* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Exploring Evolutionary Constraints Is a Task for an Integrative
Survey
Document related concepts
Sexual selection wikipedia , lookup
Dawkins vs. Gould wikipedia , lookup
Evidence of common descent wikipedia , lookup
Evolving digital ecological networks wikipedia , lookup
Catholic Church and evolution wikipedia , lookup
Natural selection wikipedia , lookup
State switching wikipedia , lookup
Evolutionary landscape wikipedia , lookup
Hologenome theory of evolution wikipedia , lookup
Punctuated equilibrium wikipedia , lookup
Population genetics wikipedia , lookup
Transcript
vol. 168, supplement the american naturalist december 2006 Exploring Evolutionary Constraints Is a Task for an Integrative Evolutionary Biology P. M. Brakefield* and J. C. Roskam Institute of Biology, Leiden University, P.O. Box 9516, 2300 RA Leiden, The Netherlands abstract: Judging by the volume of writings about evolutionary constraints, they are an important topic in evolutionary biology. However, their involvement in shaping patterns of evolutionary change from morphological stasis to adaptive radiation remains contentious. This is at least in part because of the paucity of robust analyses of potential examples of constraints, whether of a more absolute or a relative nature. Here, we argue that what is needed to explore the type of constraints and bias on evolutionary change that may emerge from the way in which phenotypic variation is generated is an integrative approach applied to systems that can be tackled at different levels of biological organization. This is illustrated using research on the evolution of patterns in butterfly wing eyespots that has applied a combination of evolutionary genetics and evo-devo to an emerging model species with the beginnings of a comparative approach to describe patterns of variability among the extant taxa of two species-rich genera. Keywords: butterfly eyespots, developmental bias, natural selection, morphospace. An integrative approach that combines evolutionary genetics and evo-devo in a comparative framework can begin to address certain issues about morphological evolution that have always been of great interest but have, until recently, been beyond the scope of direct experimental analysis. We will illustrate this approach by using the background and results of one particular experiment with butterfly wing eyespots (Beldade et al. 2002b), together with some new insights from extending the work to include a more comparative framework. The main issue is the extent to which patterns of phenotypic change in adaptive evolution are shaped not only by natural selection but also by processes that underlie the generation of phenotypic variation and that could result in what can be called, col* Corresponding author; e-mail: [email protected]. Am. Nat. 2006. Vol. 168, pp. S4–S13. 䉷 2006 by The University of Chicago. 0003-0147/2006/1680S6-41372$15.00. All rights reserved. lectively, generative constraints. In other words, what are the roles of such generative constraints in influencing what actually happens in evolution and in contributing to the patterns of how species occupy morphological space? Bridging different levels of biological organization can also more effectively make the links from genes to evolutionarily relevant phenotypes to variation in fitness in natural populations. By placing this type of integrative analysis of adaptive evolution that is now becoming possible in many emerging model organisms within a comparative framework, we can then explore open issues, including the role of generative constraints. In addition, an ability to examine not only adaptive evolution but also the processes of speciation and phylogeography within such systems will provide rich data on the extent to which evolutionary change involving adaptive responses to novel environments is associated with the processes of speciation. Spectacular patterns of adaptive radiation among species of a lineage have always enthralled biologists, but even in such cases, it remains unclear how all-powerful natural selection is. Studies of evolutionary ecology and measurements of natural selection in examples such as the Galapagos finches (Grant and Grant 2002), anolis lizards (Losos et al. 2004; Harmon et al. 2005), sticklebacks (Schluter 2000), and the hummingbird-Heliconia system (Temeles and Kress 2003) readily steer biologists toward the perception that natural selection is highly dominant, at least when vacant ecological niches and relaxed competition are involved. However, an argument for taking a more openminded position can be made. This is illustrated most forcefully in the case of the species flocks of cichlid fishes in lakes of the African Rift Valley (Fryer and Iles 1972; Kocher et al. 1993; Salzburger and Meyer 2004; Albertson and Kocher 2006). Independent radiations of the cichlids in these lakes have each led to a series of highly divergent trophic morphs in which whole suites of morphological, life-history, and behavioral traits have changed. This is dramatic enough within each lake, but what is even more striking is the degree of convergence in the trophic morphs in different lakes; the same modes of coordinated traits are apparent. Is such con- Exploring Evolutionary Constraints vergence accountable purely on the basis of closely comparable environmental and ecological opportunities leading to intense selection for certain directions of change, or are there properties of the genetical and developmental systems that underlie the suites of traits that introduce bias and thus lend themselves to evolution in the observed directions? In the latter case, the details of how the different traits eventually distill as emergent entities to produce the observed variability around parallel series of trophic morphs represent some sort of compromise between how the environments are dictating natural selection and the processes that generate the phenotypic variation