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Mating cues are often comprised of several elements, which can act independently, or in concert to attract a suitable partner. Individual elements may also function in other contexts, such as anti-predator defense or camouflage. In Heliconius butterflies, wing patterns comprise several individual color pattern elements, which advertise the butterflies' toxicity to predators. These wing patterns are also mating cues and males predominantly court females that possess the same wing pattern as their own. However, it is not known whether male preference is based on the full wing pattern or only individual pattern elements. We compared preferences of male H. erato lativitta between female models with the full wing pattern and those with some pattern elements removed. We found no differences in preference between the full wing pattern model and a model with pattern elements removed, indicating that the complete composition of all elements is not essential to the mating signal. Wing pattern preferences also contribute to pre-mating isolation between two other Heliconius taxa, H. erato cyrbia , and H. himera ; therefore, we next compared preferences for the same models in these species. H. erato cyrbia and H. himera strongly differed in preferences for the models, potentially providing a mechanism for how pre-mating isolation acts between these species. These findings suggest that contrasting levels of selective constraint act on elements across the wing pattern. [ABSTRACT FROM AUTHOR]

The activity of many animals follows recurrent patterns and foraging is one of the most important processes in their daily activity. Determining movement in the search for resources and understanding temporal and spatial patterns in foraging has therefore long been central in behavioural ecology. However, identifying and monitoring animal movements is often challenging. In this study we assess the use of camera traps to track a very specific and small‐scale interactions focused on the foraging behaviour of Heliconiini butterflies. Data on floral visitation was recorded using marked individuals of three pollen‐feeding species of Heliconius (H. erato, H. melpomene and H. sara), and two closely related, non‐pollen feeding species (Dryas iulia and Dryadula phaetusa) in a large outdoor insectary. We demonstrate that camera traps efficiently capture individual flower visitation over multiple times and locations and use our experiments to describe some features of their spatial and temporal foraging patterns. Heliconiini butterflies showed higher activity in the morning with strong temporal niche overlap. Differences in foraging activity between males and females was observed with females foraging earlier than males, mirroring published field studies. Some flowers were more explored than others, which may be explained by butterflies foraging simultaneously affecting each other's flower choices. Feeding was grouped in short periods of intense visits to the same flower, which we refer to as feeding bouts. Heliconius also consistently visits the same flower, while non‐Heliconius visited a greater number of flowers per day and their feeding bouts were shorter compared with Heliconius. This is consistent with Heliconius having more stable long‐term spatial memory and foraging preferences than outgroup genera. More broadly, our study demonstrates that camera traps can provide a powerful tool to gather information about foraging behaviour in small insects such as butterflies. © 2024 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

Pheromone communication is widespread among animals. Since it is often involved in mate choice, pheromone production is often tightly controlled. Although male sex pheromones (MSPs) and anti-aphrodisiacs have been studied in some Heliconius butterfly species, little is known about the factors affecting their production and release in these long-lived butterflies. Here, we investigate the effect of post-eclosion age on chemical blends from pheromone-emitting tissues in Heliconius atthis and Heliconius charithonia, exhibiting respectively free-mating and pupal-mating strategies that are hypothesised to differently affect the timing of their pheromone emissions. We focus on two different tissues: the wing androconia, responsible for MSPs used in courtship, and the genital tip, the production site for anti-aphrodisiac pheromones that affect post-mating behaviour. Gas chromatography-mass spectrometric analysis of tissue extracts from virgin males and females of both species from day 0 to 8 post-eclosion demonstrates the following. Some ubiquitous fatty acid precursors are already detectable at day 0. The complexity of the chemical blends increases with age regardless of tissue or sex. No obvious difference in the time course of blend production was evident between the two species, but female tissues in H. charithonia were more affected by age than in H. atthis. We suggest that compounds unique to male androconia and genitals and whose amount increases with age are potential candidates for future investigation into their roles as pheromones. While this analysis revealed some of the complexity in Heliconius chemical ecology, the effects of other factors, such as the time of day, remain unknown. [ABSTRACT FROM AUTHOR]

Anthropogenic climate change is thought to present a significant threat to biodiversity, in particular to tropical ectotherms, and the effects of long‐term developmental heat stress on this group have received relatively little research attention. Here, we studied the effects of experimentally raising developmental temperatures on a tropical butterfly. We measured survival, development time, adult body mass and wing size of Heliconius erato demophoon (Linnaeus) (Lepidoptera: Nymphalidae) across three temperature treatments. Egg survival was lower in the hotter treatments, with 84%, 73% and 49% of eggs hatching in the 20–30°C (fluctuating temperature with 12 h at 20°C followed by 12 h at 30°C), 23–33°C and 26–36°C treatments, respectively. Larval survival was three times lower in the 26–36°C treatment (8%) compared with the 20–30°C treatment (26%), and we did not detect differences in pupal survival across treatments due to high mortality in earlier stages. Under a moderately increased temperature at 23–33°C, larvae developed faster and adults had a higher body mass and wing loading, but this was not seen in the hottest treatment (26–36°C). Females were also heavier than males in the 23–33°C treatment, but there was no associated increase in wing size. This may suggest a different developmental response to moderately elevated temperatures between the sexes. In summary, high developmental temperatures are particularly lethal for eggs and less so for larvae and also affect adult morphology. This highlights the importance of understanding the effects of temperature variation across ontogeny in tropical ectotherms. [ABSTRACT FROM AUTHOR]

Abstract The activity of many animals follows recurrent patterns and foraging is one of the most important processes in their daily activity. Determining movement in the search for resources and understanding temporal and spatial patterns in foraging has therefore long been central in behavioural ecology. However, identifying and monitoring animal movements is often challenging. In this study we assess the use of camera traps to track a very specific and small‐scale interactions focused on the foraging behaviour of Heliconiini butterflies. Data on floral visitation was recorded using marked individuals of three pollen‐feeding species of Heliconius (H. erato, H. melpomene and H. sara), and two closely related, non‐pollen feeding species (Dryas iulia and Dryadula phaetusa) in a large outdoor insectary. We demonstrate that camera traps efficiently capture individual flower visitation over multiple times and locations and use our experiments to describe some features of their spatial and temporal foraging patterns. Heliconiini butterflies showed higher activity in the morning with strong temporal niche overlap. Differences in foraging activity between males and females was observed with females foraging earlier than males, mirroring published field studies. Some flowers were more explored than others, which may be explained by butterflies foraging simultaneously affecting each other's flower choices. Feeding was grouped in short periods of intense visits to the same flower, which we refer to as feeding bouts. Heliconius also consistently visits the same flower, while non‐Heliconius visited a greater number of flowers per day and their feeding bouts were shorter compared with Heliconius. This is consistent with Heliconius having more stable long‐term spatial memory and foraging preferences than outgroup genera. More broadly, our study demonstrates that camera traps can provide a powerful tool to gather information about foraging behaviour in small insects such as butterflies. © 2024 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.

