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Zoology 106 (2003): 283–290
© by Urban & Fischer Verlag
http://www.urbanfischer.de/journals/zoology
REVIEW
The power of evo-devo to explore evolutionary constraints:
experiments with butterfly eyespots**
Paul M. Brakefield*
Institute of Biology, Leiden University, Leiden, The Netherlands
Summary
Examples of adaptive radiation by Darwinian evolution will always enthral, and the theory of natural selection binds the whole of biology. But is selection all-powerful? Evolutionary developmental biology is beginning to provide the background to understanding how
the internal organisation of organisms can influence the tempo and direction of evolutionary change. Do the mechanisms that generate
the phenotype channel, bias or limit morphological evolution? The formation of eyespots on the wings of the butterfly Bicyclus anynana is a process which is becoming comparatively well understood from the genetical, developmental and ecological perspectives. We
have begun to combine this knowledge with artificial selection experiments to examine the rates at which morphological changes of a
particular eyespot pattern in different directions can be achieved. Our initial selection experiment performed over twenty-five generations has shown that in contrast to predictions based on shared genetical and developmental properties, two butterfly eyespots on the
same wing surface can be freely uncoupled from each other with respect to their pattern of relative sizes. This flexibility in development
and in response to selection may occur because our base population is part of a lineage with a long legacy of natural selection building
up genetic variation that enables independent behaviour for eyespot size, and thus it no longer reflects a tightly modular organisation of
eyespots as may well have existed close to their evolutionary origin in basal Lepidoptera. Comparisons can be made between such descriptions of the potential for short-term changes in morphology in B. anynana and observed patterns of divergence among all extant
species in this species-rich genus. Being able to map phenotypes onto genotypes via developmental processes provides a powerful basis
for exploring genetical and developmental constraints. This will eventually lead to examples of evolutionary constraints that represent
more than ‘just-so’ stories, and thus to a more balanced view of Darwinian evolution.
Key words: evolution, development, generative constraints, artificial selection, morphological stasis
Introduction
Evolutionary biologists from a variety of different backgrounds have displayed intense interest in the concept
of evolutionary constraints. Although their thoughts and
conjectures have led to a rich literature (e.g. Maynard
Smith et al., 1985; Schwenk, 1995; Schluter, 1996;
Schlichting and Pigliucci, 1998; Gould, 2002), there are
few areas of agreement and much controversy (see
Antonovics and van Tienderen, 1991; Stearns, 2002). At
least some of the problem of agreeing about constraints
stems from the difficulty in providing convincing tests
of proposed examples (Beldade and Brakefield, 2003a).
We believe that an approach of integrating evolutionary
genetics and developmental studies to analysing the origin of evolutionarily relevant phenotypes can yield insights about potential constraints in terms of the processes by which such phenotypes are generated (Brakefield et al., 2003). This endeavour forms part of the expanding field known as evolutionary developmental bi-
*
Corresponding author: Paul M. Brakefield, Institute of Biology, Leiden University, P.O. Box 9516, 2300 RA Leiden, The Netherlands; phone: ++31-71-527-4880; fax: ++31-71-527-4900; e-mail: [email protected]
**Presented at the 96th Annual Meeting of the Deutsche Zoologische Gesellschaft in Berlin, in association with the Deutsche
Gesellschaft für Parasitologie, Berlin, June 9–13, 2003
0944-2006/03/106/04-283 $ 15.00/0
P. M. Brakefield
ology or ‘evo-devo’. Development is of central importance to evolution because the processes of development translate genotypes into phenotypes, and thus
changes in development generate the variation which
natural selection can sort through.
Here I will describe the background to, and the results of
our recent experiment which has begun to build on ‘evodevo’ studies of eyespot formation on butterfly wings to
explore a proposed constraint involving the mechanisms
of generating the phenotype (Beldade et al., 2002a). Our
perspective is primarily one of evolutionary genetics, but
in general we believe that it will only be through the application of such tests that claims for constraints will become more than biological ‘just-so’ stories (Beldade and
Brakefield, 2003a). This point of view parallels in some
ways the highly influential initiative of Gould and
Lewontin (1979) that led to more rigorous and critical
thinking about the evolutionary mechanisms that could
explain what appear to be adaptive traits.
