Selection drives divergence of eye morphology in sympatric Heliconius butterflies.

When populations experience different sensory conditions, natural selection may favor sensory system divergence, affecting peripheral structures and/or downstream neural pathways. We characterized the outer eye morphology of sympatric Heliconius species from different forest types and their first-generation reciprocal hybrids to test for adaptive visual system divergence and hybrid disruption. In Panama, Heliconius cydno occurs in closed forests, whereas Heliconius melpomene resides at the forest edge. Among wild individuals, H. cydno has larger eyes than H. melpomene, and there are heritable, habitat-associated differences in the visual brain structures that exceed neutral divergence expectations. Notably, hybrids have intermediate neural phenotypes, suggesting disruption. To test for similar effects in the visual periphery, we reared both species and their hybrids in common garden conditions. We confirm that H. cydno has larger eyes and provide new evidence that this is driven by selection. Hybrid eye morphology is more H. melpomene-like despite body size being intermediate, contrasting with neural trait intermediacy. Overall, our results suggest that eye morphology differences between H. cydno and H. melpomene are adaptive, and that hybrids may suffer fitness costs due to a mismatch between the peripheral visual structures and previously described neural traits that could affect visual performance.

Adaptive introgression of a visual preference gene.

Visual preferences are important drivers of mate choice and sexual selection, but little is known of how they evolve at the genetic level. In this study, we took advantage of the diversity of bright warning patterns displayed by Heliconius butterflies, which are also used during mate choice. Combining behavioral, population genomic, and expression analyses, we show that two Heliconius species have evolved the same preferences for red patterns by exchanging genetic material through hybridization. Neural expression of regucalcin1 correlates with visual preference across populations, and disruption of regucalcin1 with CRISPR-Cas9 impairs courtship toward conspecific females, providing a direct link between gene and behavior. Our results support a role for hybridization during behavioral evolution and show how visually guided behaviors contributing to adaptation and speciation are encoded within the genome.

Divergent warning patterns influence male and female mating behaviours in a tropical butterfly.

Traits under divergent ecological selection that also function during mating can be important in maintaining species boundaries. Few studies have considered mutual mate choice, where both males and females base mating decisions on the same trait. Wing colouration in Heliconius butterflies evolved as a warning signal but also functions as a mating cue. We investigated the contribution of visual preference to assortative mating in an aposematic butterfly Heliconius cydno in the context of reproductive isolation with its sympatric, visually distinct relative Heliconius melpomene. Heliconius cydno have conspicuous white bands on their forewings, whereas those of H. melpomene are red in colour. We predicted that both sexes of H. cydno contributed to assortative mating by exhibiting visual preference towards conspecific wing colouration. We analysed published and new data from preference experiments, in which males were presented with conspecific and H. melpomene females. We also recorded female responses and mating outcomes in choice experiments, involving conspecific males with either the original white or artificially painted red forewing bands. Both sexes of H. cydno responded more positively towards the conspecific colouration, and males strongly preferred females of its own colours. In contrast, male colouration did not predict mating outcomes in female choice experiments. As courtships are initiated by males in butterflies, our findings suggest that female visual preference might be of secondary importance in H. cydno. Our data also suggest that the contribution of visual preference to reproductive isolation might be unequal between H. cydno and its sympatric relative H. melpomene.

Parallel shifts in flight-height associated with altitude across incipient Heliconius species.

Vertical gradients in microclimate, resource availability, and interspecific interactions are thought to underly stratification patterns in tropical insect communities. However, only a few studies have explored the adaptive significance of vertical space use during the early stages of reproductive isolation. We analysed flight-height variation across speciation events in Heliconius butterflies, representing parallel colonizations of high-altitude forest. We measured flight-height in wild H. erato venus and H. chestertonii, parapatric lowland and mountain specialists, respectively, and found that H. chestertonii consistently flies at a lower height. By comparing our data to previously published results for the ecologically equivalent H. e. cyrbia (lowland) and H. himera (high altitude), we found that the species flying closest to the ground are those that recently colonized high-altitude forests. We show that these repeated trends largely result from shared patterns of ecological selection producing parallel trait-shifts in H. himera and H. chestertonii. Although our results imply a signature of local adaptation, we did not find an association between resource distribution and flight-height in H. e. venus and H. chestertonii. We discuss how this pattern may be explained by variations in forest structure and microclimate. Overall, our findings underscore the importance of behavioural adjustments during early divergence mediated by altitude-shifts.

The Sensory Ecology of Speciation.

