Mimicry and the evolution of premating isolation in Heliconius melpomene Linnaeus

Ecological divergence can cause speciation if adaptive traits have pleiotropic effects on mate choice. In Heliconius butterflies, mimetic patterns play a role in mate detection between sister species, as well as signalling to predators. Here we show that male butterflies from four recently diverged parapatric populations of Heliconius melpomene are more likely to approach and court their own colour patterns as compared with those of other races. A few exceptions, where males were more attracted to patterns other than their own, suggest that some mimetic patterns are sub-optimal in mate choice. Genotype frequencies in hybrid zones between races of H. melpomene suggest that mating is random, so reinforcement is unlikely to have played a role in intra-specific divergence. In summary, co-evolved divergence of colour pattern and mate preference occurs rapidly and is likely the first step in Heliconius speciation.     

Patterns of pollen feeding and habitat preference among Heliconius species

Abstract 1. The ecological circumstances that precipitate speciation remain poorly understood. Here, a community of Heliconius butterflies in lowland Panama was studied to investigate patterns of pollen use, and more specifically the ecological changes associated with the recent divergence of Heliconius melpomene (Linnaeus) and H. cydno (Doubleday).
2. Considering the seven commonest Heliconius species in the community, 32 types of pollen or spore were encountered in pollen loads but only five pollen species were common. Systematic exploitation of pollen was therefore confined to a small proportion of the flowers visited.
3. Most of the variation in pollen load composition between individuals was explained by differences in collecting locality. The exception was Psiguria , which was used in all habitats by the melpomene / hecale clade far more than by the erato / sapho clade. This may suggest an ancestral switch within Heliconius towards increased reliance on Psiguria pollen.
4. Heliconius cydno and H. melpomene differed significantly in pollen load composition for three of the five most commonly collected pollen species. This is most probably explained by differences in habitat preference; H. melpomene and its co-mimic H. erato are found in open habitat while H. cydno and its co-mimic H. sapho are found in closed-canopy forest.
5. As melpomene and cydno are known to hybridise occasionally, such differences in adult microhabitat contribute to pre-mating isolation. Habitat divergence between H. cydno and H. melpomene , which is associated with changes in mimicry, must have played a role in their recent speciation.     

Hybrid incompatibility is consistent with a hybrid origin of Heliconius heurippa Hewitson from its close relatives, Heliconius cydno Doubleday and Heliconius melpomene Linnaeus

Abstract Shared ancestral variation and introgression complicates the reconstruction of phylogenetic relationships among closely related taxa. Here we use overall genomic compatibility as an alternative estimate of species relationships in a group where divergence is rapid and genetic exchange is common. Heliconius heurippa, a butterfly species endemic to Colombia, has a colour pattern genetically intermediate between H. cydno and H. melpomene: its hindwing is nearly indistinguishable from that of H. melpomene and its forewing band is an intermediate phenotype between both species. This observation has lead to the suggestion that the pattern of H. heurippa arose through hybridization. We present a genetic analysis of hybrid compatibility in crosses between the three taxa. Heliconius heurippa x H. cydno and female H. melpomene x male H. heurippa yield fertile and viable F1 hybrids, but male H. melpomene x female H. heurippa crosses yield sterile F1 females. In contrast, Haldane's rule has previously been detected between H. melpomene and H cydno in both directions. Therefore, H. heurippa is most closely related to H. cydno, with some evidence for introgression of genes from H. melpomene. The results are compatible with the hypothesis of a hybrid origin for H. heurippa. In addition, backcrosses using F1 hybrid males provide evidence for a large Z(X)-chromosome effect on sterility and for recessive autosomal sterility factors as predicted by Dominance Theory.     

Genetic analysis of a wild-caught hybrid between non-sister Heliconius butterfly species

Interspecific hybridization occurs regularly in wild Heliconius butterflies, although hybrid individuals are usually very rare. However, hybridization generally occurs only between the most closely related species. We report a rare naturally occurring hybrid between non-sister species and carry out the first genetic analysis of such distant hybridization. Mitochondrial and nuclear genes indicate that the specimen is an F1 hybrid between a female Heliconius ethilla and a male Heliconius melpomene, originating from a group of 13 species estimated to have diverged over 2.5 Myr ago. The presence of such distant natural hybrids, together with evidence for backcrossing, suggests that gene flow across species boundaries can take place long after speciation. Adaptive genes such as those involved in wing coloration could thus be widely shared among members of this highly mimetic genus.     

