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Darwin was right: where now for experimental
evolution?
Paul B .Rainey1,2,3, Philippe Remigi3, Andrew D Farr4 and
Peter A Lind5
Over the last two decades interest in direct realisation of
evolutionary process and the possibilities presented by real
time evolution experiments with microbes have escalated.
Long-term selection experiments with bacteria have made
increasingly transparent the process of evolution by natural
selection. In this short article we consider what next for the field
and do so by highlighting two areas of interest: the genotype-
to-phenotype map and the constraints it imposes on evolution,
and studies on major evolutionary transitions and in particular
the importance of selection working over more than one
timescale. The latter we discuss in light of new technologies
that allow imposition of Darwinian properties on populations
and communities and how this allows exploration of new
avenues of research. We conclude by commenting on
microbial communities and the operation of evolutionary
processes that are likely intrinsic — and specific — to
communities.
Addresses
1
Department of Microbial Population Biology, Max Planck Institute for
Evolutionary Biology, Plön 24306, Germany
2
Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de
Paris (ESPCI ParisTech), CNRS UMR 8231, PSL Research University,
75231 Paris Cedex 05, France
3
New Zealand Institute for Advanced Study, Massey University,
Auckland 0745, New Zealand
4
Laboratory of Genetics, Wageningen University and Research,
Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands
5
Department of Molecular Biology, Umeå University, 901 87 Umeå,
Sweden
Corresponding author: .Rainey, Paul B (rainey@evolbio.mpg.de)
Current Opinion in Genetics & Development 2017, 47:102–109
This review comes from a themed issue on Evolutionary genetics
Edited by Eric S Haag and David L Stern
http://dx.doi.org/10.1016/j.gde.2017.09.003
0959-437X/ã 2017 Elsevier Ltd. All rights reserved.
Introduction
Of Darwin’s major theoretical contributions, his reasoning
that the mechanism of evolution is natural selection was a
most spectacular philosophical leap [1]. In a single step this
Current Opinion in Genetics & Development 2017, 47:102–109
idea removed the need for teleological reasoning concern-
ing a pre-determined end point. It also did away with need
to invoke a divine entity. Despite the persuasiveness of the
arguments and the abundance of evidence from historical
and comparative analyses, direct proof that Darwin was
right has come most forcefully from studies in which the
evolution of microbes has been studied in real time.
Of the many studies that have contributed direct insight
into the mechanisms of evolution [2,3,4] there is none
that exceeds the simplicity, elegance and endurance of
Richard Lenski’s long-term selection experiment with
Escherichia coli. The experiment, begun almost thirty
years ago with a single genetic clone that founded 12 rep-
licate populations [5], has involved growth and daily
transfer of 1% of stationary phase bacteria to fresh
medium. At the time of writing, the experiment has
involved more than 10 000 transfers, with the bacteria
experiencing some 70 000 generations of evolution in a
single glucose limited broth medium. The experiment
continues to provide understanding of the moment-by-
moment workings of evolution but its greatest achieve-
ment is the unequivocal demonstration that Darwin was
right [6]. Variation, which arises solely by mutation and
whose effects are heritable, provides material for natural
selection. Natural selection eliminates deleterious muta-
tions, but also causes the periodic sweep of mutations that
improve competitive ability relative to co-occurring
types. The outcome of the process is, as Darwin foresaw,
adaptation: adaptation by natural selection [7].
In recent time (but not forgetting the past [4,8–12]) there
has been an explosion of studies in experimental evolu-
tion that have shown, and continue to show, through real
time evolution experiments with microbes (but also flies,
worms and even mice), the process of selection and its
outcome, the importance of genetic drift, historical con-
tingency, mutational, phenotypic and genetic details,
the evolutionary responses of populations to fluctuating
selection, and much more. The field has been reviewed
extensively [13–18] and there is little in this short article
that we can usefully add. Instead, we glimpse into a
crystal ball and consider new opportunities for the field.
We highlight two areas that we find particularly intriguing
and which have become tractable given conceptual and
technical advances. The first concerns the use of microbes
to understand the connection between genotype and
phenotype, both as a goal in and of itself, but also as a
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 Darwin was right: where now for experimental evolution? .Rainey et al. 103
useful complement to studies in cell biology and central
to the move to make evolution a more predictable sci-
ence. The second area concerns timescales and transi-
tions — the use of microbes to explore evolutionary tran-
sitions in Darwinian individuality and, given new
technologies for manipulating populations, the possibility
of imposing Darwinian properties on collectives of
microbes as a means of studying the evolution of higher
levels of biological organisation. We conclude with brief
mention of microbial communities and the impact of
community-level processes on evolutionary change.
The genotype-to-phenotype map
Understanding the network of regulatory and functional