In this work, we explore the potential influence of sensory ecology on speciation, including but not limited to the concept of sensory drive, which concerns the coevolution of signals and sensory systems with the local environment. The sensory environment can influence individual fitness in a variety of ways, thereby affecting the evolution of both pre- and postmating reproductive isolation. Previous work focused on sensory drive has undoubtedly advanced the field, but we argue that it may have also narrowed our understanding of the broader influence of the sensory ecology on speciation. Moreover, the clearest examples of sensory drive are largely limited to aquatic organisms, which may skew the influence of contributing factors. We review the evidence for sensory drive across environmental conditions, and in this context discuss the importance of more generalized effects of sensory ecology on adaptive behavioral divergence. Finally, we consider the potential of rapid environmental change to influence reproductive barriers related to sensory ecologies. Our synthesis shows the importance of sensory conditions for local adaptation and divergence in a range of behavioral contexts and extends our understanding of the interplay between sensory ecology and speciation.

Genetics and the Evolution of Prezygotic Isolation.

The significance of prezygotic isolation for speciation has been recognized at least since the Modern Synthesis. However, fundamental questions remain. For example, how are genetic associations between traits that contribute to prezygotic isolation maintained? What is the source of genetic variation underlying the evolution of these traits? And how do prezygotic barriers affect patterns of gene flow? We address these questions by reviewing genetic features shared across plants and animals that influence prezygotic isolation. Emerging technologies increasingly enable the identification and functional characterization of the genes involved, allowing us to test established theoretical expectations. Embedding these genes in their developmental context will allow further predictions about what constrains the evolution of prezygotic isolation. Ongoing improvements in statistical and computational tools will reveal how pre- and postzygotic isolation may differ in how they influence gene flow across the genome. Finally, we highlight opportunities for progress by combining theory with appropriate data.

Quantifying visual acuity in Heliconius butterflies.

Heliconius butterflies are well-known for their colourful wing patterns, which advertise distastefulness to potential predators and are used during mate choice. However, the relative importance of different aspects of these signals will depend on the visual abilities of Heliconius and their predators. Previous studies have investigated colour sensitivity and neural anatomy, but visual acuity (the ability to perceive detail) has not been studied in these butterflies. Here, we provide the first estimate of visual acuity in Heliconius: from a behavioural optomotor assay, we found that mean visual acuity = 0.49 cycles-per-degree (cpd), with higher acuity in males than females. We also examined eye morphology and report more ommatidia in male eyes. Finally, we estimated how visual acuity affects Heliconius visual perception compared to a potential avian predator. Whereas the bird predator maintained high resolving power, Heliconius lost the ability to resolve detail at greater distances, though colours may remain salient. These results will inform future studies of Heliconius wing pattern evolution, as well as other aspects in these highly visual butterflies, which have emerged as an important system in studies of adaptation and speciation.

Oviposition behavior is not affected by ultraviolet light in a butterfly with sexually-dimorphic expression of a UV-sensitive opsin.

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.

La visión animal cumple una función crucial guiando comportamientos complejos. Para las mariposas Heliconius, la visión juega un papel principal en comportamientos como la búsqueda de alimento, la elección de pareja y la ovoposición. La visión a color en Heliconius está compuesta por una combinación de fotoreceptores (opsinas) sensibles a rayos ultravioleta (UV), azul y ondas de longitud larga (verde­rojo). Adicionalmente, estas mariposas posen una segunda opsina sensible a rayos UV, generada por una duplicación. La expresión de estas dos opsinas UV varia ampliamente dentro del genero de Heliconius. En la especie Heliconius erato, la expresión de estas dos opsinas presenta un dimorfismo sexual donde únicamente las hembras expresan las dos opsinas sensibles a UV, lo que les permite la discriminación de ondas en el rango ultravioleta. Sin embargo, no se han estudiado las presiones ecológicas que han llevado a estas diferencias en la percepción visual entre ambos sexos. Las hembras de Heliconius invierten mucho tiempo buscando plantas hospederas para poner sus huevos y este comportamiento depende en gran medida de señales visuales. En este estudio, manipulamos la disponibilidad de rayos UV en condiciones naturales de luz, para evaluar si el comportamiento de ovoposición es guiado por la visión en UV en H. erato y su especie hermana Heliconius himera. Descubrimos que la presencia de rayos ultravioleta no influye en el número de intentos de ovoposición ni en la cantidad de huevos puestos. Además, la planta hospedera Passiflora puntata, presenta una escasa reflexión en las longitudes de onda UV. Así mismo, nuestros modelos de la visión de H. erato hembras, predicen solamente una estimulación mínima de las opsinas UV. En resumen, nuestros resultados sugieren que las ondas ultravioletas no afectan directamente la capacidad de las hembras Heliconius para encontrar sitios de oviposición adecuados. Alternativamente, la discriminación de ondas UV podría estar siendo utilizada en el contexto de la búsqueda de alimento o la elección de pareja, pero esta hipótesis sigue por ser evaluada.

