Kin discrimination increases with genetic distance in a social amoeba.
ABSTRACT In the social amoeba Dictyostelium discoideum, thousands of cells aggregate upon starvation to form a multicellular fruiting body, and approximately 20% of them die to form a stalk that benefits the others. The aggregative nature of multicellular development makes the cells vulnerable to exploitation by cheaters, and the potential for cheating is indeed high. Cells might avoid being victimized if they can discriminate among individuals and avoid those that are genetically different. We tested how widely social amoebae cooperate by mixing isolates from different localities that cover most of their natural range. We show here that different isolates partially exclude one another during aggregation, and there is a positive relationship between the extent of this exclusion and the genetic distance between strains. Our findings demonstrate that D. discoideum cells co-aggregate more with genetically similar than dissimilar individuals, suggesting the existence of a mechanism that discerns the degree of genetic similarity between individuals in this social microorganism.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Dictyostelium discoideum, a microbial model for social evolution, is known to distinguish self from non-self and show genotype-dependent behavior during chimeric development. Aside from a small number of cell-cell recognition genes, however, little is known about the genetic basis of self/non-self recognition in this species. Based on the key hypothesis that there should be differential expression of genes if D. discoideum cells were interacting with non-clone mates, we performed transcriptomic profiling study in this species during clonal vs. chimeric development. The transcriptomic profiles of D. discoideum cells in clones vs. different chimeras were compared at five different developmental stages using a customized microarray. Effects of chimerism on global transcriptional patterns associated with social interactions were observed.BMC Genomics 07/2014; 15(1):616. · 4.04 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Fusion between multicellular individuals is possible in many organisms with modular, indeterminate growth, such as marine invertebrates and fungi. Although fusion may provide various benefits, fusion usually is restricted to close relatives by allorecognition, also called heterokaryon or somatic incompatibility in fungi. A possible selective explanation for allorecognition is protection against somatic parasites. Such mutants contribute less to colony functions but more to reproduction. However, previous models testing this idea have failed to explain the high diversity of allorecognition alleles in nature. These models did not, however, consider the possible role of spatial structure. We model the joint evolution of allorecognition and somatic parasitism in a multicellular organism resembling an asexual ascomycete fungus in a spatially explicit simulation. In a 1,000-by-1,000 grid, neighboring individuals can fuse, but only if they have the same allotype. Fusion with a parasitic individual decreases the total reproductive output of the fused individuals, but the parasite compensates for this individual-level fitness reduction by a disproportional share of the offspring. Allorecognition prevents the invasion of somatic parasites, and vice versa, mutation towards somatic parasitism provides the selective conditions for extensive allorecognition diversity. On the one hand, if allorecognition diversity did not build up fast enough, somatic parasites went to fixation; conversely, once parasites had gone to fixation no allorecognition diversity built up. On the other hand, the mere threat of parasitism could select for high allorecognition diversity, preventing invasion of somatic parasites. Moderate population viscosity combined with weak global dispersal was optimal for the joint evolution of allorecognition and protection against parasitism. Our results are consistent with the widespread occurrence of allorecognition in fungi and the low degree of somatic parasitism. We discuss the implications of our results for allorecognition in other organism groups.Fungal Genetics and Biology 10/2014; · 3.26 Impact Factor
Conference Paper: The Evolution of Kin Inclusivity Levels[Show abstract] [Hide abstract]
