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.     

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.     

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.     

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.     

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.     

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.     

Evolution of a mimicry supergene from a multilocus architecture

The origin and evolution of supergenes have long fascinated evolutionary biologists. In the polymorphic butterfly Heliconius numata, a supergene controls the switch between multiple different forms, and results in near-perfect mimicry of model species. Here, we use a morphometric analysis to quantify the variation in wing pattern observed in two broods of H. numata with different alleles at the supergene locus, 'P'. Further, we genotype the broods to associate the variation we capture with genetic differences. This allows us to begin mapping the quantitative trait loci that have minor effects on wing pattern. In addition to finding loci on novel chromosomes, our data, to our knowledge, suggest for the first time that ancestral colour-pattern loci, known to have major effects in closely related species, may contribute to the wing patterns displayed by H. numata, despite the large transfer of effects to the supergene.     

Sex-linked hybrid sterility in a butterfly

Recent studies, primarily in Drosophila, have greatly advanced our understanding of Haldane's rule, the tendency for hybrid sterility or inviability to affect primarily the heterogametic sex (Haldane 1922). Although dominance theory (Turelli and Orr 1995) has been proposed as a general explanation of Haldane's rule, this remains to be tested in female-heterogametic taxa, such as the Lepidoptera. Here we describe a novel example of Haldane's rule in Heliconius melpomene (Lepidoptera; Nymphalidae). Female F1 offspring are sterile when a male from French Guiana is crossed to a female from Panama, but fertile in the reciprocal cross. Male F1s are fertile in both directions. Similar female F1 sterility occurs in crosses between French Guiana and eastern Colombian populations. Backcrosses and linkage analysis show that sterility results from an interaction between gene(s) on the Z chromosome of the Guiana race with autosomal factors in the Panama genome. Large X (or Z) effects are commonly observed in Drosophila, but to our knowledge have not been previously demonstrated for hybrid sterility in Lepidoptera. Differences in the abundance of male versus female or Z-linked versus autosomal sterility factors cannot be ruled out in our crosses as causes of Haldane's rule. Nonetheless, the demonstration that recessive Z-linked loci cause hybrid sterility in a female heterogametic species supports the contention that dominance theory provides a general explanation of Haldane's rule (Turelli and Orr 2000).     

First report of genetic hybrids between two very divergent Leishmania species: Leishmania infantum and Leishmania major

The genus Leishmania includes many pathogenic species which are genetically very distant. The possibility of genetic exchange between different strains is still an important and debated question. Very few genetic hybrids (i.e., offspring of genetically dissimilar species) have been described in Leishmania. In this study, we report the first example of genetic hybrids occurring between two divergent Leishmania species, Leishmania infantum and Leishmania major. These two species have distinct geographical distributions and are transmitted by different vector species to different mammalian reservoir hosts. These hybrid strains were isolated in Portugal from immunocompromised patients and characterized by molecular and isoenzymatic techniques. These approaches showed that these chimeric strains probably contained the complete genome of both L. major and L. infantum. We believe this is the first report of genetic hybrids between such phylogenetically and epidemiologically distant species of Leishmania. This raises questions about the frequency of such cross-species genetic exchange in natural conditions, modalities of hybrid transmission, their long term maintenance as well as the consequences of these transfers on phenotypes such as drug resistance or pathogenicity.     

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.     

Maintenance of strong morphological differentiation despite ongoing natural hybridization between sympatric species of Lomatia (Proteaceae)

Background and Aims

When species cohesion is maintained despite ongoing natural hybridization, many questions are raised about the evolutionary processes operating in the species complex. This study examined the extensive natural hybridization between the Australian native shrubs Lomatia myricoides and L. silaifolia (Proteaceae). These species exhibit striking differences in morphology and ecological preferences, exceeding those found in most studies of hybridization to date.

Methods

Nuclear microsatellite markers (nSSRs), genotyping methods and morphometric analyses were used to uncover patterns of hybridization and the role of gene flow in morphological differentiation between sympatric species.

Key Results

The complexity of hybridization patterns differed markedly between sites, however, signals of introgression were present at all sites. One site provided evidence of a large hybrid swarm and the likely presence of multiple hybrid generations and backcrosses, another site a handful of early generational hybrids and a third site only traces of admixture from a past hybridization event. The presence of cryptic hybrids and a pattern of morphological bimodality amongst hybrids often disguised the extent of underlying genetic admixture.

Conclusions

Distinct parental habitats and phenotypes are expected to form barriers that contribute to the rapid reversion of hybrid populations to their parental character state, due to limited opportunities for hybrid/intermediate advantage. Furthermore, strong genomic filters may facilitate continued gene flow between species without the danger of assimilation. Stochastic fire events facilitate temporal phenological isolation between species and may partly explain the bi-directional and site-specific patterns of hybridization observed. Furthermore, the findings suggest that F₁ hybrids are rare, and backcrosses may occur rapidly following these initial hybridization events.     

Resolving a zoological mystery: the kouprey is a real species

The kouprey is a rare and enigmatic forest ox discovered by scientists in Cambodia only in 1937. Numerous morphological hypotheses have been proposed for the origin of the kouprey: that it is a species closely related to banteng and gaur, two other wild oxen of southeast Asia; a morphologically divergent species placed in a separate genus, named Novibos; a wild species linked to aurochs and domestic cattle; a vicariant population of banteng; a feral cattle; or a hybrid of banteng with either zebu cattle, gaur or water buffalo. In a recent paper, which gained a lot of media coverage, Galbreath et al. analysed mitochondrial DNA sequences and concluded that the kouprey never existed as a wild, natural species, and that it was a feral hybrid between banteng and zebu cattle. Here we analyse eight DNA markers-three mitochondrial regions and five nuclear fragments-representing an alignment of 4582 nucleotides for the holotype of the kouprey and all related species. Our results demonstrate that the kouprey is a real and naturally occurring species, and show that Cambodian populations of banteng acquired a mitochondrial genome of kouprey by natural introgressive hybridization during the Pleistocene epoch.     

