Effects of Wolbachia on Transposable Element Expression Vary Between Drosophila melanogaster Host Genotypes.

Transposable elements (TEs) are repetitive DNA sequences capable of changing position in host genomes, thereby causing mutations. TE insertions typically have deleterious effects but they can also be beneficial. Increasing evidence of the contribution of TEs to adaptive evolution further raises interest in understanding what factors impact TE activity. Based on previous studies associating the bacterial endosymbiont Wolbachia with changes in the abundance of piRNAs, a mechanism for TE repression, and to transposition of specific TEs, we hypothesized that Wolbachia infection would interfere with TE activity. We tested this hypothesis by studying the expression of 14 TEs in a panel of 25 Drosophila melanogaster host genotypes, naturally infected with Wolbachia and annotated for TE insertions. The host genotypes differed significantly in Wolbachia titers inside individual flies, with broad-sense heritability around 20%, and in the number of TE insertions, which depended greatly on TE identity. By removing Wolbachia from the target host genotypes, we generated a panel of 25 pairs of Wolbachia-positive and Wolbachia-negative lines in which we quantified transcription levels for our target TEs. We found variation in TE expression that was dependent on Wolbachia status, TE identity, and host genotype. Comparing between pairs of Wolbachia-positive and Wolbachia-negative flies, we found that Wolbachia removal affected TE expression in 21.1% of the TE-genotype combinations tested, with up to 2.3 times differences in the median level of transcript. Our data show that Wolbachia can impact TE activity in host genomes, underscoring the importance this endosymbiont can have in the generation of genetic novelty in hosts.

Developmental Plasticity in Butterfly Eyespot Mutants: Variation in Thermal Reaction Norms Across Genotypes and Pigmentation Traits.

Developmental plasticity refers to the property by which a genotype corresponds to distinct phenotypes depending on the environmental conditions experienced during development. This dependence of phenotype expression on environment is graphically represented by reaction norms, which can differ between traits and between genotypes. Even though genetic variation for reaction norms provides the basis for the evolution of plasticity, we know little about the genes that contribute to that variation. This includes understanding to what extent those are the same genes that contribute to inter-individual variation in a fixed environment. Here, we quantified thermal plasticity in butterfly lines that differ in pigmentation phenotype to test the hypothesis that alleles affecting pigmentation also affect plasticity therein. We characterized thermal reaction norms for eyespot color rings of distinct Bicyclus anynana genetic backgrounds, corresponding to allelic variants affecting eyespot size and color composition. Our results reveal genetic variation for the slope and curvature of reaction norms, with differences between eyespots and between eyespot color rings, as well as between sexes. Our report of prevalent temperature-dependent and compartment-specific allelic effects underscores the complexity of genotype-by-environment interactions and their consequence for the evolution of developmental plasticity.

Mosaic Evolution of Molecular Pathways for Sex Pheromone Communication in a Butterfly.

Unraveling the origin of molecular pathways underlying the evolution of adaptive traits is essential for understanding how new lineages emerge, including the relative contribution of conserved ancestral traits and newly evolved derived traits. Here, we investigated the evolutionary divergence of sex pheromone communication from moths (mostly nocturnal) to butterflies (mostly diurnal) that occurred ~119 million years ago. In moths, it is the females that typically emit pheromones to attract male mates, but in butterflies males emit pheromones that are used by females for mate choice. The molecular bases of sex pheromone communication are well understood in moths, but they have remained relatively unexplored in butterflies. We used a combination of transcriptomics, real time qPCR, and phylogenetics to identify genes involved in the different steps (i.e., production, regulation, and reception) of sex pheromone communication of the butterfly Bicyclus anynana. Our results show that the biosynthesis and reception of sex pheromones relies both on moth-specific gene families (reductases) and on more ancestral insect gene families (desaturases, olfactory receptors, odorant binding proteins). Interestingly, B. anynana appears to use what was believed to be the moth-specific neuropeptide Pheromone Biosynthesis Activating Neuropeptide (PBAN) for regulating sex pheromone production. Altogether, our results suggest that a mosaic pattern best explains how sex pheromone communication evolved in butterflies, with some molecular components derived from moths, and others conserved from more ancient insect ancestors. This is the first large-scale investigation of the genetic pathways underlying sex pheromone communication in a butterfly.