Parallel evolution of behavior, physiology, and life history associated with altitudinal shifts in forest type in Heliconius butterflies.

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.

Does sexual conflict contribute to the evolution of novel warning patterns?

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.

Evolution of physical linkage between loci controlling ecological traits and mating preferences.

Coupling of multiple barriers to gene-flow, such as divergent local adaptation and reproductive isolation, facilitates speciation. However, alleles at loci that contribute to barrier effects can be dissociated by recombination. Models of linkage between diverging alleles often consider elements that reduce recombination, such as chromosomal inversions and alleles that modify recombination rate between existing loci. In contrast, here, we consider the evolution of linkage due to the close proximity of loci on the same chromosome. Examples of such physical linkage exist in several species, but in other cases, strong associations are maintained without physical linkage. We use an individual-based model to study the conditions under which the physical linkage between loci controlling ecological traits and mating preferences might be expected to evolve. We modelled a single locus controlling an ecological trait that acts also as a mating cue. Mating preferences are controlled by multiple loci, formed by mutations that are randomly placed in the "genome", within varying distances from the ecological trait locus, allowing us to examine which genomic architectures spread across the population. Our model reveals that stronger physical linkage is favoured when mating preferences and selection are weaker. Under such conditions mating among divergent phenotypes is more frequent, and matching ecological trait and mating preference alleles are more likely to become dissociated by recombination, favouring the evolution of genetic linkage. While most theoretical studies on clustering of divergent loci focus on how physical linkage influences speciation, we show how physical linkage itself can arise, establishing conditions that can favour speciation.

Homage to Felsenstein 1981, or why are there so few/many species?

If there are no constraints on the process of speciation, then the number of species might be expected to match the number of available niches and this number might be indefinitely large. One possible constraint is the opportunity for allopatric divergence. In 1981, Felsenstein used a simple and elegant model to ask if there might also be genetic constraints. He showed that progress towards speciation could be described by the build-up of linkage disequilibrium among divergently selected loci and between these loci and those contributing to other forms of reproductive isolation. Therefore, speciation is opposed by recombination, because it tends to break down linkage disequilibria. Felsenstein then introduced a crucial distinction between "two-allele" models, which are subject to this effect, and "one-allele" models, which are free from the recombination constraint. These fundamentally important insights have been the foundation for both empirical and theoretical studies of speciation ever since.

Light environment influences mating behaviours during the early stages of divergence in tropical butterflies.

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.

Neural divergence and hybrid disruption between ecologically isolated Heliconius butterflies.

The importance of behavioral evolution during speciation is well established, but we know little about how this is manifest in sensory and neural systems. A handful of studies have linked specific neural changes to divergence in host or mate preferences associated with speciation. However, the degree to which brains are adapted to local environmental conditions, and whether this contributes to reproductive isolation between close relatives that have diverged in ecology, remains unknown. Here, we examine divergence in brain morphology and neural gene expression between closely related, but ecologically distinct, Heliconius butterflies. Despite ongoing gene flow, sympatric species pairs within the melpomene-cydno complex are consistently separated across a gradient of open to closed forest and decreasing light intensity. By generating quantitative neuroanatomical data for 107 butterflies, we show that Heliconius melpomene and Heliconius cydno clades have substantial shifts in brain morphology across their geographic range, with divergent structures clustered in the visual system. These neuroanatomical differences are mirrored by extensive divergence in neural gene expression. Differences in both neural morphology and gene expression are heritable, exceed expected rates of neutral divergence, and result in intermediate traits in first-generation hybrid offspring. Strong evidence of divergent selection implies local adaptation to distinct selective optima in each parental microhabitat, suggesting the intermediate traits of hybrids are poorly matched to either condition. Neural traits may therefore contribute to coincident barriers to gene flow, thereby helping to facilitate speciation.

A novel terpene synthase controls differences in anti-aphrodisiac pheromone production between closely related Heliconius butterflies.

Plants and insects often use the same compounds for chemical communication, but not much is known about the genetics of convergent evolution of chemical signals. The terpene (E)-ß-ocimene is a common component of floral scent and is also used by the butterfly Heliconius melpomene as an anti-aphrodisiac pheromone. While the biosynthesis of terpenes has been described in plants and microorganisms, few terpene synthases (TPSs) have been identified in insects. Here, we study the recent divergence of 2 species, H. melpomene and Heliconius cydno, which differ in the presence of (E)-ß-ocimene; combining linkage mapping, gene expression, and functional analyses, we identify 2 novel TPSs. Furthermore, we demonstrate that one, HmelOS, is able to synthesise (E)-ß-ocimene in vitro. We find no evidence for TPS activity in HcydOS (HmelOS ortholog of H. cydno), suggesting that the loss of (E)-ß-ocimene in this species is the result of coding, not regulatory, differences. The TPS enzymes we discovered are unrelated to previously described plant and insect TPSs, demonstrating that chemical convergence has independent evolutionary origins.