What are evolutionary constraints?
In theory, evolutionary constraints can be of many different types. However, historical, phylogenetic and selective
constraints are not the topic of this paper (see Schlichting
and Pigliucci, 1998). Rather, I am concerned here with
types of internal constraints that involve how phenotypes
are generated by genetical and developmental mechanisms. Nearly twenty years ago, a diverse group of evolutionary and developmental biologists representing the
‘state of the art’ proposed the following definition of a
developmental constraint: “A developmental constraint
is a bias on the production of variant phenotypes or a limitation on phenotypic variability caused by the structure,
character, composition, or dynamics of the developmental system” (Maynard Smith et al., 1985). Schlichting and
Pigliucci (1998) have pointed out that substituting the
phrase ‘genetic architecture’ for ‘developmental system’
provides a good definition for genetic constraints. Indeed
this lack of specificity illustrates one of the problems
about evolutionary constraints, namely that we just do
not have hard data for sufficient examples to be able to
apply appropriate adjectives with any real conviction.
Here, I use the phrase ‘generative constraints’ to refer
to the class of constraints which potentially can have
both genetical and developmental explanations. In this
context it is worth emphasizing that for complex traits
the genetic and developmental organisations may be
very different. Thus, concerning eyespot formation on
butterfly wings a gene network has been co-opted and
elaborated in the evolution of the Lepidoptera, and during wing growth the genes of this network are involved
in the establishment of organizers, in signal generation,
in responses to the signals and in pigment synthesis at
different stages of development.
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The time frame
An important factor in considering potential constraints
on evolution is the time horizon (Table 1). Developmental biologists and analysers of the fossil record tend
to think in terms of macroevolutionary change and divergence across widely disparate taxa; in other words,
in terms of what could or could not be ‘built’ given sufficient time and mutational input. This leads to a more
absolute view of constraints. At the other end of the
spectrum, evolutionary geneticists are concerned primarily with the processes of adaptive evolution within
populations that experience environmental change
(Brakefield, 2003). The pattern of evolution will then
depend mainly on the standing genetic variation, and on
whether populations adapt and survive, or alternatively
go extinct because of a lack of time. This is a more relative view of constraints, the questions being whether
some changes in phenotype are in some sense easier or
more likely to occur than others, and how such potential differences in tempo and direction influence the
patterns of evolutionary change. In our view it is important to consider the whole continuum from absolute
through to relative constraints (Beldade and Brakefield,
2003a). However, the approach to be considered here is
concerned with the analysis of relative constraints
which could in theory bias or channel evolutionary
change.
Why are constraints important
to evolutionary theory?
Text book accounts of evolution include wonderful descriptions of so-called patterns of adaptive radiation
that involve groups of related species showing divergence in key phenotypic traits. Explanations focus on
Table 1. Some descriptive terms illustrating contrasting perspectives characterising the two ends of the continuum for potential
generative constraints.
Absolute Constraint
Relative Constraint
long-term
short(er)-term
phylogenetic relationships
local adaptation
within lineages, or groups
of lineages
among populations;
across closely related species
historical view
extant species
in spite of mutational input
standing genetic variances;
responses to selection
Zoology 106 (2003) 4
Exploring evolutionary constraints
the role of natural selection in different ecological environments leading to divergent designs in terms of form
and function. Such accounts are indeed impressive and
it is perhaps all too easy to infer that natural selection is
all-powerful. However, different types of generative
constraint could also play a role in shaping, biasing and
channeling the observed patterns of phenotypic divergence, even in such dramatic examples as the Galapagos finches (Grant, 2003), cichlid fishes in African
lakes (Fryer and Iles, 1972), and the hummingbird-Heliconia system (Temeles and Kress, 2003). Perhaps
some potential phenotypes could have been even more
successful (i.e. climbing higher adaptive peaks) in
terms of matching functional design to a specific environment, but have not become established over time because of some absolute or relative constraint associated
with the process of generating them. In genetic terms,
lineages may tend to follow genetic lines of least resistance when responding to environmental change
(Schulter, 1996). Again, we believe that insights about
such hypotheses and the role of generative constraints
will ultimately only be gained through properly integrated studies using certain systems and potential examples that lend themselves to the whole range of field,
laboratory and molecular analyses.
