THE EVOLUTIONARY GENETICS AND DEVELOPMENTAL BASIS OF WING PATTERN VARIATION IN THE BUTTERFLY BICYCLUS ANYNANA

We have studied interactions between developmental processes and genetic variation for the eyespot color pattern on the adult dorsal forewing of the nymphalid butterfly, Bicyclus anynana. Truncation selection was applied in both an upward and a downward direction to the size of a single eyespot consisting of rings with wing scales of differing color pigments. High heritabilities resulted in rapid responses to selection yielding divergent lines with very large or very small eyespots. Strong correlated responses occurred in most of the other eyespots on each wing surface. The cells at the center of a presumptive eyespot (the “focus”) act in the early pupal stage to establish the adult wing pattern. The developmental fate of the scale cells within an eyespot is specified by the “signaling” properties of the focus and the “response” thresholds of the epidermis. The individual eyespots can be envisaged as developmental homologues. Grafting experiments performed with the eyespot foci of the selected lines showed that additive genetic variance exists for both the response and, in particular, the signaling components of the developmental system. The results are discussed in the context of how constraints on the evolution of this wing pattern may be related to the developmental organization.     

The relationship between eyespot shape and wing shape in the butterfly Bicyclus anynana: A genetic and morphometrical approach

The African butterfly, Bicyclus anynana, normally possesses circular eyespots on its wings. Artificial selection lines, which express ellipsoidal eyespots on the dorsal surface of the forewing, were used to investigate correlated changes in wing shape. Morphometric analysis of linear wing measurements and wing scale counts provided evidence that eyespot shape was correlated with localised shape changes in the corresponding wing-cell, with overall shape changes in the wing, and with the density/arrangement of scales around the eyespot area.     

Developmental and genetic mechanisms for evolutionary diversification of serial repeats: eyespot size in Bicyclus anynana butterflies

Serially repeated pattern elements on butterfly wings offer the opportunity for integrating genetic, developmental, and functional aspects towards understanding morphological diversification and the evolution of individuality. We use captive populations of Bicyclus anynana butterflies, an emerging model in evolutionary developmental biology, to explore the genetic and developmental basis of compartmentalized changes in eyespot patterns. There is much variation for different aspects of eyespot morphology, and knowledge about the genetic pathways and developmental processes involved in eyespot formation. Also, despite the strong correlations across all eyespots in one butterfly, B. anynana shows great potential for independent changes in the size of individual eyespots. It is, however, unclear to what extent the genetic and developmental processes underlying eyespot formation change in a localized manner to enable such individualization. We use micromanipulations of developing wings to dissect the contribution of different components of eyespot development to quantitative differences in eyespot size on one wing surface. Reciprocal transplants of presumptive eyespot foci between artificial selection lines and controls suggest that while localized antagonistic changes in eyespot size rely mostly on localized changes in focal signal strength, concerted changes depend greatly on epidermal response sensitivities. This potentially reflects differences between the signal-response components of eyespot formation in the degrees of compartmentalization and/or the temporal pattern of selection. We also report on the phenotypic analysis of a number of mutant stocks demonstrating how single alleles can affect different eyespots in concert or independently, and thus contribute to the individualization of serially repeated traits.     

Concerted evolution and developmental integration in modular butterfly wing patterns

Developing organisms are thought to be modular in organization so that traits in different modules evolve independently whereas traits within a module change in a concerted manner. The eyespot pattern in Bicyclus anynana butterflies provides an ideal system where morphological modularity can be dissected and different levels of genetic integration analyzed. Several lines of evidence show that all eyespots in an individual butterfly are genetically integrated, suggesting that the whole pattern, rather than the separate eyespots, should be considered as a single character. However, despite the strong genetic correlations between the two eyespots on the dorsal forewing of B. anynana, there is great potential for independent changes. Here we use laboratory lines selected in different directions for the size of those eyespots to study correlated responses in the whole eyespot pattern. We show clear changes in eyespot size across all wing surfaces, which depend on eyespot position along the anterior-posterior axis. There are also changes in the number of extra eyespots and in eyespot color composition but no changes in eyespot position relative to wing margin. Our analysis of eyespot pattern modularity is discussed in the light of what is known about the cellular and genetic mechanisms of eyespot formation and the great potential for evolutionary diversification in butterfly wing patterns.     

