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.     

Multilocus analyses of admixture and introgression among hybridizing Heliconius butterflies

Introgressive hybridization is an important evolutionary process and new analytical methods provide substantial power to detect and quantify it. In this study we use variation in the frequency of 657 AFLP fragments and DNA sequence variation from 15 genes to measure the extent of admixture and the direction of interspecific gene flow among three Heliconius butterfly species that diverged recently as a result of natural selection for Miillerian mimicry, and which continue to hybridize. Bayesian clustering based on AFLP genotypes correctly delineated the three species and identified four H. cydno, three H. pachinus, and three H. melpomene individuals that were of mixed ancestry. Gene genealogies revealed substantial shared DNA sequence variation among all three species and coalescent simulations based on the Isolation with Migration (IM) model pointed to interspecific gene flow as its cause. The IM simulations further indicated that interspecific gene flow was significantly asymmetrical, with greater gene flow from H. pachinus into H. cydno (2Nm = 4.326) than the reverse (2Nm = 0.502), and unidirectional gene flow from H. cydno and H. pachinus into H. melpomene (2Nm = 0.294 and 0.252, respectively). These asymmetries are in the directions expected based on the genetics of wing patterning and the probability that hybrids of various phenotypes will survive and reproduce in different mimetic environments. This empirical demonstration of extensive interspecific gene flow is in contrast to a previous study which found little evidence of gene flow between another pair of hybridizing Heliconius species, H. himera and H. erato, and it highlights the critical role of natural selection in maintaining species diversity. Furthermore, these results lend support to the hypotheses that phenotypic diversification in the genus Heliconius has been fueled by introgressive hybridization and that reinforcement has driven the evolution of assortative mate preferences.     

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.     

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.     

Localization of Müllerian mimicry genes on a dense linkage map of Heliconius erato

We report a dense genetic linkage map of Heliconius erato, a neotropical butterfly that has undergone a remarkable adaptive radiation in warningly colored mimetic wing patterns. Our study exploited natural variation segregating in a cross between H. erato etylus and H. himera to localize wing color pattern loci on a dense linkage map containing amplified fragment length polymorphisms (AFLP), microsatellites, and single-copy nuclear loci. We unambiguously identified all 20 autosomal linkage groups and the sex chromosome (Z). The map spanned a total of 1430 Haldane cM and linkage groups varied in size from 26.3 to 97.8 cM. The average distance between markers was 5.1 cM. Within this framework, we localized two major color pattern loci to narrow regions of the genome. The first gene, D, responsible for red/orange elements, had a most likely placement in a 6.7-cM region flanked by two AFLP markers on the end of a large 87.5-cM linkage group. The second locus, Sd, affects the melanic pattern on the forewing and was found within a 6.3-cM interval between flanking AFLP loci. This study complements recent linkage analysis of H. erato's comimic, H. melpomene, and forms the basis for marker-assisted physical mapping and for studies into the comparative genetic architecture of wing-pattern mimicry in Heliconius.     

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.     

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.     

Interspecific sexual attraction because of convergence in warning colouration: is there a conflict between natural and sexual selection in mimetic species?

When species converge in their colour patterns because of mimicry, and those patterns are also used in mate recognition, there is a probability of conflicting selection pressures. Closely related species that mimic one another are particularly likely to face such confusion because of similarities in their courtship behaviour and ecology. We conducted experiments in greenhouse conditions to study interspecific attraction between two mimetic butterfly species, Heliconius erato and Heliconius melpomene. Both species spent considerable time approaching and courting females of the co-mimic species. Experiments using wing models demonstrated the importance of colour pattern in this interspecific attraction. Although males of H. melpomene were attracted to their co-mimics as much as to their own females, H. erato males were more efficient at distinguishing conspecifics, possibly using wing odours. Although preliminary, these results suggest that the use of additional cues may have evolved in H. erato to reduce the cost of convergence in visual signals with H. melpomene. Overall, our results showed that there might be a cost of mimetic convergence because of a reduction in the efficiency of species recognition. Such cost may contribute to explain the apparently stable diversity in Müllerian mimetic patterns in many tropical butterfly assemblages.     

