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Developmental and evolutionary mechanisms shaping butterfly eyespots
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Developmental and evolutionary mechanisms shaping butterfly eyespots
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Patrı´cia Beldade1,2 and Carolina M Peralta2 Butterfly eyespots are visually compelling models to study the reciprocal interactions between evolutionary and developmental processes that shape phenotypic variation. They are evolutionarily diversified, ecologically relevant, and developmentally tractable, and have made key contributions to linking genotype, development, phenotype and fitness. Advances in the availability of analytical tools (e.g. gene editing and visualization techniques) and resources (e.g. genomic and transcriptomic data) are boosting the detailed dissection of the mechanisms underlying eyespot development and evolution. Here, we review current knowledge on the ecology, development, and evolution of butterfly eyespots, with focus on recent advances. We also highlight a number of unsolved mysteries in our understanding of the patterns and processes underlying the diversification of these structures. Addresses 1 UMR5714, Universite´ Paul Sabatier – Toulouse 3, Toulouse, France 2 Instituto Gulbenkian de Cieˆncia, Oeiras, Portugal
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yet to be established about the types of changes in genotype that affect development to produce the natural phenotypic variation that fuels adaptive evolution and phenotypic diversification. To establish such principles, the eco-evo-devo community needs a broad representation of phylogenetic and morphological diversity [1,2], and the integration of detailed studies of various systems [3]. The color patterns on butterfly wings have provided much fascination and important insight. They have emerged as valuable systems for linking variation in genotypes, development, phenotypes, and fitness because they are evolutionarily diversified, ecologically relevant, and experimentally tractable [4,5]. In addition, they are a powerful tool for promoting the public understanding and appreciation of science, as well as for science education. Some butterfly species have become text-book examples of various important topics in ecoevo-devo, including mimicry and convergent evolution in Heliconius [6], and plasticity, constraint, and novelty in Bicyclus [7]. The increasing availability of genomic resources (e.g. transcriptome and whole genome sequences) and analytical tools (e.g. for visualization and testing gene function) for these and other lepidopterans are finally making it possible to properly probe the mechanisms behind these and other examples of the ‘endless forms most beautiful’ that so inspired Darwin and generations of biologists after him.
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Phenotypic diversity results from a balance between the developmental processes that translate genotype into phenotype, and the evolutionary forces that sort phenotypic variation in natural populations. Both development and evolution are shaped by interactions between organisms and their environment. The integration of concepts and approaches from ecology, evolutionary biology and developmental biology (eco-evo-devo) is, therefore, essential for a more complete understanding of the proximate and ultimate mechanisms shaping the evolution of adaptive traits. In recent years, a number of study systems have emerged in this quest, including traits from various insect groups (this issue); both in classical laboratory models (e.g. Drosophila wing spots and sex combs) and also in less studied species with exciting ecology (e.g. beetle horns, water strider legs, and butterfly wing patterns). Despite much progress, general principles are www.sciencedirect.com
Butterfly wings are covered with partly overlapping, monochromatic scales whose spatial arrangement can form exquisitely sophisticated color patterns. These scales, which inspired the name of the order of insects that includes butterflies and moths (Lepidoptera), have unique morphological and ultrastructural properties and continue to attract the attention of researchers interested in a complete understanding of butterfly wing patterns. Recent examples include the characterization of the relationship between scale size and color [8], and of scale development [9]. The arrangement of colored scales produce distinct types of pattern elements, including eyespots. Eyespots are made up of rings of contrasting colors and are one of the pattern elements described in the Nymphalid Ground Plan (NGP) [4]. The NGP is a representation of the relationships among color pattern elements on the wings of Nymphalid butterflies. It describes different groups of serially repeated pattern elements, including the eyespots Current Opinion in Insect Science 2016, 19:1–8
Please cite this article in press as: Beldade P, Peralta CM: Developmental and evolutionary mechanisms shaping butterfly eyespots, Curr Opin Insect Sci (2016), http://dx.doi.org/10.1016/ j.cois.2016.10.006
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or border ocelli, a designation that reflects their location (along the margin of the wings) and morphology (eye-like rings of color). While the NGP has been immensely useful and continues to guide comparative analyses of wing patterns between distantly and closely related species [10,11], researchers recognize some limitations. These include it being difficult to apply to diverged color patterns such as those of Heliconius butterflies, as well as the need for revision [12] and for caution with its interpretation [13]. There is documented variation for many aspects of eyespot morphology, including their number, color, size, and shape, with differences between species (examples in Figure 1a-b) and within species (geographical, seasonal, and sexual; examples in Figure 1c-d). There are also differences between wing surfaces of the same individual, and between individual eyespots on one wing surface. The spectacular diversity in butterfly eyespots is thought to be shaped by natural and sexual selection [14], and eyespot development has been characterized in different species, with focus on species such as Junonia coenia, Vanessa cardui, and Bicyclus anynana (Figure 1). Importantly, eyespots represent eco-evo-devo case studies for traits that are novel, serially repeated, and developmentally plastic. Here, we review our current understanding of ecological interactions eyespots play a role in, how they develop, and about their evolution.
Ecology of butterfly eyespots: predators, mates, and plasticity Insect pigmentation provides many visually compelling examples of adaptive evolution. Body pigmentation plays roles in thermal regulation, crypsis, and in different forms of visual communication with partners from the same or different species. Butterfly eyespots, in particular, are classically thought of as eye mimics that serve to avoid predation by either scaring off or confusing predators. There is experimental evidence consistent with both an ‘intimidation’ [15–17] and a ‘deflection’ [18,19,20] role. To fully distinguish between these alternative anti-predatory strategies, studies need to consider the eyespot pattern phenotype together with the species’ eyespotdisplay behavior. For example, otherwise-hidden pairs of eyespots flashed upon predator attack can startle and scare off predators, while series of marginal eyespots displayed in resting individuals might effectively attract the predators’ attention to the wing margin and away from the butterfly’s more vulnerable body. Experiments with manipulated eyespot phenotypes continue to shed light onto what aspects of eyespot patterns render them effective anti-predatory traits: eye mimicry or general conspicuousness [15], pairedness or different aspects of eyespot morphology [21,22]. It is also important to consider that eyespots in different species and different wing surfaces of the same species, and possibly even different eyespots on the same wing surface, might be shaped by different selection agents. This seems to be the case for the eyespots of B. anynana. While the ventral eyespots displayed Current Opinion in Insect Science 2016, 19:1–8
in resting butterflies serve as anti-predation distractions, those on the dorsal surface are displayed during courtship and the UV reflectance of their centers play a role in mate choice [14,23], by either females or males [24]. The eyespot phenotype not only affects an individual’s performance in relation to its environment, but is itself affected by the environmental conditions individuals are exposed to during development. Wing pattern formation depends on external abiotic factors such as photoperiod and temperature, which underlie striking examples of seasonal polyphenism described for different butterflies [4,25]. This type of developmental plasticity, whereby the same genotype can result in distinct phenotypes better suited to the environmental conditions adults live in, provides means for organisms to cope with environmental heterogeneity [26]. The physiological regulation of this phenomenon has been described for a number of species. In B. anynana, for example, it has been shown that temperature-induced changes in ecdysone dynamics affect different aspects of eyespot development. Manipulations of ecdysone levels during the larval wandering stage affects the size and brightness of eyespot foci [27], while manipulations during early pupal life affect the size of eyespot color rings [28]. Despite recent advances and ongoing efforts, important questions about the environmental regulation of eyespot formation remain unresolved. We do not know how changes in temperature affect ecdysone dynamics and how ecdysone dynamics affect eyespot development genes. We also know little about the evolution of plasticity in eyespot development. Studies characterizing the wing transcriptome for individuals developing in different conditions [29] can help with the former, and studies of plasticity in closely related species [30] or in differently plastic populations of the same species [31] can help with the latter.
