selection experiment. all three of these strains showed an insignificant trend to decrease in frequency over the course of fruiting body formation. since we tested the directional hypothesis that the three strains would increase, we tested whether the change was significantly greater than zero. this effect was not significant for any of the strains individually or combined. the small sample sizes could potentially prevent detection of significance, but since all results went in the opposite direction from that expected for cheating, we think it is unlikely that lack of power was a problem. this suggests that cheating did not lead to the observed increase in frequency of the three strains at low relatedness over the course of the selection experiment. higher relatedness is expected to select for altruism over a greater range of benefits and costs. as a result, we expected cheaters to be more likely to persist at low relatedness where they can gain an advantage when interacting with cooperators. to test whether some of the three strains persisting at low relatedness were able to cheat each other, we performed pair-wise cheating assays by mixing starved cells of two strains in equal proportions and counting the proportion of spores produced by each genotype. we observed cheating among the three low relatedness strains, but found no evidence that the two clones that persisted only at low relatedness did so by cheating the one that was present at both high and low relatedness. qs was cheated by both qs and qs. qs decreased in frequency when mixed with qs but not quite significantly so. we did not observe a significant change in frequency in the control mixes, nor did we observe a significant block effect in either experimental or control mix. discussion higher relatedness promotes the evolution of altruism all else being equal. spatial structure can limit interactions to neighbors and hence increase the likelihood that interactions are taking place among genetically similar individuals. to test the effect of relatedness on the evolution of altruism, we evolved genetically diverse populations for ten social generations at low and high density, which resulted in high and low relatedness, respectively. over the course of the selection experiment, we observed a highly repeatable pattern: the same three strains increased in frequency at low relatedness and one of these three strains predominated in all populations at high relatedness. the remarkably high degree of repeatability among the twelve replicated, independently evolved populations indicates that drift is not playing much of a role, even in the high-relatedness populations that started each round with only spores. it is highly unlikely that the same strain would dominate in all twelve independently evolved populations solely due to chance in twelve populations out of twelve populations ). although low relatedness populations showed higher diversity, as we expected, social advantages during the fruiting body stage did not seem to have a strong effect. the three persisting strains did not cheat the other five strains as a group when mixed head to head. we expected that cheaters would be able to persist at low relatedness where they could exploit other strains and increase in frequency during the social stage. not all of the three clones that persisted at low relatedness were equally successful in social competition with each other. however, one of the strains cheating during fruiting body formation was the most successful clone at high relatedness, indicating that it did not face a trade-off between the ability to cheat other strains and the ability to develop by itself. differences in spore production and length of the unicellular stage best explained which three strains persisted. germination rate differed only slightly among the strains and could not account for the persistence of the three strains. vegetative doubling rates did not differ measurably among the eight strains. the observed difference in genetic diversity at high and low relatedness could be the result of factors other than relatedness. to achieve conditions of high and low relatedness, we plated the populations at low and high density, respectively. while we cannot completely rule out effects due to density-dependence, we found no evidence that density-dependent effects account for the different outcomes of our selection experiment. first, the eight clones do not differ measurably in their vegetative growth rates. thus, no clone gains a significant advantage by having more cell generations in one of the treatments. the same data show that over a broad range of densities, each of our clones grows at a constant rate. we did not test the earliest stages, but at that time cells in both high-density and low-density treatments actually experience the same density, because they are all still in plaques started from single cells. thus clonal vegetative fitnesses do not change with density, and different densities do not explain the differences between treatments. the difference in population size at transfer also resulted in more vegetative doublings during the unicellular stage in the high relatedness treatments. the average number of doublings per social generations was at low relatedness and at high relatedness, which is consistent with the 100-fold increase necessary to make up the difference in plating densities. therefore, one could hypothesize that the higher genetic diversity at low relatedness is simply a transient state on a path that, if carried on farther, would yield the identical outcome as the high relatedness populations. several lines of evidence suggest that this is unlikely to be the case. we did not observe any differences in doubling rates during unicellular growth. therefore, it is unlikely that the additional doublings at high relatedness could account for the different strain compositions and diversities at high and low relatedness. in addition, over the course of the low-relatedness experiment qs and qs increased from the starting frequency of 1/ which would not be expected if they were on their way to being out-competed by the dominant one. more tellingly, the five losing clones were virtually eliminated from the low relatedness populations. if these populations were intermediates on the way to the dominance of qs seen in the high relatedness populations, we would expect to see even fewer of these loser clones at high relatedness, but in fact we see many more. this difference is significant. lastly, we calculated the average change in frequency of each strain per vegetative generation by dividing the difference between final frequency and initial frequency by the number of vegetative generations. if the difference in vegetative doublings accounted for the different outcomes at high and low relatedness, we would expect a correlation between the change in frequency at high and low relatedness