ABSTRACT: Altruism is a ubiquitous strategy among organisms ranging from microbes to mammals. Inclusive fitness theory indi-cates that altruistic strategies can be beneficial when an altruist acts to benefit organisms that share its genes. It is common for such altruistic strategies to be negatively af-fected by cheaters that do not act altruistically. A more sub-tle form of cheating involves altruists that are more selective. For example, a selective organism may benefit from a distant kin's altruistic actions without reciprocating. We consider an organism's kin inclusivity level to be the maximum num-ber of mutational differences where the other organism will be considered kin. We use evolving computer programs (dig-ital organisms) to explore competitions among organisms with different kin inclusivity levels. Using competition as-says that vary environmental parameters, we find that high mutation rates favor more inclusive colonies. When we com-peted colonies with a wide range of kin inclusivity levels, we found that moderate mutation rates and populations sizes led to intermediate inclusivity levels winning the competi-tions, indicating that extreme inclusivity levels were not al-ways optimal. However, when organisms could set their own kin inclusivity level, we found that high mutation rates se-lected for highly inclusive organisms.GECCO 2014, Vancouver, BC, Canada; 07/2014
Kin Discrimination Increases with Genetic
Distance in a Social Amoeba
Elizabeth A. Ostrowski1[*, Mariko Katoh2[, Gad Shaulsky1,2, David C. Queller1, Joan E. Strassmann1
1 Department of Ecology and Evolutionary Biology, Rice University, Houston, Texas, United States of America, 2 Department of Molecular and Human Genetics, Baylor
College of Medicine, Houston, Texas, United States of America
In the social amoeba Dictyostelium discoideum, thousands of cells aggregate upon starvation to form a multicellular
fruiting body, and approximately 20% of them die to form a stalk that benefits the others. The aggregative nature of
multicellular development makes the cells vulnerable to exploitation by cheaters, and the potential for cheating is
indeed high. Cells might avoid being victimized if they can discriminate among individuals and avoid those that are
genetically different. We tested how widely social amoebae cooperate by mixing isolates from different localities that
cover most of their natural range. We show here that different isolates partially exclude one another during
aggregation, and there is a positive relationship between the extent of this exclusion and the genetic distance between
strains. Our findings demonstrate that D. discoideum cells co-aggregate more with genetically similar than dissimilar
individuals, suggesting the existence of a mechanism that discerns the degree of genetic similarity between
individuals in this social microorganism.
Citation: Ostrowski EA, Katoh M, Shaulsky G, Queller DC, Strassmann JE (2008) Kin discrimination increases with genetic distance in a social amoeba. PLoS Biol 6(11): e287.
The ability to recognize and preferentially interact with kin
can favor the evolution of altruistic or cooperative traits [1,2].
Microorganisms exhibit complex social behaviors [3–6], but
little is known about the genetic and geographic scale of their
cooperation . Social traits, in particular, may be prone to
the emergence of incompatibilities: selection to avoid
potential costs of cooperation, including cheating, may drive
rapid evolution at discrimination or other loci and limit
cooperation to closely related strains.
The social amoeba D. discoideum (formerly known as the
cellular slime mold) offers a unique opportunity to examine
the relationship between genetic distance and discrimination
in a cooperative microbe. It is haploid, and its genome
contains numerous microsatellite loci, which permit quanti-
tative estimation of genetic differences between individuals.
It has a geographically restricted range and is found primarily
in forest soils of eastern North America and East Asia .
Upon starvation, unicellular amoebae assemble in groups of
approximately 104–105cells to form a multicellular aggregate.
The aggregate can migrate toward light and heat and
eventually develop into a fruiting body composed of a ball
of spores held aloft by a rigid cellular stalk. Approximately
70–80% of the cells in the initial aggregate will form spores,
whereas 20–30% of the cells will die and form the stalk. Stalk
formation is considered to be altruistic, because stalk cells die
to benefit the spores by lifting them above the ground, which
may increase their chances of dispersal and protect them
from hazards in the soil while they sporulate [8–11].
Aggregation in D. discoideum can occur between amoebae
that are genetically different, and so evolutionary theory
predicts selection for cheaters—genotypes that gain the
benefit of the stalk while failing to contribute their fair share
to its production [12–15]. Indeed, studies of natural isolates
have shown that genetically distinct strains of D. discoideum
can form chimeras in the laboratory that can differ in their
allocation to the prespore versus prestalk regions of the slug
. Genetic screens to examine cheating behavior in the
laboratory strain have also revealed numerous genes that,
when disrupted, lead to that mutant’s overrepresentation in
the spores .