Flight was a key innovation in the adaptive radiation of insects. However, it is a complex trait influenced by a large number of interacting biotic and abiotic factors, making it difficult to unravel the evolutionary drivers. We investigate flight patterns in neotropical heliconiine butterflies, well known for mimicry of their aposematic wing color patterns. We quantify the flight patterns (wing beat frequency and wing angles) of 351 individuals representing 29 heliconiine and 9 ithomiine species belonging to ten color pattern mimicry groupings. For wing beat frequency and up wing angles, we show that heliconiine species group by color pattern mimicry affiliation. Convergence of down wing angles to mimicry groupings is less pronounced, indicating that distinct components of flight are under different selection pressures and constraints. The flight characteristics of the Tiger mimicry group are particularly divergent due to convergence with distantly related ithomiine species. Predator-driven selection for mimicry also explained variation in flight among subspecies, indicating that this convergence can occur over relatively short evolutionary timescales. Our results suggest that the flight convergence is driven by aposematic signaling rather than shared habitat between comimics. We demonstrate that behavioral mimicry can occur between lineages that have separated over evolutionary timescales ranging from <0.5 to 70 My. [ABSTRACT FROM AUTHOR]

In Heliconius butterflies, wing colour pattern diversity and scale types are controlled by a few genes of large effect that regulate colour pattern switches between morphs and species across a large mimetic radiation. One of these genes, cortex, has been repeatedly associated with colour pattern evolution in butterflies. Here we carried out CRISPR knockouts in multiple Heliconius species and show that cortex is a major determinant of scale cell identity. Chromatin accessibility profiling and introgression scans identified cis-regulatory regions associated with discrete phenotypic switches. CRISPR perturbation of these regions in black hindwing genotypes recreated a yellow bar, revealing their spatially limited activity. In the H. melpomene/timareta lineage, the candidate CRE from yellow-barred phenotype morphs is interrupted by a transposable element, suggesting that cis-regulatory structural variation underlies these mimetic adaptations. Our work shows that cortex functionally controls scale colour fate and that its cis-regulatory regions control a phenotypic switch in a modular and pattern-specific fashion.

Abstract Background Gene duplication events are critical for the evolution of new gene functions. Aristaless is a major regulator of distinct developmental processes. It is most known for its role during appendage development across animals. However, more recently other distinct biological functions have been described for this gene and its duplicates. Butterflies and moths have two copies of aristaless, aristaless1 (al1) and aristaless2 (al2), as a result of a gene duplication event. Previous work in Heliconius has shown that both copies appear to have novel functions related to wing color patterning. Here we expand our knowledge of the expression profiles associated with both ancestral and novel functions of Al1 across embryogenesis and wing pigmentation. Furthermore, we characterize Al2 expression, providing a comparative framework between gene copies within the same species, allowing us to understand the origin of new functions following gene duplication. Results Our work shows that the expression of both Al1 and Al2 is associated with the ancestral function of sensory appendage (leg, mouth, spines, and eyes) development in embryos. Interestingly, Al1 exhibits higher expression earlier in embryogenesis while the highest levels of Al2 expression are shifted to later stages of embryonic development. Furthermore, Al1 localization appears extranuclear while Al2 co-localizes tightly with nuclei earlier, and then also expands outside the nucleus later in development. Cellular expression of Al1 and Al2 in pupal wings is broadly consistent with patterns observed during embryogenesis. We also describe, for the first time, how Al1 localization appears to correlate with zones of anterior/posterior elongation of the body during embryonic growth, showcasing a possible new function related to Aristaless’ previously described role in appendage extension. Conclusions Overall, our data suggest that while both gene copies play a role in embryogenesis and wing pigmentation, the duplicates have diverged temporally and mechanistically across those functions. Our study helps clarify principles behind sub-functionalization and gene expression evolution associated with developmental functions following gene duplication events.

Abstract Background Highly diverse butterfly wing patterns have emerged as a powerful system for understanding the genetic basis of phenotypic variation. While the genetic basis of this pattern variation is being clarified, the precise developmental pathways linking genotype to phenotype are not well understood. The gene aristaless, which plays a role in appendage patterning and extension, has been duplicated in Lepidoptera. One copy, aristaless1, has been shown to control a white/yellow color switch in the butterfly Heliconius cydno, suggesting a novel function associated with color patterning and pigmentation. Here we investigate the developmental basis of al1 in embryos, larvae, and pupae using new antibodies, CRISPR/Cas9, RNAi, qPCR assays of downstream targets, and pharmacological manipulation of an upstream activator. Results We find that Al1 is expressed at the distal tips of developing embryonic appendages consistent with its ancestral role. In developing wings, we observe Al1 accumulation within developing scale cells of white H. cydno during early pupation while yellow scale cells exhibit little Al1 at this time point. Reduced Al1 expression is also associated with yellow scale development in al1 knockouts and knockdowns. We propose that Al1 expression in future white scales might be related to an observed downregulation of the enzyme Cinnabar and other genes that synthesize and transport the yellow pigment, 3–hydroxykynurenine (3-OHK). Finally, we provide evidence that Al1 activation is under the control of Wnt signaling. Conclusions We propose a model in which high levels of Al1 during early pupation, which are mediated by Wnt, are important for melanic pigmentation and specifying white portions of the wing while reduced levels of Al1 during early pupation promote upregulation of proteins needed to move and synthesize 3-OHK, promoting yellow pigmentation. In addition, we discuss how the ancestral role of aristaless in appendage extension may be relevant in understanding the cellular mechanism behind color patterning in the context of the heterochrony hypothesis.

Gene flow between species, although usually deleterious, is an important evolutionary process that can facilitate adaptation and lead to species diversification. It also makes estimation of species relationships difficult. Here, we use the full-likelihood multispecies coalescent (MSC) approach to estimate species phylogeny and major introgression events in Heliconius butterflies from whole-genome sequence data. We obtain a robust estimate of species branching order among major clades in the genus, including the ‘melpomene-silvaniform’ group, which shows extensive historical and ongoing gene flow. We obtain chromosome-level estimates of key parameters in the species phylogeny, including species divergence times, present-day and ancestral population sizes, as well as the direction, timing, and intensity of gene flow. Our analysis leads to a phylogeny with introgression events that differ from those obtained in previous studies. We find that Heliconius aoede most likely represents the earliest-branching lineage of the genus and that ‘silvaniform’ species are paraphyletic within the melpomene-silvaniform group. Our phylogeny provides new, parsimonious histories for the origins of key traits in Heliconius, including pollen feeding and an inversion involved in wing pattern mimicry. Our results demonstrate the power and feasibility of the full-likelihood MSC approach for estimating species phylogeny and key population parameters despite extensive gene flow. The methods used here should be useful for analysis of other difficult species groups with high rates of introgression.