after correcting for vegetative generations, which is clearly not the case, except for the one clone that did well under both conditions. even so, we cannot definitively exclude a possible effect due to the different plating densities and the resulting differences in vegetative generations at high and low relatedness. gilbert et al. assessed relatedness in natural populations and observed high levels of relatedness. high relatedness among interacting individuals can be achieved in two ways, by kin discrimination or by limited dispersal, and it is worth considering what role these mechanisms may have played in our experiment. d. discoideum clones are sometimes able to discriminate against other clones. in experimental mixes with wild clones, cells from genetically distinct strains sorted more with increasing genetic distance, while genetically similar strains formed chimeric fruiting bodies. benabentos et al. recently identified high levels of polymorphism among wild isolates in members of the lag gene family which encodes trans-membrane proteins. a strong correlation between sequence divergence in these lag genes and sorting during fruiting body formation supports the hypothesis that these genes are important in kin discrimination. a preference for forming fruiting bodies with kin was also observed in other dictyostelid species. different genotypes of dictyostelium purpureum preferentially aggregated and formed fruiting bodies with their clone mates. despite some ability to discriminate, our low-relatedness treatment did generate low relatedness among spores within fruiting bodies, and generated complete mixing in 1: mixes during the cheating assays indicating that the eight strains used readily form chimeric fruiting bodies. this suggests that kin discrimination did not play an important role in our experiment as genetically different strains readily mixed and formed chimeric fruiting bodies when given the opportunity. limited dispersal could also promote interactions among genetically similar individuals. over the course of the life cycle, d. discoideum has different means of dispersal. during the unicellular stage, single cells can move short distances to explore new food sources. this kind of movement is expected to be the same in the high and low relatedness treatments. however in the high-density, low relatedness treatment single cells are more likely to encounter and compete with genetically different cells and it is this difference that we exploited to get high versus low relatedness treatments. during the multicellular stage, d. discoideum forms slugs that move towards light and away from ammonia. these slugs can move longer distances than single cells. chimeric slugs have been observed to move less far than clonal slugs of the same size and this effect would differ between our treatments. during the experiment, we minimized the effect of slug dispersal by keeping the plates in the dark without a light source. long-range dispersal of d. discoideum can occur when spores are picked up by animals or are washed away by water. in our experiment we collected all the spores after one social generation and thoroughly mixed the spores before plating them onto a fresh bacterial lawn for the next social generation. therefore uncontrolled dispersal of slugs and spores did not have any effect on the structure of the next generation or on the different outcomes at high and low relatedness. cooperating individuals are very susceptible to exploitation by cheaters. therefore it is difficult to explain the maintenance of cooperation in the light of cheating. one possibility is that cheaters have reduced fitness when they are unable to exploit cooperators and have to interact among themselves. cheating assays among the persisting strains at low relatedness confirmed the presence of cheating. one of these cheaters was unable to persist at high relatedness, suggesting such a cost to cheating. however, cheating did not seem to be costly for the other cheater, which dominated all high-relatedness populations. cheaters unable to persist at high relatedness have previously been observed in d. discoideum. a known cheater mutant was mixed with its wild type ancestor and while able to invade at low relatedness was unable to do so at high relatedness. this inability to invade when the mutant was at high levels of relatedness is a result of the high fitness cost of the cheater mutation. when grown in isolation, this cheater mutant was not able to form fruiting bodies. however, not all cheater clones experienced the same costs to cheating in our experiment. this is consistent with previous results, where we identified a large set of cheater mutants in d. discoideum. some of these cheater clones had a cost in terms of spore production when on their own, while others performed the same as the wild type, and still others produced even more spores than the wild type. all of these cheaters were able to form fruiting bodies with normal appearances on their own. our results suggest that trade-offs between cheating and the ability to form fruiting bodies alone vary among strains and that a combination of other life-history traits can partly explain the outcome of the selection experiment. even so, interactions other than the ones we tested may contribute to shaping these populations. for example, we only tested pair-wise interactions during the multicellular stage. it is possible that the cheating dynamics change when all three strains can interact. alternatively, our results may suggest that some interesting social interactions occur during the unicellular stage. since the vegetative stage is quite long, small effects early in the vegetative stage could result in a large effect over the course of one social generation.  for d. discoideum, spatial environment and the resulting differences in relatedness among interactors imposed strong selection pressures, as indicated by the highly repeatable differences in genetic diversity and strain composition between high and low relatedness. as expected, cheaters persisted better at low relatedness than at high relatedness. interestingly, the strain that predominated in all populations at high relatedness was one of the cheaters. this indicates variable trade-offs between the ability to cheat other strains and the ability to form fruiting bodies alone. the persistence of the three strains was best explained by high spore production and a short unicellular stage, both traits that are strongly affected during vegetative growth. however, none of the traits examined could account for the difference between high and low relatedness, which suggests that other possible interactions occur during the vegetative stage. our results suggest that the costs and benefits of the social interactions vary among strains and that social interactions in this social amoeba are complex and extend into the vegetative stage. 