Development of butterfly eyespots: cellular and molecular underpinnings Eyespots are arguably the wing pattern elements whose development is best understood. Classical experiments with manipulation of developing wings established that eyespot centers, or foci, are able to induce the production of rings of different colors around them. Destroying or transplanting the presumptive eyespot centers in early pupae respectively eliminates or displaces the corresponding eyespots [4]. Different successive stages in eyespot development can be recognized: 1) establishment of eyespot foci in late larval wings (Figure 2a), presumably involving signals from wing veins and the wing margin, 2) establishment of color rings in early pupae (Figure 2b), presumably involving focus-derived signals which commit surrounding cells to different color fates, and 3) pigment synthesis in late pupae, with light bright colors typically appearing before dark colors (Figure 2c). www.sciencedirect.com
Please cite this article in press as: Beldade P, Peralta CM: Developmental and evolutionary mechanisms shaping butterfly eyespots, Curr Opin Insect Sci (2016), http://dx.doi.org/10.1016/ j.cois.2016.10.006
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Eyespot diversity. Inter-species diversity and intra-specific variation in eyespot patterns. (a) Examples of Nymphalid species (genera Junonia, Vanessa, Pararge and Lasiommata) illustrate differences in the number of eyespots, as well as in number and color of eyespot rings. (b) Different species of Bicyclus and Mycalesis, two genera belonging to the Mycalesina subtribe, illustrate more micro-evolutionary differences; notably variation in relative size and position of eyespots that do not vary in number or color of rings. (c) Spontaneous mutants of B. anynana illustrate alleles of large effect on eyespot shape (Comet mutant), color composition (Frodo-Spread double mutant [64]), and size of some but not all eyespots (Pineye and 067). A number of B. anynana eyespot mutants have been mapped to chromosome sections [45]. (d) B. anynana females and males reared at different temperatures illustrate plasticity in wing patterns. Development at warmer versus cooler temperatures leads to phenotypes similar to those characteristic of the natural wet- and dry seasonal forms, respectively, with differences in eyespot size and background color. All images show the ventral surface of female hindwings, with the exception of P. aegeria (dorsal surface of a male hindwing) and male B. anynana in panel d.
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To establish the location of foci and the location of rings around them, models of eyespot formation have considered positional information via gradient (as illustrated in Figure 2b) or induction-type of mechanisms (see recent www.sciencedirect.com
discussions in [13,32]). Despite continued modeling work [33] and expansion of relevant experimental data [34,35], many issues remain unsolved. These include the nature, number, and mode of action of the signals that Current Opinion in Insect Science 2016, 19:1–8
Please cite this article in press as: Beldade P, Peralta CM: Developmental and evolutionary mechanisms shaping butterfly eyespots, Curr Opin Insect Sci (2016), http://dx.doi.org/10.1016/ j.cois.2016.10.006
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Eyespot development. Stages of eyespot development and gene functional analysis illustrated for B. anynana. (a) Determination of eyespot foci in larval wings happens in the final larval instar, presumably by the action of genes such as Antennapedia (green), Notch (yellow) and Distalless (red). Protein products of these genes are shown here in two presumptive eyespots on the ventral surface of the hindwing [41], corresponding to the section of the adult wing on the left-most panel. (b) Determination of eyespot rings around eyespot foci occurs in early pupal wings. First, there is signaling from eyespot foci, possibly by secretion of a morphogen that diffuses away to establish a concentration gradient (left panel). Epidermal cells at different distances from the focus respond to the signal by expressing different transcription factors, such as Engrailed (green) and Spalt (blue). (c) Pigment deposition occurs during late pupal wing development, with different color pigments appearing in a stereotypical order [65]. Images represent pigment deposition progression in a selected eyespot, with younger to older pupal wings shown from left to right. (d) CRISPR-Cas9-mediated knock-down of pigmentation enzymes Dopa decarboxylase (Ddc) and Ebony by injections in early embryos produces mosaic adults, with a fraction of the colored scales affected. Images represent the ventral surface of female hindwings and close-up views of areas within dotted rectangles. While Ddc knock-down removes pigment (especially visible in the eyespot black disk), Ebony knock-down converts yellow and light brown to darker brown. For comparison, we show a wildtype phenotype from an unmanipulated female reared at the same temperature.
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establish foci and of those that establish the rings of color around them. They also include the characterization of the actual connection between the ring patterning genes expressed in early pupal wings (Figure 2b) and the pigment synthesis pathways that produce the colors seen in late pupal wings (Figure 2c). Knowledge from other Current Opinion in Insect Science 2016, 19:1–8
systems, such as Drosophila wing pigmentation [36], as well as developments in methods for the analysis of butterfly wing development and of gene function [37– 40] and in availability of genomic data can help clarify these and other aspects of the cellular and molecular basis of eyespot development. www.sciencedirect.com
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In relation to the genes associated with eyespot formation, most of what is known is based on studies of expression patterns of well-known conserved genes in developing butterfly wings. These studies revealed the expression of genes such as Distal-less, Notch and Antennapedia [41] in the presumptive eyespot field, at different stages of eyespot development (Figure 2a-b) in different species [42,43]. Expansion of data on transcriptomes for developing wings [29,44,45,46] and on whole genome sequences for various butterfly species [47] is allowing a more unbiased identification of eyespot development genes. A complete understanding of the genetic basis of eyespot development will require testing the function of those genes whose expression patterns and/or levels correlate with eyespot development. Tests of gene function have had some but limited success, despite attempts with a variety of approaches, including pharmacological manipulations [48], germline transformation [49,50], ectopic expression [51], RNAi [52], and genome editing [53]. For example, functional analyses by different methods and on different species confirmed the actual involvement in eyespot formation of the candidate gene Distal-less, expressed in the presumptive eyespot field. Curiously, the results of different studies proposed opposite functions for this gene: eyespot-promoting role in B. anynana [49] and Junonia orithya [51], and eyespotrepressing role in J. coenia and V. cardui [53]. The interpretation of the results of manipulations of developmental genes such as Distal-less might be confounded by fact that it can be involved in different stages of eyespot formation (e.g. it is expressed during the establishment of foci and of color rings), as well as in other developmental processes (e.g. it is involved in appendage development). While the CRISPR-Cas9 genome editing tool holds great promise of furthering analysis of gene function in evodevo models, including butterfly wing patterns, its use is not without important limitations. For candidate eyespot development genes that also play a role in other developmental processes, for example, knock-down by injections in embryos can result in lethality before any wing patterns are formed. Studying adult mosaics having cells with and without CRISPR-induced deletions (illustrated in Figure 2d) can help overcome embryonic lethality, but it can also raise difficulties with the interpretation and validation of CRISPR-related adult phenotypes. Importantly, while CRISPR-based and other functional tests can inform about the role of candidate genes in the development of eyespots in different species, they cannot identify new candidate genes nor can they provide information about which eyespot genes contribute to variation in eyespot patterns. To those ends, we need different approaches. Analysis of transcriptomes of developing wings during color pattern formation can identify additional putative eyespot development genes [29,44], and genetic mapping can help identify loci carrying allelic www.sciencedirect.com
variation responsible for intra- and possibly inter-species differences in eyespot patterns [45,54].
