Hybrid speciation driven by multilocus introgression of ecological traits.

Hybridization allows adaptations to be shared among lineages and may trigger the evolution of new species1,2. However, convincing examples of homoploid hybrid speciation remain rare because it is challenging to demonstrate that hybridization was crucial in generating reproductive isolation3. Here we combine population genomic analysis with quantitative trait locus mapping of species-specific traits to examine a case of hybrid speciation in Heliconius butterflies. We show that Heliconius elevatus is a hybrid species that is sympatric with both parents and has persisted as an independently evolving lineage for at least 180,000 years. This is despite pervasive and ongoing gene flow with one parent, Heliconius pardalinus, which homogenizes 99% of their genomes. The remaining 1% introgressed from the other parent, Heliconius melpomene, and is scattered widely across the H. elevatus genome in islands of divergence from H. pardalinus. These islands contain multiple traits that are under disruptive selection, including colour pattern, wing shape, host plant preference, sex pheromones and mate choice. Collectively, these traits place H. elevatus on its own adaptive peak and permit coexistence with both parents. Our results show that speciation was driven by introgression of ecological traits, and that speciation with gene flow is possible with a multilocus genetic architecture.

Pervasive mimicry in flight behavior among aposematic butterflies.

Flight was a key innovation in the adaptive radiation of insects. However, it is a complex trait influenced by a large number of interacting biotic and abiotic factors, making it difficult to unravel the evolutionary drivers. We investigate flight patterns in neotropical heliconiine butterflies, well known for mimicry of their aposematic wing color patterns. We quantify the flight patterns (wing beat frequency and wing angles) of 351 individuals representing 29 heliconiine and 9 ithomiine species belonging to ten color pattern mimicry groupings. For wing beat frequency and up wing angles, we show that heliconiine species group by color pattern mimicry affiliation. Convergence of down wing angles to mimicry groupings is less pronounced, indicating that distinct components of flight are under different selection pressures and constraints. The flight characteristics of the Tiger mimicry group are particularly divergent due to convergence with distantly related ithomiine species. Predator-driven selection for mimicry also explained variation in flight among subspecies, indicating that this convergence can occur over relatively short evolutionary timescales. Our results suggest that the flight convergence is driven by aposematic signaling rather than shared habitat between comimics. We demonstrate that behavioral mimicry can occur between lineages that have separated over evolutionary timescales ranging from <0.5 to 70 My.

A polygenic explanation for Haldane's rule in butterflies.

Two robust rules have been discovered about animal hybrids: Heterogametic hybrids are more unfit (Haldane's rule), and sex chromosomes are disproportionately involved in hybrid incompatibility (the large-X/Z effect). The exact mechanisms causing these rules in female heterogametic taxa such as butterflies are unknown but are suggested by theory to involve dominance on the sex chromosome. We investigate hybrid incompatibilities adhering to both rules in Papilio and Heliconius butterflies and show that dominance theory cannot explain our data. Instead, many defects coincide with unbalanced multilocus introgression between the Z chromosome and all autosomes. Our polygenic explanation predicts both rules because the imbalance is likely greater in heterogametic females, and the proportion of introgressed ancestry is more variable on the Z chromosome. We also show that mapping traits polygenic on a single chromosome in backcrosses can generate spurious large-effect QTLs. This mirage is caused by statistical linkage among polygenes that inflates estimated effect sizes. By controlling for statistical linkage, most incompatibility QTLs in our hybrid crosses are consistent with a polygenic basis. Since the two genera are very distantly related, polygenic hybrid incompatibilities are likely common in butterflies.

Cycad phylogeny predicts host plant use of Eumaeus butterflies.