The demonstrated ubiquity and ease of social cheating in
D. discoideum pose a conundrum—what maintains the victims
in nature? One possibility is that cheaters have lower fitness
than cooperators when not in chimeras. If this is the case,
then the fitness advantage gained by cheaters might be
reduced or eliminated by mechanisms that lead to the
separation of cheaters and cooperators into distinct fruiting
bodies [14,17,18]. There are two explanations for how this
separation might occur. One possibility is that cheaters and
victims rarely interact, because population structure passively
leads to the formation of primarily clonal fruiting bodies.
Another possibility is that strains segregate from one another
before or during multicellular development, a form of kin
discrimination. Kin discrimination differs from kin recog-
nition in that the latter term refers to cognitive processes,
whereas kin discrimination describes observable behavioral
patterns [19–22]. Evidence for kin discrimination is provided
by a study in a different species (D. purpureum), which showed
that cells segregated from non-identical cells during multi-
cellular development, although no cheating was observed
Academic Editor: Nick H. Barton, University of Edinburgh, United Kingdom
Received June 23, 2008; Accepted October 10, 2008; Published November 25,
Copyright: ? 2008 Ostrowski et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: GFP, green fluorescent protein
* To whom correspondence should be addressed. E-mail: email@example.com
[ These authors contributed equally to this work.
PLoS Biology | www.plosbiology.orgNovember 2008 | Volume 6 | Issue 11 | e287 2376
P PL Lo oS S BIOLOGY
. In D. discoideum, however, evidence for discrimination is
indirect only: genetically distinct clones are found in close
proximity in the soil , but fruiting bodies are often
dominated by a single clone, at least on the rich substrate of
deer feces where the majority of wild fruiting bodies have
been found .
D. discoideum is a genetically tractable model system, and so
understanding whether it is capable of detecting and
restricting cooperation in accordance with genetic distance
is an important step toward identifying the genetic basis of
the underlying mechanisms. For example, studies of csaA
mutants of D. discoideum, which lack the cell–cell adhesion
molecule gp80, have shown that csaA–cells tend to be lost
from chimeric aggregates with wild-type cells on natural
substrates, suggesting that differences in cell adhesion among
strains could facilitate discrimination [25,26]. However,
segregation between wild isolates of D. discoideum has not
been reported, making the relevance of this finding unclear.
We examined several patterns of discrimination in D.
discoideum. First, we tested directly whether genetically differ-
ent isolates are capable of segregating from one another
during multicellular development. Second, we determined
the degree to which the genetic distance between strains
influences the extent of this exclusion. Finally, we examined
the phenotypic basis of segregation among different mixes in
light of different possible explanations for sorting based on
previous work [27,28].
To examine the relationship between the genetic similarity
of strains and the amount of segregation they exhibit during
the formation of fruiting bodies, we performed pairwise
mixes of a reference strain and a panel of natural isolates
(Table S1). To estimate the genetic distances between strains,
we genotyped them at 12 polymorphic microsatellite loci,
which were dispersed throughout the genome. We calculated
the standardized Euclidean distance between strains based on
their microsatellite allele sizes and used it as an estimate of
genetic divergence, and thus as a proxy for the probability
that strains share alleles (Table S2). Genetic distance is thus
similar to relatedness in that both measures are estimates of
identity by descent, although they differ formally, since the
latter is expressed relative to allele frequencies in a reference
population [29,30]. More important, because genetic distance
takes into account not just allelic identity but distance
between alleles, it provides greater resolution than related-
ness measures based on shared alleles for divergent strains
sampled from different geographic locations.
We first mixed the laboratory strain AX4-GFP (labeled by
transformation with the gene for green fluorescent protein)
with each of 14 natural isolates, the strain from which it was
derived (natural isolate NC4), and unlabeled AX4 (control).