to be sieved by such selection. Perhaps, if the latter processes were completely flexible and in no way limiting in either a relative or an absolute manner, the phenotypes that evolved would not be precisely those present in the lakes today and would not be so similar in the different lakes. Such hypotheses need to be tested in specific cases before any robust assessment is possible of the extent of any compromise between extrinsic and intrinsic factors in shaping patterns of evolution, whether involving change or stasis (see Gould and Lewontin 1979; Maynard Smith et al. 1985; Antonovics and van Tienderen 1991; Schwenk 1995; Schlichting and Pigliucci 1998; Gould 2002; Stearns 2002; Brakefield 2003, 2006; Blows and Hoffmann 2005; Eldredge et al. 2005). Perhaps natural selection is allpowerful but further experimental work is necessary on systems that lend themselves to examining the processes that generate phenotypic variation in the context of the ecological arena of natural selection before such a conclusion can be reached. Description of patterns of divergence among taxa within some multidimensional representation of trait space, even in the light of measurements of fitness curves, is not sufficient to exclude possible sources of bias on evolution that are introduced through either genetic variance-covariances or developmental mechanisms. Experimental tests are also needed in combination with an evo-devo understanding of the processes that map phenotypes onto genotypes (Beldade and Brakefield 2003b; Brakefield et al. 2003; Fuller et al. 2005; and see Frankino et al. 2005, 2006). With respect to genetic variation, lineages may tend to evolve in patterns that follow genetic lines of least resistance when responding to environmental change or ecological opportunities (Schluter 1996). In developmental terms, the way in which the phenotype is made or in which variation in the phenotype maps onto the genetic variances may also bias or channel evolution in particular directions (Maynard Smith et al. 1985). Here, the emphasis is not on absolute constraints, on what could never be built even with unlimited input from mutation or recombination, but rather on relative constraints by which some directions of evolution are more likely to occur than others. If natural S5 selection in a direction away from a genetic line of least resistance or a pattern of developmental bias is not sufficiently intense, stable, or prolonged, then evolution in that direction may not occur. This is the type of issue we are exploring by using an integrative approach in tropical butterflies of the speciesrich genus Bicyclus (Condamin 1973). In particular, we use artificial selection experiments in our laboratory model species, Bicyclus anynana, to ask whether, for sets of traits with a common genetic basis or a shared developmental pathway, the properties of generating the phenotype can become reflected in patterns of diversity. We work with different classes of traits in B. anynana (Wijngaarden et al. 2002; Zijlstra et al. 2002, 2004; Frankino et al. 2005, 2006; Fischer et al. 2006). Studies have included those on the evolution of the scaling of wing size to overall body size or of forewing size to hindwing size. We were able to begin to distinguish between alternative explanations for the low phenotypic variability associated with such allometric relationships in terms of either developmental constraints or stabilizing natural selection (Frankino et al. 2005, 2006). Here, we will focus on the wing eyespot pattern. Artificial selection is applied to pairs of such interacting traits to examine how they can evolve in the short term in different directions through trait or morph space. The responses in our experiments must be largely accounted for by the properties of standing genetic variation in the founder stock (contrast with long-term experimental evolution in replicated populations of E. coli; Cooper et al. 2003; Elena and Lenski 2003). The phenotypes that result from artificial selection can also be used to examine their genetic basis as well as how developmental mechanisms have been modulated during evolution. In addition, we can now begin to explore the extent to which our understanding of the shortterm evolution of correlated traits in experimental populations of B. anynana can inform us about the patterns of phenotypic variation among taxa within this genus. The Eyespot Pattern of the Butterfly Bicyclus anynana The margins of the wings of many butterflies, including Bicyclus anynana, are patterned with series of small eyespots. Each eyespot in B. anynana consists of a white central pupil surrounded by two or three concentric rings with a brown, black, or gold color. Each eyespot ring comprises many hundreds of individual epithelial scale cells that during development all become fated to synthesize a particular color pigment. The functional effectiveness of each eyespot is dependent on the process by which the different developmental fates of these scale cells in different color rings are specified. The eyespot phenotype is important in a functional con- S6 The American Naturalist text for Bicyclus butterflies. We know this from field biology and capture-recapture experiments in Malawi for the ventral wing surfaces that are exposed to potential predators in butterflies that are at rest with their wings closed (Brakefield and Frankino 2006). The eyespots on the ventral wing surfaces show striking phenotypic plasticity in the form of seasonal polyphenism (Brakefield et al. 1996). The relative fitnesses of butterflies with different eyespot patterns depend on the seasonal environments in which they have to evade predators and reproduce. Marginal eyespots are thought to function in butterflies at rest by deflecting the attacks of vertebrate predators away from the vulnerable body toward the eyespot “targets”; the margins of the wings tear away easily, enabling some butterflies at least to escape even after being grabbed by a bird