Many ways to make darker flies: Intra- and interspecific variation in Drosophila body pigmentation components.

Body pigmentation is an evolutionarily diversified and ecologically relevant trait with substantial variation within and between species, and important roles in animal survival and reproduction. Insect pigmentation, in particular, provides some of the most compelling examples of adaptive evolution, including its ecological significance and genetic bases. Pigmentation includes multiple aspects of color and color pattern that may vary more or less independently, and can be under different selective pressures. We decompose Drosophila thorax and abdominal pigmentation, a valuable eco-evo-devo model, into distinct measurable traits related to color and color pattern. We investigate intra- and interspecific variation for those traits and assess its different sources. For each body part, we measured overall darkness, as well as four other pigmentation properties distinguishing between background color and color of the darker pattern elements that decorate each body part. By focusing on two standard D. melanogaster laboratory populations, we show that pigmentation components vary and covary in distinct manners depending on sex, genetic background, and temperature during development. Studying three natural populations of D. melanogaster along a latitudinal cline and five other Drosophila species, we then show that evolution of lighter or darker bodies can be achieved by changing distinct component traits. Our results paint a much more complex picture of body pigmentation variation than previous studies could uncover, including patterns of sexual dimorphism, thermal plasticity, and interspecific diversity. These findings underscore the value of detailed quantitative phenotyping and analysis of different sources of variation for a better understanding of phenotypic variation and diversification, and the ecological pressures and genetic mechanisms underlying them.

Additive and non-additive effects of day and night temperatures on thermally plastic traits in a model for adaptive seasonal plasticity.

Developmental plasticity can match organismal phenotypes to ecological conditions, helping populations to deal with the environmental heterogeneity of alternating seasons. In contrast to natural situations, experimental studies of plasticity often use environmental conditions that are held constant during development. To explore potential interactions between day and night temperatures, we tested effects of circadian temperature fluctuations on thermally plastic traits in a seasonally plastic butterfly, Bicyclus anynana. Comparing phenotypes for four treatments corresponding to a full-factorial analysis of cooler and warmer temperatures, we found evidence of significant interaction effects between day and night temperatures. We then focused on comparing phenotypes between individuals reared under two types of temperature fluctuations (warmer days with cooler nights, and cooler days with warmer nights) and individuals reared under a constant temperature of the same daily mean. We found evidence of additive-like effects (for body size), and different types of dominance-like effects, with one particular period of the light cycle (for development time) or one particular extreme temperature (for eyespot size) having a larger impact on phenotype. Differences between thermally plastic traits, which together underlie alternative seasonal strategies for survival and reproduction, revealed their independent responses to temperature. This study underscores the value of studying how organisms integrate complex environmental information toward a complete understanding of natural phenotypic variation and of the impact of environmental change thereon.

Eco-evo-devo advances with butterfly eyespots.

Eyespots on the wings of different nymphalid butterflies have become valued models in eco-evo-devo. They are ecologically significant, evolutionarily diverse, and developmentally tractable. Their study has provided valuable insight about the genetic and developmental basis of inter-specific diversity and intra-specific variation, as well as into other key themes in evo-evo-devo: evolutionary novelty, developmental constraints, and phenotypic plasticity. Here we provide an overview of eco-evo-devo studies of butterfly eyespots, highlighting previous reviews, and focusing on both the most recent advances and the open questions expected to be solved in the future.

Genomics of Developmental Plasticity in Animals.

Developmental plasticity refers to the property by which the same genotype produces distinct phenotypes depending on the environmental conditions under which development takes place. By allowing organisms to produce phenotypes adjusted to the conditions that adults will experience, developmental plasticity can provide the means to cope with environmental heterogeneity. Developmental plasticity can be adaptive and its evolution can be shaped by natural selection. It has also been suggested that developmental plasticity can facilitate adaptation and promote diversification. Here, we summarize current knowledge on the evolution of plasticity and on the impact of plasticity on adaptive evolution, and we identify recent advances and important open questions about the genomics of developmental plasticity in animals. We give special attention to studies using transcriptomics to identify genes whose expression changes across developmental environments and studies using genetic mapping to identify loci that contribute to variation in plasticity and can fuel its evolution.

