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Evolutionary biology
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Experimental evolution of multicellularity
using microbial pseudo-organisms
David C. Queller and Joan E. Strassmann
Research
Cite this article: Queller DC, Strassmann JE.
2012 Experimental evolution of multicellularity
using microbial pseudo-organisms. Biol Lett 9:
20120636.
http://dx.doi.org/10.1098/rsbl.2012.0636
Received: 6 July 2012
Accepted: 3 September 2012
Department of Biology, Washington University in St. Louis, One Brookings Drive, Campus Box 1137,
St. Louis, MO 63130-4899, USA
In a major evolutionary transition to a new level of organization, internal conflicts must be controlled before the transition can truly be successful. One such
transition is that from single cells to multicellularity. Conflicts among cells in
multicellular organisms can be greatly reduced if they consist of genetically
identical clones. However, mutations to cheaters that experience one round
of within-individual selection could still be a problem, particularly for certain
life cycles. We propose an experimental evolution method to investigate this
issue, using micro-organisms to construct multicellular pseudo-organisms,
which can be evolved under different artificial life cycles. These experiments
can be used to test the importance of various life cycle features in maintaining
cooperation. They include structured reproduction, in which small propagule
size reduces within-individual genetic variation. They also include structured
growth, which increases local relatedness within individual bodies. Our
method provides a novel way to test how different life cycles favour
cooperation, even for life cycles that do not exist.
Subject Areas:
evolution, developmental biology
Keywords:
multicellularity, experimental evolution,
relatedness, cooperation, cell-lineage conflict,
structured growth
Author for correspondence:
David C. Queller
e-mail: [email protected]
One contribution to a Special Feature on
‘Experimental evolution’ organized by Paul
Sniegowski, Thomas Bataillon and Paul Joyce.
1. Introduction
Major evolutionary transitions are often the result of cooperation among lowerlevel units that became so great that together they could be viewed as a new,
higher-level organism [1–5]. However, for a transition to be successful, conflicts
among the lower-level units have to be resolved. In the fraternal major transitions—eusocial societies and multicellularity—kin selection plays a major
role, allowing some units to altruistically give up their reproduction for relatives
[3,6]. Eusocial insects show a rich history of kin-selected altruism, conflicts and
conflict resolution [7,8].
Leo Buss suggested that multicellular organisms have a similarly rich history
of conflict among cells [1]. Although multicellular organisms seem to be nearly
entirely conflict-free (as do the most advanced eusocial insects), selection
among variant cell lineages may have been important in two different ways.
First, successful multicellularity may have required the evolution of mechanisms
to reduce conflicts [1,4]. Second, some cell movements and induction events that
we see in the development of current organisms may be relics of competition to
become a reproductive cell, or to induce others to take up a somatic role [1].
These ideas, if true, would require a rethinking of the evolution of development as extensive as the rethinking of social insect evolution that kin selection
stimulated. However, they have been criticized by members of the social evolution community (who might have been expected to be sympathetic to this
extension of their field) because most multicellular organisms have a singlecell bottleneck in their life cycle [3,6,9,10]. Therefore, the resulting multicellular
individual is clonal. With a relatedness of 1, no cell is selected to compete with
others in its body, so there are no evolutionary conflicts of interest.
However, Buss’s ideas deserve testing for at least three reasons. First, not all
organisms have single-cell bottlenecks, or they may have them rarely [1]. They
may instead reproduce by multicellular propagules involving budding, fission
& 2012 The Author(s) Published by the Royal Society. All rights reserved.
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This paper is about a methodology that derives from a new
perspective on microbial experimental evolution experiments.
This section describes the general approach. Microbial evolution experiments typically involve iterated phases of
growth, for example on one plate, followed by dispersal, for
example to a new plate. We suggest that a useful shift in perspective can be achieved by viewing these stages as parallel
to the growth and dispersal stages in multicellular organisms.
Specifically, an experimental evolution line of micro-organisms
can be viewed as a lineage of growing and reproducing multicellular pseudo-organisms, allowing us to ask what kinds of
lineage structures best control conflicts. We will call these multicellular pseudo-organisms. We are not suggesting that they
are true organisms, which are entities with very high levels
of cooperation and very low levels of conflict [16]. But we
can use them to gain experimental leverage on how such
high-cooperation low-conflict organisms evolve.