or lizard. This hypothesis has proved difficult to evaluate, but the most recent results provide some support for it, at least when the predators concerned are naive birds (Lyytinen et al. 2003, 2004). The function of the eyespots on the dorsal surfaces of the wings has been unclear because they are not exposed to potential predators in resting butterflies. However, recent work is beginning to implicate them in mate choice and sexual selection and perhaps in species recognition (Breuker and Brakefield 2002; Robertson and Monteiro 2005). Shared Genetics and Development of Eyespots The eyespots on the wings of many butterflies and moths are ideal for exploring the flexibility of morphological change in different directions within trait space for patterns or modules made up of serial repeats of the same basic element (Brakefield 2003). The evolution of mammalian dentition and of the subsets of teeth with divergent morphologies provide further examples of this type of modular pattern for which exciting advances are being made in understanding the genetic and developmental basis of divergent morphologies both within and among species and also, in this case, in the fossil record (SalazarCiudad and Jernvall 2002; Kangas et al. 2004). Many primitive species of Lepidoptera (as well as more derived species) have a series of small undifferentiated dark spots evenly spaced along their wing margins. Each spot is positioned along an internervule between pairs of the major proximal-distal wing veins that divide the wing into socalled wing cells. This probably reflects a phenotypic module comparable with the early novelty that saw the origin of the developmental capacity to make patterns of repeated spots in each wing cell. In the Nymphalidae, and probably in several other lineages of higher Lepidoptera, such a simple pattern has become much elaborated to yield series of repeated marginal eyespots, each with concentric rings of color. The eyespots can vary in both size and color pattern, and the processes that underlie the gain in eyespot individuality and pattern complexity are the focus of our research. Thus, an initially simple module of repeated spots has been elaborated in evolution to yield a spectacular diversity of patterns across taxa that is associated with an increasingly hierarchical structure for the whole module (Brunetti et al. 2001; Beldade et al. 2002c; Arbesman et al. 2003; Monteiro et al. 2003). This type of process can also be expressed in the context of an increasing evolvability of the whole module through time. How is this achieved? In addition, what are the consequences of such a process in terms of the occupation of morphological space and of generative constraints on future evolutionary change? The results of evo-devo work on Bicyclus anynana are consistent with this general scenario. It is clear that all eyespots in this species, as well as in other members of the Nymphalidae, are formed by the same developmental pathway that is part of wing development in late larvae and early pupae (Brunetti et al. 2001; Beldade and Brakefield 2002; Reed and Serfas 2004). Thus, transplantation experiments in early pupae have shown that both of the forewing eyespots of B. anynana are formed around groups of organizing cells called foci; grafting a focus to a novel position on the wing surface results in formation of an ectopic eyespot around the grafted cell tissue (French and Brakefield 1995). The foci are established in late larvae, and then in early pupae, each focus sets up an information gradient in the surrounding epithelial cells, presumably via one or more diffusible signaling morphogens. These cells then respond to the gradient and thus, depending on their location relative to the signal source, become fated to synthesize one of a series of different color pigments just before adult eclosion. Each of the seven eyespots on the ventral surface of the hindwing of B. anynana expresses the same developmental genes, including Notch, Distal-less, engrailed, and spalt, at the same stages in eyespot formation (Brakefield et al. 1996; Brunetti et al. 2001; Beldade et al. 2002a, 2005; Reed and Serfas 2004). We have also established a series of spontaneous eyespot mutants of B. anynana. The mutant alleles typically affect all the eyespots on a particular wing surface. Furthermore, artificial selection experiments that were targeted on a particular trait (e.g., size or color composition) of a single eyespot consistently produced highly correlated responses in the other eyespots, especially on the same wing. Taken together, these observations show that the different eyespots are based on a common developmental pathway and that many eyespot genes or alleles influence all the repeated eyespot elements. This led us to design experiments to explore the consequences of the shared genetical and developmental mechanisms and to examine the potential flexibility of different eyespots to evolve in different directions of morphological space (Brakefield 1998). Exploring Evolutionary Constraints S7 Figure 1: Occupancy of morphological space for the relative size of the two dorsal forewing eyespots. The four images of the wing are representative examples of the wing pattern after 25 generations of artificial selection in Bicyclus anynana (from Beldade et al. 2002a) placed in roughly the correct position in the depicted trait space; the star shows the average wild-type pattern for this species that formed the starting point for the selection in each of the four directions. Circles show the positions of the mean patterns for different species of the genera Bicyclus (filled circles) and Mycalesis (open circles) derived from measurements of specimens in museum collections. The dashed square encloses species for which both eyespots are very small or absent and frequently difficult to measure. For species of Bicyclus and Mycalesis, total sample sizes were 1,371 and 575, respectively. Artificial Selection Experiments The wild-type