Eumaeus butterflies are obligate herbivores of Zamia, the most diverse neotropical genus of cycads. Eumaeus-Zamia interactions have been characterized mainly for species distributed in North and Central America. However, larval host plant use by the southern Eumaeus clade remains largely unknown, precluding a comprehensive study of co-evolution between the genera. Here, we combine fieldwork with museum and literature surveys to expand herbivory records for Eumaeus from 21 to 38 Zamia species. We inferred a time-calibrated phylogeny of Eumaeus to test for distinct macroevolutionary scenarios of larval host plant conservatism and co-evolution. We found a remarkable coincidence between Eumaeus and Zamia diversification, with the butterfly stem group diverging at the same time as the most recent radiation of Zamia in the Miocene. Cophylogenetic reconciliation analyses show a strong cophylogenetic signal between cycads and their butterfly herbivores. Bipartite model-based approaches indicate that this is because closely related Zamia species are used by the same Eumaeus species, suggesting larval host plant resource tracking by the butterfly herbivores. Our results highlight a case of tight evolution between Eumaeus butterflies and cycads, pointing to the generality of correlated evolution and phylogenetic tracking in plant-herbivore interactions across seed plants.

Complex basis of hybrid female sterility and Haldane's rule in Heliconius butterflies: Z-linkage and epistasis.

Hybrids between species are often sterile or inviable. Hybrid unfitness usually evolves first in the heterogametic sex-a pattern known as Haldane's rule. The genetics of Haldane's rule have been extensively studied in species where the male is the heterogametic (XX/XY) sex, but its basis in taxa where the female is heterogametic (ZW/ZZ), such as Lepidoptera and birds, is largely unknown. Here, we analyse a new case of female hybrid sterility between geographic subspecies of Heliconius pardalinus. The two subspecies mate freely in captivity, but female F1 hybrids in both directions of cross are sterile. Sterility is due to arrested development of oocytes after they become differentiated from nurse cells, but before yolk deposition. We backcrossed fertile male F1 hybrids to parental females and mapped quantitative trait loci (QTLs) for female sterility. We also identified genes differentially expressed in the ovary as a function of oocyte development. The Z chromosome has a major effect, similar to the 'large X effect' in Drosophila, with strong epistatic interactions between loci at either end of the Z chromosome, and between the Z chromosome and autosomal loci on chromosomes 8 and 20. By intersecting the list of genes within these QTLs with those differentially expressed in sterile and fertile hybrids, we identified three candidate genes with relevant phenotypes. This study is the first to characterize hybrid sterility using genome mapping in the Lepidoptera and shows that it is produced by multiple complex epistatic interactions often involving the sex chromosome, as predicted by the dominance theory of Haldane's rule.

Geographic contrasts between pre- and postzygotic barriers are consistent with reinforcement in Heliconius butterflies.

Identifying the traits causing reproductive isolation and the order in which they evolve is fundamental to understanding speciation. Here, we quantify prezygotic and intrinsic postzygotic isolation among allopatric, parapatric, and sympatric populations of the butterflies Heliconius elevatus and Heliconius pardalinus. Sympatric populations from the Amazon (H. elevatus and H. p. butleri) exhibit strong prezygotic isolation and rarely mate in captivity; however, hybrids are fertile. Allopatric populations from the Amazon (H. p. butleri) and Andes (H. p. sergestus) mate freely when brought together in captivity, but the female F1 hybrids are sterile. Parapatric populations (H. elevatus and H. p. sergestus) exhibit both assortative mating and sterility of female F1s. Assortative mating in sympatric populations is consistent with reinforcement in the face of gene flow, where the driving force, selection against hybrids, is due to disruption of mimicry and other ecological traits rather than hybrid sterility. In contrast, the lack of assortative mating and hybrid sterility observed in allopatric populations suggests that geographic isolation enables the evolution of intrinsic postzygotic reproductive isolation. Our results show how the types of reproductive barriers that evolve between species may depend on geography.

The Scent Chemistry of Heliconius Wing Androconia.

Neotropical Heliconius butterflies are members of various mimicry rings characterized by diverse colour patterns. In the present study we investigated whether a similar diversity is observed in the chemistry of volatile compounds present in male wing androconia. Recent research has shown that these androconia are used during courting of females. Three to five wild-caught male Heliconius individuals of 17 species and subspecies were analyzed by GC/MS. Most of the identified compounds originate from common fatty acids precursors, including aldehydes, alcohols, acetates or esters preferentially with a C18 and C20 chain, together with some alkanes. The compounds occurred in species-specific mixtures or signatures. For example, octadecanal is characteristic for H. melpomene, but variation in composition between the individuals was observed. Cluster analysis of compound occurrence in individual bouquets and analyses based on biosynthetic motifs such as functional group, chain length, or basic carbon-backbone modification were used to reveal structural patterns. Mimetic pairs contain different scent bouquets, but also some compounds in common, whereas sympatric species, both mimetic and non-mimetic, have more distinct compound compositions. The compounds identified here may play a role in mate choice thus helping maintain species integrity in a butterfly genus characterized by pervasive interspecific gene flow.