Evolution of butterfly eyespots: constraints, co-option, and genetics of variation Different hypotheses have been proposed for the origin of the series of marginal eyespots that decorate the wings of many Nymphalid butterflies. The NGP discussed above assumes a scenario where eyespots would have originated from the modification of ancestral marginal bands that ‘resolved’ into a series of simple ocelli that subsequently diversified [4]. Recent work based on a phylogenetic analysis of eyespots and eyespot-like pattern elements in many species proposed a scenario whereby individual eyespots would have been added independently, and originated initially as simple spots that later acquired rings around them (discussed in [13]). Whether the serially repeated eyespots originated simultaneously or independently, they share the same developmental logic and underlying genes. Studies in laboratory populations of B. anynana have explored the extent to which this shared genetic and developmental basis can bias the production of eyespot pattern variants and constrain the independent evolution of serial repeats. Artificial selection targeting pairs of eyespots in B. anynana showed that while independent changes in eyespot size were easily achieved [54], the same was not true for independent changes in eyespot color composition [55]. The likelihood of independent evolution of eyespots on the same wing surface seems to depend on the extent of the compartmentalization, or eyespot-specificity, of the effects of eyespot development genes and of the components of pattern induction [13,55,56]. The origin of eyespots, similarly to what has been proposed for other examples of novel traits, is thought to have occurred via co-option of genetic circuitries involved in the development of shared traits. Eyespots were suggested to have originated from the redeployment to specific wing locations of genes involved in the development of wing margins [57], or of genes implicated in response to wounds [58,59]. Perhaps unsurprisingly, given that pleiotropy is common for developmental genes, eyespot formation also shares genes with other processes, such as appendage and embryonic development (reviewed in [13]). The characterization of the expression of several candidate genes in the establishment of eyespot foci in different species has started to shed light onto what genes or networks were recruited at the origin of Nymphalid eyespots [42,43]. However, this type of analyses has some important limitations. First, expression in presumptive eyespots does not prove that a gene plays a role in eyespot development or that it interacts with coexpressing genes (see discussion about the need for functional studies in the ‘Development of butterfly eyespots’ section). Second, presence/absence of protein product is not sufficient for inferring homology relationships Current Opinion in Insect Science 2016, 19:1–8
Please cite this article in press as: Beldade P, Peralta CM: Developmental and evolutionary mechanisms shaping butterfly eyespots, Curr Opin Insect Sci (2016), http://dx.doi.org/10.1016/ j.cois.2016.10.006
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between structures in different lineages (see discussion about ‘developmental systems drift’ in [42]). Finally, the standard approach to identify genes involved in the origin of novel traits by focusing on the expression of conserved genes biases findings toward evidence for co-option. Future studies should also focus on candidate novel or fast evolving genes found to be expressed during eyespot formation [44,46]. Aside being models for the study of the diversification of serial repeats and of the origin of novel traits, eyespots are one of the systems successfully used in micro-evolutionary evo-devo studies exploring the genetic basis of the inter-individual variation that fuels natural selection. Studies in B. anynana have unraveled both alleles of subtle effect that allowed gradual response to artificial selection, and alleles of large effect on different aspects of eyespot morphology (Figure 1c). Attempts to identify the actual loci responsible for the different types of phenotypic variants has proceeded by genetic mapping approaches, either focused on candidate genes [54] or on polymorphic markers throughout the genome [45]. The identification of the genes and, eventually, of the nucleotide changes responsible for phenotypic variation in eyespot traits can provide answers to key questions in evo-devo for which we lack definitive answers. These include the nature of the DNA sequence changes responsible for evolutionarily relevant phenotypic variation, as well as the extent of overlap between loci that carry alleles of large effect typically studied in the lab and those that carry allelic variants of subtle effect segregating in natural populations, and those accounting for fixed differences across species.
Overview and perspectives Studies of butterfly eyespots have contributed to the ecoevo-devo community’s understanding of phenotypic diversification; all the way from genetic variation, to variation in different components of the mechanisms underlying pattern formation, to intra-specific variation in phenotype, to diversification. Eyespots are valuable models for a number of key topics in eco-evo-devo: 1) the origin and modification of novel traits, 2) developmental constraints and the diversification of serially repeated traits, 3) the regulation and evolution of adaptive developmental plasticity, and 4) the genetic and genomic basis of variation and diversity in adaptive traits. We summarized current knowledge about the ecological significance, developmental underpinnings, and evolution of butterfly eyespots, with emphasis on findings from the past couple of years. For each of the topics covered, we also highlighted a number of open questions, many of which we expect can be solved with analytical tools and resources now available. An important challenge for the future will be to fully integrate the ecological, developmental and evolutionary Current Opinion in Insect Science 2016, 19:1–8
processes that interact to shape diversity in butterfly eyespots. Toward this, we need to consider explicitly that not all eyespots, even those on the same wing, are necessarily shaped by the same mechanisms. All eyespots develop around central organizers but can have different morphologies (e.g. color, number and shape of rings), respond differently to environmental [27,28] and genetic (Figure 2c) factors, be involved in different ecological interactions, and diversify more or less independently. Even the distinct color rings of the same eyespot show some level of independence seen, for example, in how they respond to manipulations of temperature and hormone levels during development [28]. We have discussed the eyespots that decorate the margins of the wings of many Nymphalid butterflies, but eyespotlike pattern elements can be found elsewhere. They can be found in locations of Nymphalid wings other than the margins, and in wings of Lepidopterans other than Nymphalids (examples in [42]). They can also appear in caterpillars, in invertebrates other than lepidopterans, and in different vertebrates, including fish, birds, and reptiles [60–63]. It will be interesting to probe the mechanisms, proximate and ultimate, of this example of convergent evolution and reveal the extent of the overlap in underlying genes, developmental logic, and evolutionary forces shaping the diversity in these pigmentation patterns.