For each mix, we combined labeled and unlabeled amoebae
in equal proportions, deposited the mixture on damp
nitrocellulose filters, and allowed them to aggregate and
form fruiting bodies. We sampled ten fruiting bodies from
each mix and determined the ratio of fluorescent to
nonfluorescent spores in each one. We used the average
variance in this proportion across fruiting bodies, based on a
minimum of three temporally independent replicates, as an
estimate of the degree of segregation for a given pair of
The mixing experiment could have several outcomes
(Figure 1). In the absence of any discrimination, all fruiting
bodies should show identical proportions of the two clones,
resulting in low variance in that measure and no differences
between mixes of isolates at different genetic distances
(Figure 1A). Under exclusive self–nonself discrimination,
individuals would be expected to cooperate and form fruiting
bodies with genetically identical cells but segregate from all
other strains, resulting in a strongly binary response (Figure
1B). Alternatively, if the degree of discrimination depends on
the genetic similarity between the strains, we expect to see a
graded relationship between genetic distance and the degree
of sorting (Figure 1C).
The results of the mixing experiments support the third
model (Figure 2). When AX4-GFP was mixed with either
unlabelled AX4 (control) or with the parental wild isolate
NC4 (rank genetic distance 1 and 2, respectively), the
proportion of GFP-positive spores was similar between
different fruiting bodies (Figure. 2A) and the variance was
low (Figure 2B), indicating low sorting. However, mixes of
AX4-GFP with isolates of increasing genetic distance resulted
in greater segregation, reflected in the higher variance, and
mixes of the most genetically distant strains resulted in
fruiting bodies of two classes, indicating that the strains
segregated from one another (Figure 2). We observed a highly
significant correlation between the genetic distance and the
variance (Pearson correlation coefficient: r ¼ 0.773, n ¼ 16,
two-tailed p ,0.0001), indicating that segregation increased
in proportion to the genetic distance between strains.
Because the genetic distances were non-normally distributed,
we also performed a nonparametric correlation, which was
also highly significant (Spearman rank correlation: q ¼ 0.631,
n¼16, two-tailed p¼0.009). Finally, despite limited resolution
to discriminate between the more distantly related strains,
analyses in which genetic distance was estimated based on the
number of shared alleles rather than allele size differences
produced similarly significant results (Spearman rank corre-
lation: q ¼ 0.798, n ¼ 16, p ¼ 0.0002).
PLoS Biology | www.plosbiology.orgNovember 2008 | Volume 6 | Issue 11 | e287 2377
Kin Discrimination in Social Amoebae
In social amoebae such as Dictyostelium discoideum, cells aggregate
to form a multicellular slug that migrates and then forms a fruiting
body, which contains live spores (which go on to make new
amoebae) and dead stalk cells. Unlike animals where all the cells
descend from one fertilized egg, social amoeba fruiting bodies can
contain cells with different genotypes. This potential for chimerism
creates a conceptual problem in that ‘‘cheater’’ cells could arise that
preferentially become reproductive spores and force the victims to
become stalk cells and die. One way that amoebae could avoid
being cheated is if they recognize and preferentially aggregate with
genetically similar cells while avoiding genetically distant cells—a
process called kin discrimination. We tested whether cells of D.
discoideum could discriminate in this way. We mixed cells from
genetically distinct strains and found that they segregate during
multicellular development. The degree of segregation increases in a
graded fashion with the genetic distance between strains. Our
results demonstrate the existence of kin discrimination in D.
discoideum, an ability that is likely to reduce the potential for
cheating and ensure that the death of the stalk cells provides a
fitness advantage to related individuals.
To test the generality of the result, we repeated our
experiments with a different combination of strains. We
chose two natural isolates (QS32 and QS33), which mixed
poorly with AX4-GFP but were closely related to one another
(identical at all microsatellite loci we examined), and a third
strain (QS38), which was equally dissimilar to both (Table S1).
If the degree of discrimination can be predicted on the basis
of genetic similarity, then the genetically similar strains QS32
and QS33 should mix well with one another and segregate
Figure 1. Hypothetical Patterns of Discrimination
(Left panel) Deviation of individual fruiting bodies from the mean of the
population. Each symbol (þ) represents an individual fruiting body, and
mixes are plotted in order of increasing genetic distance. (Right panel)
Variance among fruiting bodies plotted as a function of genetic distance.
