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.     

What can hybrid zones tell us about speciation? The case of Heliconius erato and H. himera (Lepidoptera: Nymphalidae)

To understand speciation we need to study the genetics and ecology of intermediate cases where interspecific hybridization still occurs. Two closely related species of Heliconius butterflies meet this criterion: Heliconius himera is endemic to dry forest and thorn scrub in southern Ecuador and northern Peru, while its sister species, H. erato , is ubiquitous in wet forest throughout south and central America. In three known zones of contact, the two species remain distinct, while hybrids are found at low frequency. Collections in southern Ecuador show that the contact zone is about 5 km wide, half the width of the narrowest clines between colour pattern races of H. erato. The narrowness of this dine argues that very strong selection (s ≅ 1) is maintaining the parapatric distributions of these two species. The zone is closely related with a habitat transition from wet to dry forest, which suggests that the narrow zone of parapatry is maintained primarily by ecological adaptation. Selection on colour pattern loci, assortative mating and hybrid inviability may also be important. The genetics of hybrids between the two species shows that the major gene control of pattern elements is similar to that found in previous studies of H. erato races, and some of the loci are homologous. This suggests that similar genetic processes are involved in the morphological divergence of species and races. Evidence from related Heliconius supports a hypothesis that ecological adaptation is the driving force for speciation in the group.     

Genetic evidence for a sibling species of Heliconius charithonia (Lepidoptera; Nymphalidae)

Heliconius charithonia is a widespread species which, unlike many Heliconius, is non-mimetic and shows little racial differentiation. Only one form, 'peruvianus', which occurs in the dry forest habitats of western Ecuador and Peru, has a distinct and clearly mimetic colour pattern. Here it was shown that H. peruvianus was distinct from H. charithonia bassleri at allozyme loci (D = 0.25 over 22 loci). This differentiation was ten times greater than that between H. charithonia sampled from Ecuador and the Caribbean (D = 0.027) and was consistent with analysis of mitochondrial sequence data (3.4-4% sequence divergence between H. peruvianus and H. charithonia). One individual with a H. peruvianus colour pattern and allozyme genotype was collected in an area where H. charithonia was known to be common, demonstrating that contact between the taxa occurs in western Ecuador. Furthermore, the allozyme genotype of another individual was heterozygous for four of five diagnostic loci and was most likely an F1 hybrid between H. charithonia and H. peruvianus. These data imply that H. charithonia and H. peruvianus are distinct species which hybridize occasionally. This species pair show many similarities with H. erato and H. himera, which are similarly differentiated genetically and also show ecological and colour pattern differences. These species fulfil some of the predictions of both allopatric refugium and parapatric adaptationist models of speciation in the neotropics, suggesting that elements of both hypotheses may be true.     

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.     

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.     

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.     

Pervasive genetic associations between traits causing reproductive isolation in Heliconius butterflies

Ecological speciation proceeds through the accumulation of divergent traits that contribute to reproductive isolation, but in the face of gene flow traits that characterize incipient species may become disassociated through recombination. Heliconius butterflies are well known for bright mimetic warning patterns that are also used in mate recognition and cause both pre- and post-mating isolation between divergent taxa. Sympatric sister taxa representing the final stages of speciation, such as Heliconius cydno and Heliconius melpomene, also differ in ecology and hybrid fertility. We examine mate preference and sterility among offspring of crosses between these species and demonstrate the clustering of Mendelian colour pattern loci and behavioural loci that contribute to reproductive isolation. In particular, male preference for red patterns is associated with the locus responsible for the red forewing band. Two further colour pattern loci are associated, respectively, with female mating outcome and hybrid sterility. This genetic architecture in which ‘speciation genes’ are clustered in the genome can facilitate two controversial models of speciation, namely divergence in the face of gene flow and hybrid speciation.     

Mimicry: developmental genes that contribute to speciation

Despite renewed interest in the role of natural selection as a catalyst for the origin of species, the developmental and genetic basis of speciation remains poorly understood. Here we describe the genetics of Müllerian mimicry in Heliconius cydno and H. melpomene (Lepidoptera: Nymphalidae), sister species that recently diverged to mimic other Heliconius. This mimetic shift was a key step in their speciation, leading to pre- and postmating isolation. We identify 10 autosomal loci, half of which have major effects. At least eight appear to be homologous with genes known to control pattern differences within each species. Dominance has evolved under the influence of identifiable "modifier" loci rather than being a fixed characteristic of each locus. Epistasis is found at many levels: phenotypic interaction between specific pairs of genes, developmental canalization due to polygenic modifiers so that patterns are less sharply defined in hybrids, and overall fitness through ecological selection against nonmimetic hybrid genotypes. Most of the loci are clustered into two genomic regions or "supergenes," suggesting color pattern evolution is constrained by preexisting linked elements that may have arisen via tandem duplication rather than having been assembled by natural selection. Linkage, modifiers, and epistasis affect the strength of mimicry as a barrier to gene flow between these naturally hybridizing species and may permit introgression in genomic regions unlinked to those under disruptive selection. Müllerian mimics in Heliconius use different genetic architectures to achieve the same mimetic patterns, implying few developmental constraints. Therefore, although developmental and genomic constraints undoubtedly influence the evolutionary process, their effects are probably not strong in comparison with natural selection.     

