True UV color vision in a female butterfly with two UV opsins

ABSTRACT In true color vision, animals discriminate between light wavelengths, regardless of intensity, using at least two photoreceptors with different spectral sensitivity peaks. Heliconius butterflies have duplicate UV opsin genes, which encode ultraviolet and violet photoreceptors, respectively. In Heliconius erato, only females express the ultraviolet photoreceptor, suggesting females (but not males) can discriminate between UV wavelengths. We tested the ability of H. erato, and two species lacking the violet receptor, Heliconius melpomene and Eueides isabella, to discriminate between 380 and 390 nm, and between 400 and 436 nm, after being trained to associate each stimulus with a sugar reward. We found that only H. erato females have color vision in the UV range. Across species, both sexes show color vision in the blue range. Models of H. erato color vision suggest that females have an advantage over males in discriminating the inner UV-yellow corollas of Psiguria flowers from their outer orange petals. Moreover, previous models ( McCulloch et al., 2017) suggested that H. erato males have an advantage over females in discriminating Heliconius 3-hydroxykynurenine (3-OHK) yellow wing coloration from non-3-OHK yellow wing coloration found in other heliconiines. These results provide some of the first behavioral evidence for female H. erato UV color discrimination in the context of foraging, lending support to the hypothesis ( Briscoe et al., 2010) that the duplicated UV opsin genes function together in UV color vision. Taken together, the sexually dimorphic visual system of H. erato appears to have been shaped by both sexual selection and sex-specific natural selection.

The genetic basis of structural colour variation in mimetic Heliconius butterflies

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 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. Our results suggest that these species have followed different evolutionary trajectories in their convergent evolution of similar structural colour. This study provides a starting point for determining the genetic basis of structural colouration more broadly.

Synteny-based genome assembly for 16 species of Heliconius butterflies, and an assessment of structural variation across the genus

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 H. 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 (H. hecale, H. elevatus and H. pardalinus) differences in genome size are mainly driven by a few restricted repetitive regions. Genes within these repeat regions show an increase in exon copy number, an absence of internal stop codons, evidence of constraint on non-synonymous changes, and increased expression, all of which suggest that 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 should aid further genetic and evolutionary studies in this genus.

Perfect mimicry between Heliconius butterflies is constrained by genetics and development

Müllerian mimicry strongly exemplifies the power of natural selection. However, the exact measure of such adaptive phenotypic convergence and the possible causes of its imperfection often remain unidentified. Here, we first quantify wing colour pattern differences in the forewing region of 14 co-mimetic colour pattern morphs of the butterfly species Heliconius erato and Heliconius melpomene and measure the extent to which mimicking colour pattern morphs are not perfectly identical. Next, using gene-editing CRISPR/Cas9 KO experiments of the gene WntA , which has been mapped to colour pattern diversity in these butterflies, we explore the exact areas of the wings in which WntA affects colour pattern formation differently in H. erato and H. melpomene. We find that, while the relative size of the forewing pattern is generally nearly identical between co-mimics, the CRISPR/Cas9 KO results highlight divergent boundaries in the wing that prevent the co-mimics from achieving perfect mimicry. We suggest that this mismatch may be explained by divergence in the gene regulatory network that defines wing colour patterning in both species, thus constraining morphological evolution even between closely related species.

Wing-pattern-specific effects of experience on mating behavior in Heliconius melpomene butterflies

Many animals have the ability to learn, and some taxa have shown learned mate preference. This learning may be important for speciation in some species. The butterfly Heliconius melpomene is a model system for several areas of research, including hybridization, mate selection, and speciation, partially due to its widespread diversity of wing patterns. It remains unclear whether these butterflies can learn to prefer certain mates and if social experience shapes realized mating preferences. Here we test whether previous experience with a female influences male mate preference for two different H. melpomene subspecies, H. m. malleti and H. m. rosina. We conducted no-choice behavioral assays to determine if latency to court and whether males courted (vs no courtship) differed between naïve males and males with previous exposure to a young, sexually mature, virgin female. To test whether assortative courtship preference is learned in H. melpomene, males were either paired with a female who shared their phenotype or one who did not. Naïve H. m. malletti males courted assortatively, while naïve H.m. rosina males did not. Experienced H. m. malleti males reduced their courting relative to naïve males, suggesting that social experience with a sexually mature female that does not result in copulation may be perceived as a negative experience. In contrast, experienced H. m. rosina males exhibited similar courting rates to naïve H. m. rosina males. Our results suggest that social experience can influence male mating behavior in H. melpomene and that behavioral plasticity may differ across populations in this species.