To be concrete, suppose we seed a plate with a single
clone in the centre of the plate and allow it to grow out to
the edge of the plate. The colony is genetically like a small
flat circular clonal organism. If we then pick up a single cell
and transfer it to the centre of a new plate, we have caused
this pseudo-organism to reproduce. This artificial cycle can
be extended for as many generations as desired, monitoring
any evolved change in cooperation. This will work best
with a micro-organism that already shows some form of
multicellular cooperation, as many microbes do [17,18].
In this example, we might expect cooperation to be nicely
maintained, because this life cycle has two kinds of structure
yes
2
no (or little)
(a)
1
(b)
1
(c)
many
(d)
many
yes
structured
reproduction
(single-cell
bottleneck)
no
Figure 1. Growth and reproduction of four types of multicellular pseudoorganisms. Each pseudo-organism is initiated (black circles), grows (radial arrows,
shown only for a subset of cells in (b) and (d)), and is then reproduced by transfer
of selected cells (grey point or swath). Treatments differ in whether they have
structured growth increasing local cell relatedness within the pseudo-organism,
and structured reproduction that increases cell relatedness in offspring.
that favour it, which we call structured reproduction and structured growth. Structured reproduction is when new
individuals start out with lower genetic variation than their
parents because of small propagules or any other process
that homogenizes propagules (such as structured growth).
Single-cell bottlenecks are the most extreme form. Structured
growth occurs when cells do not move completely freely
throughout the organism or pseudo-organism. Its main effect
is clustering of similar clones, or locally high relatedness.
Under structured growth, a selfish mutant is less likely to be
successful because it cannot cheat randomly; it is more likely
to cheat others of its own type. Figure 1 shows four experimental designs of this type, varying in whether they have
structured reproduction or structured growth.
Similarly, we can increase the size of the plate to test the
prediction [19] that larger body sizes disfavour cooperation
by allowing more mutations between transfers. We can test
other forms of structured reproduction by mimicking budding, fission or runners of various lengths and widths. We
can test whether sequestered germlines reduce selfish cell
lineages by drawing propagules from a pre-defined region
near the centre of the plate. We might even test more complex
body plans where different lineages grow to different degrees
[20] by shifting from a circular plate to arenas where growth
is channelled in desired directions and amounts.
3. Past experiments
There have been many experimental evolution experiments
on social microbes, but most lack one or more important
elements for studying the joint power of mutation and
within-lineage selection.
Some experiments mix together cooperator and defector
genotypes and assess their success over one or more generations [21]. These experiments usually focus on pre-existing
variants to yield information on selection, but not on
mutation. Conversely, experiments can start from a single
clone and allow mutations to accumulate, but in an environment where the social trait is not expressed, so there can be
no direct selection on it (though there may be selection on
Biol Lett 9: 20120636
2. General methodology
structured growth
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and runners, or by aggregation. Cell-lineage selection has been
shown to work in an alga that can reproduce by fragmentation
[11]. More conflict might be expected in these life cycles [3].
Second, single-cell bottlenecks may not eliminate all celllineage competition. When a new mutation occurs during
development, that allele is unrelated to other cells and could
gain by selfishly increasing its representation in the individual’s
progeny. However, this is a weak effect because each new
mutant gets only one episode of within-individual advantage.
After that first generation, its descendants are in all-mutant
individuals where no advantage can be gained. Buss’s failure
to consider this weakness made his theory questionable, but
models have subsequently shown that a combination of selfish
within-individual selection and a sufficiently high mutation
rate could still have major effects [4,12–15].
Finally, even if cell-lineage selection is not very potent in
current multicellular organisms, we can still ask whether
other kinds of life cycles may be absent because they do
not control such conflict [1]. Current diversity of life cycles
is not just about what evolution has favoured, but also
about what it has disfavoured.
In this paper, we suggest that these ideas can be tested using
experimental evolution of artificial pseudo-organisms consisting
of many single-celled microbes allowed to grow in ways that
simulate specific multicellular life cycles. This procedure allows
a ceteris paribus comparison of life cycles, including ones that
do not exist. We can test the prediction that cooperation is
enhanced by factors that limit cell-lineage competition, including
small propagule size, small body size, low cell movement, early
sequestration of the germline and low mutation rate [1,15].
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4. Discussion
3
We thank Stuart West and other referees for helpful comments.
This material is based upon work supported by the National Science
Foundation under grant nos DEB 1201671, DEB 1204352 and
DEB-1146375.
References
1.
2.
3.
4.
5.
Buss LW. 1987 The evolution of individuality.
Princeton, NJ: Princeton University Press.