Animal vision is important for mediating multiple complex behaviors. In Heliconius butterflies, vision guides fundamental behaviors such as oviposition, foraging, and mate choice. Color vision in Heliconius involves ultraviolet (UV), blue and long‐wavelength‐sensitive photoreceptors (opsins). Additionally, Heliconius possess a duplicated UV opsin, and its expression varies widely within the genus. In Heliconius erato, opsin expression is sexually dimorphic; only females express both UV‐sensitive opsins, enabling UV wavelength discrimination. However, the selective pressures responsible for sex‐specific differences in opsin expression and visual perception remain unresolved. Female Heliconius invest heavily in finding suitable hostplants for oviposition, a behavior heavily dependent on visual cues. Here, we tested the hypothesis that UV vision is important for oviposition in H. erato and Heliconius himera females by manipulating the availability of UV in behavioral experiments under natural conditions. Our results indicate that UV does not influence the number of oviposition attempts or eggs laid, and the hostplant, Passiflora punctata, does not reflect UV wavelengths. Models of H. erato female vision suggest only minimal stimulation of the UV opsins. Overall, these findings suggest that UV wavelengths do not directly affect the ability of Heliconius females to find suitable oviposition sites. Alternatively, UV discrimination could be used in the context of foraging or mate choice, but this remains to be tested. [ABSTRACT FROM AUTHOR]

Parallel evolution of morphological traits is widely reported, providing evidence for the role of local conditions in driving adaptive divergence. Comparatively, fewer studies have tested for parallelism in behavior, and it is less clear to what extent heritable behavioral shifts contribute to adaptive divergence. We exploit repeated incipient speciation across altitudinal gradients to explore behavior and physiology in Heliconius butterflies adapted to high-elevation. We performed common garden experiments with H. chestertonii , a high-altitude specialist from the Colombian Cordillera Occidental, and H. erato venus , a low-elevation proxy for the ancestral population, and compared our results to existing data for an equivalent Ecuadorian taxa-pair. Using broad-scale climatic data, we show that both pairs diverge across similar ecological gradients, confirmed using localized data loggers in the ranges of H. chestertonii and H. e. venus. We further show that H. chestertonii and H. e. venus have divergent activity patterns, attributable to different responses to microclimate, and life histories. Finally, we provide evidence for parallelism in these traits with H. himera and H. e. cyrbia. We propose that this is a result of selection associated with independent colonizations of high-altitude forests, emphasizing the importance of heritable behavioral and physiological adaptations during population divergence and speciation. [ABSTRACT FROM AUTHOR]

Host plant specialisation by herbivorous insects is ubiquitous, especially among the Lepidoptera. Many taxa have the ability to accumulate toxic compounds from their host plants that serve as chemical defences against natural enemies. Despite common knowledge of this pattern among insect ecologists, we still have much to learn about how dietary variation affects an insect's ability to acquire toxic plant chemicals.Longwing butterfly larvae (Heliconius) accumulate toxic cyanogenic glucosides from their host plants, passion vines (Passiflora), that make them toxic to most predators.Here, we present on zebra longwing (H. charithonia) caterpillar cohorts that were reared on native P. affinis, P. biflora, P. lutea, or P. suberosa to determine how host usage affected cyanide accumulation over larval developmental stages, and ultimately the toxicity of adult butterflies. Samples were collected at third, fourth and fifth instars, and teneral adults. Cyanogenic glucosides were extracted, the cyanide molecules were captured as sodium cyanide (CN), and CN content was quantified colorimetrically.The quantity of CN acquired increased linearly over larval ontogeny and was variable among host plants. CN concentration increased over larval development for two hosts but declined on the third. There was no clear relationship between host plant and adult CN concentrations, but adults that consumed certain hosts as larvae ended up accumulating significantly more CN, and thus expressed higher CN concentrations, than others.We demonstrated that some host plants provide specialist insects with more toxins than others and that larval secondary chemical accumulation may predict which life stages are more vulnerable to natural enemies. These conclusions support the hypothesis that host choice affects the survival of specialist insect herbivores. [ABSTRACT FROM AUTHOR]

Background: Gene duplication events are critical for the evolution of new gene functions. Aristaless is a major regulator of distinct developmental processes. It is most known for its role during appendage development across animals. However, more recently other distinct biological functions have been described for this gene and its duplicates. Butterflies and moths have two copies of aristaless, aristaless1 (al1) and aristaless2 (al2), as a result of a gene duplication event. Previous work in Heliconius has shown that both copies appear to have novel functions related to wing color patterning. Here we expand our knowledge of the expression profiles associated with both ancestral and novel functions of Al1 across embryogenesis and wing pigmentation. Furthermore, we characterize Al2 expression, providing a comparative framework between gene copies within the same species, allowing us to understand the origin of new functions following gene duplication. Results: Our work shows that the expression of both Al1 and Al2 is associated with the ancestral function of sensory appendage (leg, mouth, spines, and eyes) development in embryos. Interestingly, Al1 exhibits higher expression earlier in embryogenesis while the highest levels of Al2 expression are shifted to later stages of embryonic development. Furthermore, Al1 localization appears extranuclear while Al2 co-localizes tightly with nuclei earlier, and then also expands outside the nucleus later in development. Cellular expression of Al1 and Al2 in pupal wings is broadly consistent with patterns observed during embryogenesis. We also describe, for the first time, how Al1 localization appears to correlate with zones of anterior/posterior elongation of the body during embryonic growth, showcasing a possible new function related to Aristaless' previously described role in appendage extension. Conclusions: Overall, our data suggest that while both gene copies play a role in embryogenesis and wing pigmentation, the duplicates have diverged temporally and mechanistically across those functions. Our study helps clarify principles behind sub-functionalization and gene expression evolution associated with developmental functions following gene duplication events. [ABSTRACT FROM AUTHOR]

