BUTTERFLY EYESPOTS: THE GENETICS AND DEVELOPMENT OF THE COLOR RINGS

The butterfly Bicyclus anynana has a series of distal eyespots on its wings. Each eyespot is composed of a white pupil, a black disc, and a gold outer ring. We applied artificial selection to the large dorsal eyespot on the forewing to produce a line with the gold ring reduced or absent (BLACK) and another line with a reduced black disc and a broad gold ring (GOLD). High heritabilities, coupled with a rapid response to selection, produced two lines of butterflies with very different phenotypes. Other eyespots showed a correlated change in the proportion of their color rings. Surgical experiments were performed on pupal wings from the different lines at the time of eyespot pattern specification. They showed that the additive genetic variance for this trait was in the response of the wing epidermis to signaling from the organizing cells at the eyespot center (the focus). This response was found to vary across different regions of the wing and also between the sexes. The particular eyespot color composition found for each sex, as well as the maintenance of the high genetic variation, are discussed with reference to the ecology of the butterfly, sexual selection, and visual selection by predators.     

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.     

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.     

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.     

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.     

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     

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.     

Quantitative genetics of butterfly wing color patterns

Developmental processes exert their influence on the evolution of complex morphologies through the genetic correlations they engender between traits. Butterfly wing color patterns provide a model system to examine this connection between development and evolution. In butterflies, the nymphalid groundplan is a framework used to decompose complex wing patterns into their component pattern elements. The first goal of this work has been to determine whether the components of the nymphalid groundplan are the products of independent developmental processes. To test this hypothesis, the genetic correlation matrices for two species of butterflies, Precis coenia and Precis evarete, were estimated for 27 wing pattern characters. The second purpose was to test the hypothesis that the differentiation of serial homologs lowers their genetic correlations. The “eyespots” found serially repeated across the fore- and hindwing and on the dorsal and ventral wing surfaces provided an opportunity to test this hypothesis. The genetic correlation matrices of both species were very similar. The pattern of genetic correlation measured between the different types of pattern elements and between the homologous repeats of a pattern element supported the first hypothesis of developmental independence among the elements of the groundplan. The correlation pattern among the differentiated serial homologs was similarly found to support the second hypothesis: pairs of eyespots that had differentiated had lower genetic correlations than pairs that were similar in morphology. The implications of this study are twofold: First, the apparent developmental independence among the distinct elements of wing pattern has facilitated the vast diversification in morphology found in butterflies. Second, the lower genetic correlations betweendifferentiated homologs demonstrates that developmental constraints can in fact be broken. The extent to which genetic correlations readily change, however, remains unknown. © 1994 Wiley-Liss, Inc.     

Integration of wings and their eyespots in the speckled wood butterfly Pararge aegeria

We investigated both the phenotypic and developmental integration of eyespots on the fore- and hindwings of speckled wood butterflies Pararge aegeria. Eyespots develop within a framework of wing veins, which may not only separate eyespots developmentally, but may at the same time also integrate them by virtue of being both signalling sources and barriers during eyespot development. We therefore specifically investigated the interaction between wing venation patterns and eyespot integration. Phenotypic covariation among eyespots was very high, but only eyespots in neighbouring wing cells and in homologous wing cells on different wing surfaces were developmentally integrated. This can be explained by the fact that the wing cells of these eyespots share one or more wing veins. The wing venation patterns of fore- and hindwings were highly integrated, both phenotypically and developmentally. This did not affect overall developmental integration of the eyespots. The adaptive significance of integration patterns is discussed and more specifically we stress the need to conduct studies on phenotypic plasticity of integration.     

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.     

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.     

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.     

Eyespot evolution: phylogenetic insights from Junonia and related butterfly genera (Nymphalidae: Junoniini)

SUMMARY Butterfly eyespots have been the focus of a number of developmental and evolutionary studies. However, a phylogenetic component has rarely been explicitly incorporated in these studies. In this study, I utilize a phylogeny to trace the evolution of eyespot number and position on the wing in a group of nymphalid butterflies, the subtribe Junoniini. These butterflies have two kinds of eyespot arrangements which I refer to as Serial and Individual . In the Serial arrangement, eyespots are placed in a series on compartments 1−6 (counting from the anterior wing margin). In the Individual arrangement, eyespots are isolated on specific compartments, ranging from 1 to 4 in number. This can be divided into four subtypes based on the number and positions of eyespots. I map the evolution of these five arrangements over a phylogeny of Junoniini reconstructed with ca. 3000 base pairs of sequence data from three genes. The results show that almost all arrangements have evolved at least twice, with multiple shifts between them by addition and deletion of eyespots. I propose a model involving genetic or developmental coupling between eyespots in specific compartments to explain these shifts. I discuss their evolution in light of existing knowledge about their development. I also discuss potential explanations for functional significance of the eyespot patterns found in the group. Differential selection for and against eyespots, both at different times over the phylogeny and in different regions, have driven the evolution of eyespot arrangements. The study throws open many questions about the adaptive significance of eyespots and the developmental underpinnings of the various arrangements.     

