Female choice depends on size but not symmetry of dorsal eyespots in the butterfly Bicyclus anynana

The eyespots on the ventral wings of Bicyclus anynana butterflies are exposed when at rest and interact with predators. Those on the dorsal surface are not exposed in this way, and may be involved in courtship and mate choice. In this study, we examined whether the size and fluctuating asymmetry (FA) of dorsal eyespots are reliable signals of male quality. High developmental stability is considered to result in low FA, and to be associated with high quality. Individuals of high quality are predicted to produce sexually selected traits that are large and symmetrical, at a relatively low cost. In this study, we manipulated eyespot development to uncouple eyespot size and FA in order to examine their independent roles in signalling to the female. Individual females in cages were given the choice between two or three males differing in eyespot traits. The results indicate that although size per se of the eyespots is used as a signal, FA and wing size are not. We discuss the use of FA in studies of sexual selection and aspects of sexual selection on dorsal eyespot size.     

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 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.     

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 "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.     

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.     

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.     

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.     

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.     

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.     

Prey from the eyes of predators: Color discriminability of aposematic and mimetic butterflies from an avian visual perspective

Predation exerts strong selection on mimetic butterfly wing color patterns, which also serve other functions such as sexual selection. Therefore, specific selection pressures may affect the sexes and signal components differentially. We tested three predictions about the evolution of mimetic resemblance by comparing wing coloration of aposematic butterflies and their Batesian mimics: (a) females gain greater mimetic advantage than males and therefore are better mimics, (b) due to intersexual genetic correlations, sexually monomorphic mimics are better mimics than female‐limited mimics, and (c) mimetic resemblance is better on the dorsal wing surface that is visible to predators in flight. Using a physiological model of avian color vision, we quantified mimetic resemblance from predators’ perspective, which showed that female butterflies were better mimics than males. Mimetic resemblance in female‐limited mimics was comparable to that in sexually monomorphic mimics, suggesting that intersexual genetic correlations did not constrain adaptive response to selection for female‐limited mimicry. Mimetic resemblance on the ventral wing surface was better than that on the dorsal wing surface, implying stronger natural and sexual selection on ventral and dorsal surfaces, respectively. These results suggest that mimetic resemblance in butterfly mimicry rings has evolved under various selective pressures acting in a sex‐ and wing surface‐specific manner.     

Male choice generates stabilizing sexual selection on a female fecundity correlate

We know very little about male mating preferences and how they influence the evolution of female traits. Theory predicts that males may benefit from choosing females on the basis of traits that indicate their fecundity. Here, we explore sexual selection generated by male choice on two components of female body size (wing length and body mass) in Drosophila serrata. Using a dietary manipulation to alter female size and 828 male mate choice trials, we analysed linear and nonlinear sexual selection gradients on female mass and wing length. In contrast to theoretical expectations and prevailing empirical data, males exerted stabilizing rather than directional sexual selection on female body mass, a correlate of fecundity. Sexual selection was detected only among females with access to standard resource levels as an adult, with no evidence for sexual selection among resource-depleted females. Thus the mating success of females with the same body mass differed depending upon their access to resources as an adult. This suggests that males in this species may rely on signal traits to assess body mass rather than assessing it directly. Stabilizing rather than directional sexual selection on body mass together with recent evidence for stabilizing sexual selection on candidate signal traits in this species suggests that females may trade-off resources allocated to reproduction and sexual signalling.     

Behaviour before beauty: Signal weighting during mate selection in the butterfly Papilio polytes

Mating displays often contain multiple signals. Different combinations of these signals may be equally successful at attracting a mate, as environment and signal combination may influence relative signal weighting by choosy individuals. This variation in signal weighting among choosy individuals may facilitate the maintenance of polymorphic displays and signalling behaviour. One group of animals known for their polymorphic patterning are Batesian mimetic butterflies, where the interaction of sexual selection and predation pressures is hypothesized to influence the maintenance of polymorphic wing patterning and behaviour. Males in the female‐limited polymorphic Batesian mimetic butterfly Papilio polytes use female wing pattern and female activity levels when determining whom to court. They court stationary females with mimetic wing patterns more often than stationary females with non‐mimetic, male‐like wing patterns and active females more often than inactive females. It is unclear whether females modify their behaviour to increase (or decrease) their likelihood of receiving male courtship, or whether non‐mimetic females spend more time in cryptic environments than mimetic females, to compensate for their lack of mimicry‐driven predation protection (at the cost of decreased visibility to males). In addition, relative signal weighting of female wing pattern and activity to male mate selection is unknown. To address these questions, we conducted a series of observational studies of a polymorphic P. polytes population in a large butterfly enclosure. We found that males exclusively courted active females, irrespective of female wing pattern. However, males did court active non‐mimetic females significantly more often than expected given their relative abundance in the population. Females exhibited similar activity levels, and selected similar resting environments, irrespective of wing pattern. Our results suggest that male preference for non‐mimetic females may play an active role in the maintenance of the non‐mimetic female form in natural populations, where males are likely to be in the presence of active, as well as inactive, mimetic and non‐mimetic females.     