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.     

High warning colour polymorphism in Heliconius hybrid zone roosts

1. Communal roosting behaviour has been documented among a wide range of taxa, particularly among groups of butterflies that display warning colourations. These aggregations of conspecifics and/or other species that share mimetic warning colour patterns can have a large impact on predator learning, and thus the survival of an aposematic form. Yet there has been limited investigation of communal roosting within areas where new and diverse warning colour forms are generated, such as hybrid zones. 2. Here, roosting behaviour was examined in a Heliconius erato hybrid zone in French Guiana between races with divergent warning colourations on their wings. In this hybrid zone, native individuals with nine distinct warning colourations, as well as individuals with altered forms that are not native in the French Guiana population, were marked and observed to determine if divergently coloured individuals participated in communal roosting, and if the proportions of colour pattern forms at roosts differed from the proportions that are found in the population. 3. The results demonstrated that divergently coloured individuals of the same species, including altered, non‐native forms, will readily and repeatedly participate in nocturnal communal roosting, often with extreme fidelity to specific perch locations. 4. These findings suggest that roosts composed of polymorphic warning patterns may be common in phenotypic transition zones, which could have major implications on predator training and selection dynamics in hybrid zones.     

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.     

Window of opportunity: an episode of recruitment in a Banksia hybrid zone demonstrates continuing hybridization and phenotypic plasticity

Background and Aims

In perennial plants (especially post-fire resprouters), extant populations may reflect recruitment events in the distant past. This is true of hybrid zones formed by two Banksia species of swamps and woodlands in south-eastern Australia, Banksia robur and B. oblongifolia. Both resprout after fire but recruitment is dependent on periodic fires. Although plants of intermediate morphology have also been identified as hybrids using allozyme markers, the extent of ongoing hybridization is unknown. This study investigates whether both microsatellite markers and morphological measurements can be used to distinguish between the two species and their hybrids. A recent recruitment event and microsatellite markers allow the frequency of ongoing hybridization to be estimated, and also the effects of environmental variation on the morphology of plants and seedlings to be tested.

Methods

Variation at seven microsatellite loci was scored and seven leaf characteristics within putatively pure stands and mixed stands of both species were measured, revealing that the two species were genetically and morphologically distinct and that mixed stands also contained genetically and sometimes morphologically distinct hybrids. An opportunity created by wildfires was used to analyse the genetics and morphometrics of adults and seedlings from two hybrid zones.

Key Results

Approximately 9 % of adults and 21 % of seedlings were identified as genetic hybrids in both hybrid zones. Within these sites, the genotype of mature plants correlated well with morphology, except for some hybrid plants that had parental morphology. However, seedling morphology was highly variable and insufficient to describe the composition of the hybrid zone in this cohort. Greater phenotypic plasticity was evident among seedlings growing within the hybrid zones than seedlings growing in pots.

Conclusions

The hybrid zones are complex and the range of genotypes detected in seedlings reveals both continuing hybridization and introgression.     

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.     

Do Heliconius butterfly species exchange mimicry alleles?

Hybridization has the potential to transfer beneficial alleles across species boundaries, and there are a growing number of examples in which this has apparently occurred. Recent studies suggest that Heliconius butterflies have transferred wing pattern mimicry alleles between species via hybridization, but ancestral polymorphism could also produce a signature of shared ancestry around mimicry genes. To distinguish between these alternative hypotheses, we measured DNA sequence divergence around putatively introgressed mimicry loci and compared this with the rest of the genome. Our results reveal that putatively introgressed regions show strongly reduced sequence divergence between co-mimetic species, suggesting that their divergence times are younger than the rest of the genome. This is consistent with introgression and not ancestral variation. We further show that this signature of introgression occurs at sites throughout the genome, not just around mimicry genes.     

PARALLEL RACE FORMATION AND THE EVOLUTION OF MIMICRY IN HELICONIUS BUTTERFLIES: A PHYLOGENETIC HYPOTHESIS FROM MITOCHONDRIAL DNA SEQUENCES

Mimicry has been a fundamental focus of research since the birth of evolutionary biology yet rarely has been studied from a phylogenetic perspective beyond the simple recognition that mimics are not similar due to common descent. The difficulty of finding characters to discern relationships among closely related and convergent taxa has challenged systematists for more than a century. The phenotypic diversity of wing pattens among mimetic Heliconius adds an additional twist to the problem, because single species contain more than a dozen radically different-looking geographical races even though the mimetic advantage is theoretically highest when all individuals within and between species appear the same. Mitochondrial DNA (mtDNA) offers an independent way to address these issues. In this study, Cytochrome Oxidase I and II sequences from multiple, parallel races of Heliconius erato and Heliconius melpomene are examined, to estimate intraspecific phylogeny and gauge sequence divergence and ages of clades among races within each species. Although phenotypes of sympatric races exhibit remarkable concordance between the two species, the mitochondrial cladograms show that the species have not shared a common evolutionary history. H. erato exhibits a basal split between trans- and cis-Andean groups of races, whereas H. melpomene originates in the Guiana Shield. Diverse races in either species appear to have evolved within the last 200,000 yr, and convergent phenotypes have evolved independently within as well as between species. These results contradict prior theories of the evolution of mimicry based on analysis of wing-pattern genetics.