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.     

Evolution of the wave: aerodynamic and aposematic functions of butterfly wing motion

Many unpalatable butterfly species use coloration to signal their distastefulness to birds, but motion cues may also be crucial to ward off predatory attacks. In previous research, captive passion-vine butterflies Heliconius mimetic in colour pattern were also mimetic in motion. Here, I investigate whether wing motion changes with the flight demands of different behaviours. If birds select for wing motion as a warning signal, aposematic butterflies should maintain wing motion independently of behavioural context. Members of one mimicry group (Heliconius cydno and Heliconius sapho) beat their wings more slowly and their wing strokes were more asymmetric than their sister-species (Heliconius melpomene and Heliconius erato, respectively), which were members of another mimicry group having a quick and steady wing motion. Within mimicry groups, wing beat frequency declined as its role in generating lift also declined in different behavioural contexts. In contrast, asymmetry of the stroke was not associated with wing beat frequency or behavioural context-strong indication that birds process and store the Fourier motion energy of butterfly wings. Although direct evidence that birds respond to subtle differences in butterfly wing motion is lacking, birds appear to generalize a motion pattern as much as they encounter members of a mimicry group in different behavioural contexts.     

Genomic islands of divergence in hybridizing Heliconius butterflies identified by large-scale targeted sequencing

Heliconius butterflies represent a recent radiation of species, in which wing pattern divergence has been implicated in speciation. Several loci that control wing pattern phenotypes have been mapped and two were identified through sequencing. These same gene regions play a role in adaptation across the whole Heliconius radiation. Previous studies of population genetic patterns at these regions have sequenced small amplicons. Here, we use targeted next-generation sequence capture to survey patterns of divergence across these entire regions in divergent geographical races and species of Heliconius. This technique was successful both within and between species for obtaining high coverage of almost all coding regions and sufficient coverage of non-coding regions to perform population genetic analyses. We find major peaks of elevated population differentiation between races across hybrid zones, which indicate regions under strong divergent selection. These 'islands' of divergence appear to be more extensive between closely related species, but there is less clear evidence for such islands between more distantly related species at two further points along the 'speciation continuum'. We also sequence fosmid clones across these regions in different Heliconius melpomene races. We find no major structural rearrangements but many relatively large (greater than 1 kb) insertion/deletion events (including gain/loss of transposable elements) that are variable between races.     

An analysis of the phenotypic effects of certain colour pattern genes in Heliconius (Lepidoptera: Nymphalidae)

The colour patterns of Heliconius butterflies are built up from an array of serially homologous pattern elements known as the nymphalid groundplan. An analysis of the phenotypic effects of ten genetic loci from H. melpomene and H. cydno reveals that each alters the expression either of a single element of the groundplan or of an entire row of serially homologous elements. Five of the ten loci affect the size (or presence/absence) of specific pattern elements, two affect the colour in which a pattern element is expressed, two affect pattern-inducing activity of the wing veins, and one appears to affect an overall threshold for pattern determination. Three of the ten loci have identical effects on homologues of the fore- and hindwing. We show that most of the apparently large and qualitative phenotypic effects of these genes can be readily explained by relatively small and quantitative changes in the dimensions or positions of specific pattern elements.     