connectivites that determine the mapping between geno-
type and phenotype (genetic architecture) is the goal of
many developmental biologists. A long-standing interest
for evolutionary biologists is how the network of molecu-
lar interactions influences evolutionary change. Some
have argued that it is likely to be so pervasive as to
warrant consideration as an evolutionary process in its
own right, even leading to calls for an extended evolu-
tionary synthesis [19,20]. Central to the thesis is a body
of theory that predicts that the network of interactions —
the totality of development — constrains and channels
evolution by restricting the pathways evolution takes and,
by imposing limits to phenotype space, delineates the
rubrics by which it works [19,21–24].
Although an appealing concept, the idea that genetic
evolution might conform to certain rules has a troubled
history, largely through too frequent invocation of con-
straints to explain negative data, and through lack of
experimental evidence [25–28]. Nonetheless, insights
from system-level approaches has made concrete the
notion of the genotype-to-phenotype map (GPM) [29]
which has in turn prompted new theory, much of it
focused on properties of the map such as pleiotropy,
modularity, robustness and evolvability [30]. A central
issue is to understand how genetic architecture affects the
translation of mutation into phenotypic variation. A sense
that this is a real issue, the solving of which might even
contribute toward the establishment of predictive rules
for forecasting evolution, comes from numerous observa-
tions of parallel evolution and in particular to instances of
parallel molecular evolution (the evolution of similar or
identical features in two or more lineages) [31–37].
A notable example comes from the study of flowering
time variation in wild populations of Arabidopsis thaliana
where null mutations in a single gene ( frigida) account for
early flowering in 20 independent populations [38,39]. A
similar example comes from Drosophila where, of the
numerous genes that underpin the pattern of epidermal
projections known as trichomes, mutations affecting tri-
chome patterning occur solely within the ‘input–output’
gene shavenbaby [40]. The notion that evolution follows
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the same pathway — that it might proceed via pathways
of least resistance [23] — supposes that evolution can
take multiple pathways, but these are rarely realised.
Testing such a hypothesis posses numerous challenges,
but challenges that are tractable with experimental micro-
bial populations. Lind et al. [41] used populations of
Pseudomonas fluorescens that repeatedly generate adaptive
‘wrinkly spreader’ (WS) types using a limited number of
mutational pathways [42,43]. Identification of causal
pathways allowed construction of mutants devoid of such
pathways and opportunities to replay evolution to see
whether, in the absence of commonly trod pathways,
rarely taken mutational routes were realised. Surprisingly,
WS types whose fitness was not significantly different to
those arising from commonly trod pathways were identi-
fied [41]. Genetic analysis revealed a multiplicity of
additional routes (and targets [44]) and suggested a set of
hierarchical rules consistent with the notion of ‘target
size’ — a function of genetic target size and the likeli-
hood that change generates an adaptive phenotype. Loss
of function mutations are highly likely to generate adap-
tive phenotypes when they occur in negative regulators
[45]. However, mutations are also expected in genes that
afford a smaller target size, but at a much-reduced fre-
quency. Such mutations include promoter mutations,
gene fusions — including those leading to the evolution
of new genes [46] — and activating mutations. Although
the details are fascinating (e.g. [46]), the importance lies
in the provision of unequivocal evidence that the GPM
significantly affects evolutionary trajectory. It is even
conceivable within the bounds of population size and
mutation rate, that certain high fitness phenotypes are
never realised because of genetic bias [41,47].
Recent work has taken the importance of bias in muta-
tional process a step further by showing that E. coli
populations propagated in chemostat culture under dif-
ferent limiting conditions of carbon, iron, nitrogen, phos-
phate and oxygen affect the rate and spectra of mutations
[48]. An independent experiment examining mutation
under aerobic versus anaerobic conditions also showed
differences in the spectra of mutations under the two
conditions [49]. Thus, not only does genetic architecture
bias the spectrum of mutations seen by selection — and
thus the capacity to respond to selective challenges —
but the environment also contributes in surprising and
significant ways to the availability of particular kinds of
mutations, and the total number of mutations [50].
Another underexplored question concerns how heteroge-
neity in mutation rate within a gene or genome can lead to
mutation-biased adaptation [51]. If some genes experi-
ence mutation at substantially higher rates, due to intrin-
sic properties or interactions with DNA replication and
transcription machineries [52], this will further bias the
range of adaptive routes taken and contribute to parallel
evolution.
Current Opinion in Genetics & Development 2017, 47:102–109
104 Evolutionary genetics
The idea of using mutants severely compromised in key
components of cell biology as the starting point for
selection experiments designed to determine how evo-
lution compensates for defects has a long history
