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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The ecological genetics of homoploid hybrid speciation

Our understanding of homoploid hybrid speciation has advanced substantially since this mechanism of species formation was codified 50 years ago. Early theory and research focused almost exclusively on the importance of chromosomal rearrangements, but it later became evident that natural selection, specifically ecological selection, might play a major role as well. In light of this recent shift, we present an evaluation of ecology's role in homoploid hybrid speciation, with an emphasis on the genetics underlying ecological components of the speciation process. We briefly review new theoretical developments related to the ecology of homoploid hybrid speciation; propose a set of explicit, testable questions that must be answered to verify the role of ecological selection in homoploid hybrid speciation; discuss published work with reference to these questions; and also report new data supporting the importance of ecological selection in the origin of the homoploid hybrid sunflower species Helianthus deserticola. Overall, theory and empirical evidence gathered to date suggest that ecological selection is a major factor promoting homoploid hybrid speciation, with the strongest evidence coming from genetic studies.     

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.     

Hybrid sterility,Haldane's rule and speciation in Heliconius cydno and H. melpomene

Most genetic studies of Haldane's rule, in which hybrid sterility or inviability affects the heterogametic sex preferentially, have focused on Drosophila. It therefore remains unclear to what extent the conclusions of that work apply more generally, particularly in female-heterogametic taxa such as birds and Lepidoptera. Here we present a genetic analysis of Haldane's rule in Heliconius butterflies. Female F(1) hybrids between Heliconius melpomene and H. cydno are completely sterile, while males have normal to mildly reduced fertility. In backcrosses of male F(1) hybrids, female offspring range from completely sterile to fully fertile. Linkage analysis using the Z-linked triose-phosphate isomerase locus demonstrates a "large X" (Z) effect on sterility. Expression of female sterility varies among crosses in this and a previous study of Heliconius. Sterility may result from the production of normal but infertile eggs, production of small infertile eggs, or from a complete failure to develop ovarioles, which suggests multiple routes to the evolution of hybrid sterility in these Heliconius species. These results conform to the expectations of the "dominance" rather than "faster male" theories of Haldane's rule and suggest that relatively few loci are responsible. The two species are broadly sympatric and hybridize in the wild, so that female hybrid sterility forms one of several strong but incomplete barriers to gene flow in nature. The effect of female sterility is comparable to that of selection against non-mimetic hybrids, while mate choice forms a much stronger barrier to gene transfer.     

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.     

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.     

Genetic analysis of a wild-caught hybrid between non-sister Heliconius butterfly species

Interspecific hybridization occurs regularly in wild Heliconius butterflies, although hybrid individuals are usually very rare. However, hybridization generally occurs only between the most closely related species. We report a rare naturally occurring hybrid between non-sister species and carry out the first genetic analysis of such distant hybridization. Mitochondrial and nuclear genes indicate that the specimen is an F1 hybrid between a female Heliconius ethilla and a male Heliconius melpomene, originating from a group of 13 species estimated to have diverged over 2.5 Myr ago. The presence of such distant natural hybrids, together with evidence for backcrossing, suggests that gene flow across species boundaries can take place long after speciation. Adaptive genes such as those involved in wing coloration could thus be widely shared among members of this highly mimetic genus.     

Sharp genetic discontinuity across a unimodal Heliconius hybrid zone

Hybrid zones are powerful natural systems to study evolutionary processes to gain an understanding of adaptation and speciation. In the Cauca Valley (Colombia), two butterfly races, Heliconius cydno cydnides and Heliconius cydno weymeri, meet and hybridize. We characterized this hybrid zone using a combination of mitochondrial DNA (mtDNA) sequences, amplified fragment length polymorphisms (AFLPs), microsatellites and sequences for nuclear loci within and outside of the genomic regions that cause differences in wing colour pattern. The hybrid zone is largely composed of individuals of mixed ancestry. However, there is strong genetic discontinuity between the hybridizing races in mtDNA and, to a lesser extent, in all nuclear markers surveyed. The mtDNA clustering of H. c. cydnides with the H. cydno race from the Magdalena Valley and H. c. weymeri with the H. cydno race from the pacific coast suggests that H. c. cydnides colonized the Cauca Valley from the north, whereas H. c. weymeri did so by crossing the Andes in the southern part, implying a secondary contact origin. Colonization of the valley by H. cydno was accompanied by mimicry shift. Strong ecological isolation, driven by locally adaptive differences in mimetic wing patterns, is playing an important role in maintaining the hybrid zone. However, selection on wing pattern alone is not sufficient to explain the genetic discontinuity observed. There is evidence for differences in male mating preference, but the contribution of additional barriers needs further investigation. Overall, our results support the idea that speciation is a cumulative process, where the combination of multiple isolation barriers, combined with major phenotypic differences, facilitates population divergence in face of gene flow.     

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 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.     

ECOLOGICAL SPECIATION IN STICKLEBACKS: ENVIRONMENT-DEPENDENT HYBRID FITNESS

“Ecological” speciation occurs when reproductive isolation evolves as a consequence of divergent selection between populations exploiting different resources or environments. We tested this hypothesis of speciation in a young stickleback species pair by measuring the direct contribution of ecological selection pressures to hybrid fitness. The two species (limnetic and benthic) are strongly differentiated morphologically and ecologically, whereas hybrids are intermediate. Fitness of hybrids is high in the laboratory, especially F₁ and F₂ hybrids (backcrosses may show some breakdown). We transplanted F₁ hybrids to enclosures in the two main habitats in the wild to test whether the distribution of resources available in the environment generates a hybrid disadvantage not detectable in the laboratory. Hybrids grew more slowly than limnetics in the open water habitat and more slowly than benthics in the littoral zone. Growth of F₁ hybrids was inferior to the average of the parent species across both habitats, albeit not significantly. The contrast between laboratory and field results supports the hypothesis that mechanisms of F₁ hybrid fitness in the wild are primarily ecological and do not result from intrinsic genetic incompatibilities. Direct selection on hybrids contributes to the maintenance of sympatric stickleback species and may have played an important role in their origin.