Open circles represent the control mix between genetically identical
labeled and unlabeled cells. Full circles represent mixes between
genetically distinct cells. We consider three hypotheses:
(A) No discrimination. The left panel shows each fruiting body contains
similar proportions of the two clones. The right panel shows the resulting
variance among fruiting bodies is low and there is no difference between
self-mixes (open circle) and non-self mixes (full circles).
(B) Exclusive self–nonself discrimination. The left panel shows the labeled
strain mixes well with genetically identical cells but poorly with other
clones. The right panel shows there is a difference between the variance
of the self mix and the nonself mixes, but no difference among the
(C) Discrimination according to genetic similarity. The left panel shows
mixes of genetically identical cells produce well-mixed fruiting bodies,
but segregation into distinct fruiting bodies is observed as the genetic
distance between clones increases. The right panel shows increasing
variance is proportional to the genetic distance between strains.
Figure 2. Segregation Increases with Genetic Distance in Mixed Fruiting
Reference cells (AX4-GFP) were mixed in equal proportions with test cells
of various genetic distances, and the mixes were allowed to form fruiting
bodies. The numbers of GFP-positive and negative spores were
determined in ten individual fruiting bodies for each of three or four
(A) Combined data from replicate mixes showing the proportion of GFP-
positive spores in each fruiting body (þ), centered around the mean and
plotted as a function of the rank genetic distance from the reference
(B) The average variance in the proportion of GFP-positive spores for
each of 16 strains, plotted as above, based on three or four independent
mix experiments for each strain pair. The correlation between the
variance and the genetic distance was positive and statistically
significant (Spearman’s correlation q ¼ 0.631, n ¼ 16, p ¼ 0.009),
indicating greater segregation with increased genetic distance. Data for
strains QS33 and QS32 are plotted separately (rank genetic distances 11
and 12, respectively) but were assigned tied ranks for the purposes of
calculating the Spearman rank correlation coefficient.
PLoS Biology | www.plosbiology.org November 2008 | Volume 6 | Issue 11 | e287 2378
Kin Discrimination in Social Amoebae
from the genetically distant strain QS38. To test this
prediction, we labeled the strain QS32 with a vital fluorescent
dye and developed it in pairwise mixtures with the other two
strains and with unlabeled QS32 cells as a control. We
observed little segregation in the control mix and in the mix
of the genetically similar strains QS32 and QS33 (Figure 3). By
contrast, mixing QS32 with the genetically distant strain
QS38 resulted in fruiting bodies with more variable propor-
tions of labeled spores, indicating stronger segregation. These
results are consistent with the studies performed with the
labeled laboratory strain, suggesting that the property of
genetically related segregation is transitive  and robust to
Segregation could result from differential aggregation or
from post-aggregative segregation. To distinguish between
these possibilities, we transformed one of the natural isolates
with a GFP-expression vector (QS44-GFP) and mixed it with a
genetically dissimilar strain, labeled with a DsRed expression
vector (AX4-DsRed, Figure 4A). As a control, we also mixed
AX4-GFP cells with AX4-DsRed cells (Figure 4B). In both the
experiment and control mixes, the labeled and unlabeled
cells were well mixed when initially plated on agar to induce
development (Figure 4A and 4B, 0 h). As development
progressed, both mixes initiated aggregation and all the cells
moved toward the same aggregation centers regardless of
their genetic similarity (Figure 4A and 4B, 9 h). However,
clusters of differentially labeled cells became increasingly
evident in mixes of the genetically dissimilar strains (Figure
4A, 9 h) whereas the genetically identical cells remained
intermixed (Figure 4B, 9 h). Segregation of the genetically
dissimilar strains continued throughout the aggregation
Figure 3. The Property of Segregation Is Transitive and Robust to Strain
Labeled reference cells (QS32) were mixed at equal proportions with
unlabeled test cells: QS32 (genetically identical), QS33 (identical by 12
genetic markers but isolated from a different geographic location), and
QS38 (identical at one genetic marker). The mixed cells were allowed to
form fruiting bodies and the numbers of fluorescent and nonfluorescent
spores were determined in at least ten fruiting bodies. The proportion of
fluorescent spores in each fruiting body (þ) is plotted as a function of the
rank genetic distance from the reference strain QS32.
