The deflection hypothesis: eyespots on the margins of butterfly wings do not influence predation by lizards

Feeding experiments with lizards are used to examine the function of small eyespot markings found along the wing margins of many butterfly species. Such eyespots are frequently suggested to function by deflecting the attacks of vertebrate predators away from the vulnerable body towards the wing margins, which can tear easily; the eyespots are considered to mislead predators and to act as targets for their attacks. Such misdirected attacks give the butterflies a chance to evade capture, albeit sometimes losing pieces of wing tissue. As a model prey species, we used fruit-feeding individuals of the tropical butterfly Bicyclus anynana that were attacked in a standard way in laboratory cages by the anolis lizard, Anolis carolinensis . We also manipulated the butterflies' wing patterns by pasting eyespots on different parts of the wings to examine the deflection hypothesis in more detail. Our results indicate no influence on the lizard attacks either of the presence of eyespots, or of their position on the wings. The lizards attacked butterflies in a highly stereotyped manner both when the prey were presented on matching or on contrasting backgrounds. We thus found no support for the deflection hypothesis for attacks by insectivorous lizards. Indeed, our only support to date has been obtained for naïve flycatcher birds, but even this requires further corroboration. Although effective deflection may occur rather infrequently, except perhaps under certain ecological conditions such as high-density feeding of butterflies on fallen fruit, it may still be sufficiently consistent over time to have contributed to shaping the evolution of marginal eyespot patterns.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 661–667.     

Does predation maintain eyespot plasticity in Bicyclus anynana?

The butterfly Bicyclus anynana exhibits phenotypic plasticity involving the wet-season phenotype, which possesses marginal eyespots on the ventral surface of the wings, and the dry-season form, which lacks these eyespots. We examined the adaptive value of phenotypic plasticity of B. anynana in relation to the defence mechanisms of crypsis and deflection. We assessed the visibility differences between spotless and spotted butterflies against backgrounds of brown (dry season) or green (wet season) leaves. Spotless butterflies were highly cryptic and less predated by adult bird predators than were spotted ones when presented against brown leaf litter. However, the advantage of crypsis disappeared in the wet-season habitat as both forms were equally visible. In later experiments, naive birds presented with resting butterflies in the wet-season habitat tended to learn more rapidly to capture spotless butterflies, suggesting a slight selective advantage of possessing eyespots. Moreover, marginal eyespots increased significantly the escape probability of butterflies that were attacked by naive birds compared to those attacked by adult birds, although there were no differences in prey capture success within naive predators. Our results show that natural selection acts against eyespots in the dry season, favouring crypsis, whereas in the wet season it may favour eyespots as deflective patterns.     

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.     

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.     

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.     

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.     

Conserved developmental processes and the formation of evolutionary novelties: examples from butterfly wings

The origin and diversification of evolutionary novelties-lineage-specific traits of new adaptive value-is one of the key issues in evolutionary developmental biology. However, comparative analysis of the genetic and developmental bases of such traits can be difficult when they have no obvious homologue in model organisms. The finding that the evolution of morphological novelties often involves the recruitment of pre-existing genes and/or gene networks offers the potential to overcome this challenge. Knowledge about shared developmental processes obtained from extensive studies in model organisms can then be used to understand the origin and diversification of lineage-specific structures. Here, we illustrate this approach in relation to eyespots on the wings of Bicyclus anynana butterflies. A number of spontaneous mutations isolated in the laboratory affect eyespots, lepidopteran-specific features, and also processes that are shared by most insects. We discuss how eyespot mutants with disturbed embryonic development may help elucidate the genetic pathways involved in eyespot formation, and how venation mutants with altered eyespot patterns might shed light on mechanisms of eyespot development.     

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.     

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.     

Predator mimicry,not conspicuousness,explains the efficacy of butterfly eyespots

Large conspicuous eyespots on butterfly wings have been shown to deter predators. This has been traditionally explained by mimicry of vertebrate eyes, but recently the classic eye-mimicry hypothesis has been challenged. It is proposed that the conspicuousness of the eyespot, not mimicry, is what causes aversion due to sensory biases, neophobia or sensory overloads. We conducted an experiment to directly test whether the eye-mimicry or the conspicuousness hypothesis better explain eyespot efficacy. We used great tits (Parus major) as model predator, and tested their reaction towards animated images on a computer display. Birds were tested against images of butterflies without eyespots, with natural-looking eyespots, and manipulated spots with the same contrast but reduced resemblance to an eye, as well as images of predators (owls) with and without eyes. We found that mimetic eyespots were as effective as true eyes of owls and more efficient in eliciting an aversive response than modified, less mimetic but equally contrasting eyespots. We conclude that the eye-mimicry hypothesis explains our results better than the conspicuousness hypothesis and is thus likely to be an important mechanism behind the evolution of butterfly eyespots.     