In terms of rates of change and the dynamics of evolution, the opposite end of the spectrum in relation to patterns of continuing evolutionary divergence and adaptive radiation is morphological stasis (Jackson and
Cheetham, 1999). Evolutionary geneticists and biologists, especially those interested in natural selection and
the process of adaptation, tend to focus on patterns of
phenotypic divergence within groups of related species.
However, in many respects lineages that show little or
no change over long periods of time in the fossil record
are of equal fascination. There are now several case
studies where thorough and careful sampling, sometimes in combination with ecological surveys of extant
taxa, provide more rigorous descriptions of patterns of
stasis occurring in many (but not all) lineages. In the
case of marine bryozoans (reviewed by Jackson and
Cheetham, 1999), studies of extant species have revealed the life history consequences of changes in morphology, and also demonstrate the presence of heritable
variation for the morphological traits within species.
Why do such taxa show patterns of stasis? Generative
constraints could play a role, although it seems more
likely that explanations are to be found in the processes
of ecology and population structure that determine the
chance of novel phenotypes which have become established in local populations to spread throughout the
species range, and thus eventually to become detectable
in the fossil record (Eldredge et al., 2003).
Zoology 106 (2003) 4
Eyespot formation and ‘evo-devo’
Some years ago, we began to integrate studies on evolutionary genetics and development using a system of eyespot formation on the wings of butterflies (reviewed in
Brakefield and French 1999; Beldade and Brakefield
2002). The work was built on the classic experiments by
Fred Nijhout on eyespot development in Precis
(= Junonia) coenia (Nijhout 1980). Our research used an
African butterfly, Bicyclus anynana, which can be readily reared in the laboratory with a short generation time.
Butterfly wing colour patterns are attractive from a developmental perspective because, compared to many
more complex morphological features, they are structurally simple and amenable to developmental characterisation. Wing patterns are created by spatially ordered
arrangements of monochromatic scale cells in a single
epithelial cell layer. Studying development is about understanding the way in which these scale cells gain the
necessary information during growth to make the appropriate decisions that determine which one of a limited set
of colour pigments will be synthesized by each cell just
before adult eclosion from the pupa. An eyespot will
only appear as a functional morphological unit if the
many individual scale cells within each of its concentric
rings of colour all contain the same pigment having all
made the same decision in the specification of cell fate.
These decisions are made in the growing wing disc at
around the time of pupation, and sometimes many
months before pigment synthesis and adult eclosion.
Nijhout (1980) used surgical manipulations on the
growing wing in the early pupa to show that an eyespot
is formed around a central group of cells that acts as a
developmental organizer. He postulated a model for
eyespot formation based on this organizer, or eyespot
focus, establishing a cone-shaped information gradient
in the surrounding cell layer, perhaps through diffusion
of a morphogen (Nijhout 1980, 1991). Following focal
signalling in early pupae the surrounding cells interpret
the gradient and, depending on the concentration of the
morphogen, become fated to synthesize a particular
colour pigment. The more recent results remain broadly
consistent with this type of model although the morphogen itself remains elusive (see reviews: Beldade
and Brakefield, 2002; McMillan et al., 2002). Application of molecular tools, and in particular examination of
gene expression patterns, has identified a number of
genes involved in eyespot formation both at the early
stages of focal determination and later, at the end of
focal signalling (Brunetti et al., 2001). A recent association study has also shown that one of these genes, Distal-less, contributes to inter-individual variation in eyespot size in B. anynana (Beldade et al., 2002b).
Surveys of wing patterns across many species of butterflies inspired the concept of the Nymphalid groundplan
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P. M. Brakefield
(Nijhout, 1991). This is composed of different types of
pattern elements: chevrons and bands as well as eyespots. The eyespots are present as a repeated series,
with one element along the midline of each marginal
wing cell (a wing cell is a compartment bounded by
veins or trachea). Together, the eyespots can be viewed
as a single module that has some independence from
other modules comprised of band or chevron elements.
We have found genetic variation in a number of the features of the eyespots in B. anynana, namely in their
size, colour composition, shape and position (Brakefield, 1998). Each of these traits responds to artificial
selection targetted at a single eyespot; only eyespot
shape shows a low heritability with slow and comparatively subtle changes in phenotypes (Monterio et al.,
1997a).