Butterfly Wings Are Three-Dimensional: Pupal Cuticle Focal Spots and Their Associated Structures in Junonia Butterflies

Butterfly wing color patterns often contain eyespots, which are developmentally determined at the late larval and early pupal stages by organizing activities of focal cells that can later form eyespot foci. In the pupal stage, the focal position of a future eyespot is often marked by a focal spot, one of the pupal cuticle spots, on the pupal surface. Here, we examined the possible relationships of the pupal focal spots with the underneath pupal wing tissues and with the adult wing eyespots using Junonia butterflies. Large pupal focal spots were found in two species with large adult eyespots, J. orithya and J. almana, whereas only small pupal focal spots were found in a species with small adult eyespots, J. hedonia. The size of five pupal focal spots on a single wing was correlated with the size of the corresponding adult eyespots in J. orithya. A pupal focal spot was a three-dimensional bulge of cuticle surface, and the underside of the major pupal focal spot exhibited a hollowed cuticle in a pupal case. Cross sections of a pupal wing revealed that the cuticle layer shows a curvature at a focal spot, and a positional correlation was observed between the cuticle layer thickness and its corresponding cell layer thickness. Adult major eyespots of J. orithya and J. almana exhibited surface elevations and depressions that approximately correspond to the coloration within an eyespot. Our results suggest that a pupal focal spot is produced by the organizing activity of focal cells underneath the focal spot. Probably because the focal cell layer immediately underneath a focal spot is thicker than that of its surrounding areas, eyespots of adult butterfly wings are three-dimensionally constructed. The color-height relationship in adult eyespots might have an implication in the developmental signaling for determining the eyespot color patterns.     

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.     

The regulation of phenotypic plasticity of eyespots in the butterfly Bicyclus anynana

We use an outcrossed stock and selected lines of Bicyclus anynana in combination with measurements and manipulations of ecdysteroid hormones in early pupae to examine the regulation of eyespot size in adult butterflies. The eyespots on the ventral wing surfaces express adaptive phenotypic plasticity in response to the dry-wet seasonal environments of the butterflies. Larvae reared at low or high temperatures produce adults with small or large ventral eyespots, respectively. Our experiments examine the role of ecdysteroids in mediating this phenotypic plasticity. Higher titers of ecdysteroids shortly after pupation yield eclolarger ventral wing eyespots. There is an uncoupling of the ventral eyespots and those on the dorsal forewing. The latter do not show phenotypic plasticity. They show very little response to rearing temperature, and variation in their size is not associated with differences in the dynamics of ecdysteroids in early pupae. A testable hypothesis in terms of the distribution of hormone receptors in the developmental "organizers" or foci of the eyespots is proposed to account for how some eyespots express plasticity while others do not.     

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.     

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.     

A simulation study of the genetic regulatory hierarchy for butterfly eyespot focus determination

The color patterns on the wings of butterflies have been an important model system in evolutionary developmental biology. Two types of models have been used to study these patterns. The first type of model employs computational techniques and generalized mechanisms of pattern formation to make predictions about how color patterns will vary as parameters of the model are changed. These generalized mechanisms include diffusion gradient, reaction-diffusion, lateral inhibition, and threshold responses. The second type of model uses known genetic interactions from Drosophila melanogaster and patterns of candidate gene expression in one of several butterfly species (most often Junonia (Precis) coenia or Bicyclus anynana) to propose specific genetic regulatory hierarchies that appear to be involved in color pattern formation. This study combines these two approaches using computational techniques to test proposed genetic regulatory hierarchies for the determination of butterfly eyespot foci (also known as border ocelli foci). Two computer programs, STELLA 8.1 and Delphi 2.0, were used to simulate the determination of eyespot foci. Both programs revealed weaknesses in a genetic model previously proposed for eyespot focus determination. On the basis of these simulations, we propose two revised models for eyespot focus determination and identify components of the genetic regulatory hierarchy that are particularly sensitive to changes in model parameter values. These components may play a key role in the evolution of butterfly eyespots. Simulations like these may be useful tools for the study of other evolutionary developmental model systems and reveal similar sensitive components of the relevant genetic regulatory hierarchies.