Eyespots divert attacks by fish

Eyespots (colour patterns consisting of concentric rings) are found in a wide range of animal taxa and are often assumed to have an anti-predator function. Previous experiments have found strong evidence for an intimidating effect of eyespots against passerine birds. Some eyespots have been suggested to increase prey survival by diverting attacks towards less vital body parts or a direction that would facilitate escape. While eyespots in aquatic environments are widespread, their function is extremely understudied. Therefore, we investigated the protective function of eyespots against attacking fish. We used artificial prey and predator-naive three-spined sticklebacks (Gasterosteus aculeatus) as predators to test both the diversion (deflection) and the intimidation hypothesis. Interestingly, our results showed that eyespots smaller than the fish’ own eye very effectively draw the attacks of the fish towards them. Furthermore, our experiment also showed that this was not due to the conspicuousness of the eyespot, because attack latency did not differ between prey items with and without eyespots. We found little support for an intimidating effect by larger eyespots. Even though also other markings might misdirect attacks, we can conclude that the misdirecting function may have played an important role in the evolution of eyespots in aquatic environments.     

The role of eyespots as anti-predator mechanisms, principally demonstrated in the Lepidoptera

Eyespots are found in a variety of animals, in particular lepidopterans. The role of eyespots as antipredator mechanisms has been discussed since the 19th Century, with two main hypotheses invoked to explain their occurrence. The first is that large, centrally located eyespots intimidate predators by resembling the eyes of the predators' own enemies; the second, though not necessarily conflicting, hypothesis is that small, peripherally located eyespots function as markers to deflect the attacks of predators to non-vital regions of the body. A third possibility is also proposed; that eyespots intimidate predators merely because they are novel or rarely encountered salient features. These hypotheses are reviewed, with special reference given to avian predators, since these are likely to be the principal visually hunting predators of the lepidopterans considered. Also highlighted is the necessity to consider the potential influence of sexual selection on lepidopteran wing patterns, and the genetics and development of eyespot formation.     

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.     

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.     

Body size determines eyespot size and presence in coral reef fishes

Numerous organisms display conspicuous eyespots. These eye‐like patterns have been shown to effectively reduce predation by either deflecting strikes away from nonvital organs or by intimidating potential predators. While investigated extensively in terrestrial systems, determining what factors shape eyespot form in colorful coral reef fishes remains less well known. Using a broadscale approach we ask: How does the size of the eyespot relate to the actual eye, and at what size during ontogeny are eyespots acquired or lost? We utilized publicly available images to generate a dataset of 167 eyespot‐bearing reef fish species. We measured multiple features relating to the size of the fish, its eye, and the size of its eyespot. In reef fishes, the area of the eyespot closely matches that of the real eye; however, the eyespots “pupil” is nearly four times larger than the real pupil. Eyespots appear at about 20 mm standard length. However, there is a marked decrease in the presence of eyespots in fishes above 48 mm standard length; a size which is tightly correlated with significant decreases in documented mortality rates. Above 75–85 mm, the cost of eyespots appears to outweigh their benefit. Our results identify a “size window” for eyespots in coral reef fishes, which suggests that eyespot use is strictly body size‐dependent within this group.     

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 ‘sparkle’ in fake eyes – the protective effect of mimic eyespots in lepidoptera

Many species of lepidoptera bear conspicuous circular patterns on their wings, known as eyespots, that are hypothesised to protect their bearers against predatory birds. In this study, we focus on a small but ubiquitous feature occurring naturally in lepidopteran eyespots, namely the so‐called ‘sparkle’. The ‘pupil’ in an eyespot is often highlighted by a ‘sparkle’, which is hypothesised to mimic a natural corneal total light reflection evident as a highlight, twinkle, or sparkle in the vertebrate eye. In a study exploring the presence of such sparkles, we found that 53% of lepidopteran eyespots exceeding 1 mm in diameter have a central, pinpoint‐like ‘sparkle’, 12% have a marginal, crescent‐shaped ‘sparkle’, 13% have a semi‐circular ‘sparkle’, and 22% have an intermediate semi‐circular to crescent‐shaped ‘sparkle’. In the lepidopterans’ natural resting position, the marginal ‘sparkles’ are positioned in the upper part of the eyespots’‘pupil’ and thus may create the illusion of a spherical eyeball. The ‘sparkles’ in lepidopteran eyespots do not only appear white to humans, but also reflect ultraviolet light. White and UV‐reflecting ‘sparkles’ also appear ‘white’ for UV‐sensitive viewers such as birds, and thus may effectively mimic the natural highlight in vertebrate eyes as an area of total light reflection. In field experiments using lepidopteran dummies baited with a mealworm, we show that the ‘sparkle’ is one of several components of eyespots eliciting a deterrent effect and that eyespots with a ‘sparkle’ in a natural position have a stronger deterrent effect than those with a ‘sparkle’ in an unnatural position. These findings support the eye mimicry hypothesis that better vertebrate eye mimicry improves the deterrent effect of eyespots.     

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.