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.     

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.     

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.     

COMPARATIVE GENOMIC AND POPULATION GENETIC ANALYSES INDICATE HIGHLY POROUS GENOMES AND HIGH LEVELS OF GENE FLOW BETWEEN DIVERGENT HELIANTHUS SPECIES

While speciation can be found in the presence of gene flow, it is not clear what impact this gene flow has on genome- and range-wide patterns of differentiation. Here we examine gene flow across the entire range of the common sunflower, H. annuus , its historically allopatric sister species H. argophyllus and a more distantly related, sympatric relative H. petiolaris . Analysis of genotypes at 26 microsatellite loci in 1015 individuals from across the range of the three species showed substantial introgression between geographically proximal populations of H. annuus and H. petiolaris , limited introgression between H. annuus and H. argophyllus , and essentially no gene flow between the allopatric pair, H. argophyllus and H. petiolaris. Analysis of sequence divergence levels among the three species in 1420 orthologs identified from EST databases identified a subset of loci showing extremely low divergence between H. annuus and H. petiolaris and extremely high divergence between the sister species H. annuus and H. argophyllus , consistent with introgression between H. annuus and H. petiolaris at these loci. Thus, at many loci, the allopatric sister species are more genetically divergent than the more distantly related sympatric species, which have exchanged genes across much of the genome while remaining morphologically and ecologically distinct.     

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.     

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.     

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.     

A set of microsatellite markers for Heliconius melpomene and closely related species

The butterflies in the genus Heliconius offer an exceptional opportunity for the study of the ecology and genetics of an adaptive radiation due to their extensive intra‐ and interspecific variation in wing colour patterns and mimetic associations. Here, we characterize 22 polymorphic microsatellite loci in Heliconius melpomene that have been shown to be useful for linkage mapping and population studies in this and other species. Levels of variation were high, although heterozygosity deficiencies were found in most loci, probably due to null alleles. The loci showed broad amplification success on six other species across the genus.     

Behavioral and Physiological Differences between Two Parapatric Heliconius Species1

The behavior and physiology of two parapatric sibling species, i Heliconius erato cyrbia Godt. and H. himera Hew., were investigated to assess if environmental adaptation enabled stable morphological, genetic, and ecological differences to exist in the face of hybridization. Morning and evening activity, egg production, and larval development time of H. himera and H. erato in insectaries were recorded; individuals were collected in allopatry and in sympatry from a hybrid zone in which the species overlapped. Studies were performed at ambient conditions within the natural range of H. himera. H. himera was considerably more active than H, erato flying earlier in the morning and later in the evening, even when both species were collected in sympatry. Similarly, H. himera laid more eggs, and the hatched larvae developed more rapidly. The results suggest that physiological constraints are an important selective force that may have been important in speciation and counteracts hybridization in the maintenance of the H. himeral H. erato contact zone. Ecological selection, arising from adaptation to low temperatures, may help explain the competitive exclusion of H. erato by H. himera in the drier, cooler montane habitat favored by the latter species.     

Contrasting genomic and phenotypic outcomes of hybridization between pairs of mimetic butterfly taxa across a suture zone

Hybrid zones, whereby divergent lineages come into contact and eventually hybridize, can provide insights on the mechanisms involved in population differentiation and reproductive isolation, and ultimately speciation. Suture zones offer the opportunity to compare these processes across multiple species. In this paper we use reduced‐complexity genomic data to compare the genetic and phenotypic structure and hybridization patterns of two mimetic butterfly species, Ithomia salapia and Oleria onega (Nymphalidae: Ithomiini), each consisting of a pair of lineages differentiated for their wing colour pattern and that come into contact in the Andean foothills of Peru. Despite similarities in their life history, we highlight major differences, both at the genomic and phenotypic level, between the two species. These differences include the presence of hybrids, variations in wing phenotype, and genomic patterns of introgression and differentiation. In I. salapia, the two lineages appear to hybridize only rarely, whereas in O. onega the hybrids are not only more common, but also genetically and phenotypically more variable. We also detected loci statistically associated with wing colour pattern variation, but in both species these loci were not over‐represented among the candidate barrier loci, suggesting that traits other than wing colour pattern may be important for reproductive isolation. Our results contrast with the genomic patterns observed between hybridizing lineages in the mimetic Heliconius butterflies, and call for a broader investigation into the genomics of speciation in Ithomiini ‐ the largest radiation of mimetic butterflies.