Some early works on heliconiine butterflies and their biology (Lepidoptera, Nymphalidae)

In the course of a survey of the history of the study of Heliconiinae, particularly their biology, in the eighteenth and nineteenth centuries it is shown that Heliconius nigromarginatus (Goeze) and H. pallescens (Goeze) are nomina oblita , and that H. cinereofuscus (Goeze) is not, as always supposed, a species in its own right. A new name is proposed for H. melpomene cybele (Cramer). The origin and application of some of Linnaeus' names is discussed.
Illustrations of Heliconius by Seba, Clerck, Petiver and Cramer show that three species, melpomene, erato and doris have been polymorphic at least since the middle of the eighteenth century.
The Indo-Australian genus Cethosia should probably be included in the Heliconiinae.     

Hybrid sterility,Haldane's rule and speciation in Heliconius cydno and H. melpomene

Most genetic studies of Haldane's rule, in which hybrid sterility or inviability affects the heterogametic sex preferentially, have focused on Drosophila. It therefore remains unclear to what extent the conclusions of that work apply more generally, particularly in female-heterogametic taxa such as birds and Lepidoptera. Here we present a genetic analysis of Haldane's rule in Heliconius butterflies. Female F(1) hybrids between Heliconius melpomene and H. cydno are completely sterile, while males have normal to mildly reduced fertility. In backcrosses of male F(1) hybrids, female offspring range from completely sterile to fully fertile. Linkage analysis using the Z-linked triose-phosphate isomerase locus demonstrates a "large X" (Z) effect on sterility. Expression of female sterility varies among crosses in this and a previous study of Heliconius. Sterility may result from the production of normal but infertile eggs, production of small infertile eggs, or from a complete failure to develop ovarioles, which suggests multiple routes to the evolution of hybrid sterility in these Heliconius species. These results conform to the expectations of the "dominance" rather than "faster male" theories of Haldane's rule and suggest that relatively few loci are responsible. The two species are broadly sympatric and hybridize in the wild, so that female hybrid sterility forms one of several strong but incomplete barriers to gene flow in nature. The effect of female sterility is comparable to that of selection against non-mimetic hybrids, while mate choice forms a much stronger barrier to gene transfer.     

Genomic tools and cDNA derived markers for butterflies

The Lepidoptera have long been used as examples in the study of evolution, but some questions remain difficult to resolve due to a lack of molecular genetic data. However, as technology improves, genomic tools are becoming increasingly available to tackle unanswered evolutionary questions. Here we have used expressed sequence tags (ESTs) to develop genetic markers for two Müllerian mimic species, Heliconius melpomene and Heliconius erato. In total 1363 ESTs were generated, representing 330 gene objects in H. melpomene and 431 in H. erato. User-friendly bioinformatic tools were used to construct a nonredundant database of these putative genes (available at http://www.heliconius.org), and annotate them with blast similarity searches, InterPro matches and Gene Ontology terms. This database will be continually updated with EST sequences for the Papilionideae as they become publicly available, providing a tool for gene finding in the butterflies. Alignments of the Heliconius sequences with putative homologues derived from Bombyx mori or other public data sets were used to identify conserved PCR priming sites, and develop 55 markers that can be amplified from genomic DNA in both H. erato and H. melpomene. These markers will be used for comparative linkage mapping in Heliconius and will have applications in other phylogenetic and genomic studies in the Lepidoptera.     

Adaptive introgression across species boundaries in Heliconius butterflies

It is widely documented that hybridisation occurs between many closely related species, but the importance of introgression in adaptive evolution remains unclear, especially in animals. Here, we have examined the role of introgressive hybridisation in transferring adaptations between mimetic Heliconius butterflies, taking advantage of the recent identification of a gene regulating red wing patterns in this genus. By sequencing regions both linked and unlinked to the red colour locus, we found a region that displays an almost perfect genotype by phenotype association across four species, H. melpomene, H. cydno, H. timareta, and H. heurippa. This particular segment is located 70 kb downstream of the red colour specification gene optix, and coalescent analysis indicates repeated introgression of adaptive alleles from H. melpomene into the H. cydno species clade. Our analytical methods complement recent genome scale data for the same region and suggest adaptive introgression has a crucial role in generating adaptive wing colour diversity in this group of butterflies.     