Figure 4. Sorting of Strains during Multicellular Development
Cells expressing either GFP or DsRed were mixed at equal proportions and allowed to develop on agar plates. Pictures were taken at the indicated
developmental time points and the merged image of the two fluorophores is shown.
(A) A mix of the genetically dissimilar strains AX4-DsRed and QS44-GFP shows increased segregation with time.
(B) A mix of the genetically identical strains AX4-DsRed and AX4-GFP shows no segregation.
PLoS Biology | www.plosbiology.orgNovember 2008 | Volume 6 | Issue 11 | e287 2379
Kin Discrimination in Social Amoebae
stage, at which point there was partial separation of labeled
and unlabeled cells into different aggregates, as well as
segregation within aggregates (Figure 4A, 13 h). Thus,
genetically different strains segregate, but they do so
imperfectly. The control mixes showed no segregation at
that stage or at any later time (Figure 4B, 13 h, and
unpublished data). We observed similar segregation in mixes
of AX4 with the genetically different isolates QS32 and QS38
(unpublished data). The post-aggregative nature of segrega-
tion suggests that the sorting does not result from differences
in developmental timing or the use of different chemo-
attractants, which are known to reduce interspecific chimer-
Our findings demonstrate that social amoebae discriminate
between genetically similar and dissimilar cells in a graded
manner, mixing more with the former and segregating from
the latter during multicellular development. The similarity of
the segregation patterns among a number of different isolates
suggests a common underlying mechanism and is consistent
with differences that could arise from differential cell
adhesion . For example, cell-cell adhesion is required
for cell streaming, and the spatial segregation of the prestalk
and prespore cells, which also occurs in the mound following
aggregation, is attributed to differences in the relative
adhesiveness of these different cell types [26,32–35].
We propose two evolutionary explanations for these
discrimination patterns, which are not mutually exclusive
[31,36,37]. One possibility is that genetic drift or adaptation
to different components of the environment drives genetic
divergence at the loci that cause discrimination, ultimately
resulting in a diminished ability to form chimeric fruiting
bodies, a process analogous to allopatric speciation .
Alternatively, divergence could result from selection to avoid
the costs of chimerism, including cheating. Previous studies
in D. discoideum have shown that cheaters are abundant in
nature [8,39] and that its genome contains numerous genes
that confer cheating behavior when mutated . Knowledge
of the distribution of genetic variation in nature may help to
determine how often foreign individuals encounter one
another and the importance of selection in driving the
discrimination patterns we show here . Nevertheless, our
findings provide a possible explanation for the high levels of
clonality observed in fruiting bodies in natural populations
, as well as the co-existence of cheaters and victims in
close proximity in nature .
Although we observed a strong correlation between genetic
distance and the degree of segregation, most fruiting bodies
retained some representation of both clones, indicating some
ability to coaggregate and form multicellular structures that
extends across nearly the entire species range. Moreover, the
demonstrated ability of these strains to segregate, but their
failure to do so completely, suggests that an important
component of elucidating the role of selection on discrim-
ination ability will involve quantifying not only the costs to
forming chimeras, such as cheating, but also the benefits,
including increased aggregate size [31,40]. Our results also
differ from most other examples of discriminatory behaviors
in microbes, such as toxin production in bacteria  and
vegetative incompatibility in fungi . In those cases,
discrimination behaviors show strong self–nonself recogni-
tion, and the discrimination phenotypes, which often involve
cell death, are strongly binary.