Background: Highly diverse butterfly wing patterns have emerged as a powerful system for understanding the genetic basis of phenotypic variation. While the genetic basis of this pattern variation is being clarified, the precise developmental pathways linking genotype to phenotype are not well understood. The gene aristaless, which plays a role in appendage patterning and extension, has been duplicated in Lepidoptera. One copy, aristaless1, has been shown to control a white/yellow color switch in the butterfly Heliconius cydno, suggesting a novel function associated with color patterning and pigmentation. Here we investigate the developmental basis of al1 in embryos, larvae, and pupae using new antibodies, CRISPR/Cas9, RNAi, qPCR assays of downstream targets, and pharmacological manipulation of an upstream activator. Results: We find that Al1 is expressed at the distal tips of developing embryonic appendages consistent with its ancestral role. In developing wings, we observe Al1 accumulation within developing scale cells of white H. cydno during early pupation while yellow scale cells exhibit little Al1 at this time point. Reduced Al1 expression is also associated with yellow scale development in al1 knockouts and knockdowns. We propose that Al1 expression in future white scales might be related to an observed downregulation of the enzyme Cinnabar and other genes that synthesize and transport the yellow pigment, 3–hydroxykynurenine (3-OHK). Finally, we provide evidence that Al1 activation is under the control of Wnt signaling. Conclusions: We propose a model in which high levels of Al1 during early pupation, which are mediated by Wnt, are important for melanic pigmentation and specifying white portions of the wing while reduced levels of Al1 during early pupation promote upregulation of proteins needed to move and synthesize 3-OHK, promoting yellow pigmentation. In addition, we discuss how the ancestral role of aristaless in appendage extension may be relevant in understanding the cellular mechanism behind color patterning in the context of the heterochrony hypothesis. [ABSTRACT FROM AUTHOR]

Heliconius butterflies are an ideal organism for studying ecology, behavior, adaptation, and speciation. These butterflies can be found in various locations and habitats in Central and South America, where they encounter and interact with different sources of pollen, nectar, and host plants. However, there is limited knowledge on how geographic and habitat variations affect the microbiota of these insects, and whether microbial associates play a role in their ability to exploit different habitats. To date, research on the microbial communities associated with Heliconius has mainly focused on host phylogenetic signal in microbiomes or microbiome characterization in specific communities of butterflies. In this study, we characterized the microbiomes of several species and populations of Heliconius from distant locations that represent contrasting environments. We found that the microbiota of different Heliconius species is taxonomically similar but vary in abundance. Notably, this variation is associated with a major geographic barrier—the Central Cordillera of Colombia. Additionally, we confirmed that this microbiota is not associated with pollen-feeding. Therefore, it seems likely that geography shapes the abundance of microbiota that the butterfly carries, but not the taxonomic diversity of the microbial community. Based on the current evidence, the bacterial microbiota associated with Heliconius does not appear to play a beneficial role for these butterflies. [ABSTRACT FROM AUTHOR]

Abstract Animal vision is important for mediating multiple complex behaviors. In Heliconius butterflies, vision guides fundamental behaviors such as oviposition, foraging, and mate choice. Color vision in Heliconius involves ultraviolet (UV), blue and long‐wavelength‐sensitive photoreceptors (opsins). Additionally, Heliconius possess a duplicated UV opsin, and its expression varies widely within the genus. In Heliconius erato, opsin expression is sexually dimorphic; only females express both UV‐sensitive opsins, enabling UV wavelength discrimination. However, the selective pressures responsible for sex‐specific differences in opsin expression and visual perception remain unresolved. Female Heliconius invest heavily in finding suitable hostplants for oviposition, a behavior heavily dependent on visual cues. Here, we tested the hypothesis that UV vision is important for oviposition in H. erato and Heliconius himera females by manipulating the availability of UV in behavioral experiments under natural conditions. Our results indicate that UV does not influence the number of oviposition attempts or eggs laid, and the hostplant, Passiflora punctata, does not reflect UV wavelengths. Models of H. erato female vision suggest only minimal stimulation of the UV opsins. Overall, these findings suggest that UV wavelengths do not directly affect the ability of Heliconius females to find suitable oviposition sites. Alternatively, UV discrimination could be used in the context of foraging or mate choice, but this remains to be tested.

Why warning patterns are so diverse is an enduring evolutionary puzzle. Because predators associate particular patterns with unpleasant experiences, an individual's predation risk should decrease as the local density of its warning pattern increases, promoting pattern monomorphism. Distasteful Heliconius butterflies are known for their diversity of warning patterns. Here, we explore whether interlocus sexual conflict can contribute to their diversification. Male Heliconius use warning patterns as mating cues, but mated females may suffer costs if this leads to disturbance, favouring novel patterns. Using simulations, we show that under our model conditions drift alone is unlikely to cause pattern diversification, but that sexual conflict can assist such a process. We also find that genetic architecture influences the evolution of male preferences, which track changes in warning pattern due to sexual selection. When male attraction imposes costs on females, this affects the speed at which novel pattern alleles increase. In two experiments, females laid fewer eggs with males present. However, although males in one experiment showed less interest in females with manipulated patterns, we found no evidence that female colouration mitigates sex‐specific costs. Overall, male attraction to conspecific warning patterns may impose an unrecognized cost on Heliconius females, but further work is required to determine this experimentally. [ABSTRACT FROM AUTHOR]

Over a century after the first description of a polymorphism controlled by a supergene, these genetic architectures still puzzle biologists. Supergenes are groups of tightly linked loci facilitating the co-segregation of combinations of alleles underlying alternative, complex adaptive strategies. The suppression of recombination at supergenes is generally caused by polymorphic chromosomal rearrangements, such as inversions. The existence of inversion polymorphisms and supergene raises theoretical and empirical questions. Why do these architectures evolve? How can alternative combinations of alleles be formed? How and why is polymorphism maintained? The purpose of this paper is to provide answers to these questions by reviewing recent advances in the study of Heliconius numata, an Amazonian butterfly displaying a striking diversity of wing color patterns. In a broad context, this review highlights mechanisms that play an important role in the evolution of new genomic architecture and in the adaptation of species.

Multi-omic approaches promise to supply the power to detect genes underlying disease and fitness-related phenotypes. Optimal use of the resulting profusion of data requires detailed investigation of individual candidate genes, a challenging proposition. Here, we combine transcriptomic and genomic data with molecular modelling of candidate enzymes to characterize the evolutionary history and function of the serine protease cocoonase. Heliconius butterflies possess the unique ability to feed on pollen; recent work has identified cocoonase as a candidate gene in pollen digestion. Cocoonase was first described in moths, where it aids in eclosure from the cocoon and is present as a single copy gene. In heliconiine butterflies it is duplicated and highly expressed in the mouthparts of adults. At least six copies of cocoonase are present in Heliconius melpomene and copy number varies across H. melpomene sub-populations. Most cocoonase genes are under purifying selection, however branch-site analyses suggest cocoonase 3 genes may have evolved under episodic diversifying selection. Molecular modelling of cocoonase proteins and examination of their predicted structures revealed that the active site region of each type has a similar structure to trypsin, with the same predicted substrate specificity across types. Variation among heliconiine cocoonases instead lies in the outward-facing residues involved in solvent interaction. Thus, the neofunctionalization of cocoonase duplicates appears to have resulted from the need for these serine proteases to operate in diverse biochemical environments. We suggest that cocoonase may have played a buffering role in feeding during the diversification of Heliconius across the neotropics by enabling these butterflies to digest protein from a range of biochemical milieux.