Seasonal plasticity in anti-predatory strategies: Matching of color and color preference for effective crypsis.

Effective anti-predatory strategies typically require matching appearance and behavior in prey, and there are many compelling examples of behavioral repertoires that enhance the effectiveness of morphological defenses. When protective adult morphology is induced by developmental environmental conditions predictive of future predation risk, adult behavior should be adjusted accordingly to maximize predator avoidance. While behavior is typically strongly affected by the adult environment, developmental plasticity in adult behavior-mediated by the same pre-adult environmental cues that affect morphology-could ensure an effective match between anti-predatory morphology and behavior. The coordination of environmentally induced responses may be especially important in populations exposed to predictable environmental fluctuations (e.g., seasonality). Here, we studied early and late life environmental effects on a suite of traits expected to work together for effective crypsis. We focused on wing color and background color preference in Bicyclus anynana, a model of developmental plasticity that relies on crypsis as a seasonal strategy for predator avoidance. Using a full-factorial design, we disentangled effects of developmental and adult ambient temperature on both appearance and behavior. We showed that developmental conditions affect both adult color and color preference, with temperatures that simulate natural dry season conditions leading to browner butterflies with a perching preference for brown backgrounds. This effect was stronger in females, especially when butterflies were tested at lower ambient temperatures. In contrast to the expectation that motionlessness enhances crypsis, we found no support for our hypothesis that the browner dry-season butterflies would be less active. We argue that the integration of developmental plasticity for morphological and behavioral traits might improve the effectiveness of seasonal anti-predatory strategies.

Genetic basis of thermal plasticity variation in Drosophila melanogaster body size.

Body size is a quantitative trait that is closely associated to fitness and under the control of both genetic and environmental factors. While developmental plasticity for this and other traits is heritable and under selection, little is known about the genetic basis for variation in plasticity that can provide the raw material for its evolution. We quantified genetic variation for body size plasticity in Drosophila melanogaster by measuring thorax and abdomen length of females reared at two temperatures from a panel representing naturally segregating alleles, the Drosophila Genetic Reference Panel (DGRP). We found variation between genotypes for the levels and direction of thermal plasticity in size of both body parts. We then used a Genome-Wide Association Study (GWAS) approach to unravel the genetic basis of inter-genotype variation in body size plasticity, and used different approaches to validate selected QTLs and to explore potential pleiotropic effects. We found mostly "private QTLs", with little overlap between the candidate loci underlying variation in plasticity for thorax versus abdomen size, for different properties of the plastic response, and for size versus size plasticity. We also found that the putative functions of plasticity QTLs were diverse and that alleles for higher plasticity were found at lower frequencies in the target population. Importantly, a number of our plasticity QTLs have been targets of selection in other populations. Our data sheds light onto the genetic basis of inter-genotype variation in size plasticity that is necessary for its evolution.

Developmental and evolutionary mechanisms shaping butterfly eyespots.

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.

Adaptive developmental plasticity: compartmentalized responses to environmental cues and to corresponding internal signals provide phenotypic flexibility.

BACKGROUND: The environmental regulation of development can result in the production of distinct phenotypes from the same genotype and provide the means for organisms to cope with environmental heterogeneity. The effect of the environment on developmental outcomes is typically mediated by hormonal signals which convey information about external cues to the developing tissues. While such plasticity is a wide-spread property of development, not all developing tissues are equally plastic. To understand how organisms integrate environmental input into coherent adult phenotypes, we must know how different body parts respond, independently or in concert, to external cues and to the corresponding internal signals. RESULTS: We quantified the effect of temperature and ecdysone hormone manipulations on post-growth tissue patterning in an experimental model of adaptive developmental plasticity, the butterfly Bicyclus anynana. Following a suite of traits evolving by natural or sexual selection, we found that different groups of cells within the same tissue have sensitivities and patterns of response that are surprisingly distinct for the external environmental cue and for the internal hormonal signal. All but those wing traits presumably involved in mate choice responded to developmental temperature and, of those, all but the wing traits not exposed to predators responded to hormone manipulations. On the other hand, while patterns of significant response to temperature contrasted traits on autonomously-developing wings, significant response to hormone manipulations contrasted neighboring groups of cells with distinct color fates. We also showed that the spatial compartmentalization of these responses cannot be explained by the spatial or temporal compartmentalization of the hormone receptor protein. CONCLUSIONS: Our results unravel the integration of different aspects of the adult phenotype into developmental and functional units which both reflect and impact evolutionary change. Importantly, our findings underscore the complexity of the interactions between environment and physiology in shaping the development of different body parts.