wing pattern of the dorsal forewing of Bicyclus anynana consists of a small anterior eyespot and a large posterior eyespot. The wing cells within which these eyespots lie also differ in size in the same direction as the eyespots. This pattern can be considered to be somewhere in the middle of trait space for this pair of eyespots. Our laboratory stock of B. anynana was established from more than 80 gravid females collected at a single locality in Malawi and was cultured to maintain high levels of standing genetic variation. An early selection experiment on the size of the posterior eyespot revealed a moderate realized heritability with rapid upward and downward responses to selection (Monteiro et al. 1994). After only five generations of selection, both eyespots were substantially larger in a high line and smaller in a low line, thus demonstrating a positive genetic correlation. The color composition of the eyespots changed very little, indicating low genetic correlations between the two eyespot traits of size and color (see also Monteiro et al. 1997a). Transplantation experiments on early pupae involving the organizing foci of both lines traced the phenotypic difference in eyespot size mainly to differences in the activity of the signaling cells of the eyespot foci. There was some evidence for differences at the level of the threshold responses of the epithelial cells to the focal signals, but foci donated by pupae of the high line consistently yielded larger ectopic eyespots in adults, whether grafted into pupal hosts of the high or the low line (Monteiro et al. 1994). A second experiment was then designed to explore whether a phenotype in which one eyespot was smaller and the other larger could be as readily produced by artificial selection as the phenotypes in which both eyespots were either small or large. Replicated lines were established from the same founder population and selected toward each of the four corners of trait space (see fig. 1), that is, along both directions of the coupled axis, following the shared genetics and development, and along both directions of the uncoupled axis, against the proposed genetic line of least resistance and plane of developmental bias. Selection took place over 25 generations, although this included a period of relaxed selection (Beldade et al. 2002b). As expected, selection in both directions along the coupled axis produced rapid responses, with butterflies after 25 generations of selection having either no eyespots at all or two very large eyespots, phenotypes completely dif- S8 The American Naturalist ferent from any present in the unselected population. However, the populations along the other uncoupled axis also responded in a dramatic way to selection, eventually producing phenotypes in which one eyespot was very large and the other absent or very small (see fig. 1). We concluded that this pattern in relative size of the two eyespots behaved in a highly flexible manner, with no evidence of any generative constraints (Beldade et al. 2002b). However, this does not necessarily mean that there is no developmental bias or effect of the genetic correlation between the two eyespots on patterns of diversity among different taxa. An initial examination of photographs in a monograph on the genus Bicyclus (Condamin 1973; Roskam and Brakefield 1999) suggested that although species occurred with a large anterior eyespot and no posterior eyespot, there were none showing the reversed pattern (Beldade et al. 2002b). We have now begun to investigate the occupancy of this morphological space more carefully by using measurements of specimens from museum collections not only for 69 species of Bicyclus from sub-Saharan Africa but also for 40 species of the closely related genus Mycalesis that extends throughout Asia into Australia. Representative dorsal wing surfaces of four species of each genus are shown in figure 2. Comparisons of Eyespot Patterns among Species The morphometric data for species of Bicyclus confirm the occurrence of a rather wide range of variation among species for the relative size of the two eyespots on the dorsal forewing. The pattern of occupation of morphological space within the genus can then be compared with the results of the artificial selection based on the standing genetic variation at a single locality for one of the species (fig. 1). A complete analysis of the data on the size of both dorsal and ventral eyespots using a molecular phylogeny for species of Bicyclus (Monteiro and Pierce 2001) will be published elsewhere (J. C. Roskam, K. van der Linde, and P. M. Brakefield, unpublished manuscript). Many species of Bicyclus, including Bicyclus anynana, clearly lie along the coupled axis that represents a positive genetic correlation among the two eyespots together with a common developmental pathway. There are, however, species that extend toward having a large anterior eyespot but a small Figure 2: Representative dorsal surfaces of the right forewing and hindwing of (A) four species of Bicyclus and (B) four species of Mycalesis. Note among species in each genus the variability in the size of the anterior and posterior eyespots as well as in the patterns of relative eyespot size. Species names in clockwise order from top left in each group are (A) taenias, danckelmani, smithi, and simulacris and (B) sirius, splendens, mineus, and oculus. Exploring Evolutionary Constraints posterior eyespot. The rest of the morphological space, the region covering the opposite pattern not only in relative size but also in the direction of both eyespots being very large, is not occupied by any of the surveyed (extant) species. Because artificial selection on a single laboratory population has yielded replicate lines that have readily crossed this morphological space (see fig. 1, superimposed wings), we must conclude that such patterns are absent in extant present-day taxa because of the way in which natural selection has operated in the past rather than because