Abstract The evolution of mimicry in similarly defended prey is well described by the Müllerian mimicry theory, which predicts the convergence of warning patterns in order to gain the most protection from predators. However, despite this prediction, we can find great diversity of color patterns among Müllerian mimics such as Heliconius butterflies in the neotropics. Furthermore, some species have evolved the ability to maintain multiple distinct warning patterns in single populations, a phenomenon known as polymorphic mimicry. The adaptive benefit of these polymorphisms is questionable since variation from the most common warning patterns is expected to be disadvantageous as novel signals are punished by predators naive to them. In this study, we use artificial butterfly models throughout Central and South America to characterize the selective pressures maintaining polymorphic mimicry in Heliconius doris. Our results highlight the complexity of positive frequency‐dependent selection, the principal selective pressure driving convergence among Müllerian mimics, and its impacts on interspecific variation of mimetic warning coloration. We further show how this selection regime can both limit and facilitate the diversification of mimetic traits.

Structural colours, produced by the reflection of light from ultrastructures, have evolved multiple times in butterflies. Unlike pigmentary colours and patterns, little is known about the genetic basis of these colours. Reflective structures on wing-scale ridges are responsible for iridescent structural colour in many butterflies, including the Müllerian mimics Heliconius erato and Heliconius melpomene. Here, we quantify aspects of scale ultrastructure variation and colour in crosses between iridescent and non-iridescent subspecies of both of these species and perform quantitative trait locus (QTL) mapping. We show that iridescent structural colour has a complex genetic basis in both species, with offspring from crosses having a wide variation in blue colour (both hue and brightness) and scale structure measurements. We detect two different genomic regions in each species that explain modest amounts of this variation, with a sex-linked QTL in H. erato but not H. melpomene. We also find differences between species in the relationships between structure and colour, overall suggesting that these species have followed different evolutionary trajectories in their evolution of structural colour. We then identify genes within the QTL intervals that are differentially expressed between subspecies and/or wing regions, revealing likely candidates for genes controlling structural colour formation. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'. [ABSTRACT FROM AUTHOR]

Aposematic animals advertise their toxicity or unpalatability with bright warning coloration. However, acquiring and maintaining chemical defenses can be energetically costly, and consequent associations with other important traits could shape chemical defense evolution. Here, we have tested whether chemical defenses are involved in energetic trade‐offs with other traits, or whether the levels of chemical defenses are condition dependent, by studying associations between biosynthesized cyanogenic toxicity and a suite of key life‐history and fitness traits in a Heliconius butterfly under a controlled laboratory setting. Heliconius butterflies are well known for the diversity of their warning color patterns and widespread mimicry and can both sequester the cyanogenic glucosides of their Passiflora host plants and biosynthesize these toxins de novo. We find energetically costly life‐history traits to be either unassociated or to show a general positive association with biosynthesized cyanogenic toxicity. More toxic individuals developed faster and had higher mass as adults and a tendency for increased lifespan and fecundity. These results thus indicate that toxicity level of adult butterflies may be dependent on individual condition, influenced by genetic background or earlier conditions, with maternal effects as one strong candidate mechanism. Additionally, toxicity was higher in older individuals, consistent with previous studies indicating accumulation of toxins with age. As toxicity level at death was independent of lifespan, cyanogenic glucoside compounds may have been recycled to release resources relevant for longevity in these long‐living butterflies. Understanding the origins and maintenance of variation in defenses is necessary in building a more complete picture of factors shaping the evolution of aposematic and mimetic systems. [ABSTRACT FROM AUTHOR]

Abstract The degree to which loci promoting reproductive isolation cluster in the genome—that is, the genetic architecture of reproductive isolation—can influence the tempo and mode of speciation. Tight linkage between these loci can facilitate speciation in the face of gene flow. Pheromones play a role in reproductive isolation in many Lepidoptera species, and the role of endogenously produced compounds as secondary metabolites decreases the likelihood of pleiotropy associated with many barrier loci. Heliconius butterflies use male sex pheromones to both court females (aphrodisiac wing pheromones) and ward off male courtship (male‐transferred antiaphrodisiac genital pheromones), and it is likely that these compounds play a role in reproductive isolation between Heliconius species. Using a set of backcross hybrids between H. melpomene and H. cydno, we investigated the genetic architecture of putative male pheromone compound production. We found a set of 40 significant quantitative trait loci (QTL) representing 33 potential pheromone compounds. QTL clustered significantly on two chromosomes, chromosome 8 for genital compounds and chromosome 20 for wing compounds, and chromosome 20 was enriched for potential pheromone biosynthesis genes. There was minimal overlap between pheromone QTL and known QTL for mate choice and color pattern. Nonetheless, we did detect linkage between a QTL for wing androconial area and optix, a color pattern locus known to play a role in reproductive isolation in these species. This tight clustering of putative pheromone loci might contribute to coincident reproductive isolating barriers, facilitating speciation despite ongoing gene flow.

Abstract Aposematic animals advertise their toxicity or unpalatability with bright warning coloration. However, acquiring and maintaining chemical defenses can be energetically costly, and consequent associations with other important traits could shape chemical defense evolution. Here, we have tested whether chemical defenses are involved in energetic trade‐offs with other traits, or whether the levels of chemical defenses are condition dependent, by studying associations between biosynthesized cyanogenic toxicity and a suite of key life‐history and fitness traits in a Heliconius butterfly under a controlled laboratory setting. Heliconius butterflies are well known for the diversity of their warning color patterns and widespread mimicry and can both sequester the cyanogenic glucosides of their Passiflora host plants and biosynthesize these toxins de novo. We find energetically costly life‐history traits to be either unassociated or to show a general positive association with biosynthesized cyanogenic toxicity. More toxic individuals developed faster and had higher mass as adults and a tendency for increased lifespan and fecundity. These results thus indicate that toxicity level of adult butterflies may be dependent on individual condition, influenced by genetic background or earlier conditions, with maternal effects as one strong candidate mechanism. Additionally, toxicity was higher in older individuals, consistent with previous studies indicating accumulation of toxins with age. As toxicity level at death was independent of lifespan, cyanogenic glucoside compounds may have been recycled to release resources relevant for longevity in these long‐living butterflies. Understanding the origins and maintenance of variation in defenses is necessary in building a more complete picture of factors shaping the evolution of aposematic and mimetic systems.