Maynard Smith J, Szathmáry E. 1995 The major
transitions in evolution. Oxford, UK: W. H. Freeman.
Queller DC. 2000 Relatedness and the fraternal
major transitions. Phil. Trans. R. Soc. Lond. B 355,
1647–1655. (doi:10.1098/rstb.2000.0727)
Michod RE. 1996 Cooperation and conflict in the
evolution of individuality. II. Conflict mediation.
Proc. R. Soc. Lond. B 263, 813–822. (doi:10.1098/
rspb.1996.0121)
Grosberg RK, Strathmann RR. 2007 The evolution of
multicellularity: a minor major transition? Annu.
Rev. Ecol. Evol. Syst. 38, 621 –654. (doi:10.1146/
annurev.ecolsys.36.102403.114735)
6.
7.
8.
9.
Queller DC, Strassmann JE. 2010 Complex societies:
eusociality and multicellularity. In Evolutionary
behavioral ecology (eds D Westneat, C Fox), pp. 327–
340. Oxford, UK: Oxford University Press.
Strassmann JE, Queller DC. 2007 Insect societies as
divided organisms: the complexities of purpose
and cross-purpose. Proc. Natl Acad. Sci. USA 104,
8619 –8626. (doi:10.1073/pnas.0701285104)
Ratnieks FLW, Foster KR, Wenseleers T. 2006 Conflict
resolution in insect societies. Annu. Rev. Entomol.
51, 581– 608. (doi:10.1146/annurev.ento.51.
110104.151003)
Seger J. 1988 Review of L. W. Buss: the evolution
of individuality. Q. Rev. Biol. 63, 336 –337. (doi:10.
1086/415964)
10. Maynard Smith J. 1989 Evolutionary progress and
the levels of selection. In Evolutionary progress (ed.
MH Nitecki), pp. 219– 230. Chicago, IL: University of
Chicago Press.
11. Monro K, Poore AGB. 2009 The potential
for evolutionary responses to cell-lineage
selection on growth form and its plasticity in a red
seaweed. Am. Nat. 173, 151– 163. (doi:10.1086/
595758)
12. Michod RE, Roze D. 1999 Cooperation and conflict in
the evolution of multicellularity. Heredity 86, 1 –7.
(doi:10.1046/j.1365-2540.2001.00808.x)
13. Michod RE, Roze D. 1997 Transitions in individuality.
Proc. R. Soc. Lond. B 264, 853 –857. (doi:10.1098/
rspb.1997.0119)
Biol Lett 9: 20120636
Microbes are already widely used in studies of cooperation
among cells [16,17,21]. We suggest that experimental evolution
using social microbes offers a unique way to investigate questions of considerable importance to the evolution of
multicellularity. By viewing experimental growth and transfer
of microbial populations as parallels to growth and reproduction of multicellular organisms, we can test the effectiveness
of different life cycles in maintaining cooperation.
The key question for Buss’s hypotheses about conflict and
multicellularity [1], the power of mutation and within-lineage
selection, can be studied using experimental evolution. Typically, experimental evolution allows each lineage to continue,
with no competition between lineages. Thus there is no equivalent to selection among multicellular organisms, but the
experiments can address how strong this selection would
need to be to counter within-organism effects. In addition to
the experiments sketched out in figure 1, related experiments
could explore the effects of body size, alternative reproductive
modes such as fission or runners, formation of individuals by
aggregation, germline sequestration and cancer.
The chief limitation of this approach is that it uses cooperative traits of the test microbe, rather than the traits of current
multicellular organisms. This limitation is reduced if eukaryotic
microbes are used because at least they have similar kinds of
genes and gene families, but it is still true that the proximate
details will differ from true multicellular organisms.
However, there are some strong advantages of this
approach compared with working on real multicellular organisms. First, most multicellular organisms have life cycles that
are not easily manipulated. Comparisons of life cycle traits in
different, often distant, taxa are complicated by their many
other differences. Secondly, we have little way of measuring
the effects of cell-lineage conflicts in multicellular organisms,
but this crucial variable can be measured using cooperative
traits of microbes. Finally, as illustrated by the Dictyostelium
experiment, using artificial life cycles allows one to test life
cycles that do not exist, and perhaps discover why they do not.