Eyespots and exploring constraints
Why use butterfly eyespots to explore the potential of
generative constraints in the evolution of wing patterns? The answer lies in the notion of a nymphalid
groundplan in combination with several developmental
and genetical properties of groups of eyespots. The
groundplan shows a series of marginal eyespots each of
which is more or less of similar size and colour pattern.
How do differences evolve across repeated eyespots, or
in other words, how can the different eyespots within
specific wing cells gain individuality during the course
of evolution and thus diverge in phenotype? A very
useful framework for examining how such patterns
may evolve is that of modularity. The potential to produce a simple, undifferentiated pattern element or spot
along the midline of each of the marginal wing cells is
considered the evolutionary origin of butterfly eyespots
(see Brunetti et al., 2001). Further evolution involving
genetical and developmental elaboration of eyespot formation has led to the more complex compositions of individual eyespots with rings or patterns of different
colours as found in many extant species. An initial,
tightly coupled or integrated module may thus have
changed over evolutionary time with respect to the
complexity of the underlying genetical and developmental organisation, perhaps to the extent of interacting
levels of modularity or different sub-modules (Beldade
et al., 2002c; Beldade and Brakefield, 2003b; Monteiro
et al., 2003). Such questions concerning the different
individual eyespots are comparable to considering how
different teeth on a jaw bone, or segments along a body,
may become differentiated (Beldade et al., 2002c;
Beldade and Brakefield, 2003b). In the case of butterfly
eyespots, we know that all eyespots on a wing surface
share a common sequence regarding the expression of
developmental genes during their formation (Brake286
field et al., 1996; Brunetti et al., 2001). Furthermore,
nearly all single gene mutants established in laboratory
stocks influence all eyespots in a similar way (see
Fig. 1). Finally, in artificial selection experiments targetted at either the size or the colour composition of a
single eyespot, other eyespots, especially those on the
same wing surface, respond to the selection in a highly
concerted way (Monteiro et al., 1994, 1997b). These
observations indicate both a genetical and a developmental coupling of the eyespots; they indeed form serial repeated units within a single module.
These various properties of the eyespot pattern in
B. anynana led to the prediction that directions of evolutionary change that involved uncoupling of individual
eyespots might be more ‘difficult’ to achieve than others involving parallel changes, and thus, that the processes of pattern differentiation might have biased the
evolutionary trajectories in this speciose genus (Brakefield, 1998). The forewings of B. anynana have two
eyespots, a small anterior eyespot and a large posterior
one. An artificial selection experiment (Fig. 2) was designed to test the predictions about evolutionary change
in the pattern of eyespot changes, and more specifically
to explore the tempo of evolutionary change in different
directions of the potential morphological space regarding the pattern of relative eyespot size on the forewing.
The results of the experiment have been published in
detail elsewhere (Beldade et al., 2002a) so that here I
Fig. 1. The ventral wing pattern of a gynandromorph individual
of the butterfly Bicyclus anynana reared at 27 °C. The left side is
male, and the right side, female. The left forewing is about 20 mm
long. The female wings show the wild-type pattern while the
male wings have larger marginal eyespots due to expression of
the Bigeye allele. Note the pattern of fusion that occurs in some
adjacent eyespots on the left hindwing due to Bigeye.
Zoology 106 (2003) 4
Exploring evolutionary constraints
Fig. 2. Design of the artificial selection experiment using Bicyclus anynana to explore the rate of
response to selection in different directions of morphological space regarding the forewing pattern of
eyespot size (from Brakefield, 1998). The centrally
positioned forewing shows the wild-type pattern of
this species with a small anterior eyespot and a
large posterior eyespot. Selection attempted to
change this pattern of eyespot sizes along both an
axis of coupled change (i.e., both smaller or both
larger) and of uncoupled change (one smaller and
the other larger). Selection experiments targetted at
the larger, posterior eyespot had already demonstrated positive genetic correlations and thus we
predicted that movement along the ‘coupled’ axis
(solid lines) with concerted changes in both eyespots would be rapid and unconstrained (√ ). In
contrast, selection along the ‘uncoupled’ axis (dotted lines) was expected to be more limited (?).