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 evolution of wing color: male mate choice opposes adaptive wing color divergence in Colias butterflies

Abstract Correlated evolution of mate signals and mate preference may be constrained if selection pressures acting on mate preference differ from those acting on mate signals. In particular, opposing selection pressures may act on mate preference and signals when traits have sexual as well as nonsexual functions. In the butterfly Colias philodice eriphyle , divergent selection on wing color across an elevational gradient in response to the thermal environment has led to increasing wing melanization at higher elevations. Wing color is also a long-range signal used by males in mate searching. We conducted experiments to test whether sexual selection on wing melanization via male mate choice acts in the same direction as natural selection on mate signals due to the thermal environment. We performed controlled mate choice experiments in the field over an elevational range of 1500 meters using decoy butterflies with different melanization levels. Also, we obtained a more direct estimate of the relation between wing color and sexual selection by measuring mating success in wild-caught females. Both our experiments showed that wing melanization is an important determinant of female mating success in C. p. eriphyle . However, a lack of elevational variation in male mate preference prevents coevolution of mate signals and mate preference, as males at all elevations prefer less-melanized females. We suggest that this apparently maladaptive mate choice may be maintained by differences in detectability between the morphs or by preservation of species recognition.     

Size Dependent Sexual Selection in Drosophila ananassae

Mate choice based on body size is widespread and can have numerous consequences. We present data, which show the effect of male and female body size on sexual selection in Drosophila ananassae. The relationships between wing size, locomotor activity, mating latency, courtship pattern, fertility and mating success were studied. Mating latency was negatively correlated with wing length and with locomotor activity, while wing length and locomotor activity was positively correlated in males as well as in females. In female- and male-choice, we found that mate choice influenced size-assortative mating by: (1) large and small males preferring to mate with large females, (2) large males successfully competing for large females, leaving small males to mate with small females. Males increased their reproductive success by mating with large and more fecund females. In addition, in pairs of long/short winged flies, long winged flies courted and mated more successfully than short winged flies and they also have longer duration of copulation and more progeny than short winged flies. We found sterile mating in pairs of small winged males and females.     

Deflective and intimidating eyespots: a comparative study of eyespot size and position in Junonia butterflies

Eyespots are conspicuous circular features found on the wings of several lepidopteran insects. Two prominent hypotheses have been put forth explaining their function in an antipredatory role. The deflection hypothesis posits that eyespots enhance survival in direct physical encounters with predators by deflecting attacks away from vital parts of the body, whereas the intimidation hypothesis posits that eyespots are advantageous by scaring away a potential predator before an attack. In the light of these two hypotheses, we investigated the evolution of eyespot size and its interaction with position and number within a phylogenetic context in a group of butterflies belonging to the genus Junonia. We found that larger eyespots tend to be found individually, rather than in serial dispositions. Larger size and conspicuousness make intimidating eyespots more effective, and thus, we suggest that our results support an intimidation function in some species of Junonia with solitary eyespots. Our results also show that smaller eyespots in Junonia are located closer to the wing margin, thus supporting predictions of the deflection hypothesis. The interplay between size, position, and arrangement of eyespots in relation to antipredation and possibly sexual selection, promises to be an interesting field of research in the future. Similarly, further comparative work on the evolution of absolute eyespot size in natural populations of other butterfly groups is needed.     

Differential Involvement of Hedgehog Signaling in Butterfly Wing and Eyespot Development

Butterfly eyespots may have evolved from the recruitment of pre-existent gene circuits or regulatory networks into novel locations on the wing. Gene expression data suggests one such circuit, the Hedgehog (Hh) signaling pathway and its target gene engrailed (en), was recruited from a role in patterning the anterior-posterior insect wing axis to a role patterning butterfly eyespots. However, while Junonia coenia expresses hh and en both in the posterior compartment of the wing and in eyespot centers, Bicyclus anynana lacks hh eyespot-specific expression. This suggests that Hh signaling may not be functioning in eyespot development in either species or that it functions in J. coenia but not in B. anynana. In order to test these hypotheses, we performed functional tests of Hh signaling in these species. We investigated the effects of Hh protein sequestration during the larval stage on en expression levels, and on wing size and eyespot size in adults. Hh sequestration led to significantly reduced en expression and to significantly smaller wings and eyespots in both species. But while eyespot size in B. anynana was reduced proportionately to wing size, in J. coenia, eyespots were reduced disproportionately, indicating an independent role of Hh signaling in eyespot development in J. coenia. We conclude that while Hh signaling retains a conserved role in promoting wing growth across nymphalid butterflies, it plays an additional role in eyespot development in some, but not all, lineages of nymphalid butterflies. We discuss our findings in the context of alternative evolutionary scenarios that led to the differential expression of hh and other Hh pathway signaling members across nymphalid species.     

Heat shock in the developmentally sensitive period of butterfly eyespots fails to increase fluctuating asymmetry

Fluctuating asymmetry (FA) is considered to provide a means of evaluating developmental stability and to reflect an individual's quality or the stress experienced during development. Stress is predicted to increase the phenotypic variation of both FA and trait size. In this study we examined the effect of a particular heat shock on both FA and size of eyespots in the butterfly, Bicyclus anynana. We also examined whether those eyespots thought to be involved in partner choice and sexual selection were particularly sensitive to stress. We applied a heat shock of 39.5 degrees C for 3 h before, during, and after a sensitive period in eyespot development. We examined the FA, variation in FA, size, and variation in size of five eyespots, two on the dorsal forewing (sexually selected traits), two on the ventral forewing, and one on the ventral hindwing (nonsexually selected traits). For each sex and treatment, the heat shock did not result in significant changes in mean trait size and FA nor in the variation of size and FA. There were no differences in the response to the heat shock between sexually and nonsexually selected traits. We discuss how the increased production of heat shock proteins, including HSP60, may have stabilized development and how this might explain the results.