Pollen feeding in Heliconius butterflies: the singular evolution of an adaptive suite

Major evolutionary transitions can be triggered by behavioural novelty, and are often associated with ‘adaptive suites’, which involve shifts in multiple co-adapted traits subject to complex interactions. Heliconius butterflies represent one such example, actively feeding on pollen, a behaviour unique among butterflies. Pollen feeding permits a prolonged reproductive lifespan, and co-occurs with a constellation of behavioural, neuroanatomical, life history, morphological and physiological traits that are absent in closely related, non-pollen-feeding genera. As a highly tractable system, supported by considerable ecological and genomic data, Heliconius are an excellent model for investigating how behavioural innovation can trigger a cascade of adaptive shifts in multiple diverse, but interrelated, traits. Here, we synthesize current knowledge of pollen feeding in Heliconius, and explore potential interactions between associated, putatively adaptive, traits. Currently, no physiological, morphological or molecular innovation has been explicitly linked to the origin of pollen feeding, and several hypothesized links between different aspects of Heliconius biology remain poorly tested. However, resolving these uncertainties will contribute to our understanding of how behavioural innovations evolve and subsequently alter the evolutionary trajectories of diverse traits impacting resource acquisition, life history, senescence and cognition.     

Characterization of microsatellite loci in neotropical Heliconius butterflies

The Heliconius butterflies offer exceptional opportunities for the study of the ecology and evolution of mimicry. Despite previous reports of difficulties in the development of microsatellite loci in Lepidoptera, we characterize 15 polymorphic loci in H. erato that show promise for genetic mapping and population studies in this and other species. Levels of variation were high, in both numbers and size ranges of alleles. The loci showed broad amplification success across the genus and in two other genera. All loci that amplified in a population of H. melpomene were polymorphic.     

Beyond magic traits: Multimodal mating cues in Heliconius butterflies

Species coexistence involves the evolution of reproductive barriers opposing gene flow. Heliconius butterflies display colorful patterns affecting mate choice and survival through warning signaling and mimicry. These patterns are called “magic traits” for speciation because divergent natural selection may promote mimicry shifts in pattern whose role as mating cue facilitates reproductive isolation. By contrast, between comimetic species, natural selection promotes pattern convergence. We addressed whether visual convergence interferes with reproductive isolation by testing for sexual isolation between two closely related species with similar patterns, H. timareta thelxinoe and H. melpomene amaryllis. Experiments with models confirmed visual attraction based on wing phenotype, leading to indiscriminate approach. Nevertheless, mate choice experiments showed assortative mating. Monitoring male behavior toward live females revealed asymmetry in male preference, H. melpomene males courting both species equally while H. timareta males strongly preferred conspecifics. Experiments with hybrid males suggested an important genetic component for such asymmetry. Behavioral observations support a key role for short‐distance cues in determining male choice in H. timareta. Scents extracts from wings and genitalia revealed interspecific divergence in chemical signatures, and hybrid female scent composition was significantly associated with courtship intensity by H. timareta males, providing candidate chemical mating cues involved in sexual isolation.     

Hybrid zones and the speciation continuum in Heliconius butterflies

Tropical butterflies in the genus Heliconius have long been models in the study of the stages of speciation. Heliconius are unpalatable to predators, and many species are notable for multiple geographic populations with striking warning colour pattern differences associated with Müllerian mimicry. A speciation continuum is evident in Heliconius hybrid zones. Examples range from hybrid zones across which (a) there is little genetic differentiation other than at mimicry loci, but where hybrids are common, (b) to ‘bimodal‘ hybrid zones with strong genetic divergence and few hybrids, (c) through to ‘good’ sympatric species, with hybridization extremely rare or absent. Now, in this issue of Molecular Ecology, Arias et al. ( 2012 ) have found an intermediate case in Colombian Heliconius cydno showing evidence for assortative mating and molecular differences, but where hybrids are abundant.     

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.     

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.     

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.     

Reinforcement of mate preference among hybridizing Heliconius butterflies

Recent models of mate preference evolution suggest that direct selection on alleles at preference loci and correlated evolution of preference with locally adapted mating cues are more likely to drive the evolution of assortative mate preference than reinforcement. Mate preference evolution in mimetic Heliconius butterflies has been attributed to all three forms of selection, but here we show that reinforcement has been critical. By examining geographical variation in assortative mating and male mate preference among seven populations of three hybridizing Heliconius species from Costa Rica, we found pronounced character displacement of preference such that sexual isolation was enhanced in areas of interspecific contact. Of the different explanations for the evolution of assortative mate preference, only reinforcement is dependent on interspecific contact in this system. Thus, the observed pattern of reproductive character displacement of mate preference is best explained as a product of indirect selection generated by natural selection against nonmimetic hybrids.     

The population genetics of mimetic diversity in Heliconius butterflies

Theory predicts strong stabilizing selection on warning patterns within species and convergent evolution among species in Müllerian mimicry systems yet Heliconius butterflies exhibit extreme wing pattern diversity. One potential explanation for the evolution of this diversity is that genetic drift occasionally allows novel warning patterns to reach the frequency threshold at which they gain protection. This idea is controversial, however, because Heliconius butterflies are unlikely to experience pronounced population subdivision and local genetic drift. To examine the fine-scale population genetic structure of Heliconius butterflies we genotyped 316 individuals from eight Costa Rican Heliconius species with 1428 AFLP markers. Six species exhibited evidence of population subdivision and/or isolation by distance indicating genetic differentiation among populations. Across species, variation in the extent of local genetic drift correlated with the roles different species have played in generating pattern diversity: species that originally generated the diversity of warning patterns exhibited striking population subdivision while species that later radiated onto these patterns had intermediate levels of genetic diversity and less genetic differentiation among populations. These data reveal that Heliconius butterflies possess the coarse population genetic structure necessary for local populations to experience pronounced genetic drift which, in turn, could explain the origin of mimetic diversity.     