Acknowledgements The authors wish to acknowledge Erik van Bergen and Elvira Lafuente for comments on the manuscript, and Yara Rodrigues (Figure 1d), Suzanne V Saenko (Figure 2b), Marcel J Dix (Figure 2b), Nicolien Pul (Figure 2c) for images. The work was funded by grant PTDC/BIA-EVF/0017/2014 awarded to PB by the Portuguese funding agency, ‘Fundac¸a˜o para a Cieˆncia e Tecnologia’.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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10. Suzuki TK, Tomita S, Sezutsu H: Gradual and contingent evolutionary emergence of leaf mimicry in butterfly wing patterns. BMC Evol Biol 2014, 14:229. 11. Abbasi R, Marcus JM: Colour pattern homology and evolution in Vanessa butterflies (Nymphalidae: Nymphalini): eyespot characters. J Evol Biol 2015, 28:2009-2026. 12. Otaki JM: Color pattern analysis of nymphalid butterfly wings: revision of the nymphalid groundplan. Zoolog Sci 2012, 29:568576. 13. Monteiro A: Origin, development, and evolution of butterfly eyespots. Annu Rev Entomol 2015, 60:253-271. Critical review of our knowledge about the origin, development and evolution of butterfly eyespots, with discussion of alternative scenarios to explain the origin of eyespots and of eyespot patterns, as well as constraints on the diversification of different aspects of eyespot morphology. 14. Oliver JC, Robertson KA, Monteiro A: Accommodating natural and sexual selection in butterfly wing pattern evolution. Proc Biol Sci 2009, 276:2369-2375. 15. Stevens M, Hardman CJ, Stubbins CL: Conspicuousness, not eye mimicry, makes ‘‘eyespots’’ effective antipredator signals. Behav Ecol 2008, 19:525-531. 16. Kodandaramaiah U, Vallin A, Wiklund C: Fixed eyespot display in a butterfly thwarts attacking birds. Anim Behav 2009, 77:1415-1419. 17. Merilaita S, Vallin A, Kodandaramaiah U, Dimitrova M, Ruuskanen S, Laaksonen T: Number of eyespots and their intimidating effect on naive predators in the peacock butterfly. Behav Ecol 2011, 22:1326-1331. 18. Olofsson M, Vallin A, Jakobsson S, Wiklund C: Marginal eyespots on butterfly wings deflect bird attacks under low light intensities with UV wavelengths. PLoS One 2010, 5:e10798. 19. Vallin A, Dimitrova M, Kodandaramaiah U, Merilaita S: Deflective effect and the effect of prey detectability on anti-predator function of eyespots. Behav Ecol Sociobiol 2011, 65:1629-1636. 20. Prudic KL, Stoehr AM, Wasik BR, Monteiro A: Eyespots deflect predator attack increasing fitness and promoting the evolution of phenotypic plasticity. Proc Biol Sci 2015, 282:20141531. The authors use praying mantis (predator) and live butterflies representing alternative seasonal forms of Bicyclus anynana (prey) to test the antipredatory value of the larger conspicuous eyespots in wet season butterflies. They confirm the old hypothesis that such eyespots deflect predator attack to the wing margin and make it more likely for the butterfly to escape alive. 21. Mukherjee R, Kodandaramaiah U: What makes eyespots intimidating-the importance of pairedness. BMC Evol Biol 2015, 15:34. The authors use paper models of Junonia almana (prey) and domestic chickens (predator) in indoor experiments to test whether eyespots intimidate predators because of eye-mimicry or conspicuousness. The use of paper models with manipulated eyespot phenotypes offers many possibilities for the study of how eyespots minimize predation.
28. Mateus ARA, Marques-Pita M, Oostra V, Lafuente E, Brakefield PM, Zwaan BJ, Beldade P: Adaptive developmental plasticity: compartmentalized responses to environmental cues and to corresponding internal signals provide phenotypic flexibility. BMC Biol 2014, 12:97. By quantifying the effect of temperature during pre-adult development and of injections of ecdysone during pupal life, this study shows that different eyespots and eyespot color rings are differently plastic. The compartmentalization of effects of the external cue and corresponding internal signal providing information to developing organs was likely shaped by past selection and impacts future change. 29. Daniels EV, Murad R, Mortazavi A, Reed RD: Extensive transcriptional response associated with seasonal plasticity of butterfly wing patterns. Mol Ecol 2014, 23:6123-6134. 30. Oostra V, Brakefield PM, Hiltemann Y, Zwaan BJ, Brattstro¨m O: On the fate of seasonally plastic traits in a rainforest butterfly under relaxed selection. Ecol Evol 2014, 4:2654-2667. 31. Daniels EV, Mooney KA, Reed RD: Seasonal wing colour plasticity varies dramatically between buckeye butterfly populations in different climatic zones. Ecol Entomol 2012, 37:155-159. 32. Otaki JM: Color-pattern analysis of eyespots in butterfly wings: a critical examination of morphogen gradient models. Zoolog Sci 2011, 28:403-413. 33. Sekimura T, Venkataraman C, Madzvamuse A: A model for selection of eyespots on butterfly wings. PLoS One 2015, 10:e0141434. 34. Ohno Y, Otaki JM: Spontaneous long-range calcium waves in developing butterfly wings. BMC Dev Biol 2015, 15:17. The authors describe the occurrence of spontaneous low-frequency waves of calcium travelling over long distances in developing pupal wings. This discovery opens the door for studies of this and possibly other ions in the development of butterfly wing color patterns, including eyespots. 35. Taira W, Otaki JM: Butterfly wings are three-dimensional: pupal cuticle focal spots and their associated structures in Junonia butterflies. PLoS One 2016, 11:e0146348. 36. Wittkopp PJ, Beldade P: Development and evolution of insect pigmentation: genetic mechanisms and the potential consequences of pleiotropy. Semin Cell Dev Biol 2009, 20:65-71. 37. Monteiro A, Prudic KM: Multiple approaches to study color pattern evolution in butterflies. Trends Evol Biol 2010, 2:2. 38. Iwata M, Ohno Y, Otaki JM: Real-time in vivo imaging of butterfly wing development: revealing the cellular dynamics of the pupal wing tissue. PLoS One 2014, 9:e89500.
22. Ho S, Schachat SR, Piel WH, Monteiro A: Attack risk for butterflies changes with eyespot number and size. R Soc Open Sci 2016, 3:150614.
39. Adhikari K, Otaki JM: A single-wing removal method to assess correspondence between gene expression and phenotype in butterflies: the case of distal-less. Zoolog Sci 2016, 33:13-20.
23. Robertson KA, Monteiro A: Female Bicyclus anynana butterflies choose males on the basis of their dorsal UV-reflective eyespot pupils. Proc Biol Sci 2005, 272:1541-1546.