of any strong generative constraints. Thus, while there is clustering of species of Bicyclus around the axis of genetic and developmental coupling that appears to reflect a genetic line of least resistance, in Schluter’s (1996) terminology, the gross pattern of occupancy must be accounted for by environments found in nature in combination with the legacies of natural selection. This interpretation is given more weight by a comparison with the closely related genus Mycalesis (fig. 1). Here there is a (weaker) trend toward following a parallel genetic line of least resistance, but many species are then displaced toward the pattern with a large posterior eyespot and a small anterior eyespot (fig. 1, bottom right) rather than the opposite pattern, as in Bicyclus (fig. 1, top left). Again, considering all of this in the context of the results of artificial selection in B. anynana suggests that there is some factor in terms of the environment and selection that has influenced the different patterns of occupancy of morphological space shown by the two genera. Some recent experimental work suggests that these eyespots are involved in sexual selection and mate choice (Breuker and Brakefield 2002; Robertson and Monteiro 2005). During courtship in B. anynana, the wings of males are opened and closed very rapidly during a specific flickering phase. The precise positioning of the male relative to the female at this stage of courtship suggests that at least the anterior eyespot on the apical tip of the forewing is then visible to the female. Furthermore, preliminary data from mate competition experiments (see Joron and Brakefield 2003) using free-flying males from the selected lines of Beldade et al. (2002b) suggest that males with the larger anterior eyespots have a higher mating success than those in which this eyespot is small, while the posterior eyespot has no affect on fitness (J. de Visser and P. M. Brakefield, unpublished data). These observations need to be followed up in more detail, but they may indicate some fundamental difference in the structure of courtship between the two genera that accounts for the striking difference in occupancy of morphological space (fig. 1). Whatever the details of environment and selective history, the results to date strongly suggest that the explanation for the difference between the two genera is unlikely to have any substantial developmental component. There may be relative con- S9 straints, in the sense of a genetic line of least resistance, that are sufficient to account for some clustering of species along an axis of coupling for the two eyespots. However, the high degree of flexibility found in the experiments with 25 generations of artificial selection in B. anynana indicates that any such generative constraint is likely to have very weak consequences, especially if natural selection is prolonged and strong. The Potential for Generative Constraints for Other Eyespot Traits We have also examined the evolutionary and developmental genetics of an additional trait for the posterior forewing eyespot, namely, color composition. Artificial selection yielded either gold butterflies, in which this eyespot had a broad outer gold ring, or black butterflies with a narrow gold ring (Monteiro et al. 1997a, 1997b). The estimate for realized heritability of eyespot color was similar to that for size, and again the other dorsal eyespots showed highly coordinated responses. There is also little, if any, evidence for correlated responses for the two traits, size and color (see also Beldade et al. 2002c; Beldade and Brakefield 2003a). Although these observations might suggest comparable predictions for evolutionary responses to a given intensity of natural selection, caution is needed because the two traits have a different developmental basis. Thus, while the transplant experiments using pupae from the selected lines with divergent eyespot size showed that large eyespots result primarily from strong focal signals, comparable trials using the gold and black lines traced no effect to the focus but rather traced effects only to the threshold responses to the focal signal of the host cells that surround the graft and eventually produce the color rings (see Beldade and Brakefield 2002). These observations can be interpreted as revealing two divergent developmental aspects of these traits: (1) that eyespot color is more of a wing-level property (threshold responses of the whole epithelial cell layer), while size is dependent on the localized focal signal in the wing cell between each pair of wing veins, and (2) that because size is to some degree also influenced by the response thresholds, there are more developmental options for changing size than color. What are the evolutionary consequences of such differences, and how did such differences in evolvability arise? Figure 3 illustrates the ventral wing surfaces of four representative species of each genus, Bicyclus and Mycalesis. These wings tend to have more complete series of the marginal eyespots. In each genus there are clearly species characterized by eyespots with narrow gold rings and others characterized by broader gold rings. However, it appears that a mixture of eyespots that span a substantial proportion of this overall range in color composition is S10 The American Naturalist not found in any of the species, especially within the surface of one of the wings. Thus, this trait contrasts sharply with eyespot size, for which the pattern of relative size can differ dramatically across species (fig. 2). Although such patterns of variability in eyespot color among species remain to be properly quantified, they do suggest that it may in some way be more difficult to uncouple the color composition of two eyespots on the same wing (i.e., one gold and the other black) by artificial selection on the standing genetic variation of a single species. Such experiments are underway and appear to be yielding different results to eyespot size (C. E. Allen, unpublished data). Responses to Artificial Selection and Revealing Variation in Development It is worthwhile considering how, in general, the responses observed in artificial selection experiments inform us about the role of development in generating variation in the phenotype that is relevant to natural selection and the ecological arena. The answer is probably very little when no further analysis is available. It is rather only when such observations are combined with analyses in developmental terms of what has happened—or, indeed, of what has not happened—that relevant information will be forthcoming. The phenotypes at the end of selection, whether in singleor in double-trait experiments, can be used to explore the morphological changes in terms of the mechanisms of development; they may indeed also reveal more about the developmental process itself. Thus, follow-up studies can identify which of the potential modes of variability in development or options for change have yielded the observed changes in phenotype. Moreover, because artificial selection experiments usually target standing genetic variation derived from one or more natural populations, they are likely to provide more relevant information about the (short-term) potential for phenotypic change in nature than descriptions of variation based on mutagenic screens. Thus, combining information from artificial selection experiments about the developmental and genetical options for change in a complex morphology will inform us about evolvability with respect to responses to short-term selection in different directions of trait space. Furthermore, matching experimental studies using a model species in Figure 3: Representative ventral surfaces of the left wings of (A) four species of Bicyclus and (B) four species of Mycalesis. Species names in clockwise order from top left in each group are (A) simulacris, evadne, taenias, and xeneas and (B) anaxias, mucia, duponcheli, and terminus. Note the consistently narrow or broader outer gold rings of the marginal eyespots within species in contrast to the variability among species in each genus. Exploring Evolutionary Constraints S11 Figure 4: Diagram illustrating a scenario of the evolution of the eyespot pattern on the ventral hindwing in the genera Bicyclus and Mycalesis. Evolution of the key innovation of making wing spots probably occurred in basal Lepidoptera. This was followed by a period of elaboration leading to a modular pattern with serial repeats of similar eyespots having both larger size and color ring composition (below is a representative species showing a comparable pattern extending over both hindwing and forewing, Mycalesis horsfeldi). For eyespot size, species in different selective environments have diversified through a process of evolutionary tinkering. This has led to a high evolvability of the whole eyespot module and an absence of any strong generative constraints. In contrast, the pattern of eyespot color composition apparently still shows low evolvability and the presence of (strong) relative constraints. the laboratory with a more comparative descriptive approach for the whole lineage will reveal the extent to which the responses in short-term selection experiments can inform us about patterns of evolutionary change over long timescales. To us, the most exciting prospect of this type of integrative approach for amenable systems will be the ability to compare patterns of evolvability across different sets of traits involved in the evolution of complex morphologies. This will, in turn, provide more general insights about how the properties of change in developmental processes contribute to observed patterns of evolution and the occupancy of morphological space. However, more of a challenge than making such descriptions of patterns of change in developmental pathways will be disentangling cause and effect. Why is there more standing genetic and developmental variation along certain axes of morphological variation than along others? Putting it another way, why do genetic covariances among developmentally related traits have the properties they do? Is this because of some fundamental ways in which the development processes work, or, rather, does it reflect the legacy of how natural selection has influenced the morphologies concerned? Such questions will be answered only when an integrated approach is taken in certain key systems that lend themselves to studies at different levels of biological organization, including how natural selection works in the eco- logical arena. Again, this can be illustrated by referring to the evolution in patterns of butterfly eyespots. Perspectives: Fusing Micro- and Macroscales of Evolutionary Change Developmental studies on Bicyclus eyespots have already suggested differences in the mechanisms of pattern determination for eyespot size and color composition (Monteiro et al. 1994, 1997a). Artificial selection experiments in which two eyespots on the same wing surface have been the target of selection are starting to reveal differences in the evolvability of the pattern of two or more eyespots for these different traits that may be traced back to the properties of eyespot development. If the early results from a combination of the artificial selection experiments and morphometric analyses across extant species are confirmed, a relative lack of genetic variation to facilitate an uncoupling response for eyespot color in contrast to size could be accounted for by (1) the absence of history of selection in different environments for butterflies with a combination of some eyespots with narrow and others with broad rings, (2) a more absolute developmental constraint for eyespot color, perhaps relating to the wing-level property of threshold responses to focal signals of different eyespots, or (3) a combination of these two effects working S12 The American Naturalist in harness to make any substantial evolution of evolvability in color less