Site fidelity plays an important role in increasing foraging efficiency, particularly when food resources are reliable. In insects, site fidelity has largely been studied in Hymenopteran species, which consistently return to their nest site after foraging bouts.In butterflies, evidence of foraging site fidelity is limited but may be present in species with specific foraging specialisations, such as Heliconius, which have a derived foraging behaviour centred around active pollen feeding. Unlike many Hymenoptera, Heliconius are neither eusocial nor nesting species, positioning them as a peculiar case where foraging site fidelity may occur in the absence of any central nest‐like structure. However, to date, existing studies do not consider low dispersal as an alternative to site fidelity.In this study, we use a mark–release–recapture experiment to test whether individuals of two Heliconius species exhibit true site fidelity. We further test this fidelity by measuring flight orientation during a translocation experiment, and by recapturing translocated butterflies to identify whether individuals return to their site of origin.We found that non‐translocated butterflies display extreme stability in site choice, and translocated butterflies consistently return to their site of origin, rapidly orientating towards their home site upon release. This suggests site fidelity in Heliconius is not solely explained by low dispersal, but is a response to the distribution and stability in ecological resources.We further note that the ability to return to home sites when artificially dispersed suggests a sophisticated ability to navigate to specific spatial goals. Our study provides insights into the ecological drivers of site fidelity, a novel example distinct from nest building or group living, and opens an avenue of new research on navigational mechanisms in insects. A free Plain Language Summary can be found within the Supporting Information of this article. [ABSTRACT FROM AUTHOR]

Integrating and weighting sensory perception across modalities is crucial to how animals adapt to their environment. Divergence in brain structure is often in sensory processing regions, suggesting that investment reflects ecological needs. Here, we use two parapatric closely related species, Heliconius erato cyrbia and Heliconius himera , to test the hypothesis that divergence in sensory brain regions affects foraging decisions. These butterflies are isolated across an ecological gradient, which is linked to differences in brain morphology, with H. e. cyrbia investing more in visual centres and H. himera investing in olfactory centres. Here, we demonstrate that these two species vary in how they associate visual and olfactory cues with positive food rewards. We found that when individuals were trained on paired olfactory and visual stimuli, then presented with these stimuli in conflict, they showed distinct behavioural responses. Heliconius himera was more likely to favour positive olfactory cues than H. e. cyrbia , which favoured visual cues regardless of the paired stimulus. This suggests that these species have diverged in the emphasis placed on these different sensory domains during foraging, consistent with observed differences in brain morphology. This result strengthens evidence that speciation initiated by local adaptation is partly facilitated by changes in the neural basis of key behavioural functions. • Divergence in brain sensory processing regions may reflect ecological need. • We tested whether this divergence affects foraging in two Heliconius butterflies. • Visual/olfactory cue association with positive rewards differed between species. • This sensory domain divergence is consistent with brain morphology divergence. [ABSTRACT FROM AUTHOR]

Abstract Background Heliconius butterflies are widely distributed across the Neotropics and have evolved a stunning array of wing color patterns that mediate Müllerian mimicry and mating behavior. Their rapid radiation has been strongly influenced by hybridization, which has created new species and allowed sharing of color patterning alleles between mimetic species pairs. While these processes have frequently been observed in widespread species with contiguous distributions, many Heliconius species inhabit patchy or rare habitats that may strongly influence the origin and spread of species and color patterns. Here, we assess the effects of historical population fragmentation and unique biology on the origins, genetic health, and color pattern evolution of two rare and sparsely distributed Brazilian butterflies, Heliconius hermathena and Heliconius nattereri. Results We assembled genomes and re-sequenced whole genomes of eight H. nattereri and 71 H. hermathena individuals. These species harbor little genetic diversity, skewed site frequency spectra, and high deleterious mutation loads consistent with recent population bottlenecks. Heliconius hermathena consists of discrete, strongly isolated populations that likely arose from a single population that dispersed after the last glacial maximum. Despite having a unique color pattern combination that suggested a hybrid origin, we found no genome-wide evidence that H. hermathena is a hybrid species. However, H. hermathena mimicry evolved via introgression, from co-mimetic Heliconius erato, of a small genomic region upstream of the color patterning gene cortex. Conclusions Heliconius hermathena and H. nattereri population fragmentation, potentially driven by historical climate change and recent deforestation, has significantly reduced the genetic health of these rare species. Our results contribute to a growing body of evidence that introgression of color patterning alleles between co-mimetic species appears to be a general feature of Heliconius evolution.

Abstract Evolutionary convergence of color pattern in mimetic species is tightly linked with the evolution of chemical defenses. Yet, the evolutionary forces involved in natural variations of chemical defenses in aposematic species are still understudied. Herein, we focus on the evolution of chemical defenses in the butterfly tribe Heliconiini. These neotropical butterflies contain large concentrations of cyanogenic glucosides, cyanide‐releasing compounds acting as predator deterrent. These compounds are either de novo synthesized or sequestered from their Passiflora host plant, so that their concentrations may depend on host plant specialization and host plant availability. We sampled 375 wild Heliconiini butterflies across Central and South America, covering 43% species of this clade, and quantify individual variations in the different CGs using liquid chromatography coupled with tandem mass spectrometry. We detected new compounds and important variations in chemical defenses both within and among species. Based on the most recent and well‐studied phylogeny of Heliconiini, we show that ecological factors such as mimetic interactions and host plant specialization have a significant association with chemical profiles, but these effects are largely explained by phylogenetic relationships. Our results therefore suggest that shared ancestries largely contribute to chemical defense variation, pointing out at the interaction between historical and ecological factors in the evolution of Müllerian mimicry.

The evolution of mimicry in similarly defended prey is well described by the Müllerian mimicry theory, which predicts the convergence of warning patterns in order to gain the most protection from predators. However, despite this prediction, we can find great diversity of color patterns among Müllerian mimics such as Heliconius butterflies in the neotropics. Furthermore, some species have evolved the ability to maintain multiple distinct warning patterns in single populations, a phenomenon known as polymorphic mimicry. The adaptive benefit of these polymorphisms is questionable since variation from the most common warning patterns is expected to be disadvantageous as novel signals are punished by predators naive to them. In this study, we use artificial butterfly models throughout Central and South America to characterize the selective pressures maintaining polymorphic mimicry in Heliconius doris. Our results highlight the complexity of positive frequency‐dependent selection, the principal selective pressure driving convergence among Müllerian mimics, and its impacts on interspecific variation of mimetic warning coloration. We further show how this selection regime can both limit and facilitate the diversification of mimetic traits. [ABSTRACT FROM AUTHOR]

