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.     

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.     

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.     

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.     

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.     

Subtle variation in size and shape of the whole forewing and the red band among co‐mimics revealed by geometric morphometric analysis in Heliconius butterflies

Heliconius are unpalatable butterflies that exhibit remarkable intra‐ and interspecific variation in wing color pattern, specifically warning coloration. Species that have converged on the same pattern are often clustered in Müllerian mimicry rings. Overall, wing color patterns are nearly identical among co‐mimics. However, fine‐scale differences exist, indicating that factors in addition to natural selection may underlie wing phenotype. Here, we investigate differences in shape and size of the forewing and the red band in the Heliconius postman mimicry ring (H. erato phyllis and the co‐mimics H. besckei, H. melpomene burchelli, and H. melpomene nanna) using a landmark‐based approach. If phenotypic evolution is driven entirely by predation pressure, we expect nonsignificant differences among co‐mimics in terms of wing shape. Also, a reinforcement of wing pattern (i.e., greater similarity) could occur when co‐mimics are in sympatry. We also examined variation in the red forewing band because this trait is critical for both mimicry and sexual communication. Morphometric results revealed significant but small differences among species, particularly in the shape of the forewing of co‐mimics. Although we did not observe greater similarity when co‐mimics were in sympatry, nearly identical patterns provided evidence of convergence for mimicry. In contrast, mimetic pairs could be distinguished based on the shape (but not the size) of the red band, suggesting an “advergence” process. In addition, sexual dimorphism in the red band shape (but not size) was found for all lineages. Thus, we infer that natural selection due to predation by birds might not be the only mechanism responsible for variation in color patterns, and sexual selection could be an important driver of wing phenotypic evolution in this mimicry ring.     

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.     

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.     

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.     

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.     

Estimates of Selection and Gene Flow from Measures of Cline Width and Linkage Disequilibrium in Heliconius Hybrid Zones

Hybrid zones can yield estimates of natural selection and gene flow. The width of a cline in gene frequency is approximately proportional to gene flow (sigma) divided by the square root of per-locus selection (square root of s). Gene flow also causes gametic correlations (linkage disequilibria) between genes that differ across hybrid zones. Correlations are stronger when the hybrid zone is narrow, and rise to a maximum roughly equal to s. Thus cline width and gametic correlations combine to give estimates of gene flow and selection. These indirect measures of sigma and s are especially useful because they can be made from collections, and require no field experiments. The method was applied to hybrid zones between color pattern races in a pair of Peruvian Heliconius butterfly species. The species are Müllerian mimics of one another, and both show the same changes in warning color pattern across their respective hybrid zones. The expectations of cline width and gametic correlation were generated using simulations of clines stabilized by strong frequency-dependent selection. In the hybrid zone in Heliconius erato, clines at three major color pattern loci were between 8.5 and 10.2 km wide, and the pairwise gametic correlations peaked at R approximately 0.35. These measures suggest that s approximately 0.23 per locus, and that sigma approximately 2.6 km. In erato, the shapes of the clines agreed with that expected on the basis of dominance. Heliconius melpomene has a nearly coincident hybrid zone. In this species, cline widths at four major color pattern loci varied between 11.7 and 13.4 km. Pairwise gametic correlations peaked near R approximately 1.00 for tightly linked genes, and at R approximately 0.40 for unlinked genes, giving s approximately 0.25 per locus and sigma approximately 3.7 km. In melpomene, cline shapes did not perfectly fit theoretical shapes based on dominance; this deviation might be explained by long-distance migration and/or strong epistasis. Compared with erato, sample sizes in melpomene are lower and the genetics of its color patterns are less well understood. In spite of these problems, selection and gene flow are clearly of the same order of magnitude in the two species. The relatively high per locus selection coefficients agree with "major gene" theories for the evolution of Müllerian mimicry, but the genetic architecture of the color patterns does not. These results show that the genetics and evolution of mimicry are still only sketchily understood.     

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 hybrid zone provides evidence for incipient ecological speciation in Heliconius butterflies

In Heliconius butterflies, it has been proposed that speciation occurs through a combination of divergence in ecological habitat preferences and mimetic colour patterns. Here we test this hypothesis by investigating a parapatric form of the widespread species Heliconius erato. Mendelian (colour patterns) and molecular genetic data permit us to address hypotheses about introgression and genetic differentiation between different populations. Combined analysis of colour pattern, microsatellite loci and mitochondrial DNA showed that Heliconius erato venus and Heliconius erato chestertonii form a bimodal hybrid zone implying partial reproductive isolation. In a sample of 121 individuals collected in sympatry, 25% were hybrids representing a significant deficit of heterozygotes compared to the Hardy-Weinberg expectation. Seven microsatellite loci, analysed for a subset of these individuals, showed marked differentiation between the parental taxa, and unambiguously identified two genotypic clusters concordant with our phenotypic classification of individuals. Mitochondrial DNA analysis showed H. erato venus as a monophyletic group well differentiated from H. erato chestertonii, implying a lack of historical introgression between the populations. Heliconius erato chestertonii is therefore an incipient species that maintains its integrity despite high levels of hybridization. Moreover, H. erato chestertonii is found at higher altitudes than other races of H. erato and has a distinct colour pattern and mimetic relationship. Hence, there are now two examples of parapatric incipient species related to H. erato, H. himera and H. erato chestertonii, both of which are associated with higher altitudes, more arid habitats and distinct mimetic relationships. This implies that parapatric habitat adaptation is a likely cause of speciation in this group.     

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.     

First-generation linkage map of the warningly colored butterfly Heliconius erato

We report the first genetic linkage map of Heliconius erato, a species that shows remarkable variation in its warningly colored wing patterns. We use crosses between H. erato and its sister species, H. himera, to place two major color pattern genes, D and Cr, on a linkage map containing AFLP, allozyme, microsatellite and single-copy nuclear loci. We identified all 21 linkage groups in an initial genetic screen of 22 progeny from an F1 female x male H. himera family. Of the 229 markers, 87 used to identify linkage groups were also informative in 35 progeny from a sibling backcross (H. himera female x F1 male). With these, and an additional 33 markers informative in the second family, we constructed recombinational maps for 19 of the 21 linkage groups. These maps varied in length from 18.1 to 431.1 centimorgans (cM) and yielded an estimated total length of 2400 cM. The average distance between markers was 23 cM, and eight of the 19 linkage groups, including the sex chromosome (Z) and the chromosome containing the Cr locus, contained two or more codominant anchor loci. Of the three potential candidate genes mapped here, Cubitus interruptus (Ci), Decapentaplegic (Dpp) and Wingless (Wg), only Ci was linked, although loosely, to a known Heliconius color pattern locus. This work is an important first step for constructing a denser genetic map of the H. erato color pattern radiation and for a comparative genomic study of the architecture of mimicry in Heliconius butterflies.     

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.     

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.