A new mimetic species of Heliconius (Lepidoptera: Nymphalidae), from southeastern Colombia, revealed by cladistic analysis of mitochondrial DNA sequences

A new species of Heliconius and a new geographical race of Heliconius melpomene are described from the vicinity of Mocoa, Dpto. Putumayo, Colombia, based on molecular and morphological characters. The new species, H. tristero , is a close relative of H. cydno , a geographically differentiated species which lacks red coloration and engages in Müllerian mimicry with other blue and yellow Heliconius species in Central and northwestern South America. H. tristero has switched mimetic associations, instead mimicking the local, sympatric forms of two widespread mimetic species, H. erato and H. melpomene. This discovery provides evidence that the splinter species H. heurippa, H. tristero and H. timareta represent phenotypically divergent members of the H. cydno group that are endemic to successive river valleys on the eastern slope of the northern Andean Cordillera. The nominal taxon Heliconius amaryllis bellula Stichel, currently misapplied to both H. tristero and H. melpomene populations from the Mocoa region of Colombia, is considered here to represent a hybrid between H. heurippa and H. tristero. The Mocoa melpomene race is formally named Heliconius melpomene mocoa , new subspecies.     

ASSORTATIVE MATING PREFERENCES AMONG HYBRIDS OFFERS A ROUTE TO HYBRID SPECIATION

Homoploid speciation generates species without a change in chromosome number via introgressive hybridization and has been considered rare in animals. Heliconius butterflies exhibit bright aposematic color patterns that also act as cues in assortative mating. Heliconius heurippa has a color pattern that can be recreated by introgression of the H. melpomene red band into an H. cydno genetic background. Wild H. heurippa males show assortative mating based on color pattern and we here investigate the origin of this preference by studying first-generation backcross hybrids between H. melpomene and H. cydno that resemble H. heurippa . These hybrids show assortative mating preferences, showing a strong preference for their own color pattern over that of either parental species. This is consistent with a genetic basis to wing pattern preference and implies, first, that assortative mating preferences would facilitate the initial establishment of a homozygous hybrid color pattern by increasing the likelihood that early generation hybrids mate among themselves. Second, once established such a lineage would inherit assortative mating preferences that would lead to partial reproductive isolation from parental lineages.     

Review. Hybrid trait speciation and Heliconius butterflies

Homoploid hybrid speciation (HHS) is the establishment of a novel species through introgressive hybridization without a change in chromosome number. We discuss different routes by which this might occur and propose a novel term, 'hybrid trait speciation', which combines the idea that hybridization can generate adaptive novelty with the 'magic trait' model of ecological speciation. Heliconius butterflies contain many putative examples of hybrid colour patterns, but only recently has the HHS hypothesis been tested explicitly in this group. Molecular data has shown evidence for gene flow between many distinct species. Furthermore, the colour pattern of Heliconius heurippa can be recreated in laboratory crosses between Heliconius melpomene and Heliconius cydno and, crucially, plays a role in assortative mating between the three species. Nonetheless, although the genome of H. heurippa shows evidence for hybridization, it is not a mosaic of the two parental species. Instead, ongoing hybridization has likely blurred any signal of the original speciation event. We argue that where hybridization leads to novel adaptive traits that also cause reproductive isolation, it is likely to trigger speciation.     

Correlations between scale structure and pigmentation in butterfly wings

SUMMARY We examined the correlation between color and structure of wing scales in the nymphalid butterflies Bicyclus anynana and Heliconius melpomene . All scales in B. anynana are rather similar in comparison to the clear structural differences of differently pigmented scales in H. melpomene . Where scale structural differences in H. melpomene are qualitative, they seem to be quantitative in B. anynana . There is a "gradient" in the density of some structural elements, the cross ribs, in the scales of B. anynana : black, gold, and brown scales show progressively lower cross rib density within an individual. There is, however, high individual variation in the absolute cross rib densities (i.e., scales with a particular color and cross rib density in one individual may have a different color but similar density in another individual). By ectopically inducing color pattern during early pupal development, we examined whether a scale's color and its microstructure could be uncoupled. The effect of these manipulations appears to be different in B. anynana and H. melpomene . In Bicyclus , "black" scales induced by wing damage at an ectopic location normally containing brown scales acquire both an intermediate structure and color between that of brown and normal black scales. In Heliconius , however, intermediate colors or scale structure were never observed, and scales with an altered color (due to damage) always have the same structure as normal scales with that color. The results are discussed on the basis of gene expression patterns, variability in rates of scale development and pigment, and scale sclerotization pathways.     