40. Fujiwara H, Nishikawa H: Functional analysis of genes involved in color pattern formation in Lepidoptera. Curr Opin Insect Sci 2016, 17:16-23.
24. Prudic KL, Jeon C, Cao H, Monteiro A: Developmental plasticity in sexual roles of butterfly species drives mutual sexual ornamentation. Science 2011, 331:73-75.
41. Saenko SV, Marialva MSP, Beldade P: Involvement of the conserved Hox gene Antennapedia in the development and evolution of a novel trait. Evodevo 2011, 2:9.
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42. Shirai LT, Saenko SV, Keller RA, Jero´nimo MA, Brakefield PM, Descimon H, Wahlberg N, Beldade P: Evolutionary history of the recruitment of conserved developmental genes in association to the formation and diversification of a novel trait. BMC Evol Biol 2012, 12:21. 43. Oliver JC, Tong XL, Gall LF, Piel WH, Monteiro A: A single origin for nymphalid butterfly eyespots followed by widespread loss of associated gene expression. PLoS Genet 2012, 8:e1002893. 44. Beldade P, Rudd S, Gruber JD, Long AD: A wing expressed sequence tag resource for Bicyclus anynana butterflies, an evo-devo model. BMC Genomics 2006, 7:130. 45. Beldade P, Saenko SV, Pul N, Long AD: A gene-based linkage map for Bicyclus anynana butterflies allows for a comprehensive analysis of synteny with the lepidopteran reference genome. PLoS Genet 2009, 5:e1000366. 46. Connahs H, Rhen T, Simmons RB: Transcriptome analysis of the painted lady butterfly, Vanessa cardui during wing color pattern development. BMC Genomics 2016, 17:270. Using next-generation sequencing, the authors characterize the transcriptome of developing wings of V. cardui. They highlight a list of transcripts that are expressed in developing wings of other butterflies, but have no clear homolog in other metazoans. It will be interesting to study the function in wing pattern evo-devo of candidate novel genes such as these. 47. Challis R, Kumar S, Dasmahapatra K, Jiggins CD, Blaxter M: Lepbase: the Lepidopteran genome database. bioRxiv 2016 http://dx.doi.org/10.1101/056994. 48. Martin A, Reed RD: Wnt signaling underlies evolution and development of the butterfly wing pattern symmetry systems. Dev Biol 2014, 395:367-378. 49. Monteiro A, Chen B, Ramos DM, Oliver JC, Tong X, Guo M, Wang WK, Fazzino L, Kamal F: Distal-less regulates eyespot patterns and melanization in Bicyclus butterflies. J Exp Zool B Mol Dev Evol 2013, 320:321-331. 50. Tong X, Hrycaj S, Podlaha O, Popadic A, Monteiro A: Overexpression of Ultrabithorax alters embryonic body plan and wing patterns in the butterfly Bicyclus anynana. Dev Biol 2014, 394:357-366. 51. Dhungel B, Ohno Y, Matayoshi R, Iwasaki M, Taira W, Adhikari K, Gurung R, Otaki JM: Distal-less induces elemental color patterns in Junonia butterfly wings. Zool Lett 2016, 2:4. Using a baculovirus-mediated gene transfer technique, the authors tested the role of Distal-less in eyespot pattern development in J. orithya by ectopically expressing it in pupal wings. Ectopic expression can test gene sufficiency and is a valuable complementary approach to the tests of gene necessity achieved with knowck-down tools. 52. Terenius O, Papanicolaou A, Garbutt JS, Eleftherianos I, Huvenne H, Kanginakudru S, Albrechtsen M, An C, Aymeric JL, Barthel A et al.: RNA interference in Lepidoptera: an overview of
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successful and unsuccessful studies and implications for experimental design. J Insect Physiol 2011, 57:231-245. 53. Zhang L, Reed RD: Genome editing in butterflies reveals that spalt promotes and Distal-less represses eyespot colour patterns. Nat Commun 2016, 7:11769. 54. Beldade P, Brakefield PM, Long AD: Contribution of Distal-less to quantitative variation in butterfly eyespots. Nature 2002, 415:315-318. Taking advantage of the CRISPR-Cas9 genome editing tool, the authors confirmed a role for transcription factors Spalt and Distal-less in the development of the eyespots of V. cardui and J. coenia. This is the first use of CRISPR-Cas9 to study the function of genes expressed in association with developing eyespots. Many more such studies are expected. 55. Allen CE, Beldade P, Zwaan BJ, Brakefield PM: Differences in the selection response of serially repeated color pattern characters: standing variation, development, and evolution. BMC Evol Biol 2008, 8:94. 56. Monteiro A, Prijs J, Bax M, Hakkaart T, Brakefield PM: Mutants highlight the modular control of butterfly eyespot patterns. Evol Dev 2003, 5:180-187. 57. Held LI: Rethinking butterfly eyespots. Evol Biol 2012, 40:158168. 58. Monteiro A, Glaser G, Stockslager S, Glansdorp N, Ramos D: Comparative insights into questions of lepidopteran wing pattern homology. BMC Dev Biol 2006, 6:52. 59. Saenko SV, French V, Brakefield PM, Beldade P: Conserved developmental processes and the formation of evolutionary novelties: examples from butterfly wings. Philos Trans R Soc Lond B Biol Sci 2008, 363:1549-1555. 60. Skelhorn J, Holmes GG, Hossie TJ, Sherratt TN: Eyespots. Curr Biol 2016, 26:R52-R54. 61. Castellano S, Cermelli P: Preys’ exploitation of predators’ fear: when the caterpillar plays the Gruffalo. Proc Biol Sci 2015, 282:20151786. 62. Ohno Y, Otaki JM: Eyespot colour pattern determination by serial induction in fish: Mechanistic convergence with butterfly eyespots. Sci Rep 2012, 2:290. 63. Sun K, Meiklejohn KA, Faircloth BC, Glenn TC, Braun EL, Kimball RT: The evolution of peafowl and other taxa with ocelli (eyespots): a phylogenomic approach. Proc Biol Sci 2014:281. 64. Saenko SV, Brakefield PM, Beldade P: Single locus affects embryonic segment polarity and multiple aspects of an adult evolutionary novelty. BMC Biol 2010, 8:111. 65. Koch PB, Lorenz U, Brakefield PM, ffrench-Constant RH: Butterfly wing pattern mutants: developmental heterochrony and coordinately regulated phenotypes. Dev Genes Evol 2000, 210:536-544.