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correlated traits) [22]. This limitation is shared by the one
mutation accumulation experiment we know of that focused
on a microbial social trait, because transfer between generations preceded expression of the social trait [23]. But
mutation accumulation experiments do mimic the singlecelled bottlenecks of multicellular life cycles. To convert
them to our ends, we need to allow social mutations to be
expressed. Instead of including bottlenecks as frequently as
possible in order to minimize selection (to measure
mutation), they can be spaced out to any desired degree to
measure the joint effects of mutation and selection as body
size increases.
Perhaps the most similar study to those we propose was a
recent study of the social amoeba Dictyostelium discoideum
[23]. These amoebas, when starved, aggregate and form fruiting bodies in which about 20 per cent of cells altruistically
form a somatic stalk that enhances the dispersal of the
remaining 80 per cent, which differentiate as reproductive
spores [24]. We put them through the odd artificial life
cycle in figure 1d, which is the opposite of the one discussed
earlier (figure 1a). This life cycle minimizes both structured
reproduction (by transferring cells from a wide swath
across the plate) and structured growth (by initiating
pseudo-organisms from many locations, so that lineages
mix as they grow). Not surprisingly, in the absence of these
features, cheater mutants thrived. This included an increase
in most lines of parasitic cheaters that could not fruit on
their own, so that lines declined in vigour [23].
The corresponding experiments with structured reproduction and/or structured growth (figure 1a–c) have not
been done, but a separate mutation accumulation experiment
showed that the mutation rate to these parasitic cheaters is
low [23]. This allowed a calculation that single-cell bottlenecks would generally be sufficient to keep these cheaters
from increasing significantly, not just for the plate-sized
pseudo-organisms, but also for pseudo-organisms as large
as a blue whale.
The role of mutation rate also can be studied. An experiment using Pseudomonas aeruginosa similarly used
unstructured growth and reproduction (liquid culture, transfer of many cells) but compared mutator and non-mutator
clones [25]. As expected, there were greater declines in cooperative siderophore production in the high-mutation lines.
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19.
20.
21.
xanthus. Nature 404, 598–601. (doi:10.1038/
35007066)
23. Kuzdzal-Fick JJ, Fox SA, Strassmann JE, Queller DC.
2011 High relatedness is necessary and sufficient
to maintain multicellularity in Dictyostelium.
Science 334, 1548– 1551. (doi:10.1126/science.
1213272)
24. Strassmann JE, Queller DC. 2011 Evolution of
cooperation and control of cheating in a
social microbe. Proc. Natl Acad. Sci. USA
108, 10 855 –10 862. (doi:10.1073/pnas.
1102451108)
25. Harrison F, Buckling A. 2005 Hypermutatbility
impedes cooperation in pathogenic bacteria.
Curr. Biol. 15, 1968 –1971. (doi:10.1016/j.cub.
2005.09.048)
4
Biol Lett 9: 20120636
22.
ecology and evolution (eds T Szekely, A Moore, J
Komdeur), pp. 331 –356. Cambridge, UK:
Cambridge University Press.
Roze D, Michod R. 2001 Mutation, multilevel
seleciton, and the evolution of propagule size
during the evolution of multicellularity. Am. Nat.
158, 638 –654. (doi:10.1086/323590)
Azevedo RBR et al. 2005 The simplicity of metazoan cell
lineages. Nature 433, 152–156. (doi:10.1038/
nature03178)
Griffin AS, West SA, Buckling A. 2004
Cooperation and competition in pathogenic
bacteria. Nature 430, 1024 –1027. (doi:10.1038/
nature02744)
Velicer GJ, Kroos L, Lenski RE. 2000 Developmental
cheating in the social bacterium Myxococcus
rsbl.royalsocietypublishing.org
14. Michod RE. 1997 Cooperation and conflict in the
evolution of individuality. I. Multi-level selection
of the organism. Am. Nat. 149, 607 –645. (doi:10.
1086/286012)
15. Michod RE. 1999 Darwinian dynamics: evolutionary
transitions in fitness and individuality. Princeton, NJ:
Princeton University Press.
16. Queller DC, Strassmann JE. 2009 Beyond society: the
evolution of organismality. Phil. Trans. R. Soc. B
364, 3143 –3155. (doi:10.1098/rstb.2009.0095)
17. West SA, Diggle SP, Buckling A, Gardner A, Griffins
AS. 2007 The social lives of microbes. Annu. Rev.
Ecol. Evol. Syst. 38, 53– 77. (doi:10.1146/annurev.
ecolsys.38.091206.095740)
18. Foster KF. 2011 Social behavior in
microorganisms. In Social behaviour: genes,