Fig. 3. Representative female forewings from the populations of Bicyclus anynana selected in the direction of each corner of morphological space for the two eyespots: anterior (A, a) and posterior (P, p) (see Fig. 2). The butterflies of each generation are arranged in the
same pattern as in Fig. 2, without the wild-type pattern in the centre. Butterfly wings are shown from generations 11 and 25 (from Beldade et al., 2002a). Note the more extreme phenotypes of the later generation, and that highly divergent phenotypes were obtained along
both the leading axis of concerted change in eyespot size (both smaller or both larger), and also along the uncoupled directions in which
one eyespot became smaller and the other larger.
will only summarize them. Figure 3 shows representative butterflies in the middle of the experiment (generation 11) and at the end of the experiment (generation
25) placed in the same arrangement regarding the directions of selection as shown in Fig. 2. They illustrate
Zoology 106 (2003) 4
clearly that the standing genetic variation within our
laboratory stock, in combination with a flexibility in
the developmental mechanisms underlying specification of eyespot size, was sufficient to produce responses to selection that were effectively uncon287
P. M. Brakefield
strained. This extreme flexibility in the response to artificial selection occurred in spite of the prior predictions about the likelihood of more limited responses in
those directions involving the necessity of uncoupling
the shared genetical and developmental properties of
the individual eyespots. In terms of size, the anterior
and posterior forewing eyespots did not behave as
members of a tightly integrated single module. Final
phenotypes after twenty-five generations of selection
were widely divergent in all directions of selection
whether involving parallel (‘coupled’) or opposing
(‘uncoupled’) changes in the two eyespots. This was
especially impressive in one of the uncoupled directions (small posterior, large anterior eyespot), since
none of the eighty or so species of Bicyclus butterflies
show this phenotype (Fig. 4). Thus, although this eyespot pattern might have been suspected to represent a
‘forbidden morphology’, the experimental result
strongly suggests that its absence in nature is due to a
lack of appropriate natural selection in any lineage.
Perhaps this pattern has not proved advantageous in
any natural environment either in terms of potential
natural selection by visual predators (see Lyytinen et
al., 2003a, b) or of sexual selection involving mate
choice (Breuker and Brakefield, 2002).
Fig. 4. Distribution of species of Bicyclus in the morphological
space for the two forewing eyespots (see also Fig 2). Representative species names are given. Note the absence of any extant
species that shows a pattern of a large posterior eyespot in combination with no anterior eyespot. This pattern was, however, obtained in B. anynana following 25 generations of artificial selection in that direction (see Fig. 3) (Fig. redrawn by Steve Thurston
from Beldade et al., 2002a).
288
Discussion
This artificial selection experiment shows that although
eyespots share a common developmental mechanism,
the relative size of the dorsal forewing eyespots can
readily move through morphospace under appropriate
selection regimes. This result does not mean that there
are no generative constraints on evolutionary change in
the eyespot patterns of B. anynana and other butterflies.
Indeed, we have recently analysed the responses
recorded in our selection experiment with regard to the
full module of marginal eyespots on each wing, and on
both dorsal and ventral wing surfaces (Beldade et al.,
2002c; Beldade and Brakefield, 2003b). The response
on the ventral surface of the forewing was straightforward: the pattern of change in the anterior and posterior
eyespots in each direction of selection closely paralleled the response on the dorsal surface, although it was
less extreme. For the ventral hindwing, the pattern of
relative sizes within the full series of seven eyespots
(one within each marginal wing cell) was, however,
more illuminating (Fig. 5). The eyespots could be classified into two groups: the four more anterior eyespots
tended to respond in parallel with the anterior forewing
eyespot, while the three more posterior eyespots (especially numbers 6 and 7) changed in accordance with the
posterior forewing eyespot. Thus, there may be two domains or subcompartments of the wing leading to some
subdivision of the eyespot module into submodules
(see also Monteiro et al., 2003). Perhaps the eyespots
within each of these groups are more tightly coupled
than are the two forewing eyespots (in wing cells 2 and
5) or the eyespots in different submodules of the hindwing. We will test this hypothesis and study the consequences of the organisation of the hindwing eyespots in
a follow-up artificial selection experiment (see Fig. 5).