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Developmental and evolutionary mechanisms shaping butterfly eyespots
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Patrı´cia Beldade1,2 and Carolina M Peralta2 Butterfly eyespots are visually compelling models to study the reciprocal interactions between evolutionary and developmental processes that shape phenotypic variation. They are evolutionarily diversified, ecologically relevant, and developmentally tractable, and have made key contributions to linking genotype, development, phenotype and fitness. Advances in the availability of analytical tools (e.g. gene editing and visualization techniques) and resources (e.g. genomic and transcriptomic data) are boosting the detailed dissection of the mechanisms underlying eyespot development and evolution. Here, we review current knowledge on the ecology, development, and evolution of butterfly eyespots, with focus on recent advances. We also highlight a number of unsolved mysteries in our understanding of the patterns and processes underlying the diversification of these structures. Addresses 1 UMR5714, Universite´ Paul Sabatier – Toulouse 3, Toulouse, France 2 Instituto Gulbenkian de Cieˆncia, Oeiras, Portugal
Current Opinion in Insect Science 2016, 19:xx–yy This review comes from a themed issue on Development and regulation Edited by Haruhiko Fujiwara and Yoshinori Tomoyasu
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yet to be established about the types of changes in genotype that affect development to produce the natural phenotypic variation that fuels adaptive evolution and phenotypic diversification. To establish such principles, the eco-evo-devo community needs a broad representation of phylogenetic and morphological diversity [1,2], and the integration of detailed studies of various systems [3]. The color patterns on butterfly wings have provided much fascination and important insight. They have emerged as valuable systems for linking variation in genotypes, development, phenotypes, and fitness because they are evolutionarily diversified, ecologically relevant, and experimentally tractable [4,5]. In addition, they are a powerful tool for promoting the public understanding and appreciation of science, as well as for science education. Some butterfly species have become text-book examples of various important topics in ecoevo-devo, including mimicry and convergent evolution in Heliconius [6], and plasticity, constraint, and novelty in Bicyclus [7]. The increasing availability of genomic resources (e.g. transcriptome and whole genome sequences) and analytical tools (e.g. for visualization and testing gene function) for these and other lepidopterans are finally making it possible to properly probe the mechanisms behind these and other examples of the ‘endless forms most beautiful’ that so inspired Darwin and generations of biologists after him.
Butterfly eyespots and eyespot patterns 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
Phenotypic diversity results from a balance between the developmental processes that translate genotype into phenotype, and the evolutionary forces that sort phenotypic variation in natural populations. Both development and evolution are shaped by interactions between organisms and their environment. The integration of concepts and approaches from ecology, evolutionary biology and developmental biology (eco-evo-devo) is, therefore, essential for a more complete understanding of the proximate and ultimate mechanisms shaping the evolution of adaptive traits. In recent years, a number of study systems have emerged in this quest, including traits from various insect groups (this issue); both in classical laboratory models (e.g. Drosophila wing spots and sex combs) and also in less studied species with exciting ecology (e.g. beetle horns, water strider legs, and butterfly wing patterns). Despite much progress, general principles are www.sciencedirect.com
Butterfly wings are covered with partly overlapping, monochromatic scales whose spatial arrangement can form exquisitely sophisticated color patterns. These scales, which inspired the name of the order of insects that includes butterflies and moths (Lepidoptera), have unique morphological and ultrastructural properties and continue to attract the attention of researchers interested in a complete understanding of butterfly wing patterns. Recent examples include the characterization of the relationship between scale size and color [8], and of scale development [9]. The arrangement of colored scales produce distinct types of pattern elements, including eyespots. Eyespots are made up of rings of contrasting colors and are one of the pattern elements described in the Nymphalid Ground Plan (NGP) [4]. The NGP is a representation of the relationships among color pattern elements on the wings of Nymphalid butterflies. It describes different groups of serially repeated pattern elements, including the eyespots Current Opinion in Insect Science 2016, 19:1–8
Please cite this article in press as: Beldade P, Peralta CM: Developmental and evolutionary mechanisms shaping butterfly eyespots, Curr Opin Insect Sci (2016), http://dx.doi.org/10.1016/ j.cois.2016.10.006
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or border ocelli, a designation that reflects their location (along the margin of the wings) and morphology (eye-like rings of color). While the NGP has been immensely useful and continues to guide comparative analyses of wing patterns between distantly and closely related species [10,11], researchers recognize some limitations. These include it being difficult to apply to diverged color patterns such as those of Heliconius butterflies, as well as the need for revision [12] and for caution with its interpretation [13]. There is documented variation for many aspects of eyespot morphology, including their number, color, size, and shape, with differences between species (examples in Figure 1a-b) and within species (geographical, seasonal, and sexual; examples in Figure 1c-d). There are also differences between wing surfaces of the same individual, and between individual eyespots on one wing surface. The spectacular diversity in butterfly eyespots is thought to be shaped by natural and sexual selection [14], and eyespot development has been characterized in different species, with focus on species such as Junonia coenia, Vanessa cardui, and Bicyclus anynana (Figure 1). Importantly, eyespots represent eco-evo-devo case studies for traits that are novel, serially repeated, and developmentally plastic. Here, we review our current understanding of ecological interactions eyespots play a role in, how they develop, and about their evolution.
Ecology of butterfly eyespots: predators, mates, and plasticity Insect pigmentation provides many visually compelling examples of adaptive evolution. Body pigmentation plays roles in thermal regulation, crypsis, and in different forms of visual communication with partners from the same or different species. Butterfly eyespots, in particular, are classically thought of as eye mimics that serve to avoid predation by either scaring off or confusing predators. There is experimental evidence consistent with both an ‘intimidation’ [15–17] and a ‘deflection’ [18,19,20] role. To fully distinguish between these alternative anti-predatory strategies, studies need to consider the eyespot pattern phenotype together with the species’ eyespotdisplay behavior. For example, otherwise-hidden pairs of eyespots flashed upon predator attack can startle and scare off predators, while series of marginal eyespots displayed in resting individuals might effectively attract the predators’ attention to the wing margin and away from the butterfly’s more vulnerable body. Experiments with manipulated eyespot phenotypes continue to shed light onto what aspects of eyespot patterns render them effective anti-predatory traits: eye mimicry or general conspicuousness [15], pairedness or different aspects of eyespot morphology [21,22]. It is also important to consider that eyespots in different species and different wing surfaces of the same species, and possibly even different eyespots on the same wing surface, might be shaped by different selection agents. This seems to be the case for the eyespots of B. anynana. While the ventral eyespots displayed Current Opinion in Insect Science 2016, 19:1–8
in resting butterflies serve as anti-predation distractions, those on the dorsal surface are displayed during courtship and the UV reflectance of their centers play a role in mate choice [14,23], by either females or males [24]. The eyespot phenotype not only affects an individual’s performance in relation to its environment, but is itself affected by the environmental conditions individuals are exposed to during development. Wing pattern formation depends on external abiotic factors such as photoperiod and temperature, which underlie striking examples of seasonal polyphenism described for different butterflies [4,25]. This type of developmental plasticity, whereby the same genotype can result in distinct phenotypes better suited to the environmental conditions adults live in, provides means for organisms to cope with environmental heterogeneity [26]. The physiological regulation of this phenomenon has been described for a number of species. In B. anynana, for example, it has been shown that temperature-induced changes in ecdysone dynamics affect different aspects of eyespot development. Manipulations of ecdysone levels during the larval wandering stage affects the size and brightness of eyespot foci [27], while manipulations during early pupal life affect the size of eyespot color rings [28]. Despite recent advances and ongoing efforts, important questions about the environmental regulation of eyespot formation remain unresolved. We do not know how changes in temperature affect ecdysone dynamics and how ecdysone dynamics affect eyespot development genes. We also know little about the evolution of plasticity in eyespot development. Studies characterizing the wing transcriptome for individuals developing in different conditions [29] can help with the former, and studies of plasticity in closely related species [30] or in differently plastic populations of the same species [31] can help with the latter.