Understanding how organisms adapt to their local environment is central to evolution. With new whole‐genome sequencing technologies and the explosion of data, deciphering the genomic basis of complex traits that are ecologically relevant is becoming increasingly feasible. Here, we studied the genomic basis of wing shape in two Neotropical butterflies that inhabit large geographical ranges. Heliconius butterflies at high elevations have been shown to generally have rounder wings than those in the lowlands. We reared over 1,100 butterflies from 71 broods of H. erato and H. melpomene in common‐garden conditions and showed that wing aspect ratio, that is, elongatedness, is highly heritable in both species and that elevation‐associated wing aspect ratio differences are maintained. Genome‐wide associations with a published data set of 666 whole genomes from across a hybrid zone, uncovered a highly polygenic basis to wing aspect ratio variation in the wild. We identified several genes that have roles in wing morphogenesis or wing aspect ratio variation in Drosophila flies, making them promising candidates for future studies. There was little evidence for molecular parallelism in the two species, with only one shared candidate gene, nor for a role of the four known colour pattern loci, except for optix in H. erato. Thus, we present the first insights into the heritability and genomic basis of within‐species wing aspect ratio in two Heliconius species, adding to a growing body of evidence that polygenic adaptation may underlie many ecologically relevant traits. [ABSTRACT FROM AUTHOR]

In Heliconius butterflies, wing colour pattern diversity and scale types are controlled by a few genes of large effect that regulate colour pattern switches between morphs and species across a large mimetic radiation. One of these genes, cortex, has been repeatedly associated with colour pattern evolution in butterflies. Here we carried out CRISPR knockouts in multiple Heliconius species and show that cortex is a major determinant of scale cell identity. Chromatin accessibility profiling and introgression scans identified cis-regulatory regions associated with discrete phenotypic switches. CRISPR perturbation of these regions in black hindwing genotypes recreated a yellow bar, revealing their spatially limited activity. In the H. melpomene/timareta lineage, the candidate CRE from yellow-barred phenotype morphs is interrupted by a transposable element, suggesting that cis-regulatory structural variation underlies these mimetic adaptations. Our work shows that cortex functionally controls scale colour fate and that its cis-regulatory regions control a phenotypic switch in a modular and pattern-specific fashion.

Heliconius butterflies (Lepidoptera: Nymphalidae) are a group of 48 neotropical species widely studied in evolutionary research. Despite the wealth of genomic data generated in past years, chromosomal level genome assemblies currently exist for only two species, Heliconius melpomene and Heliconius erato , each a representative of one of the two major clades of the genus. Here, we use these reference genomes to improve the contiguity of previously published draft genome assemblies of 16 Heliconius species. Using a reference-assisted scaffolding approach, we place and order the scaffolds of these genomes onto chromosomes, resulting in 95.7–99.9% of their genomes anchored to chromosomes. Genome sizes are somewhat variable among species (270–422 Mb) and in one small group of species (Heliconius hecale , Heliconius elevatus , and Heliconius pardalinus) expansions in genome size are driven mainly by repetitive sequences that map to four small regions in the H. melpomene reference genome. Genes from these repeat regions show an increase in exon copy number, an absence of internal stop codons, evidence of constraint on nonsynonymous changes, and increased expression, all of which suggest that at least some of the extra copies are functional. Finally, we conducted a systematic search for inversions and identified five moderately large inversions fixed between the two major Heliconius clades. We infer that one of these inversions was transferred by introgression between the lineages leading to the erato / sara and burneyi / doris clades. These reference-guided assemblies represent a major improvement in Heliconius genomic resources that enable further genetic and evolutionary discoveries in this genus. [ABSTRACT FROM AUTHOR]

Abstract Background Many traits evolve by cis-regulatory modification, by which changes to noncoding sequences affect the binding affinity for available transcription factors and thus modify the expression profile of genes. Multiple examples of cis-regulatory evolution have been described at pattern switch genes responsible for butterfly wing pattern polymorphism, including in the diverse neotropical genus Heliconius, but the identities of the factors that can regulate these switch genes have not been identified. Results We investigated the spatial transcriptomic landscape across the wings of three closely related butterfly species, two of which have a convergently evolved co-mimetic pattern and the other having a divergent pattern. We identified candidate factors for regulating the expression of wing patterning genes, including transcription factors with a conserved expression profile in all three species, and others, including both transcription factors and Wnt pathway genes, with markedly different profiles in each of the three species. We verified the conserved expression profile of the transcription factor homothorax by immunofluorescence and showed that its expression profile strongly correlates with that of the selector gene optix in butterflies with the Amazonian forewing pattern element ‘dennis.’ Conclusion Here we show that, in addition to factors with conserved expression profiles like homothorax, there are also a variety of transcription factors and signaling pathway components that appear to vary in their expression profiles between closely related butterfly species, highlighting the importance of genome-wide regulatory evolution between species.

Chemical defences against predators underlie the evolution of aposematic coloration and mimicry, which are classic examples of adaptive evolution. Surprisingly little is known about the roles of ecological and evolutionary processes maintaining defence variation, and how they may feedback to shape the evolutionary dynamics of species. Cyanogenic Heliconius butterflies exhibit diverse warning color patterns and mimicry, thus providing a useful framework for investigating these questions. We studied intraspecific variation in de novo biosynthesized cyanogenic toxicity and its potential ecological and evolutionary sources in wild populations of Heliconius erato along environmental gradients, in common-garden broods and with feeding treatments. Our results demonstrate substantial intraspecific variation, including detectable variation among broods reared in a common garden. The latter estimate suggests considerable evolutionary potential in this trait, although predicting the response to selection is likely complicated due to the observed skewed distribution of toxicity values and the signatures of maternal contributions to the inheritance of toxicity. Larval diet contributed little to toxicity variation. Furthermore, toxicity profiles were similar along steep rainfall and altitudinal gradients, providing little evidence for these factors explaining variation in biosynthesized toxicity in natural populations. In contrast, there were striking differences in the chemical profiles of H. erato from geographically distant populations, implying potential local adaptation in the acquisition mechanisms and levels of defensive compounds. The results highlight the extensive variation and potential for adaptive evolution in defense traits for aposematic and mimetic species, which may contribute to the high diversity often found in these systems. [ABSTRACT FROM AUTHOR]

Speciation is facilitated when traits under divergent selection also act as mating cues. Fluctuations in sensory conditions can alter signal perception independently of adaptation to the broader sensory environment, but how this fine-scale variation may constrain or promote behavioural isolation has received little attention. The warning patterns of Heliconius butterflies are under selection for aposematism and act as mating cues. Using computer vision, we extracted behavioural data from 1481 h of video footage, for 387 individuals. We show that the putative hybrid species H. heurippa and its close relative H. timareta linaresi differ in their response to divergent warning patterns, but that these differences are strengthened with increased local illuminance. Trials with live individuals reveal low-level assortative mating that is sufficiently explained by differences in visual attraction. Finally, results from hybrid butterflies are consistent with linkage between a major warning pattern gene and the corresponding behaviour, though the differences in behaviour we observe are unlikely to cause rapid reproductive isolation as predicted under a model of hybrid trait speciation. Overall, our results reveal that the contribution of ecological mating cues to reproductive isolation may depend on the immediate sensory conditions during which they are displayed to conspecifics. [ABSTRACT FROM AUTHOR]