Ecdysteroid hormones link the juvenile environment to alternative adult life histories in a seasonal insect.

The conditional expression of alternative life strategies is a widespread feature of animal life and a pivotal adaptation to life in seasonal environments. To optimally match suites of traits to seasonally changing ecological opportunities, animals living in seasonal environments need mechanisms linking information on environmental quality to resource allocation decisions. The butterfly Bicyclus anynana expresses alternative adult life histories in the alternating wet and dry seasons of its habitat as endpoints of divergent developmental pathways triggered by seasonal variation in preadult temperature. Pupal ecdysteroid hormone titers are correlated with the seasonal environment, but whether they play a functional role in coordinating the coupling of adult traits in the alternative life histories is unknown. Here, we show that manipulating pupal ecdysteroid levels is sufficient to mimic in direction and magnitude the shifts in adult reproductive resource allocation normally induced by seasonal temperature. Crucially, this allocation shift is accompanied by changes in ecologically relevant traits, including timing of reproduction, life span, and starvation resistance. Together, our results support a functional role for ecdysteroids during development in mediating strategic reproductive investment decisions in response to predictive indicators of environmental quality. This study provides a physiological mechanism for adaptive developmental plasticity, allowing organisms to cope with variable environments.

Evolution of thorax architecture in ant castes highlights trade-off between flight and ground behaviors.

The concerted evolution of morphological and behavioral specializations has compelling examples in ant castes. Unique to ants is a marked divergence between winged queens and wingless workers, but morphological specializations for behaviors on the ground have been overlooked. We analyzed thorax morphology of queens and workers in species from 21 of the 25 ant subfamilies. We uncovered unique skeletomuscular modifications in workers that presumably increase power and flexibility of head-thorax articulation, emphasizing that workers are not simply wingless versions of queens. We also identified two distinct types of queens and showed repeated evolutionary associations with strategies of colony foundation. Solitary founding queens that hunt have a more worker-like thorax. Our results reveal that ants invest in the relative size of thorax segments according to their tasks. Versatility of head movements allows for better manipulation of food and objects, which arguably contributed to the ants' ecological and evolutionary success. DOI: http://dx.doi.org/10.7554/eLife.01539.001.

Evolutionary history of the recruitment of conserved developmental genes in association to the formation and diversification of a novel trait.

BACKGROUND: The origin and modification of novel traits are important aspects of biological diversification. Studies combining concepts and approaches of developmental genetics and evolutionary biology have uncovered many examples of the recruitment, or co-option, of genes conserved across lineages for the formation of novel, lineage-restricted traits. However, little is known about the evolutionary history of the recruitment of those genes, and of the relationship between them -for example, whether the co-option involves whole or parts of existing networks, or whether it occurs by redeployment of individual genes with de novo rewiring. We use a model novel trait, color pattern elements on butterfly wings called eyespots, to explore these questions. Eyespots have greatly diversified under natural and sexual selection, and their formation involves genetic circuitries shared across insects. RESULTS: We investigated the evolutionary history of the recruitment and co-recruitment of four conserved transcription regulators to the larval wing disc region where circular pattern elements develop. The co-localization of Antennapedia, Notch, Distal-less, and Spalt with presumptive (eye)spot organizers was examined in 13 butterfly species, providing the largest comparative dataset available for the system. We found variation between families, between subfamilies, and between tribes. Phylogenetic reconstructions by parsimony and maximum likelihood methods revealed an unambiguous evolutionary history only for Antennapedia, with a resolved single origin of eyespot-associated expression, and many homoplastic events for Notch, Distal-less, and Spalt. The flexibility in the (co-)recruitment of the targeted genes includes cases where different gene combinations are associated with morphologically similar eyespots, as well as cases where identical protein combinations are associated with very different phenotypes. CONCLUSIONS: The evolutionary history of gene (co-)recruitment is consistent with both divergence from a recruited putative ancestral network, and with independent co-option of individual genes. The diversity in the combinations of genes expressed in association with eyespot formation does not parallel diversity in characteristics of the adult phenotype. We discuss these results in the context of inferring homology. Our study underscores the importance of widening the representation of phylogenetic, morphological, and genetic diversity in order to establish general principles about the mechanisms behind the evolution of novel traits.