A conserved supergene locus controls colour pattern diversity in Heliconius butterflies

We studied whether similar developmental genetic mechanisms are involved in both convergent and divergent evolution. Mimetic insects are known for their diversity of patterns as well as their remarkable evolutionary convergence, and they have played an important role in controversies over the respective roles of selection and constraints in adaptive evolution. Here we contrast three butterfly species, all classic examples of Müllerian mimicry. We used a genetic linkage map to show that a locus, Yb, which controls the presence of a yellow band in geographic races of Heliconius melpomene, maps precisely to the same location as the locus Cr, which has very similar phenotypic effects in its co-mimic H. erato. Furthermore, the same genomic location acts as a "supergene", determining multiple sympatric morphs in a third species, H. numata. H. numata is a species with a very different phenotypic appearance, whose many forms mimic different unrelated ithomiine butterflies in the genus Melinaea. Other unlinked colour pattern loci map to a homologous linkage group in the co-mimics H. melpomene and H. erato, but they are not involved in mimetic polymorphism in H. numata. Hence, a single region from the multilocus colour pattern architecture of H. melpomene and H. erato appears to have gained control of the entire wing-pattern variability in H. numata, presumably as a result of selection for mimetic "supergene" polymorphism without intermediates. Although we cannot at this stage confirm the homology of the loci segregating in the three species, our results imply that a conserved yet relatively unconstrained mechanism underlying pattern switching can affect mimicry in radically different ways. We also show that adaptive evolution, both convergent and diversifying, can occur by the repeated involvement of the same genomic regions.     

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.     

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.     

Evolution of a mimicry supergene from a multilocus architecture

The origin and evolution of supergenes have long fascinated evolutionary biologists. In the polymorphic butterfly Heliconius numata, a supergene controls the switch between multiple different forms, and results in near-perfect mimicry of model species. Here, we use a morphometric analysis to quantify the variation in wing pattern observed in two broods of H. numata with different alleles at the supergene locus, 'P'. Further, we genotype the broods to associate the variation we capture with genetic differences. This allows us to begin mapping the quantitative trait loci that have minor effects on wing pattern. In addition to finding loci on novel chromosomes, our data, to our knowledge, suggest for the first time that ancestral colour-pattern loci, known to have major effects in closely related species, may contribute to the wing patterns displayed by H. numata, despite the large transfer of effects to the supergene.     

Ecological and genetic associations across a Heliconius hybrid zone

Differences in habitat use can bridge early and late stages of speciation by initiating assortative mating. Heliconius colour pattern races might select habitats over which each pattern confers a relative fitness advantage because signal efficacy of wing patterns can vary by environment. Thus habitat preferences could serve to promote the evolution of mimetic colour patterns for mate choice. Here I compare colour pattern genotype and phenotype frequencies to environmental variation across the H. erato hydara x H. erato erato hybrid zone in French Guiana to determine whether races exhibit habitat preferences. I found that genotype and phenotype frequencies correspond to differences in land cover moreso than to other environmental factors. Temporal shifts in colour pattern genotypes, phenotypes and land cover also were associated at individual sample sites, which further suggests that H. erato races differ in habitat use and that habitat preferences may promote speciation among Heliconius butterflies.     

CASE STUDIES AND MATHEMATICAL MODELS OF ECOLOGICAL SPECIATION. 4. HYBRID SPECIATION IN BUTTERFLIES IN A JUNGLE

We build a spatial individual-based multilocus model of homoploid hybrid speciation tailored for a tentative case of hybrid origin of Heliconius heurippa from H. melpomene and H. cydno in South America. Our model attempts to account for empirical patterns and data on genetic incompatibility, mating preferences and selection by predation (both based on coloration patterns), habitat preference, and local adaptation for all three Heliconius species. Using this model, we study the likelihood of recombinational speciation and identify the effects of various ecological and genetic parameters on the dynamics, patterns, and consequences of hybrid ecological speciation. Overall, our model supports the possibility of hybrid origin of H. heurippa under certain conditions. The most plausible scenario would include hybridization between H. melpomene and H. cydno in an area geographically isolated from the rest of both parental species with subsequent long-lasting geographic isolation of the new hybrid species, followed by changes in the species ranges, the secondary contact, and disappearance of H. melpomene -type ecomorph in the hybrid species. However, much more work (both empirical and theoretical) is necessary to be able to make more definite conclusions on the importance of homoploid hybrid speciation in animals.