Interestingly, social incompatibilities also occur in the soil
bacterium Myxococcus xanthus, which also forms multicellular
fruiting structures in response to starvation. In that system,
strong antagonism is observed between geographically
distinct isolates, with mixes causing reductions in sporulation
or even population extinction . By contrast, we observe
little evidence of antagonism but increasing avoidance of
sociality with genetically dissimilar strains, a behavior that
should limit the fitness advantages afforded by cheating and
help to explain the maintenance of altruism in this species.
More generally, the differences between M. xanthus and D.
discoideum in their response to foreign individuals illustrate
that different microbes, despite broad similarities in their
social life history traits, may find different solutions to the
problem of ensuring cooperation [44,45].
Materials and Methods
Strains and culture conditions. In mixes with AX4-GFP, we grew all
competitor strains, including AX4, on SM-agar plates (per liter: 10 g
glucose, 10 g Bacto Peptone (Oxoid), 1 g yeast extract (Oxoid), 1g
MgSO4, 1.9 g KH2PO4, 0.6 g K2HPO4, 20 g agar) in association with
Klebsiella pneumoniae at room temperature. We grew the reference
strain AX4-GFP axenically in HL5 medium supplemented with 5 lg/
ml G418 with shaking at 22 8C to maintain GFP expression .
Mixing experiments. We harvested each strain during the mid-
exponential phase of growth, washed the cells twice with cold KK2
buffer (14.0 mM K2HPO4 and 3.4 mM K2HPO4, pH ¼ 6.4), and
resuspended them at a density of 6 3 107cells/ml in KK2 buffer. For
each mix, we combined the two strains in equal proportions and
deposited an aliquot of 1.5 3 107cells on a nitrocellulose filter at a
density of 3.53106cells/cm2. As a control, we also plated each strain
individually at the same cell density as the mixes. We placed the filters
in Petri dishes atop a single filter pad (Pall), which was soaked in 1.5
ml of PDF buffer (20.1 mM KCl, 5.3 mM MgCl2?6H2O, 9.2 mM
K2HPO4, 13.2 mM KH2PO4, 0.5 g/l streptomycin sulfate, pH ¼ 6.4),
placed them in a humid chamber, and incubated them at 22 8C in the
dark for development. Following fruiting body formation (;24 h), we
picked at least ten individual fruiting bodies randomly from each mix
filter. We resuspended the spores from each fruiting body in 10 ll
detergent to eliminate amoebae and counted the spores using phase
contrast and fluorescent microscopy to determine the proportion of
GFP-positive spores. We counted approximately 400 spores for each
Cell tracker staining. We grew each wild isolate to mid-exponential
phase in association with K. pneumoniae on SM-agar plates, washed the
cells twice in cold KK2 buffer, and resuspended them at a density of 1
3 107cells/ml. We stained the cells with CellTracker Green CMFDA
(Molecular Probes) according to the manufacturer’s recommended
protocol with the following modifications. We added the cell tracker
reagent at a concentration of 50 lM, incubated the cells for 30 min,
washed them twice with cold KK2 buffer, and incubated them for
another 30 min in KK2 buffer to allow the cells to efflux the excess
dye. Following staining, we resuspended the cells at a density of 6 3
107cells/ml, mixed them in equal proportions with unlabeled cells,
and deposited the mix on filters, as described above. Following
fruiting body formation, we harvested the spores from individual
fruiting bodies in detergent and analyzed the proportion of
fluorescent spores on a BD LSRII flow cytometer.