Shortcuts in biodiversity research: What determines the performance of higher taxa as surrogates for species?

Biodiversity research is often impeded by the time and resources required to identify species. One possible solution is to use higher taxa to predict species richness and community composition. However, previous studies have shown that the performance of higher taxa as surrogates for species is highly variable, making it difficult to predict whether the method will be reliable for a particular objective. Using 8 independent datasets, I tested whether higher taxa accurately characterize the responses of beetle and ant communities to environmental drivers. For each dataset, ordinations were carried out using species and higher taxa, and the two compared using the Procrustes m² statistic (a scale-independent variant of Procrustes sum of squares). I then modelled the relationship between five hypothesised explanatory variables and 1) Procrustes m², and 2) the coefficient of determination (R²) for the correlation between richness of species and higher taxa. The species to higher taxon ratio, community structure, beta diversity, completeness of sampling, and taxon (beetles or ants) were all significant predictors of m², together explaining 88% of the variance. The only significant predictor of R² was the species to higher taxon ratio, which explained 45% of the variance. When using higher taxa to predict community composition, better performance is expected when the ratio of species to higher taxa is low, in communities with high evenness and high species turnover, and when there is niche conservation within higher taxa. When using higher taxa to predict species richness, effective surrogacy can be expected when the species to higher taxon ratio is very low. When it is not, surrogacy performance may be strongly influenced by stochastic factors, making predictions of performance difficult.

Variation in cyanogenic compounds concentration within a Heliconius butterfly community: does mimicry explain everything?

BACKGROUND: Aposematic species advertise their unpalatability using warning signals such as striking coloration. Given that predators need to sample aposematic prey to learn that they are unprofitable, prey with similar warning signals share the cost of predator learning. This reduction in predation risk drives evolutionary convergence of warning signals among chemically defended prey (Müllerian mimicry). Whether such warning signal convergence is associated to similar defence levels among co-mimics is still an open question that has rarely been tested in wild populations. We quantified variation in cyanide-based (CN) chemical protection in wild caught individuals of eight aposematic Heliconius butterfly species belonging to four sympatric mimicry rings. We then tested for correlations between chemical protection and ecological species-specific traits. RESULTS: We report significant differences in CN concentrations both within and between sympatric species, even when accounting for the phylogeny, and within and between mimicry rings, even after considering inter-specific variation. We found significant correlations between CN concentration and both hostplant specialization and gregarious behaviour in adults and larvae. However, differences in CN concentrations were not significantly linked to mimicry ring abundance, although the two most toxic species did belong to the rarest mimicry ring. CONCLUSIONS: Our results suggest that mimicry can explain the variation in the levels of chemical defence to a certain extent, although other ecological factors are also relevant to the evolution of such variability.

Crossing fitness valleys: empirical estimation of a fitness landscape associated with polymorphic mimicry.

Characterizing fitness landscapes associated with polymorphic adaptive traits enables investigation of mechanisms allowing transitions between fitness peaks. Here, we explore how natural selection can promote genetic mechanisms preventing heterozygous phenotypes from falling into non-adaptive valleys. Polymorphic mimicry is an ideal system to investigate such fitness landscapes, because the direction of selection acting on complex mimetic colour patterns can be predicted by the local mimetic community composition. Using more than 5000 artificial butterflies displaying colour patterns exhibited by the polymorphic Müllerian mimic Heliconius numata, we directly tested the role of wild predators in shaping fitness landscapes. We compared predation rates on mimetic phenotypes (homozygotes at the supergene controlling colour pattern), intermediate phenotypes (heterozygotes), exotic morphs (absent from the local community) and palatable cryptic phenotypes. Exotic morphs were significantly more attacked than local morphs, highlighting predators' discriminatory capacities. Overall, intermediates were attacked twice as much as local homozygotes, suggesting the existence of deep fitness valleys promoting strict dominance and reduced recombination between supergene alleles. By including information on predators' colour perception, we also showed that protection on intermediates strongly depends on their phenotypic similarity to homozygous phenotypes and that ridges exist between similar phenotypes, which may facilitate divergence in colour patterns.