Discrimination of flying mimetic,passion-vine butterflies Heliconius

Wing-beat frequency and the degree of asymmetry in wing motion were more similar among mimics than among sister species of passion-vine butterflies in the genus Heliconius. Asymmetry in wing motion is not attributed to lift production, and serves as the first clear example of a mimetic behavioural signal for a flying organism. Because the similarities in wing motion are too subtle for humans to observe with the naked eye, they serve as a previously unexplored mimetic signal.     

Primers for the amplification of nuclear introns in Heliconius butterflies

I described the development of degenerate polymerase chain reaction (PCR) primers for intron‐containing regions of nine candidate wing patterning and pigmentation genes in Heliconius butterflies. Primers were developed by comparing sequence data from Drosophila melanogaster, Precis coenia, and a variety of other insects so they are likely to be applicable widely among the butterfly family Nymphalidae and perhaps Lepidoptera in general. The amplified regions are highly variable and should be useful for inferring relationships among closely related species and estimating the phylogeographical and population genetic structure of individual species.     

Quantified reproductive isolation in Heliconius butterflies: Implications for introgression and hybrid speciation

Heliconius butterflies have become a model for the study of speciation with gene flow. For adaptive introgression to take place, there must be incomplete barriers to gene exchange that allow interspecific hybridization and multiple generations of backcrossing. The recent publication of estimates of individual components of reproductive isolation between several species of butterflies in the Heliconius melpomene–H. cydno clade allowed us to calculate total reproductive isolation estimates for these species. According to these estimates, the butterflies are not as promiscuous as has been implied. Differences between species are maintained by intrinsic mechanisms, while reproductive isolation of geographical races within species is mainly due to allopatry. We discuss the implications of this strong isolation for basic aspects of the hybrid speciation with introgression hypothesis.     

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.     

Conservatism and novelty in the genetic architecture of adaptation in Heliconius butterflies

Understanding the genetic architecture of adaptive traits has been at the centre of modern evolutionary biology since Fisher; however, evaluating how the genetic architecture of ecologically important traits influences their diversification has been hampered by the scarcity of empirical data. Now, high-throughput genomics facilitates the detailed exploration of variation in the genome-to-phenotype map among closely related taxa. Here, we investigate the evolution of wing pattern diversity in Heliconius, a clade of neotropical butterflies that have undergone an adaptive radiation for wing-pattern mimicry and are influenced by distinct selection regimes. Using crosses between natural wing-pattern variants, we used genome-wide restriction site-associated DNA (RAD) genotyping, traditional linkage mapping and multivariate image analysis to study the evolution of the architecture of adaptive variation in two closely related species: Heliconius hecale and H. ismenius. We implemented a new morphometric procedure for the analysis of whole-wing pattern variation, which allows visualising spatial heatmaps of genotype-to-phenotype association for each quantitative trait locus separately. We used the H. melpomene reference genome to fine-map variation for each major wing-patterning region uncovered, evaluated the role of candidate genes and compared genetic architectures across the genus. Our results show that, although the loci responding to mimicry selection are highly conserved between species, their effect size and phenotypic action vary throughout the clade. Multilocus architecture is ancestral and maintained across species under directional selection, whereas the single-locus (supergene) inheritance controlling polymorphism in H. numata appears to have evolved only once. Nevertheless, the conservatism in the wing-patterning toolkit found throughout the genus does not appear to constrain phenotypic evolution towards local adaptive optima.     

Molecular evolution of a long wavelength-sensitive opsin in mimetic Heliconius butterflies (Lepidoptera: Nymphalidae)

This study examines the pattern of opsin nucleotide and amino acid substitution among mimetic species 'rings' of Heliconius butterflies that are characterized by divergent wing colour patterns. A long wavelength opsin gene, OPS1 , was sequenced from each of seven species of Heliconius and one species of Dryas (Lepidoptera: Nymphalidae). A parsimony analysis of OPS1 nucleotide and amino acid sequences resulted in a phylogeny that was consistent with that presented by Brower & Egan in 1997, which was based on mitochondrial cytochrome oxidase I and II as well as nuclear wingless genes. Nodes in the OPS1 phylogeny were well supported by bootstrap analysis and decay indices. An analysis of specific sites within the gene indicates that the accumulation of amino acid substitutions has occurred independently of the morphological diversification of Heliconius wing colour patterns. Amino acid substitutions were examined with respect to their location within the opsin protein and their possible interactions with the chromophore and the G-protein. Of the 15 amino acid substitutions identified among the eight species, one nonconservative replacement (A226Q) was identified in a position that may be involved in binding with the G-protein.     

Parallel genetic architecture of parallel adaptive radiations in mimetic Heliconius butterflies

It is unknown whether homologous loci underlie the independent and parallel wing pattern radiations of Heliconius butterflies. By comparing the locations of color patterning genes on linkage maps we show that three loci that act similarly in the two radiations are in similar positions on homologous chromosomes.