Genomic sequence around butterfly wing development genes: annotation and comparative analysis.

BACKGROUND: Analysis of genomic sequence allows characterization of genome content and organization, and access beyond gene-coding regions for identification of functional elements. BAC libraries, where relatively large genomic regions are made readily available, are especially useful for species without a fully sequenced genome and can increase genomic coverage of phylogenetic and biological diversity. For example, no butterfly genome is yet available despite the unique genetic and biological properties of this group, such as diversified wing color patterns. The evolution and development of these patterns is being studied in a few target species, including Bicyclus anynana, where a whole-genome BAC library allows targeted access to large genomic regions. METHODOLOGY/PRINCIPAL FINDINGS: We characterize ∼1.3 Mb of genomic sequence around 11 selected genes expressed in B. anynana developing wings. Extensive manual curation of in silico predictions, also making use of a large dataset of expressed genes for this species, identified repetitive elements and protein coding sequence, and highlighted an expansion of Alcohol dehydrogenase genes. Comparative analysis with orthologous regions of the lepidopteran reference genome allowed assessment of conservation of fine-scale synteny (with detection of new inversions and translocations) and of DNA sequence (with detection of high levels of conservation of non-coding regions around some, but not all, developmental genes). CONCLUSIONS: The general properties and organization of the available B. anynana genomic sequence are similar to the lepidopteran reference, despite the more than 140 MY divergence. Our results lay the groundwork for further studies of new interesting findings in relation to both coding and non-coding sequence: 1) the Alcohol dehydrogenase expansion with higher similarity between the five tandemly-repeated B. anynana paralogs than with the corresponding B. mori orthologs, and 2) the high conservation of non-coding sequence around the genes wingless and Ecdysone receptor, both involved in multiple developmental processes including wing pattern formation.

Involvement of the conserved Hox gene Antennapedia in the development and evolution of a novel trait.

BACKGROUND: Hox proteins specify segment identity during embryogenesis and have typical associated expression patterns. Changes in embryonic expression and activity of Hox genes were crucial in the evolution of animal body plans, but their role in the post-embryonic development of lineage-specific traits remains largely unexplored. Here, we focus on the insect Hox genes Ultrabithorax (Ubx) and Antennapedia (Antp), and implicate the latter in the formation and diversification of novel, butterfly-specific wing patterns. RESULTS: First, we describe a conserved pattern of Ubx expression and a novel pattern of Antp expression in wing discs of Bicyclus anynana butterflies. The discrete, reiterated domains of Antp contrast with the typical expression of Hox genes in single continuous regions in arthropod embryos. Second, we show that this pattern is associated with the establishment of the organizing centres of eyespots. Antp upregulation is the earliest event in organizer development described to date, and in contrast to all genes implicated in eyespot formation, is exclusive to those centres. Third, our comparative analysis of gene expression across nymphalids reveals unexpected differences in organizer determination. CONCLUSIONS: We show that the Antp's recruitment for the formation of novel traits in butterfly wing discs involved the evolution of new expression domains, and is restricted to a particular lineage. This study contributes novel insights into the evolution of Antp expression, as well as into the genetic mechanisms underlying morphological diversification. Our results also underscore how a wider representation of morphological and phylogenetic diversity is essential in evolutionary developmental biology.

Evolution and molecular mechanisms of adaptive developmental plasticity.