altruism can be favored by high relatedness among interactants. we found that at high relatedness a single clone prevailed in all twelve populations. a combination of high spore production and short unicellular stage most successfully explained the three successful clones at low relatedness, but not why one of them fared better at high relatedness. after one and ten social generations we assessed the strain composition of the populations. in addition, we expected that low relatedness might allow the persistence of social cheaters as it provides opportunity to exploit other strains. we mixed eight genetically distinct wild isolates and maintained twelve replicated populations at a high and a low relatedness treatment. at low relatedness three clones predominated in all twelve populations. furthermore, the dominant clone at high-relatedness was not cheated by the other two that persisted at low relatedness. the high degree of repeatability of our results indicates that these effects are strong and points to the importance of new approaches to studying interactions in d. discoideum. differences in density did not account for the results, as the clones did not differ in vegetative growth rates nor did they change the growth rates over relevant densities. these results suggest that social competition and something beyond solitary growth differences occurs during the vegetative stage when amoebae eat bacteria and divide by binary fission. interestingly, exploitation of some clones by others in the social stage did not explain the results. we tested the effect of relatedness in experimental populations of the social amoeba dictyostelium discoideum, where altruism occurs in a starvation-induced social stage when some amoebae die to form a stalk that lifts the fertile spores above the soil facilitating dispersal. the single cells that aggregate during the social stage can be genetically diverse, which can lead to conflict over spore and stalk allocation.
 altruism can be favored by high relatedness among interactants. we tested the effect of relatedness in experimental populations of the social amoeba dictyostelium discoideum, where altruism occurs in a starvation-induced social stage when some amoebae die to form a stalk that lifts the fertile spores above the soil facilitating dispersal. the single cells that aggregate during the social stage can be genetically diverse, which can lead to conflict over spore and stalk allocation. we mixed eight genetically distinct wild isolates and maintained twelve replicated populations at a high and a low relatedness treatment. after one and ten social generations we assessed the strain composition of the populations. we expected that some strains would be out-competed in both treatments. in addition, we expected that low relatedness might allow the persistence of social cheaters as it provides opportunity to exploit other strains.  we found that at high relatedness a single clone prevailed in all twelve populations. at low relatedness three clones predominated in all twelve populations. interestingly, exploitation of some clones by others in the social stage did not explain the results. when we mixed each winner against the pool of five losers, the winner did not prevail in the spores because all contributed fairly to the stalk and spores. furthermore, the dominant clone at high-relatedness was not cheated by the other two that persisted at low relatedness. a combination of high spore production and short unicellular stage most successfully explained the three successful clones at low relatedness, but not why one of them fared better at high relatedness. differences in density did not account for the results, as the clones did not differ in vegetative growth rates nor did they change the growth rates over relevant densities.  these results suggest that social competition and something beyond solitary growth differences occurs during the vegetative stage when amoebae eat bacteria and divide by binary fission. the high degree of repeatability of our results indicates that these effects are strong and points to the importance of new approaches to studying interactions in d. discoideum. 