Butterfly wing pattern mutants: developmental heterochrony and co-ordinately regulated phenotypes

Butterfly wings are colored late in development, when pigments are synthesized in specialized wing scale cells in a fixed developmental succession. In this succession, colored pigments are deposited first and the remaining areas are later melanized black or brown. Here we studied the developmental changes underlying two wing pattern mutants, firstly melanic mutants of the swallowtail Papilio glaucus, in which the yellow background is turned black, and secondly a Spotty mutant of the satyrid Bicyclus anynana, which carries two additional eyespots. Despite the very different pattern changes in these two mutants, they are both associated with changes in rates of scale development and correspondingly, the final color pattern. In the melanic swallowtail, background scales originally destined to become yellow (normally developing early and synthesizing papiliochrome) show delayed development, fail to make papiliochrome, and subsequently melanize at the same time as scales in the wild-type black pattern. In the B. anynana eyespot, scale maturation begins with the central white focus, then progresses to the surrounding gold ring and later finishes with melanization of the black center. Mutants showing additional eyespots display accelerated rates of scale development (corresponding to new eyespots) in wing cells not normally occupied by eyespots. Thus by either delaying or accelerating rates of scale development, the final color, or position, of a wing pattern element can be changed. We propose that this heterochrony of scale development is a basic mechanism of color pattern formation on which developmental mutants act to change lepidopteran color patterns. Received: 20 April 2000 / Accepted: 19 July 2000     

Butterfly Eyespot Patterns: Evidence for Specification by a Morphogen Diffusion Gradient

In this paper we describe a test for Nijhout's (1978, 1980a) hypothesis that the eyespot patterns on butterfly wings are the result of a threshold reaction of the epidermal cells to a concentration gradient of a diffusing degradable morphogen produced by focal cells at the centre of the future eyespot. The wings of the nymphalid butterfly, Bicyclus anynana, have a series of eyespots, each composed of a white pupil, a black disc and a gold outer ring. In earlier extirpation and transplantation experiments (Nijhout 1980a; French and Brakefield, 1995) it has been established that these eyespots are indeed organised around groups of signalling cells active during the first hours of pupal development. If these cells were to supply the positional information for eyespot formation in accordance with Nijhout's diffusion-degradation gradient model, then, when two foci are close together, the signals should sum, and this effect should be apparent in the detailed shape of the resulting pigment pattern. We give an equation for the form of the contours that would be obtained in this manner. We use this to test the morphogen gradient hypothesis on measurements of the outlines of fused eyespots obtained either by grafting focal cells close together, or by using a mutation (Spotty) that produces adjacent fused eyespots. The contours of the fused patterns were found to satisfy our equation, thus corroborating Nijhout's hypothesis to the extent possible with this particular type of experiment.     

Automatic recognition and measurement of butterfly eyespot patterns

A favorite wing pattern element in butterflies that has been the focus of intense study in evolutionary and developmental biology, as well as in behavioral ecology, is the eyespot. Because the pace of research on these bull's eye patterns is accelerating we sought to develop a tool to automatically detect and measure butterfly eyespot patterns in digital images of the wings. We used a machine learning algorithm with features based on circularity and symmetry to detect eyespots on the images. The algorithm is first trained with examples from a database of images with two different labels (eyespot and non-eyespot), and subsequently is able to provide classification for a new image. After an eyespot is detected the radius measurements of its color rings are performed by a 1D Hough Transform which corresponds to histogramming. We trained software to recognize eyespot patterns of the nymphalid butterfly Bicyclus anynana but eyespots of other butterfly species were also successfully detected by the software.     