likely. Figure 4 illustrates these scenarios. We argue that early in the radiation of the Lepidoptera, an evolutionary novelty that involved co-option of the hedgehog signaling pathway, perhaps in a new functional context, as well as novel tissue, late in development yielded the pattern of serial repeats of small undifferentiated spots (see Brunetti et al. 2001). Such patterns are found in many Lepidoptera, including basal groups. There followed a period of evolutionary elaboration with further co-opting and evolution of gene regulation to yield the module of repeated structures, each with a potentially larger and more structured color pattern element. This pattern is illustrated in the central column of figure 4; the species Mycalesis horsfeldi represents a striking candidate for possession of an eyespot pattern closely similar to such a prototype module. Finally, species of Bicyclus and Mycalesis in different environments of predator pressure, of light and resting backgrounds, and of communities of conspecifics evolved the ability for independent evolution in the serial eyespot elements, at least for eyespot size. For some reason, perhaps accounted for by a combination of selection history and developmental bias, this process is less advanced for eyespot color composition. The important point here is that an integration of experimental work using a model species that is amenable to a broad evo-devo framework with a more comparative approach among many species can open up issues of the roles of generative constraints to analysis. Such an integrative evolutionary biology will also enable links to be made from the processes of adaptive evolution to patterns of diversity among species. In addition, the basis of differences in evolvability can be investigated and eventually related to differences in histories of selection as well as in the genetical variances and developmental mechanisms that generate the phenotype. Acknowledgments We are indebted to the various museums that provided J.C.R. with access to their collections. We would also like to thank the whole community of researchers on Bicyclus anynana for its support. D. Schluter provided very useful comments on the manuscript. Some of the ideas discussed here were elaborated on during discussions of a National Center for Ecological Analysis and Synthesis working group organized by N. Eldredge and J. Thompson, to whom P.M.B. is most grateful. P.M.B. also thanks M. McPeek for his invitation to contribute to his symposium. Literature Cited Albertson, R. C., and T. D. Kocher. 2006. Genetic and developmental basis of cichlid trophic diversity. Heredity 97:211–221. Antonovics, J., and P. H. van Tienderen. 1991. Ontoecogenophyloconstraints? the chaos of constraint terminology. Trends in Ecology & Evolution 6:166–168. Arbesman, S., L. Enthoven, and A. Monteiro. 2003. Ancient wings: animating the evolution of butterfly wing patterns. Biosystems 71: 289–295. Beldade, P., and P. M. Brakefield. 2002. The genetics and evo-devo of butterfly wing patterns. Nature Reviews Genetics 3:442–452. ———. 2003a. Concerted evolution and developmental integration in modular butterfly wing patterns. Evolution and Development 5:169–179. ———. 2003b. The difficulty of agreeing about constraints. Evolution and Development 5:119–120. Beldade, P., P. M. Brakefield, and A. D. Long. 2002a. Contribution of Distal-less to quantitative variation in butterfly eyespots. Nature 415:315–318. Beldade, P., K. Koops, and P. M. Brakefield. 2002b. Developmental constraints versus flexibility in morphological evolution. Nature 416:844–847. ———. 2002c. Modularity, individuality, and evo-devo in butterfly wings. Proceedings of the National Academy of Sciences of the USA 99:14262–14267. Beldade, P., P. M. Brakefield, and A. D. Long. 2005. Generating phenotypic variation: prospects from “evo-devo” research on Bicyclus anynana wing patterns. Evolution and Development 7:101–107. Blows, M. W., and A. A. Hoffmann. 2005. A reassessment of genetic limits to evolutionary change. Ecology 86:1371–1384. Brakefield, P. M. 1998. The evolution-development interface and advances with the eyespot patterns of Bicyclus butterflies. Heredity 80:265–272. ———. 2003. The power of evo-devo to explore evolutionary constraints: experiments with butterfly eyespots. Zoology 106:283–290. ———. 2006. Evo-devo and constraints on selection. Trends in Ecology & Evolution 21:362–368. Brakefield, P. M., and W. A. Frankino. 2006. Polyphenisms in Lepidoptera: multidisciplinary approaches to studies of evolution. Pages 121–151 in D. W. Whitman and T. N. Ananthakrishnan, eds. Phenotypic plasticity in insects: mechanisms and consequences. Science, Plymouth. Brakefield, P. M., J. Gates, D. Keys, F. Kesbeke, P. J. Wijngaarden, A. Monteiro, V. French, and S. B. Carroll. 1996. Development, plasticity and evolution of butterfly eyespot patterns. Nature 384:236– 242. Brakefield, P. M., V. French, and B. J. Zwaan. 2003. Development and the genetics of evolutionary change within species. Annual Review of Ecology, Evolution, and Systematics 34:633–660. Breuker, C. J., and P. M. Brakefield. 2002. Female choice depends on size but not symmetry of dorsal eyespots in the butterfly Bicyclus anynana. Proceedings of the Royal Society B: Biological Sciences 269:1233–1239. Brunetti, C. R., J. E. Selegue, A. Monteiro, V. French, P. M. Brakefield, and S. B. Carroll. 2001. The generation and diversification of butterfly eyespot color patterns. Current Biology 11:1578–1585. Condamin, M. 1973. Monographie du genre Bicyclus (Lepidoptera, Satyridae). IFAN, Dakar. Cooper, T. F., D. E. Rozen, and R. E. Lenski. 2003. Parallel changes in gene expression after 20,000 generations of evolution in Escherichia coli. Proceedings of the National Academy of Sciences of the USA 100:1072–1077. Eldredge, N., J. N. Thompson, P. M. Brakefield, S. Gavrilets, D. Exploring Evolutionary Constraints Jablonski, J. B. C. Jackson, R. E. Lenski, B. S. Lieberman, M. A. McPeek, and W. Miller III. 2005. The dynamics of evolutionary stasis. Paleobiology 31(suppl.):133–145. Elena, S. F., and R. E. Lenski. 