Limited genetic parallels underlie convergent evolution of quantitative pattern variation in mimetic butterflies

Mimetic systems allow us to address the question of whether the same genes control similar phenotypes in different species. Although widespread parallels have been found for major effect loci, much less is known about genes that control quantitative trait variation. In this study, we identify and compare the loci that control subtle changes in the size and shape of forewing pattern elements in two Heliconius butterfly co-mimics. We use quantitative trait locus (QTL) analysis with a multivariate phenotyping approach to map the variation in red pattern elements across the whole forewing surface of Heliconius erato and Heliconius melpomene. These results are compared to a QTL analysis of univariate trait changes, and show that our resolution for identifying small effect loci is improved with the multivariate approach. QTL likely corresponding to the known patterning gene optix were found in both species but otherwise, a remarkably low level of genetic parallelism was found. This lack of similarity indicates that the genetic basis of convergent traits may not be as predictable as assumed from studies that focus solely on Mendelian traits.

Perfect mimicry between Heliconius butterflies is constrained by genetics and development

Müllerian mimicry strongly exemplifies the power of natural selection. However, the exact measure of such adaptive phenotypic convergence and the possible causes of its imperfection often remain unidentified. The butterfly species Heliconius erato and Heliconius melpomene have a large diversity of co-mimicking geographic races with remarkable resemblance in melanic patterning across the mid-forewing that has been linked to expression patterns of the gene WntA. Recent CRISPR/Cas9 experiments have informed us on the exact areas of the wings in which WntA affects color pattern formation in both H. erato and H. melpomene, thus providing a unique comparative dataset to explore the functioning of a gene and its potential effect on phenotypic evolution. We therefore quantified wing color pattern differences in the mid-forewing region of 14 co-mimetic races of H. erato and H. melpomene and measured the extent to which mimicking races are not perfectly identical. While the relative size of the mid-forewing pattern is generally nearly identical, our results highlight the areas of the wing that prevent these species from achieving perfect mimicry and demonstrate that this mismatch can be largely explained by constraints imposed by divergence in the gene regulatory network that define wing color patterning. Divergence in the developmental architecture of a trait can thus constrain morphological evolution even between relatively closely related species.

A novel terpene synthase produces an anti-aphrodisiac pheromone in the butterfly Heliconius melpomene

Terpenes, a group of structurally diverse compounds, are the biggest class of secondary metabolites. While the biosynthesis of terpenes by enzymes known as terpene synthases (TPSs) has been described in plants and microorganisms, few TPSs have been identified in insects, despite the presence of terpenes in multiple insect species. Indeed, in many insect species, it remains unclear whether terpenes are sequestered from plants or biosynthesised de novo. No homologs of plant TPSs have been found in insect genomes, though insect TPSs with an independent evolutionary origin have been found in Hemiptera and Coleoptera. In the butterfly Heliconius melpomene, the monoterpene (E)-β-ocimene acts as an anti-aphrodisiac pheromone, where it is transferred during mating from males to females to avoid re-mating by deterring males. To date only one insect monoterpene synthase has been described, in Ips pini (Coleoptera), and is a multifunctional TPS and isoprenyl diphosphate synthase (IDS). Here, we combine linkage mapping and expression studies to identify candidate genes involved in the biosynthesis of (E)-β-ocimene. We confirm that H. melpomene has two enzymes that exhibit TPS activity, and one of these, HMEL037106g1 is able to synthesise (E)-β-ocimene in vitro. Unlike the enzyme in Ips pini, these enzymes only exhibit residual IDS activity, suggesting they are more specialised TPSs, akin to those found in plants. Phylogenetic analysis shows that these enzymes are unrelated to previously described plant and insect TPSs. The distinct evolutionary origin of TPSs in Lepidoptera suggests that they have evolved multiple times in insects.Significance statementTerpenes are a diverse class of natural compounds, used by both plants and animals for a variety of functions, including chemical communication. In insects it is often unclear whether they are synthesised de novo or sequestered from plants. Some plants and insects have converged to use the same compounds. For instance, (E)-β-ocimene is a common component of floral scent and is also used by the butterfly Heliconius melpomene as an anti-aphrodisiac pheromone. We describe two novel terpene synthases, one of which synthesises (E)-β-ocimene in H. melpomene, unrelated not only to plant enzymes but also other recently identified insect terpene synthases. This provides the first evidence that the ability to synthesise terpenes has arisen multiple times independently within the insects.