Mutants highlight the modular control of butterfly eyespot patterns

SUMMARY The eyespots on butterfly wings are thought to be serially homologous pattern elements. Yet eyespots differ greatly in number, shape, color, and size, within and among species. To what extent do these serially homologues have separate developmental identities, upon which selection acts to create diversity? We examined x‐ray–induced mutations for the eyespots of the nymphalid butterfly Bicyclus anynana that highlight the modular control of these serially homologous wing pattern elements. These mutations reduce or eliminate individual eyespots, or groups of eyespots, with no further effect on the wing color pattern. The collection of mutants highlights a greater potential developmental repertoire than that observed across the genus Bicyclus. We studied in detail one such mutation, of codominant effect, that causes the elimination of two adjacent eyespots on the ventral hindwing. By analyzing the expression of genes known to be involved in eyespot formation, we found an alteration in the differentiation of the “organizing” cells at the eyespot's center. No such cells differentiate in the wing subdivisions lacking the two eyespots in the mutants. We propose several developmental models, based on wing compartmentalization in Drosophila, that provide the first framework for thinking about the molecular evolution of butterfly wing pattern modularity.     

Significance of butterfly eyespots as an anti-predator device in ground-based and aerial attacks

Many butterfly genera are characterised by the presence of marginal eyespots on their wings. One hypothesis to account for an occurrence of eyespots is that these wing pattern elements are partly the outcome of visual selection by predators. Bicyclus anynana (Satyrinae) has underside spotting on its wings but there is also a seasonal form in which the eyespots are reduced in size or totally absent. This natural variation gives us a useful tool to test the hypothesis that marginal eyespot patterns can decoy the attacking predator by, at least sometimes, diverting attack from vital body parts to the edges of the wings. We used lizards, Anolis carolinensis , and pied flycatchers, Ficedula hypoleuca , as predators for living spotted and spotless B. anynana . The presence of eyespots did not increase the escape probability of resting butterflies once captured (even a form with enlarged eyespots did not add to effective deflection of attacks). There was also no evidence that eyespots influenced the location of strikes by the predators. This study thus provides no support that marginal eyespot patterns can act as an effective deflection mechanism to avoid lizard or avian predation.     

Butterfly wing patterns

This paper integrates genetical studies of variation in the wing patterns of Lepidoptera with experimental investigations of developmental mechanisms. Research on the tropical butterfly,Bicyclus anynana, is described. This work includes artificial selection of lines with different patterns of wing eyespots followed by grafting experiments on the lines to examine the phenotypic and genetic differences in terms of developmental mechanisms. The results are used to show how constraints on the evolution of this wing pattern may be related to the developmental organisation. The eyespot pattrn can be envisaged as a set of developmental homologues; a common developmental mechanism is associated with a quantitative genetic system involving high genetic correlations. However, individual genes which influence only subsets of the eyespots, thus uncoupling the interdependence of the eyespots, may be important in evolutionary change. The postulated evolutionary constraints are illustrated with respect to differences in wing pattern found among other species ofBicyclus.     

The Genetics and Development of an Eyespot Pattern in the Butterfly Bicyclus Anynana: Response to Selection for Eyespot Shape

The normally circular eyespots on the wing of the butterfly Bicyclus anynana were selected to become elliptical in two divergent lines, with antero-posterior elongation of the eyespots in one line and proximodistal elongation in the other. Selection was continued for nine generations, and symmetrical realized heritabilities of ~15% were achieved initially. The elliptical eyespot shapes characteristic of each line were still produced when the signaling center of the eyespot (the focus) was surgically rotated by 90 or 180° or when an eyespot was induced ectopically by localized damage. We conclude that selection changed general properties of the epidermis that responds to signals emanating from the eyespot focus but did not affect the mechanism of focal signaling.     