[4,12,53], but there exists further opportunity to shed
new light on even well studied processes — as well as
elucidating the rules evolution follows. Recent work on
BEM1 in yeast, a central determinant of cell polarity,
showed how analysing mutants that compensated for
BEM1 defects could generate new understanding of
seemingly well-studied aspects of cell biology [54].
Blank et al. [55] identified mutations compensating for
deleterious metabolic reactions in an extensive set of
E. coli mutants and showed that compensation occurred
in either structural or regulatory genes depending on
cellular function: structural mutations were likely when
compensation occurred in genetic pathways unrelated to
a specific defect, whereas regulatory mutations were
more likely when novel function arose from changes in
related gene sets.
Studies such as these give hope to the possibility of
improved evolutionary forecasting. Significant recent
advances have come from application of coarse-grained
top-down approaches to phenotypically diverse asexual
populations subject to strong selection [56,57], but molec-
ular details obtained from bottom-up studies are likely to
prove useful [58]. The challenge is to combine top-
down approaches with bottom up mechanistic studies in
order to define a minimal information set for the con-
struction of robust predictive models of evolutionary
change.
Transitions and timescales
Thinking about major evolutionary transitions in individ-
uality, such as those responsible for evolution of the
eukaryotic cell, for multicellularity and for eusociality
in certain insect species [59] has, until recently, largely
fallen within the domain of theorists and philosophers
[60,61]. Over the last few years there have been bold
attempts to explore early phases in these transitions
through real time evolution experiments, and in particular
the transition from single cells to multicellular life [62–
64]. Such thinking goes well beyond the evolution of
cooperation with focus on kin selection [65] and trait
group models [66] and to the heart of the evolution of
Darwinian individuality.
A central issue for each transition concerns the emergence
of Darwinian properties of variation, reproduction and
heredity without which the process of evolution by natu-
ral selection cannot occur [67]. With exception of the
transition from non-life to life, these properties manifest
at the lower level, but their existence at, for example, the
level of the individual cell, does not mean that these same
properties automatically appear at the level of collectives
of cells [68–71]. Consider a nascent multicellular
Current Opinion in Genetics & Development 2017, 47:102–109
collective that arises from a mutation that causes cell
adhesion. The collective that results grows by virtue of
the reproductive capacity of individual cells, but in the
absence of a means of collective level reproduction, the
collective — like soma — is an evolutionary dead end.
This leads to a very particular dilemma, namely, the need
to explain the evolution of Darwinian properties, in non-
Darwinian entities (e.g. the first collective), by non-Dar-
winian means (it is not possible to invoke the thing one
wants to explain (the evolution or reproduction) as the
cause of its own evolution).
One potential solution comes from recognising the role
that the environment can play in exogenously imposing
Darwinian properties on otherwise ‘unwitting’ particles.
The idea, in general form, we refer to as ‘ecological
scaffolding’ and will be more fully elaborated elsewhere,
but a specific example has been put forward based on
experimental observations of Pseudomonas populations
[68,72,73] and subsequently realised in a lengthy, elabo-
rate, and on-going multilevel selection experiment on the
evolution of multicellularity [64].
The essence of the idea requires patchily distributed
resources and a means of migration between patches.
For Pseudomonas growing in unshaken liquid, the primary
limiting resource is oxygen but this can be obtained at
the air–liquid interface. This requires types that can
colonise this environment (it is largely inaccessible to
the ancestral type), which in turn requires mutations
that cause overproduction of adhesive polymers leading
to the formation of WS mats. However mats are not
buoyant and cannot remain at the air–liquid interface
without a physical structure to which attachment is
possible.
Consider a pond with randomly placed reeds, each of
which constitutes a scaffold around which a mat can form.
Mats eventually collapse and go extinct due to their
increasing mass, but provided a mat can re-establish at
the original reed, or around one or more new reeds, then a
process akin to collective-level reproduction occurs. With
this comes the possibility of a Darwinian process at the
level of mat collectives. Importantly when this occurs,
selection works with potency over a timescale that is
longer than the doubling time of cells. Just how the
reproductive event occurs is not trivial [74], but possi-
bilities range from simple fragmentation (although this
is unlikely to be successful over the long term), to the
possibility that cheating types arising within mats act as
dispersing agents — rather like a germ line — with capac-
ity to regenerate the mat-forming type (Figure 1) [68].
Under a scenario like this, ecology is everything: the
structure of the environment permits realisation of
Darwinian properties at the collective level even in the
absence of these properties being endogenously
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 Darwin was right: where now for experimental evolution? .Rainey et al. 105