Butterfly wing colours: scale beads make white pierid wings brighter

The wing-scale morphologies of the pierid butterflies Pieris rapae (small white) and Delias nigrina (common jezabel), and the heliconine Heliconius melpomene are compared and related to the wing-reflectance spectra. Light scattering at the wing scales determines the wing reflectance, but when the scales contain an absorbing pigment, reflectance is suppressed in the absorption wavelength range of the pigment. The reflectance of the white wing areas of P. rapae, where the scales are studded with beads, is considerably higher than that of the white wing areas of H. melpomene, which has scales lacking beads. The beads presumably cause the distinct matt-white colour of the wings of pierids and function to increase the reflectance amplitude. This will improve the visual discrimination between conspecific males and females.     

Phylogenetic codivergence supports coevolution of mimetic Heliconius butterflies

The unpalatable and warning-patterned butterflies Heliconius erato and Heliconius melpomene provide the best studied example of mutualistic Müllerian mimicry, thought-but rarely demonstrated-to promote coevolution. Some of the strongest available evidence for coevolution comes from phylogenetic codivergence, the parallel divergence of ecologically associated lineages. Early evolutionary reconstructions suggested codivergence between mimetic populations of H. erato and H. melpomene, and this was initially hailed as one of the most striking known cases of coevolution. However, subsequent molecular phylogenetic analyses found discrepancies in phylogenetic branching patterns and timing (topological and temporal incongruence) that argued against codivergence. We present the first explicit cophylogenetic test of codivergence between mimetic populations of H. erato and H. melpomene, and re-examine the timing of these radiations. We find statistically significant topological congruence between multilocus coalescent population phylogenies of H. erato and H. melpomene. Cophylogenetic historical reconstructions support repeated codivergence of mimetic populations, from the base of the sampled radiations. Pairwise distance correlation tests, based on our coalescent analyses plus recently published AFLP and wing colour pattern gene data, also suggest that the phylogenies of H. erato and H. melpomene show significant topological congruence. Divergence time estimates, based on a Bayesian coalescent model, suggest that the evolutionary radiations of H. erato and H. melpomene occurred over the same time period, and are compatible with a series of temporally congruent codivergence events. Our results suggest that differences in within-species genetic divergence are the result of a greater overall effective population size for H. erato relative to H. melpomene and do not imply incongruence in the timing of their phylogenetic radiations. Repeated codivergence between Müllerian co-mimics, predicted to exert mutual selection pressures, strongly suggests coevolution. Our results therefore support a history of reciprocal coevolution between Müllerian co-mimics characterised by phylogenetic codivergence and parallel phenotypic change.     

Multiple Mechanisms of Photoreceptor Spectral Tuning in Heliconius Butterflies

The evolution of color vision is often studied through the lens of receptor gain relative to an ancestor with fewer spectral classes of photoreceptor. For instance, in Heliconius butterflies, a genus-specific UVRh opsin duplication led to the evolution of UV color discrimination in Heliconius erato females, a rare trait among butterflies. However, color vision evolution is not well understood in the context of loss. In Heliconius melpomene and Heliconius ismenius lineages, the UV2 receptor subtype has been lost, which limits female color vision in shorter wavelengths. Here, we compare the visual systems of butterflies that have either retained or lost the UV2 photoreceptor using intracellular recordings, ATAC-seq, and antibody staining. We identify several ways these butterflies modulate their color vision. In H. melpomene, chromatin reorganization has downregulated an otherwise intact UVRh2 gene, whereas in H. ismenius, pseudogenization has led to the truncation of UVRh2. In species that lack the UV2 receptor, the peak sensitivity of the remaining UV1 photoreceptor cell is shifted to longer wavelengths. Across Heliconius, we identify the widespread use of filtering pigments and co-expression of two opsins in the same photoreceptor cells. Multiple mechanisms of spectral tuning, including the molecular evolution of blue opsins, have led to the divergence of receptor sensitivities between species. The diversity of photoreceptor and ommatidial subtypes between species suggests that Heliconius visual systems are under varying selection pressures for color discrimination. Modulating the wavelengths of peak sensitivities of both the blue- and remaining UV-sensitive photoreceptor cells suggests that Heliconius species may have compensated for UV receptor loss.     

Synteny and chromosome evolution in the lepidoptera: evidence from mapping in Heliconius melpomene

The extent of conservation of synteny and gene order in the Lepidoptera has been investigated previously only by comparing a small subset of linkage groups between the moth Bombyx mori and the butterfly Heliconius melpomene. Here we report the mapping of 64 additional conserved genes in H. melpomene, which contributed 47 markers to a comparative framework of 72 orthologous loci spanning all 21 H. melpomene chromosomes and 27 of the 28 B. mori chromosomes. Comparison of the maps revealed conserved synteny across all chromosomes for the 72 loci, as well as evidence for six cases of chromosome fusion in the Heliconius lineage that contributed to the derived 21-chromosome karyotype. Comparisons of gene order on these fused chromosomes revealed two instances of colinearity between H. melpomene and B. mori, but also one instance of likely chromosomal rearrangement. B. mori is the first lepidopteran species to have its genome sequenced, and the finding that there is conserved synteny and gene order among Lepidoptera indicates that the genomic tools developed in B. mori will be broadly useful in other species.     