The "eyespot module" and eyespots as modules: development, evolution, and integration of a complex phenotype

Organisms are inherently modular, yet modules also evolve in response to selection for functional integration or functional specialization of traits. For serially repeated homologous traits, there is a clear expectation that selection on the function of individual traits will reduce the integration between traits and subdivide a single ancestral module. The eyespots on butterfly wings are one example of serially repeated morphological traits that share a common developmental mechanism but are subject to natural and sexual selection for divergent functions. Here, I test two hypotheses about the organization of the eyespot pattern into independent dorsal-ventral and anterior-posterior modules, using a graphical modeling technique to examine patterns of eyespot covariation among and within wing surfaces in the butterfly Bicyclus anynana. Although there is a hierarchical and complex pattern of integration among eyespots, the results show a surprising mismatch between patterns of eyespot integration and the developmental and evolutionary eyespot units identified in previous empirical studies. These results are discussed in light of the relationships between developmental, functional, and evolutionary modules, and they suggest that developmental sources of independent trait variation are often masked by developmental sources of trait integration.     

Modelling butterfly wing eyespot patterns

Eyespots are concentric motifs with contrasting colours on butterfly wings. Eyespots have intra- and interspecific visual signalling functions with adaptive and selective roles. We propose a reaction-diffusion model that accounts for eyespot development. The model considers two diffusive morphogens and three non-diffusive pigment precursors. The first morphogen is produced in the focus and determines the differentiation of the first eyespot ring. A second morphogen is then produced, modifying the chromatic properties of the wing background pigment precursor, inducing the differentiation of a second ring. The model simulates the general structural organization of eyespots, their phenotypic plasticity and seasonal variability, and predicts effects from microsurgical manipulations on pupal wings as reported in the literature.     

Artificial selection on the shape of reaction norms for eyespot size in the butterfly Bicyclus anynana: direct and correlated responses

The tropical butterfly Bicyclus anynana shows phenotypic plasticity in its ventral wing pattern as an adaptive response to wet‐dry seasonality. Wet season form individuals have large eyespots, whereas individuals of the dry season generation have small eyespots. In the laboratory these forms can be obtained by rearing larvae at high and low temperatures, respectively. To study the extent to which the shape of the nearly linear reaction norms for eyespot size can be changed we applied four generations of sib selection by rearing full‐sib families across three temperatures. In addition, we measured ecdysteroid titres shortly after pupation in the final generation. Although phenotypic variation in shape was present initially, the experiment yielded lines with reaction norms with similar shapes but different elevations. High, positive genetic correlation across temperatures can explain this lack of response. Differences in ecdysteroid titres did not readily relate to differences in eyespot size.     

Simultaneous selection on two fitness-related traits in the butterfly Bicyclus anynana

Abstract. Theory about the role of constraints in evolution is abundant, but few empirical data exist to describe the consequences a bias in phenotypic variation has for micro evolution. Responses to natural selection can be severely hampered by a genetic correlation among a suite of traits. Constraints can be studied using antagonistic selection experiments, that is, two-trait selection in opposition to this correlation. The two traits studied here were development time and wing pattern (eyespot size) in the butterfly Bicyclus anynana , both of which have a clear adaptive significance. Rates of response were higher for eyespot size than for development time, but were independent of the concurrent selection (either in the same direction as the correlation or perpendicular to it). Regimes differed in both traits in all directions after 11 generations of selection. The uncoupling lines had higher relative responses than the synergistic lines in development time and equal relative responses in eyespot size. The patterns for eyespot size (reaction norms) were consistent across different rearing temperatures. Differences in lines selected for fast and slow development time were more pronounced at lower temperatures, irrespective of the direction of joint wing pattern selection. Furthermore, correlated responses in pupal weight and growth rate were observed; lines selected for a slower development had higher pupal weights, especially at lower temperatures. The response of the uncoupling lines was not hampered by a lack of selectable genetic variation, and the relative response in the development time was larger than expected based on response in the coupled direction and quantitative genetic predictions. This suggests that the structure of the genetic architecture does not constrain the short-term, independent evolution of both wing pattern and development time.     