Figure 1

Current Opinion in Genetics & Development

Pond scum acquires Darwinian properties via ecological scaffolding. The illustration shows six reeds in a pond. Surrounding each reed is a set of
different microbial mat forming types. Reeds are sufficiently widely spaced as to prevent confluent growth of mats, thus ensuring variation at the
level of mats. Consider that the yellow mat occupying the reed marked with the solid arrow collapses. Death of this mat provides opportunity for
birth of a new mat, provided there exists a means of dispersal (by biotic or abiotic means) between reeds. In this example, cells from the red mat
recolonise the vacant reed. The dispersal and recolonisation event is akin to mat-level reproduction and, because the cells founding the new mat
came from the old mat, the offspring mat resembles the parental mat (there is heredity). Mats begin to take part in the process of evolution by
natural selection by virtue of Darwinian properties that are exogenously determined. Additionally, selection sees two time scales: the doubling time
of individual cells and the doubling time of mats. Continued selection under such ecological conditions allows the possibility that Darwinian
properties become endogenised, that is, they come to be determined by the activity of the collectives themselves with no need for scaffolding. An
early stage might be the evolution of a developmentally determined life cycle.

determined. As Hammerschmidt et al. [64] showed, there
exists ample possibility for Darwinian properties to
become endogenous. A first step observed through exper-
imentation was evolution of a simple but effective genetic
switch that allowed reliable transition between soma-like
and germ-like phases of a two-stage life cycle that marks a
first step in the evolution of development.
Our aim here is not to dwell on the finer points of recent
explorations of the origins of multicellularity, but to draw
attention to new understanding, particularly with regard
to timescales and the possibilities afforded through eco-
logical scaffolding realisable on an unprecedented scale
through new technologies.
When Darwinian properties manifest over time scales
that exceed that of the doubling time of individual cells
a process of special evolutionary significance begins to
unfold [61]. Required are conditions that ensure that
particle-level variation is partitioned into discrete collec-
tive-level packages and that these packages manifest
heritable variance in fitness. Once achieved, then selec-
tion working over the longer timescale, defined by the
birth (and associated death) of collectives [75], stands to
trump selection working on the short timescale (for
example, the doubling time of particles). This causes
particle fitness to increasingly align with the fitness of
collectives. Because particle growth rate is no longer the
focus of selection, drift becomes an important factor
allowing individual particles (and thus collectives) to
explore a much greater range of phenotypic possibility.
For example, the process allows evolution of phenotypes
that are altogether new and that emerge from the action
of collective-level selection. The possibilities arising
from implementation of a second collective-level time-
scale extend beyond research in evolutionary transitions
and opens new ways of thinking about how experi-
mental evolution might be used to engineer communities
to generate new functions, chemistries and processes
with application in biotechnology, medicine and agri-
culture [76].
At the core of these possibilities are emulsion-based
technologies known as microfluidics and millifluidics
[77,78]. By dividing a community of cells into discrete
parcels by means of enclosure within a droplet sur-
rounded by oil, variation at a level above that of the
individual cell is achieved [79]. Droplets, harbouring up
to 106 to 107 cells, can be manipulated in a myriad of ways:
the activity of contents can be monitored by fluorescent-
based assay (in real time), contents replenished, sampled,
and substrates added; droplets can be diluted, sorted,
merged, and they can be split. The latter being akin to a
droplet-level reproduction event and immediately affords
the possibility of implementing a birth/death process
based on some community level readout of performance,