Shared and divergent expression domains on mimetic Heliconius wings

SUMMARY Heliconius butterfly wing patterns show repeated convergence between species and have adaptive value in mimicry and mate choice, offering an opportunity to connect adaptive changes in phenotype with their underlying genotypes. Here we study forewing ommochrome pigmentation in Heliconius melpomene . We clone two new ommochrome pathway genes for the Lepidoptera, karmoisin and kynurenine formamidase ( kf  ), and analyze the expression patterns of all known ommochrome genes across pupal wing development. In combination with published work, this generates the first comparative gene expression data for the co-mimics Heliconius erato and H. melpomene . In both species cinnabar expression correlates with the forewing band, but the expression pattern of vermillion differs significantly between the mimics. This demonstrates that both shared and divergent expression patterns are associated with mimetic phenotypes between Heliconius species. Two genes not studied in H. erato, scarlet and possibly kf , also show enhanced expression in the forewing band of H. melpomene , implying co-ordinated upregulation of several members of this biosynthetic pathway during pattern formation.     

Comparative population genetics of a mimicry locus among hybridizing Heliconius butterfly species

The comimetic Heliconius butterfly species pair, H. erato and H. melpomene, appear to use a conserved Mendelian switch locus to generate their matching red wing patterns. Here we investigate whether H. cydno and H. pachinus, species closely related to H. melpomene, use this same switch locus to generate their highly divergent red and brown color pattern elements. Using an F2 intercross between H. cydno and H. pachinus, we first map the genomic positions of two novel red/brown wing pattern elements; the G locus, which controls the presence of red vs brown at the base of the ventral wings, and the Br locus, which controls the presence vs absence of a brown oval pattern on the ventral hind wing. The results reveal that the G locus is tightly linked to markers in the genomic interval that controls red wing pattern elements of H. erato and H. melpomene. Br is on the same linkage group but approximately 26 cM away. Next, we analyze fine-scale patterns of genetic differentiation and linkage disequilibrium throughout the G locus candidate interval in H. cydno, H. pachinus and H. melpomene, and find evidence for elevated differentiation between H. cydno and H. pachinus, but no localized signature of association. Overall, these results indicate that the G locus maps to the same interval as the locus controlling red patterning in H. melpomene and H. erato. This, in turn, suggests that the genes controlling red pattern elements may be homologous across Heliconius, supporting the hypothesis that Heliconius butterflies use a limited suite of conserved genetic switch loci to generate both convergent and divergent wing patterns.     

The ecology and social behaviour of the Neotropical butterfly Heliconius xanthocles Bates in Colombia

The mimicry, population ecology¹, adult behaviour and life cycle of Heliconius xanthocles were studied at two sites in Colombian tropical forest. The results were compared with the known biology of other Heliconius species. H. xanthocles is probably unpalatable and is a Mullerian comimic of some other species. In the Amazon basin H. xanthocles belongs to the 'dennis ray' mimicry ring of the Heliconiini, which includes Eueides tales, H. bumeyi, H. elevatus, H. melpomene and H. erato. In the Rio Negro valley, where the latter species either do not exist or have different mimetic allegiances, the 'clennis-ray' pattern of H. xanthocles breaks down. The adults are pollen-feeders, like other Heliconius. but they also visit other food sources such as bird droppings. During a mark-recapture programme in the Rio Negro, adults were found to be almost entirely restricted to small areas near their foodplant Passiflorapraeacuta. Females were rarer than males, but this was probably due to a behavioural difference between the sexes. The males had a Fisher-Ford survival rate of 0.95 per day and a life expectancy of 19 days. Population size estimates of males were between 11 and 17 individuals. These estimates are lower than, but comparable to those for other Heliconius. Males as well as females visit the larval toodplant, and an adult male was observed hovering over a small group of larvae. The females lav batches of 12–41 eggs. The largest batch (41 eggs) was laid cooperatively by two females. The larvae are gregarious feeders on shoot-tips of Passiflora praeacuta. and all lile stages are described for the first time. Predation on the larvae by a bug is described. The pupa has a distinctive morphology unlike that of other Heliconius. The adult population ecology, mimicry and larval behaviour are discussed.