Microsatellite genotyping. We genotyped the strains at 12 micro-
satellite loci, which were dispersed throughout the genome. These
microsatellite loci were designed based on the AX4 sequence (our
focal strain) and described previously . To extract genomic DNA,
we collected spores from 5–10 fruiting bodies and incubated them in
a mixture of 150 ll of 5% Bio-Rad Chelex-10 and 10 ll of 20 mg/ml
proteinase K for 4 h at 56 8C, followed by 30 min at 98 8C. Each
microsatellite locus was amplified by PCR using fluorescently labeled
primers (Table S2). The resulting product was analyzed on an ABI
3100 sequencer, and the programs GeneScan 3.7 and GENOTYPER
were used to determine the fragment size. To estimate the genetic
distances, we calculated the standardized Euclidean distance between
PLoS Biology | www.plosbiology.orgNovember 2008 | Volume 6 | Issue 11 | e287 2380
Kin Discrimination in Social Amoebae
strains, using the PCR product size as a quantitative variable (Table
S1). Relatedness and genetic similarity based on multi-locus, multi-
variate clustering techniques incorporating Euclidean distances
between haplotypes have been described previously [48–51]. We used
a standardized Euclidean distance metric, which scales each locus by
its variance, such that they contribute equally . Clustering based
on allele size rather than number of shared alleles offers greater
resolution of genetic differences, particularly for more distantly
related individuals. It is analogous to a stepwise mutational model
(SMM). SMM have been shown to perform well [53–56], particularly
when microsatellite mutation rates are low and there is not a strong
directional bias in the allele length changes, both of which have been
shown for these loci (described in ). Analyses were repeated where
distances were calculated based on the presence of shared alleles,
where alleles were considered identical if the estimated size was
within 3 bp of the allele for AX4.
Strain construction. We inoculated spores of natural isolate QS44
from fruiting bodies into Petri dishes containing HL5 medium
supplemented with 10% fetal bovine serum and grew the resulting
cells in submerged culture until they fully covered the surface. We
transformed the cells with an expression vector containing the S65T-
GFP reporter driven by the act15 promoter. Transformation
conditions were modified from protocols described previously .
We harvested the cells after washing twice in ice-cold KK2 buffer and
resuspended them at 1 3 108cells/ml in ice-cold H-50 buffer. We
mixed 100 ll of cell suspension with 5–15 lg of plasmid in a 0.1-cm-
gap electroporation cuvette and electroporated the mixture at 0.95
KV and 25 mF three times at approximately 5-s intervals. The
transformants were grown in submerged culture with HL5 supple-
mented with 8 lg/ml G418, and selected clones were tested for GFP-
expression using fluorescent microscopy. We verified the genetic
background using microsatellite genotyping.
Statistical methods. Proportions were arcsine square root trans-
formed to ensure that the variance was statistically independent of
the mean, and thus to account for differences among mixes in the
overall proportion of fluorescent spores . The transformation
does not affect the statistical significance of the results. The genetic
distances were non-normally distributed, so we also performed a
nonparametric (Spearman’s rank) correlation. All pairwise mixes
against the reference strain AX4-GFP were performed a minimum of
three times and used to calculate an average variance. Three of the
mixes (NC4, QS43, and QS45) were replicated four times. In such
cases, we computed the average variance across all four replicates,
although the inclusion of the fourth replicate did not affect the
statistical significance of our results.
Table S1. Dictyostelium Strains Used in This Study
Found at doi:10.1371/journal.pbio.0060287.st001 (82 KB DOC).
Table S2. PCR Primers for Amplification of 12 Microsatellite Loci
Found at doi:10.1371/journal.pbio.0060287.st002 (58 KB DOC).
We thank J. Landolt for providing several of the strains and T.
Cooper, K. Foster, O. Gilbert, and several anonymous reviewers for
helpful discussions, advice, and comments on earlier versions of this
Author contributions. EAO and MK conducted the experimental
work. EAO, MK, GS, DCQ, and JES conceived of the study, discussed
the results, and wrote the manuscript.
Funding. This material is based on work supported by the National
Science Foundation (NSF) Program under grant EF-0626963. EAO
was supported by a postdoctoral fellowship from the Keck Center for
Interdisciplinary Bioscience Training of the Gulf Coast Consortia
(NLM grant number 5T15LM07093).
Competing interests. The authors have declared that no competing
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Note Added in Proof
Similar findings have been reported in Dictyostelium purpureum by Mehdia-
badi et al. .
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