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.     

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.     

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.     

Spectral sensitivity of the compound eye in butterflies (Heliconius)

Summary The spectral sensitivity of the compound eye in three butterfly species (Heliconius erato, H. numata, H, sara) was tested electrophysiologically in the wavelength region 310 to 650 nm. Sensitivity maxima were found at 370 to 390 nm, 450 to 470 nm, and 550 to 570 nm, for all species. The three sensitivity maxima are suggested to be due to different photoreceptor types effecting wave-length discrimination. An interspecies difference in spectral sensitivity was also found. The difference is suggested to be due to the relative number of photoreceptors of each type. In some of the present experiments a small discontinuity in sensitivity was found at 610 or 630 nm. It is probably caused by a selective reflection of these wavelengths from a tapetum.     

The evolution of ecologically important characters in Heliconius butterflies (Lepidoptera: Nymphalidae): a cladistic review

The biology of Heliconius butterflies has provided a rich source of data to test theories of ecological genetics, coevolution and community ecology. Many putatively adaptive characters have been discussed with reference to a phylogenetic hypothesis based on a variety of morphological and life-history traits interpreted from an evolutionary taxonomic perspective. Here, alternate interpretations of characters on the traditional tree and a more recent mitochondrial DNA cladogram with a substantially different topology are compared and contrasted. It is shown that many characters ostensibly providing support for the traditional phylogenetic hypothesis are almost equally parsimoniously distributed and in some cases more parsimoniously distributed on the mtDNA tree than on the tree inferred from those characters. Discussion of alternate evolutionary scenarios based on the mDNA-based topology is presented for pupal mating, pollen feeding, foodplant coevolution, and other ecologically significant features.     

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.     

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 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.     

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.     

Mechanical damage to pollen aids nutrient acquisition in Heliconius butterflies (Nymphalidae)

Neotropical Heliconius and Laparus butterflies actively collect pollen onto the proboscis and extract nutrients from it. This study investigates the impact of the processing behaviour on the condition of the pollen grains. Pollen samples (n = 72) were collected from proboscides of various Heliconius species and Laparus doris in surrounding habitats of the Tropical Research Station La Gamba (Costa Rica). Examination using a light microscope revealed that pollen loads contained 74.88 ± 53.67% of damaged Psychotria pollen, 72.04 ± 23.4% of damaged Psiguria/Gurania pollen, and 21.35 ± 14.5% of damaged Lantana pollen (numbers represent median ± first quartile). Damaged pollen grains showed deformed contours, inhomogeneous and/or leaking contents, or they were empty. Experiments with Heliconius and Laparus doris from a natural population in Costa Rica demonstrated that 200 min of pollen processing behaviour significantly increased the percentage of damaged pollen of Psychotria compared to pollen from anthers (P = 0.015, Z = ?2.44, Mann–Whitney U-test). Examination of pollen loads from green house reared Heliconius butterflies resulted in significantly greater amounts of damaged Psiguria pollen after 200 min of processing behaviour compared to pollen from flowers (P < 0.001, Z = ?4.583, Mann–Whitney U-test). These results indicate that pollen processing functions as extra oral digestion whereby pollen grains are ruptured to make the content available for ingestion.     

Sex-specific chemical cues from immatures facilitate the evolution of mate guarding in Heliconius butterflies

Competition for mates has substantial effects on sensory systems and often leads to the evolution of extraordinary mating behaviours in nature. The ability of males to find sexually immature females and associate with them until mating is a remarkable example. Although several aspects of such pre-copulatory mate guarding have been investigated, little is known about the mechanisms used by males to locate immature females and assess their maturity. These are not only key components of the origin and maintenance of this mating strategy, but are also necessary for inferring the level to which females cooperate and thus the incidence of sexual conflict. We investigated the cues involved in recognition of immature females in Heliconius charithonia, a butterfly that exhibits mate guarding by perching on pupae. We found that males recognized female pupae using sex-specific volatile monoterpenes produced by them towards the end of pupal development. Considering the presumed biosynthetic pathways of such compounds and the reproductive biology of Heliconius, we propose that these monoterpenes are coevolved signals and not just sex-specific cues exploited by males. Their maintenance, despite lack of female mate choice, may be explained by variation in cost that females pay with this male behaviour under heterogeneous ecological conditions.     

Phylogenetic discordance at the species boundary: comparative gene genealogies among rapidly radiating Heliconius butterflies

Recent adaptive radiations provide excellent model systems for understanding speciation, but rapid diversification can cause problems for phylogenetic inference. Here we use gene genealogies to investigate the phylogeny of recent speciation in the heliconiine butterflies. We sequenced three gene regions, intron 3 ( approximately 550 bp) of sex-linked triose-phosphate isomerase (Tpi), intron 3 ( approximately 450 bp) of autosomal mannose-phosphate isomerase (Mpi), and 1,603 bp of mitochondrial cytochrome oxidase subunits I and II (COI and COII), for 37 individuals from 25 species of Heliconius and related genera. The nuclear intron sequences evolved at rates similar to those of mitochondrial coding sequences, but the phylogenetic utility of introns was restricted to closely related geographic populations and species due to high levels of indel variation. For two sister species pairs, Heliconius erato-Heliconius himera and Heliconius melpomene-Heliconius cydno, there was highly significant discordance between the three genes. At mtDNA and Tpi, the hypotheses of reciprocal monophyly and paraphyly of at least one species with respect to its sister could not be distinguished. In contrast alleles sampled from the third locus, Mpi, showed polyphyletic relationships between both species pairs. In all cases, recent coalescence of mtDNA lineages within species suggests that polyphyly of nuclear genes is not unexpected. In addition, very similar alleles were shared between melpomene and cydno, implying recent gene flow. Our finding of discordant genealogies between genes is consistent with models of adaptive speciation with ongoing gene flow and highlights the need for multiple locus comparisons to resolve phylogeny among closely related species.     

Convergent, modular expression of ebony and tan in the mimetic wing patterns of Heliconius butterflies

The evolution of pigmentation in vertebrates and flies has involved repeated divergence at a small number of genes related to melanin synthesis. Here, we study insect melanin synthesis genes in Heliconius butterflies, a group characterised by its diversity of wing patterns consisting of black (melanin), and yellow and red (ommochrome) pigmented scales. Consistent with their respective biochemical roles in Drosophila melanogaster, ebony is upregulated in non-melanic wing regions destined to be pigmented red whilst tan is upregulated in melanic regions. Wing regions destined to be pigmented yellow, however, are downregulated for both genes. This pattern is conserved across multiple divergent and convergent phenotypes within the Heliconii, suggesting a conserved mechanism for the development of black, red and yellow pattern elements across the genus. Linkage mapping of five melanin biosynthesis genes showed that, in contrast to other organisms, these genes do not control pattern polymorphism. Thus, the pigmentation genes themselves are not the locus of evolutionary change but lie downstream of a wing pattern regulatory factor. The results suggest a modular system in which particular combinations of genes are switched on whenever red, yellow or black pattern elements are favoured by natural selection for diverse and mimetic wing patterns.