Color-pattern analysis of eyespots in butterfly wings: a critical examination of morphogen gradient models

Butterfly wing color patterns consist of many color-pattern elements such as eyespots. It is believed that eyespot patterns are determined by a concentration gradient of a single morphogen species released by diffusion from the prospective eyespot focus in conjunction with multiple thresholds in signal-receiving cells. As alternatives to this single-morphogen model, more flexible multiple-morphogen model and induction model can be proposed. However, the relevance of these conceptual models to actual eyespots has not been examined systematically. Here, representative eyespots from nymphalid butterflies were analyzed morphologically to determine if they are consistent with these models. Measurement of ring widths of serial eyespots from a single wing surface showed that the proportion of each ring in an eyespot is quite different among homologous rings of serial eyespots of different sizes. In asymmetric eyespots, each ring is distorted to varying degrees. In extreme cases, only a portion of rings is expressed remotely from the focus. Similarly, there are many eyespots where only certain rings are deleted, added, or expanded. In an unusual case, the central area of an eyespot is composed of multiple "miniature eyespots," but the overall macroscopic eyespot structure is maintained. These results indicate that each eyespot ring has independence and flexibility to a certain degree, which is less consistent with the single-morphogen model. Considering a "periodic eyespot", which has repeats of a set of rings, damage-induced eyespots in mutants, and a scale-size distribution pattern in an eyespot, the induction model is the least incompatible with the actual eyespot diversity.     

Spatial patterns of correlated scale size and scale color in relation to color pattern elements in butterfly wings

Complex butterfly wing color patterns are coordinated throughout a wing by unknown mechanisms that provide undifferentiated immature scale cells with positional information for scale color. Because there is a reasonable level of correspondence between the color pattern element and scale size at least in Junonia orithya and Junonia oenone, a single morphogenic signal may contain positional information for both color and size. However, this color–size relationship has not been demonstrated in other species of the family Nymphalidae. Here, we investigated the distribution patterns of scale size in relation to color pattern elements on the hindwings of the peacock pansy butterfly Junonia almana, together with other nymphalid butterflies, Vanessa indica and Danaus chrysippus. In these species, we observed a general decrease in scale size from the basal to the distal areas, although the size gradient was small in D. chrysippus. Scales of dark color in color pattern elements, including eyespot black rings, parafocal elements, and submarginal bands, were larger than those of their surroundings. Within an eyespot, the largest scales were found at the focal white area, although there were exceptional cases. Similarly, ectopic eyespots that were induced by physical damage on the J. almana background area had larger scales than in the surrounding area. These results are consistent with the previous finding that scale color and size coordinate to form color pattern elements. We propose a ploidy hypothesis to explain the color–size relationship in which the putative morphogenic signal induces the polyploidization (genome amplification) of immature scale cells and that the degrees of ploidy (gene dosage) determine scale color and scale size simultaneously in butterfly wings.     

Generation of butterfly wing eyespot patterns: a model for morphological determination of eyespot and parafocal element

The determination of color patterns of butterfly wing eyespots has been explained by the morphogen concentration gradient model. The induction model has been proposed recently as a more realistic alternative, in which the eyespot-specifying signal does not depend entirely on focal activity. However, this model requires further elaboration and supporting evidence to be validated. Here, I examined various color patterns of nymphalid butterflies to propose the mechanics of the induction model. Based on cases in which an eyespot light ring is identical to the background in color, I propose that eyespots are fundamentally composed of dark rings and non-dark "background" spaces between them. In the induction model, the dark-ring-inducing signal that is released from a prospective eyespot focus (the primary organizing center) as a slow-moving wave effects both selfenhancement and peripheral induction of the dark-ring-inhibitory signal at the secondary organizing centers, resulting in an eyespot that has alternate dark and light rings. Moreover, there are cases in which an unseen "imaginary light ring" surrounds an eyespot proper and in which PFEs are integrated into the eyespot. It appears that PFEs constitute a periodic continuum of eyespot dark rings; thus, a background space between the eyespot and a PFE is mechanistically equivalent to eyespot light rings. The eyespot dark-ring-inducing signals and PFE-inducing signal are likely to be identical in quality, but released at different times from the same organizing center. Computer simulations based on the reaction-diffusion system support the feasibility of the induction model.