Development of butterfly eyespots: cellular and molecular underpinnings Eyespots are arguably the wing pattern elements whose development is best understood. Classical experiments with manipulation of developing wings established that eyespot centers, or foci, are able to induce the production of rings of different colors around them. Destroying or transplanting the presumptive eyespot centers in early pupae respectively eliminates or displaces the corresponding eyespots [4]. Different successive stages in eyespot development can be recognized: 1) establishment of eyespot foci in late larval wings (Figure 2a), presumably involving signals from wing veins and the wing margin, 2) establishment of color rings in early pupae (Figure 2b), presumably involving focus-derived signals which commit surrounding cells to different color fates, and 3) pigment synthesis in late pupae, with light bright colors typically appearing before dark colors (Figure 2c). www.sciencedirect.com
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Eyespot diversity. Inter-species diversity and intra-specific variation in eyespot patterns. (a) Examples of Nymphalid species (genera Junonia, Vanessa, Pararge and Lasiommata) illustrate differences in the number of eyespots, as well as in number and color of eyespot rings. (b) Different species of Bicyclus and Mycalesis, two genera belonging to the Mycalesina subtribe, illustrate more micro-evolutionary differences; notably variation in relative size and position of eyespots that do not vary in number or color of rings. (c) Spontaneous mutants of B. anynana illustrate alleles of large effect on eyespot shape (Comet mutant), color composition (Frodo-Spread double mutant [64]), and size of some but not all eyespots (Pineye and 067). A number of B. anynana eyespot mutants have been mapped to chromosome sections [45]. (d) B. anynana females and males reared at different temperatures illustrate plasticity in wing patterns. Development at warmer versus cooler temperatures leads to phenotypes similar to those characteristic of the natural wet- and dry seasonal forms, respectively, with differences in eyespot size and background color. All images show the ventral surface of female hindwings, with the exception of P. aegeria (dorsal surface of a male hindwing) and male B. anynana in panel d.
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To establish the location of foci and the location of rings around them, models of eyespot formation have considered positional information via gradient (as illustrated in Figure 2b) or induction-type of mechanisms (see recent www.sciencedirect.com
discussions in [13,32]). Despite continued modeling work [33] and expansion of relevant experimental data [34,35], many issues remain unsolved. These include the nature, number, and mode of action of the signals that Current Opinion in Insect Science 2016, 19:1–8
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Eyespot development. Stages of eyespot development and gene functional analysis illustrated for B. anynana. (a) Determination of eyespot foci in larval wings happens in the final larval instar, presumably by the action of genes such as Antennapedia (green), Notch (yellow) and Distalless (red). Protein products of these genes are shown here in two presumptive eyespots on the ventral surface of the hindwing [41], corresponding to the section of the adult wing on the left-most panel. (b) Determination of eyespot rings around eyespot foci occurs in early pupal wings. First, there is signaling from eyespot foci, possibly by secretion of a morphogen that diffuses away to establish a concentration gradient (left panel). Epidermal cells at different distances from the focus respond to the signal by expressing different transcription factors, such as Engrailed (green) and Spalt (blue). (c) Pigment deposition occurs during late pupal wing development, with different color pigments appearing in a stereotypical order [65]. Images represent pigment deposition progression in a selected eyespot, with younger to older pupal wings shown from left to right. (d) CRISPR-Cas9-mediated knock-down of pigmentation enzymes Dopa decarboxylase (Ddc) and Ebony by injections in early embryos produces mosaic adults, with a fraction of the colored scales affected. Images represent the ventral surface of female hindwings and close-up views of areas within dotted rectangles. While Ddc knock-down removes pigment (especially visible in the eyespot black disk), Ebony knock-down converts yellow and light brown to darker brown. For comparison, we show a wildtype phenotype from an unmanipulated female reared at the same temperature.
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establish foci and of those that establish the rings of color around them. They also include the characterization of the actual connection between the ring patterning genes expressed in early pupal wings (Figure 2b) and the pigment synthesis pathways that produce the colors seen in late pupal wings (Figure 2c). Knowledge from other Current Opinion in Insect Science 2016, 19:1–8
systems, such as Drosophila wing pigmentation [36], as well as developments in methods for the analysis of butterfly wing development and of gene function [37– 40] and in availability of genomic data can help clarify these and other aspects of the cellular and molecular basis of eyespot development. www.sciencedirect.com
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In relation to the genes associated with eyespot formation, most of what is known is based on studies of expression patterns of well-known conserved genes in developing butterfly wings. These studies revealed the expression of genes such as Distal-less, Notch and Antennapedia [41] in the presumptive eyespot field, at different stages of eyespot development (Figure 2a-b) in different species [42,43]. Expansion of data on transcriptomes for developing wings [29,44,45,46] and on whole genome sequences for various butterfly species [47] is allowing a more unbiased identification of eyespot development genes. A complete understanding of the genetic basis of eyespot development will require testing the function of those genes whose expression patterns and/or levels correlate with eyespot development. Tests of gene function have had some but limited success, despite attempts with a variety of approaches, including pharmacological manipulations [48], germline transformation [49,50], ectopic expression [51], RNAi [52], and genome editing [53]. For example, functional analyses by different methods and on different species confirmed the actual involvement in eyespot formation of the candidate gene Distal-less, expressed in the presumptive eyespot field. Curiously, the results of different studies proposed opposite functions for this gene: eyespot-promoting role in B. anynana [49] and Junonia orithya [51], and eyespotrepressing role in J. coenia and V. cardui [53]. The interpretation of the results of manipulations of developmental genes such as Distal-less might be confounded by fact that it can be involved in different stages of eyespot formation (e.g. it is expressed during the establishment of foci and of color rings), as well as in other developmental processes (e.g. it is involved in appendage development). While the CRISPR-Cas9 genome editing tool holds great promise of furthering analysis of gene function in evodevo models, including butterfly wing patterns, its use is not without important limitations. For candidate eyespot development genes that also play a role in other developmental processes, for example, knock-down by injections in embryos can result in lethality before any wing patterns are formed. Studying adult mosaics having cells with and without CRISPR-induced deletions (illustrated in Figure 2d) can help overcome embryonic lethality, but it can also raise difficulties with the interpretation and validation of CRISPR-related adult phenotypes. Importantly, while CRISPR-based and other functional tests can inform about the role of candidate genes in the development of eyespots in different species, they cannot identify new candidate genes nor can they provide information about which eyespot genes contribute to variation in eyespot patterns. To those ends, we need different approaches. Analysis of transcriptomes of developing wings during color pattern formation can identify additional putative eyespot development genes [29,44], and genetic mapping can help identify loci carrying allelic www.sciencedirect.com
variation responsible for intra- and possibly inter-species differences in eyespot patterns [45,54].