2003. Evolution experiments with microorganisms: the dynamics and genetic bases of adaptation. Nature Review Genetics 4:457–469. Fischer, K., A. N. M. Bot, P. M. Brakefield, and B. J. Zwaan. 2006. Do mothers producing large offspring have to sacrifice fecundity? Journal of Evolutionary Biology 19:380–391. Frankino, W. A., B. J. Zwaan, D. L. Stern, and P. M. Brakefield. 2005. Natural selection and developmental constraints in the evolution of allometries. Science 307:718–720. ———. 2006. Internal and external constraints in the evolution of a forewing-hindwing allometry. Evolution (forthcoming). French, V., and P. M. Brakefield. 1995. Eyespot development on butterfly wings: the focal signal. Developmental Biology 168:112–123. Fryer, G., and T. D. Iles. 1972. The cichlid fishes of the great lakes of Africa: their biology and evolution. Oliver & Boyd, London. Fuller, R. C., C. F. Baer, and J. Travis. 2005. How and when selection experiments might actually be useful. Integrative and Comparative Biology 45:391–404. Gould, S. J. 2002. The structure of evolutionary theory. Harvard University Press, Cambridge, MA. Gould, S. J., and R. C. Lewontin. 1979. The spandrels of San Marco and the Panglossian paradigm: a critique of the adaptationist programme. Proceedings of the Royal Society B: Biological Sciences 205:581–598. Grant, P. R., and B. R. Grant. 2002. Unpredictable evolution in a 30year study of Darwin’s finches. Science 296:707–711. Harmon, L. J., J. J. Kolbe, J. M. Cheverud, and J. B. Losos. 2005. Convergence and the multidimensional niche. Evolution 59:409– 421. Joron, M., and P. M. Brakefield. 2003. Captivity masks inbreeding effects on male mating success in butterflies. Nature 424:191–194. Kangas, A. T., A. R. Evans, I. Thesleff, and J. Jernvall. 2004. Nonindependence of mammalian dental characters. Nature 432:211– 214. Kocher, T. D., J. A. Conroy, K. R. McKaye, and J. R. Stauffer. 1993. Similar morphologies of cichlids in lakes Tanganyika and Malawi are due to convergence. Molecular Phylogenetics and Evolution 2: 158–165. Losos, J. B., T. W. Schoener, and D. A. Spiller. 2004. Predator-induced behaviour shifts and natural selection in field-experimental lizard populations. Nature 432:505–508. Lyytinen, A., P. M. Brakefield, and J. Mappes. 2003. Significance of butterfly eyespots as an anti-predator device in ground-based and aerial attacks. Oikos 100:373–379. Lyytinen, A., P. M. Brakefield, L. Lindström, and J. Mappes. 2004. Does predation maintain eyespot plasticity in Bicyclus anynana? Proceedings of the Royal Society B: Biological Sciences 271:279– 283. Maynard Smith, J., R. Burian, S. Kaufman, P. Alberch, J. Campbell, B. Goodwin, R. Lande, D. Raup, and L. Wolpert. 1985. Developmental constraints and evolution. Quarterly Review of Biology 60:265–287. Monteiro, A., and N. E. Pierce. 2001. Phylogeny of Bicyclus (Lepi- S13 doptera: Nymphalidae) inferred from COI, COII and EF-1a gene sequences. Molecular Phylogenetics and Evolution 18:264–281. Monteiro, A., P. M. Brakefield, and V. French. 1997a. Butterfly eyespots: the genetics and development of the color rings. Evolution 51:1207–1216. ———. 1997b. The genetics and development of an eyespot pattern in the butterfly Bicyclus anynana: response to selection for eyespot shape. Genetics 146:287–294. Monteiro, A., J. Prijs, M. Bax, T. Hakkart, and P. M. Brakefield. 2003. Mutants highlight the modular control of butterfly eyespot patterns. Evolution and Development 5:180–187. Monteiro, A. F., P. M. Brakefield, and V. French. 1994. The evolutionary genetics and developmental basis of wing pattern variation in the butterfly Bicyclus anynana. Evolution 48:1147–1157. Reed, R. D., and M. S. Serfas. 2004. Butterfly wing pattern evolution is associated with changes in a Notch/Distal-less temporal pattern formation process. Current Biology 14:1159–1166. Robertson, K. A., and A. Monteiro. 2005. Female Bicyclus anynana butterflies choose males on the basis of their dorsal UV-reflective eyespot pupils. Proceedings of the Royal Society B: Biological Sciences 272:1541–1546. Roskam, J. C., and P. M. Brakefield. 1999. Seasonal polyphenism in Bicyclus (Lepidoptera: Satyridae) butterflies: different climates need different cues. Biological Journal of the Linnean Society 66: 345–356. Salazar-Ciudad, I., and J. Jernvall. 2002. A gene network model accounting for development and evolution of mammalian teeth. Proceedings of the National Academy of Sciences of the USA 99: 8116–8120. Salzburger, W., and A. Meyer. 2004. The species flocks of East African cichlid fishes: recent advances in molecular phylogenetics and population genetics. Naturwissenschaften 91:277–290. Schlichting, C. D., and M. Pigliucci. 1998. Phenotypic evolution: a reaction norm perspective. Sinauer, Sunderland, MA. Schluter, D. 1996. Adaptive radiation along genetic lines of least resistance. Evolution 50:1766–1774. ———. 2000. The ecology of adaptive radiation. Oxford University Press, Oxford. Schwenk, K. 1995. A utilitarian approach to evolutionary constraints. Zoology 98:251–262. Stearns, S. C. 2002. Less would have been more. Evolution 56:2339– 2345. Temeles, E. J., and W. J. Kress. 2003. Adaptation in a planthummingbird association. Science 300:630–633. Wijngaarden, P. J., P. B. Koch, and P. M. Brakefield. 2002. Artificial selection on the shape of reaction norms for eyespot size in the butterfly Bicyclus anynana: direct and correlated responses. Journal of Evolutionary Biology 15:290–300. Zijlstra, W. G., F. Kesbeke, B. J. Zwaan, and P. M. Brakefield. 2002. Protandry in the butterfly Bicyclus anynana. Evolutionary Ecology Research 4:1229–1240. Zijlstra, W. G., M. J. Steigenga, P. B. Koch, B. J. Zwaan, and P. M. Brakefield. 2004. Butterfly selected lines explore the hormonal basis of interactions between life histories and morphology. American Naturalist 163:E76–E87. Symposium Editor: Mark A. McPeek