Extensive range overlap between heliconiine sister species: evidence for sympatric speciation in butterflies?

BACKGROUND: Sympatric speciation is today generally viewed as plausible, and some well-supported examples exist, but its relative contribution to biodiversity remains to be established. We here quantify geographic overlap of sister species of heliconiine butterflies, and use age-range correlations and spatial simulations of the geography of speciation to infer the frequency of sympatric speciation. We also test whether shifts in mimetic wing colour pattern, host plant use and climate niche play a role in speciation, and whether such shifts are associated with sympatry. RESULTS: Approximately a third of all heliconiine sister species pairs exhibit near complete range overlap, and analyses of the observed patterns of range overlap suggest that sympatric speciation contributes 32%-95% of speciation events. Müllerian mimicry colour patterns and host plant choice are highly labile traits that seem to be associated with speciation, but we find no association between shifts in these traits and range overlap. In contrast, climatic niches of sister species are more conserved. CONCLUSIONS: Unlike birds and mammals, sister species of heliconiines are often sympatric and our inferences using the most recent comparative methods suggest that sympatric speciation is common. However, if sister species spread rapidly into sympatry (e.g. due to their similar climatic niches), then assumptions underlying our methods would be violated. Furthermore, although we find some evidence for the role of ecology in speciation, ecological shifts did not show the associations with range overlap expected under sympatric speciation. We delimit species of heliconiines in three different ways, based on "strict and " "relaxed" biological species concepts (BSC), as well as on a surrogate for the widely-used "diagnostic" version of the phylogenetic species concept (PSC). We show that one reason why more sympatric speciation is inferred in heliconiines than in birds may be due to a different culture of species delimitation in the two groups. To establish whether heliconiines are exceptional will require biogeographic comparative studies for a wider range of animal taxa including many more invertebrates.

Stable Heliconius butterfly hybrid zones are correlated with a local rainfall peak at the edge of the Amazon basin.

Multilocus clines between Müllerian mimetic races of Heliconius butterflies provide a classic example of the maintenance of hybrid zones and their importance in speciation. Concordant hybrid zones in the mimics Heliconius erato and H. melpomene in northern Peru were carefully documented in the 1980s, and this prior work now permits a historical analysis of the movement or stasis of the zones. Previous work predicted that these zones might be moving toward the Andes due to selective asymmetry. Extensive deforestation and climate change might also be expected to affect the positions and widths of the hybrid zones. We show that the positions and shapes of these hybrid zones have instead remained remarkably stable between 1985 and 2012. The stability of this interaction strongly implicates continued selection, rather than neutral mixing following secondary contact. The stability of cline widths and strong linkage disequilibria (gametic correlation coefficients Rmax = 0.35-0.56 among unlinked loci) over 25 years suggest that mimetic selection pressures on each color pattern locus have remained approximately constant (s ≈ 0.13-0.40 per locus in both species). Exceptionally high levels of precipitation at the edge of the easternmost Andes may act as a population density trough for butterflies, trapping the hybrid zones at the foot of the mountains, and preventing movement. As such, our results falsify one prediction of the Pleistocene Refugium theory: That the ranges of divergent species or subspecies should be centered on regions characterized by maxima of rainfall, with hybrid zones falling in more arid regions between them.

Nest inheritance is the missing source of direct fitness in a primitively eusocial insect.

Animals that cooperate with nonrelatives represent a challenge to inclusive fitness theory, unless cooperative behavior is shown to provide direct fitness benefits. Inheritance of breeding resources could provide such benefits, but this route to cooperation has been little investigated in the social insects. We show that nest inheritance can explain the presence of unrelated helpers in a classic social insect model, the primitively eusocial wasp Polistes dominulus. We found that subordinate helpers produced more direct offspring than lone breeders, some while still subordinate but most after inheriting the dominant position. Thus, while indirect fitness obtained through helping relatives has been the dominant paradigm for understanding eusociality in insects, direct fitness is vital to explain cooperation in P. dominulus.