Evolution of butterfly eyespots: constraints, co-option, and genetics of variation Different hypotheses have been proposed for the origin of the series of marginal eyespots that decorate the wings of many Nymphalid butterflies. The NGP discussed above assumes a scenario where eyespots would have originated from the modification of ancestral marginal bands that ‘resolved’ into a series of simple ocelli that subsequently diversified [4]. Recent work based on a phylogenetic analysis of eyespots and eyespot-like pattern elements in many species proposed a scenario whereby individual eyespots would have been added independently, and originated initially as simple spots that later acquired rings around them (discussed in [13]). Whether the serially repeated eyespots originated simultaneously or independently, they share the same developmental logic and underlying genes. Studies in laboratory populations of B. anynana have explored the extent to which this shared genetic and developmental basis can bias the production of eyespot pattern variants and constrain the independent evolution of serial repeats. Artificial selection targeting pairs of eyespots in B. anynana showed that while independent changes in eyespot size were easily achieved [54], the same was not true for independent changes in eyespot color composition [55]. The likelihood of independent evolution of eyespots on the same wing surface seems to depend on the extent of the compartmentalization, or eyespot-specificity, of the effects of eyespot development genes and of the components of pattern induction [13,55,56]. The origin of eyespots, similarly to what has been proposed for other examples of novel traits, is thought to have occurred via co-option of genetic circuitries involved in the development of shared traits. Eyespots were suggested to have originated from the redeployment to specific wing locations of genes involved in the development of wing margins [57], or of genes implicated in response to wounds [58,59]. Perhaps unsurprisingly, given that pleiotropy is common for developmental genes, eyespot formation also shares genes with other processes, such as appendage and embryonic development (reviewed in [13]). The characterization of the expression of several candidate genes in the establishment of eyespot foci in different species has started to shed light onto what genes or networks were recruited at the origin of Nymphalid eyespots [42,43]. However, this type of analyses has some important limitations. First, expression in presumptive eyespots does not prove that a gene plays a role in eyespot development or that it interacts with coexpressing genes (see discussion about the need for functional studies in the ‘Development of butterfly eyespots’ section). Second, presence/absence of protein product is not sufficient for inferring homology relationships Current Opinion in Insect Science 2016, 19:1–8
Please cite this article in press as: Beldade P, Peralta CM: Developmental and evolutionary mechanisms shaping butterfly eyespots, Curr Opin Insect Sci (2016), http://dx.doi.org/10.1016/ j.cois.2016.10.006
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between structures in different lineages (see discussion about ‘developmental systems drift’ in [42]). Finally, the standard approach to identify genes involved in the origin of novel traits by focusing on the expression of conserved genes biases findings toward evidence for co-option. Future studies should also focus on candidate novel or fast evolving genes found to be expressed during eyespot formation [44,46]. Aside being models for the study of the diversification of serial repeats and of the origin of novel traits, eyespots are one of the systems successfully used in micro-evolutionary evo-devo studies exploring the genetic basis of the inter-individual variation that fuels natural selection. Studies in B. anynana have unraveled both alleles of subtle effect that allowed gradual response to artificial selection, and alleles of large effect on different aspects of eyespot morphology (Figure 1c). Attempts to identify the actual loci responsible for the different types of phenotypic variants has proceeded by genetic mapping approaches, either focused on candidate genes [54] or on polymorphic markers throughout the genome [45]. The identification of the genes and, eventually, of the nucleotide changes responsible for phenotypic variation in eyespot traits can provide answers to key questions in evo-devo for which we lack definitive answers. These include the nature of the DNA sequence changes responsible for evolutionarily relevant phenotypic variation, as well as the extent of overlap between loci that carry alleles of large effect typically studied in the lab and those that carry allelic variants of subtle effect segregating in natural populations, and those accounting for fixed differences across species.
Overview and perspectives Studies of butterfly eyespots have contributed to the ecoevo-devo community’s understanding of phenotypic diversification; all the way from genetic variation, to variation in different components of the mechanisms underlying pattern formation, to intra-specific variation in phenotype, to diversification. Eyespots are valuable models for a number of key topics in eco-evo-devo: 1) the origin and modification of novel traits, 2) developmental constraints and the diversification of serially repeated traits, 3) the regulation and evolution of adaptive developmental plasticity, and 4) the genetic and genomic basis of variation and diversity in adaptive traits. We summarized current knowledge about the ecological significance, developmental underpinnings, and evolution of butterfly eyespots, with emphasis on findings from the past couple of years. For each of the topics covered, we also highlighted a number of open questions, many of which we expect can be solved with analytical tools and resources now available. An important challenge for the future will be to fully integrate the ecological, developmental and evolutionary Current Opinion in Insect Science 2016, 19:1–8
processes that interact to shape diversity in butterfly eyespots. Toward this, we need to consider explicitly that not all eyespots, even those on the same wing, are necessarily shaped by the same mechanisms. All eyespots develop around central organizers but can have different morphologies (e.g. color, number and shape of rings), respond differently to environmental [27,28] and genetic (Figure 2c) factors, be involved in different ecological interactions, and diversify more or less independently. Even the distinct color rings of the same eyespot show some level of independence seen, for example, in how they respond to manipulations of temperature and hormone levels during development [28]. We have discussed the eyespots that decorate the margins of the wings of many Nymphalid butterflies, but eyespotlike pattern elements can be found elsewhere. They can be found in locations of Nymphalid wings other than the margins, and in wings of Lepidopterans other than Nymphalids (examples in [42]). They can also appear in caterpillars, in invertebrates other than lepidopterans, and in different vertebrates, including fish, birds, and reptiles [60–63]. It will be interesting to probe the mechanisms, proximate and ultimate, of this example of convergent evolution and reveal the extent of the overlap in underlying genes, developmental logic, and evolutionary forces shaping the diversity in these pigmentation patterns.
Acknowledgements The authors wish to acknowledge Erik van Bergen and Elvira Lafuente for comments on the manuscript, and Yara Rodrigues (Figure 1d), Suzanne V Saenko (Figure 2b), Marcel J Dix (Figure 2b), Nicolien Pul (Figure 2c) for images. The work was funded by grant PTDC/BIA-EVF/0017/2014 awarded to PB by the Portuguese funding agency, ‘Fundac¸a˜o para a Cieˆncia e Tecnologia’.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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