The degree to which loci promoting reproductive isolation cluster in the genome—that is, the genetic architecture of reproductive isolation—can influence the tempo and mode of speciation. Tight linkage between these loci can facilitate speciation in the face of gene flow. Pheromones play a role in reproductive isolation in many Lepidoptera species, and the role of endogenously produced compounds as secondary metabolites decreases the likelihood of pleiotropy associated with many barrier loci. Heliconius butterflies use male sex pheromones to both court females (aphrodisiac wing pheromones) and ward off male courtship (male‐transferred antiaphrodisiac genital pheromones), and it is likely that these compounds play a role in reproductive isolation between Heliconius species. Using a set of backcross hybrids between H. melpomene and H. cydno, we investigated the genetic architecture of putative male pheromone compound production. We found a set of 40 significant quantitative trait loci (QTL) representing 33 potential pheromone compounds. QTL clustered significantly on two chromosomes, chromosome 8 for genital compounds and chromosome 20 for wing compounds, and chromosome 20 was enriched for potential pheromone biosynthesis genes. There was minimal overlap between pheromone QTL and known QTL for mate choice and color pattern. Nonetheless, we did detect linkage between a QTL for wing androconial area and optix, a color pattern locus known to play a role in reproductive isolation in these species. This tight clustering of putative pheromone loci might contribute to coincident reproductive isolating barriers, facilitating speciation despite ongoing gene flow. [ABSTRACT FROM AUTHOR]

Neural circuits govern the interface between the external environment, internal cues and outwardly directed behaviours. To process multiple environmental stimuli and integrate these with internal state requires considerable neural computation. Expansion in neural network size, most readily represented by whole brain size, has historically been linked to behavioural complexity, or the predominance of cognitive behaviours. Yet, it is largely unclear which aspects of circuit variation impact variation in performance. A key question in the field of evolutionary neurobiology is therefore how neural circuits evolve to allow improved behavioural performance or innovation. We discuss this question by first exploring how volumetric changes in brain areas reflect actual neural circuit change. We explore three major axes of neural circuit evolution-replication, restructuring and reconditioning of cells and circuits-and discuss how these could relate to broader phenotypes and behavioural variation. This discussion touches on the relevant uses and limitations of volumetrics, while advocating a more circuit-based view of cognition. We then use this framework to showcase an example from the insect brain, the multi-sensory integration and internal processing that is shared between the mushroom bodies and central complex. We end by identifying future trends in this research area, which promise to advance the field of evolutionary neurobiology.

Developmental modularity has long been viewed as a hierarchical organization that facilitates evolution through modification or reuse of preexisting modules. More recently, developmental modularity has been proposed as a mechanism capable of driving rapid evolution of novel color pattern phenotypes between closely related taxa. In this scenario, recombination between modular cis-regulatory elements (CREs) generates novel phenotypes by shuffling genetic variation at preexisting color pattern modules into new arrangements. Recent functional evidence from Drosophila flies and Heliconius butterflies, however, provides a series of examples in which CREs function in multiple developmental contexts and are thus highly pleiotropic. The potential prevalence of pleiotropy in CRE function could be a barrier to the proposed importance of CRE modules as a mechanism for rapid evolutionary change. Here we review the concept of developmental modularity, some examples that suggest developmental modularity underlies pattern evolution, and recent evidence that indicates modular CREs may be less common than previously expected. This leads us to suggest that alternative, non-modular hypotheses should be considered alongside proposals of modular CREs. We then propose the concept of evolutionary modularity as a specific alternative to developmental modularity when discrete, seemingly modular, phenotypes occur in hybridizing taxa. We suggest that evolutionary modularity provides a potentially important pathway for exchange of phenotypic elements between hybridizing taxa independent of the underlying developmental architecture.

Butterfly wing patterns have emerged as exceptional model systems with which to link the developmental and genetic processes that generate morphological variation with the ecological and evolutionary processes that shape this variation in natural populations. Among butterflies, research on species within the genus Heliconius has provided remarkable opportunities to explore how phenotypic diversity is generated within the context of an extraordinary adaptive radiation. Wing pattern diversity among the 48 species and hundreds of intraspecific variants arose within the last 12–14 million years and includes striking pattern convergence between distantly related species, as well as marked pattern divergence between closely related populations and species. Here, we synthesize recent research aimed at gaining a mechanistic understanding of how this variation is generated. This research integrates decades of controlled crossing experiments, and the discovery of major wing patterning genes (optix, aristaless1, WntA and cortex) with recent functional genetic manipulation using CRISPR/Cas9 targeted mutagenesis. The emerging data provides a rich framework with which to explore the repeatability of evolution, particularly within the context of how natural selection acts on divergent gene regulatory networks to generate both highly convergent, as well as highly divergent phenotypes. Overall, the functional data show that the gene regulatory networks underlying pattern variation diverge rapidly in Heliconius; yet these networks retain enough flexibility so that natural selection can drive the evolution of nearly identical patterns from different developmental genetic starting points. Moreover, for the first time this research is starting to illuminate the links between the genetic changes modulating pattern variation and how they influence the larger gene networks that are ultimately responsible for patterning a butterfly wing. There are still large gaps in our understanding, but current research priorities are well laid out and experimental methodologies are in place to address them. The challenge is to synthesize diverse research strategies into a cohesive picture of morphological evolution.

A major driving force behind the evolution of species-specific traits and novel structures is alterations in gene regulatory networks (GRNs). Comprehending evolution therefore requires an understanding of the nature of changes in GRN structure and the responsible mechanisms. Here, we review two insect pigmentation GRNs in order to examine common themes in GRN evolution and to reveal some of the challenges associated with investigating changes in GRNs across different evolutionary distances at the molecular level. The pigmentation GRN in Drosophila melanogaster and other drosophilids is a well-defined network for which studies from closely related species illuminate the different ways co-option of regulators can occur. The pigmentation GRN for butterflies of the Heliconius species group is less fully detailed but it is emerging as a useful model for exploring important questions about redundancy and modularity in cis-regulatory systems. Both GRNs serve to highlight the ways in which redeployment of trans-acting factors can lead to GRN rewiring and network co-option. To gain insight into GRN evolution, we discuss the importance of defining GRN architecture at multiple levels both within and between species and of utilizing a range of complementary approaches.