Our selection experiment detected no important developmental constraints on the evolution of the pattern of
relative sizes of the two forewing eyespots in B. anynana. This result demonstrated sufficient variation in
genes that differentially modify the developmental process that underlies eyespot formation to give the anterior and posterior eyespots individuality in size within
the overall wing pattern. We now believe that our selection experiment was actually based on a stock of a
species from a lineage that had already experienced a
long period of natural selection involving an accumulation of individuality in the specification of size of the
anterior and posterior eyespots. Thus the notion of a
groundplan with a module of tightly coupled, serial
eyespots is probably more relevant to the genetic and
developmental architecture nearer the time of the evolutionary origin of eyespots in basal Lepidoptera (Beldade et al., 2002c; Beldade and Brakefield, 2003b;
Monteiro et al., 2003).
Zoology 106 (2003) 4
Exploring evolutionary constraints
Fig. 5. Analysis of the modularity of eyespots in the butterfly Bicyclus anynana. The specimen to the left shows the ventral forewing
and hindwing patterns for the wild type (wet season phenotype) with small anterior (A) and large posterior (P) forewing eyespots, and a
series of seven hindwing eyespots, one in each wing cell (1–7) that show a characteristic pattern of relative sizes. Analysis of butterflies
from the selection experiment of Beldade et al. (2002a) as illustrated in Figs. 2 and 3 showed that hindwing eyespots1–4 responded in
concert with the direction of selection of the anterior eyespot of the forewing (A), while 5–7 responded in line with the posterior eyespot
(P) (Beldade et al., 2002c; Beldade and Brakefield, 2003). Thus they tended to shift in two blocks, and in opposing directions if selection for size on the two dorsal forewing eyespots was in opposite directions. This suggests a substructure of two domains or submodules
within the hindwing (note that these do not reflect the anterior-posterior compartments known from the Drosophila wing). The righthand side of the diagram illustrates a protocol to be used in a follow-up selection experiment on pairs of hindwing eyespots. It is designed to test the consequences of such a substructuring of modularity and to compare patterns of responses to selection regarding two
different features of the eyespots, namely their size and colour composition. Different predictions can be made depending on the developmental basis of variation of each feature and whether the pairs of targetted eyespots are within the same submodule or not (for further
information see the text).
A future experiment (see Fig. 5) will explore whether
the same degree of flexibility as we have found for the
forewing eyespots is also evident within each of the
apparent sub-compartments on the hindwing, and also
regarding a different feature of eyespots, their colour
composition (i.e. the relative dimensions of the different concentric colour rings). The latter trait appears to
show less variation among eyespots within a species
than does size; all eyespots on a wing tend to show a
similar colour composition, although the overall pattern can differ across species (see Condamin, 1973).
This might have an explanation in terms of natural selection but could also reflect the existence of stronger
constraints on colour composition than on size. The
latter hypothesis may be consistent with the results
from an earlier comparison (Monteiro et al., 1994,
1997b) of the responses of the posterior forewing eyespot to artificial selection regarding size or colour
composition. In terms of realized heritability and rate
of responses these two features of the eyespot behaved in a closely similar manner. However, the underlying development was found to be very different.
While divergence in eyespot size was largely accounted for by the strength of the focal signal, differences in colour were explained solely through the inZoology 106 (2003) 4
terpretation of the focal signal by surrounding cells,
and thus presumably by variation in threshold responses (see Beldade and Brakefield, 2002). The latter may tend to be a more or less uniform property
across the whole distal portion of the wing, while the
former may be more tightly coupled to establishment
of the individual focal signals in each marginal wing
cell. This sort of reasoning underlies the qualitative
predictions regarding the responses in a new selection
experiment targetted at both these features in the
hindwing eyespots (see Fig. 5).
We believe that our experiments to date already illustrate the potential power of an evolutionary genetics approach in combination with ‘evo-devo’ insights about
developmental processes to explore the likelihood of
generative constraints, and to understand the underlying mechanisms (see also Brakefield et al., 2003). A
new selection experiment should reveal more about just
how successful this approach is likely to be in helping
to unravel the role of constraints in shaping patterns of
evolution. A more balanced view of Darwinian evolution will result from a better understanding of how the
processes of development and physiology that generate
variation in the phenotype influence the patterns of
evolution.
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P. M. Brakefield
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