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106 Evolutionary genetics

Figure 2

(a)

(b)

(c)
Current Opinion in Genetics & Development

Ecological scaffolding in a droplet-based evolution machine ensures droplets are units of selection. Parallel horizontal lines are walls of Teflon
tubes containing an emulsion of oil and regularly spaced droplets harbouring bacterial cells. (a) At the start of operation all droplets are founded
by identical types. (b) A period of cellular growth occurs within droplets. During this stage mutations within individual cells arise that affect the
colour of each droplet. Droplets whose colour is not sufficiently bright are marked for extinction allowing the possibility that when the contents of
the droplets are diluted in order to establish a new round of selection, the brightest droplets are split into two offspring droplets. (c) Selection thus
works over two time scales — the doubling time of cells, and the doubling time of droplets. As in Figure 1, Darwinian properties are imposed
(scaffolded) on droplets causing droplets to function as units of selection in their own right.

over a longer time scale than that of the doubling time
of individual cells, with potentially dramatic effect
(Figure 2).
We anticipate the combination of technology and its uses
to explore evolutionary process over two or more time-
scales to provoke a wave of new theory and experimental
possibility. Such approaches have already been used to
investigate the importance of spatial structure and migra-
tion on the evolutionary dynamics of bacterial populations
[80]. But there exist — through ability to miniaturise and
parallelise — numerous ordinary uses beyond the impo-
sition of Darwinian properties on populations and collec-
tives, including for example, ability to re-run Lenski’s
experiment with thousands of replicate populations and
with automated serial transfer occurring the moment cells
reach late exponential phase thus significantly decreasing
the time required to pass through thousands of genera-
tions. Using standard serial transfer approaches, a train of
droplets can be constructed so that in a single experiment
the evolutionary response to multiple different environ-
ments can be studied. Additional possibilities exist for
analysis of evolution in small populations, for testing ideas
concerning the evolution of new genes, for systematic
analysis of the effects of drift, migration and environmen-
tal fluctuation and, if coupled to genomic and phenotypic
assays studies that connect genotype to phenotype can
move to a new high throughput era [14]. But emulsion
technologies are not the only possibility for manipulation
of experimental populations. Interesting opportunities
are set to emerge from electrowetting that allows the
same set of possibilities afforded by emulsion-based
technologies, but without the need to package droplets
within a matrix of oil [81].
Conclusion
The field of experimental evolution has a long history
[6], but has particularly flourished over the last two
decades. Much still remains possible and our focus on
the GPM and timescales necessarily leaves untouched
much else that stands as new. In concluding we particu-
larly emphasise opportunities for investigation of evolu-
tionary process within the context of microbial commu-
nities. Elegant work has begun [82,83], but given
technical advances in DNA sequencing coupled with
imaginative applications of techniques such as chromo-
somal conformational capture [84,85], which allows
interactions between DNA molecules to be determined,
it is possible to conceive of selection experiments involv-
ing communities of microbes, with manipulations that
fuel or retard the extent of lateral gene transfer, that can,
despite immense challenges, be investigated mechanis-
tically. Here there is much to learn about the community-
level impact on the genomic and phenotypic dynamic of
adaptive evolution at the level of individual strains.
Advances in this field will likely make understandable
changes in patterns of diversity recorded from myriad
community/microbe metagenomic studies.
The latter is particularly important given the overarching
goal of much experimental evolution, which is to simplify
in order to understand processes too complex to fathom in
real world situations. But experimental evolution should
equally contribute toward understanding of evolution in

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 Darwin was right: where now for experimental evolution? .Rainey et al. 107
more complex real world systems [86,87,88]. While there
are possibilities for selection experiments in the wild
using appropriately marked strains, our hope is that as
experimental studies of evolution move to more complex
and yet tractable systems, the gap between evolution in
controlled systems and evolution in the wild is bridged.
Conflicts of interest
PBR is a founder of MilliDrop Instruments.
Acknowledgements
The authors are grateful for support from the Marsden Fund Council from
New Zealand Government funding, administered by the Royal Society of
New Zealand.
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Current Opinion in Genetics & Development 2017, 47:102–109