Clustering of loci controlling species differences in male chemical bouquets of sympatric Heliconius butterflies.

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.

Visual mate preference evolution during butterfly speciation is linked to neural processing genes.

Many animal species remain separate not because their individuals fail to produce viable hybrids but because they "choose" not to mate. However, we still know very little of the genetic mechanisms underlying changes in these mate preference behaviours. Heliconius butterflies display bright warning patterns, which they also use to recognize conspecifics. Here, we couple QTL for divergence in visual preference behaviours with population genomic and gene expression analyses of neural tissue (central brain, optic lobes and ommatidia) across development in two sympatric Heliconius species. Within a region containing 200 genes, we identify five genes that are strongly associated with divergent visual preferences. Three of these have previously been implicated in key components of neural signalling (specifically an ionotropic glutamate receptor and two regucalcins), and overall our candidates suggest shifts in behaviour involve changes in visual integration or processing. This would allow preference evolution without altering perception of the wider environment.

Species specificity and intraspecific variation in the chemical profiles of Heliconius butterflies across a large geographic range.

In many animals, mate choice is important for the maintenance of reproductive isolation between species. Traits important for mate choice and behavioral isolation are predicted to be under strong stabilizing selection within species; however, such traits can also exhibit variation at the population level driven by neutral and adaptive evolutionary processes. Here, we describe patterns of divergence among androconial and genital chemical profiles at inter- and intraspecific levels in mimetic Heliconius butterflies. Most variation in chemical bouquets was found between species, but there were also quantitative differences at the population level. We found a strong correlation between interspecific chemical and genetic divergence, but this correlation varied in intraspecific comparisons. We identified "indicator" compounds characteristic of particular species that included compounds already known to elicit a behavioral response, suggesting an approach for identification of candidate compounds for future behavioral studies in novel systems. Overall, the strong signal of species identity suggests a role for these compounds in species recognition, but with additional potentially neutral variation at the population level.

Hybridization and transgressive exploration of colour pattern and wing morphology in Heliconius butterflies.

Hybridization can generate novel phenotypes distinct from those of parental lineages, a phenomenon known as transgressive trait variation. Transgressive phenotypes might negatively or positively affect hybrid fitness, and increase available variation. Closely related species of Heliconius butterflies regularly produce hybrids in nature, and hybridization is thought to play a role in the diversification of novel wing colour patterns despite strong stabilizing selection due to interspecific mimicry. Here, we studied wing phenotypes in first- and second-generation hybrids produced by controlled crosses between either two co-mimetic species of Heliconius or between two nonmimetic species. We quantified wing size, shape and colour pattern variation and asked whether hybrids displayed transgressive wing phenotypes. Discrete traits underlain by major-effect loci, such as the presence or absence of colour patches, generate novel phenotypes. For quantitative traits, such as wing shape or subtle colour pattern characters, hybrids only exceed the parental range in specific dimensions of the morphological space. Overall, our study addresses some of the challenges in defining and measuring phenotypic transgression for multivariate traits and our data suggest that the extent to which transgressive trait variation in hybrids contributes to phenotypic diversity depends on the complexity and the genetic architecture of the traits.

A major locus controls a biologically active pheromone component in Heliconius melpomene.

Understanding the production, response, and genetics of signals used in mate choice can inform our understanding of the evolution of both intraspecific mate choice and reproductive isolation. Sex pheromones are important for courtship and mate choice in many insects, but we know relatively little of their role in butterflies. The butterfly Heliconius melpomene uses a complex blend of wing androconial compounds during courtship. Electroantennography in H. melpomene and its close relative Heliconius cydno showed that responses to androconial extracts were not species specific. Females of both species responded equally strongly to extracts of both species, suggesting conservation of peripheral nervous system elements across the two species. Individual blend components provoked little to no response, with the exception of octadecanal, a major component of the H. melpomene blend. Supplementing octadecanal on the wings of octadecanal-rich H. melpomene males led to an increase in the time until mating, demonstrating the bioactivity of octadecanal in Heliconius. Using quantitative trait locus (QTL) mapping, we identified a single locus on chromosome 20 responsible for 41% of the parental species' difference in octadecanal production. This QTL does not overlap with any of the major wing color or mate choice loci, nor does it overlap with known regions of elevated or reduced FST . A set of 16 candidate fatty acid biosynthesis genes lies underneath the QTL. Pheromones in Heliconius carry information relevant for mate choice and are under simple genetic control, suggesting they could be important during speciation.