Aside from its selective role in filtering inter-individual variation during evolution by natural selection, the environment also plays an instructive role in producing variation during development. External environmental cues can influence developmental rates and/or trajectories and lead to the production of distinct phenotypes from the same genotype. This can result in a better match between adult phenotype and selective environment and thus represents a potential solution to problems posed by environmental fluctuation. The phenomenon is called adaptive developmental plasticity. The study of developmental plasticity integrates different disciplines (notably ecology and developmental biology) and analyses at all levels of biological organization, from the molecular regulation of changes in organismal development to variation in phenotypes and fitness in natural populations. Here, we focus on recent advances and examples from morphological traits in animals to provide a broad overview covering (i) the evolution of developmental plasticity, as well as its relevance to adaptive evolution, (ii) the ecological significance of alternative environmentally induced phenotypes, and the way the external environment can affect development to produce them, (iii) the molecular mechanisms underlying developmental plasticity, with emphasis on the contribution of genetic, physiological and epigenetic factors, and (iv) current challenges and trends, including the relevance of the environmental sensitivity of development to studies in ecological developmental biology, biomedicine and conservation biology.

Single locus affects embryonic segment polarity and multiple aspects of an adult evolutionary novelty.

BACKGROUND: The characterization of the molecular changes that underlie the origin and diversification of morphological novelties is a key challenge in evolutionary developmental biology. The evolution of such traits is thought to rely largely on co-option of a toolkit of conserved developmental genes that typically perform multiple functions. Mutations that affect both a universal developmental process and the formation of a novelty might shed light onto the genetics of traits not represented in model systems. Here we describe three pleiotropic mutations with large effects on a novel trait, butterfly eyespots, and on a conserved stage of embryogenesis, segment polarity. RESULTS: We show that three mutations affecting eyespot size and/or colour composition in Bicyclus anynana butterflies occurred in the same locus, and that two of them are embryonic recessive lethal. Using surgical manipulations and analysis of gene expression patterns in developing wings, we demonstrate that the effects on eyespot morphology are due to changes in the epidermal response component of eyespot induction. Our analysis of morphology and of gene expression in mutant embryos shows that they have a typical segment polarity phenotype, consistent with the mutant locus encoding a negative regulator of Wingless signalling. CONCLUSIONS: This study characterizes the segregation and developmental effects of alleles at a single locus that controls the morphology of a lineage-specific trait (butterfly eyespots) and a conserved process (embryonic segment polarity and, specifically, the regulation of Wingless signalling). Because no gene with such function was found in the orthologous, highly syntenic genomic regions of two other lepidopterans, we hypothesize that our locus is a yet undescribed, possibly lineage-specific, negative regulator of the conserved Wnt/Wg pathway. Moreover, the fact that this locus interferes with multiple aspects of eyespot morphology and maps to a genomic region containing key wing pattern loci in different other butterfly species suggests it might correspond to a 'hotspot' locus in the diversification of this novel trait.

A gene-based linkage map for Bicyclus anynana butterflies allows for a comprehensive analysis of synteny with the lepidopteran reference genome.

Lepidopterans (butterflies and moths) are a rich and diverse order of insects, which, despite their economic impact and unusual biological properties, are relatively underrepresented in terms of genomic resources. The genome of the silkworm Bombyx mori has been fully sequenced, but comparative lepidopteran genomics has been hampered by the scarcity of information for other species. This is especially striking for butterflies, even though they have diverse and derived phenotypes (such as color vision and wing color patterns) and are considered prime models for the evolutionary and developmental analysis of ecologically relevant, complex traits. We focus on Bicyclus anynana butterflies, a laboratory system for studying the diversification of novelties and serially repeated traits. With a panel of 12 small families and a biphasic mapping approach, we first assigned 508 expressed genes to segregation groups and then ordered 297 of them within individual linkage groups. We also coarsely mapped seven color pattern loci. This is the richest gene-based map available for any butterfly species and allowed for a broad-coverage analysis of synteny with the lepidopteran reference genome. Based on 462 pairs of mapped orthologous markers in Bi. anynana and Bo. mori, we observed strong conservation of gene assignment to chromosomes, but also evidence for numerous large- and small-scale chromosomal rearrangements. With gene collections growing for a variety of target organisms, the ability to place those genes in their proper genomic context is paramount. Methods to map expressed genes and to compare maps with relevant model systems are crucial to extend genomic-level analysis outside classical model species. Maps with gene-based markers are useful for comparative genomics and to resolve mapped genomic regions to a tractable number of candidate genes, especially if there is synteny with related model species. This is discussed in relation to the identification of the loci contributing to color pattern evolution in butterflies.