The roles of hybridization and habitat fragmentation in the evolution of Brazil's enigmatic longwing butterflies, Heliconius nattereri and H. hermathena.

BACKGROUND: Heliconius butterflies are widely distributed across the Neotropics and have evolved a stunning array of wing color patterns that mediate Müllerian mimicry and mating behavior. Their rapid radiation has been strongly influenced by hybridization, which has created new species and allowed sharing of color patterning alleles between mimetic species pairs. While these processes have frequently been observed in widespread species with contiguous distributions, many Heliconius species inhabit patchy or rare habitats that may strongly influence the origin and spread of species and color patterns. Here, we assess the effects of historical population fragmentation and unique biology on the origins, genetic health, and color pattern evolution of two rare and sparsely distributed Brazilian butterflies, Heliconius hermathena and Heliconius nattereri. RESULTS: We assembled genomes and re-sequenced whole genomes of eight H. nattereri and 71 H. hermathena individuals. These species harbor little genetic diversity, skewed site frequency spectra, and high deleterious mutation loads consistent with recent population bottlenecks. Heliconius hermathena consists of discrete, strongly isolated populations that likely arose from a single population that dispersed after the last glacial maximum. Despite having a unique color pattern combination that suggested a hybrid origin, we found no genome-wide evidence that H. hermathena is a hybrid species. However, H. hermathena mimicry evolved via introgression, from co-mimetic Heliconius erato, of a small genomic region upstream of the color patterning gene cortex. CONCLUSIONS: Heliconius hermathena and H. nattereri population fragmentation, potentially driven by historical climate change and recent deforestation, has significantly reduced the genetic health of these rare species. Our results contribute to a growing body of evidence that introgression of color patterning alleles between co-mimetic species appears to be a general feature of Heliconius evolution.

Genomic evidence for gene flow between monarchs with divergent migratory phenotypes and flight performance.

Monarch butterflies are known for their spectacular annual migration in eastern North America, with millions of monarchs flying up to 4,500 km to overwintering sites in central Mexico. Monarchs also live west of the Rocky Mountains, where they travel shorter distances to overwinter along the Pacific Coast. It is often assumed that eastern and western monarchs form distinct evolutionary units, but genomic studies to support this notion are lacking. We used a tethered flight mill to show that migratory eastern monarchs have greater flight performance than western monarchs, consistent with their greater migratory distances. However, analysing more than 20 million SNPs in 43 monarch genomes, we found no evidence for genomic differentiation between eastern and western monarchs. Genomic analysis also showed identical and low levels of genetic diversity, and demographic analyses indicated similar effective population sizes and ongoing gene flow between eastern and western monarchs. Gene expression analysis of a subset of candidate genes during active flight revealed differential gene expression related to nonmuscular motor activity. Our results demonstrate that eastern and western monarchs maintain migratory differences despite ongoing gene flow, and suggest that migratory differences between eastern and western monarchs are not driven by select major-effects alleles. Instead, variation in migratory distance and destination may be driven by environmentally induced differential gene expression or by many alleles of small effect.

Frequency dependence shapes the adaptive landscape of imperfect Batesian mimicry.

Despite more than a century of biological research on the evolution and maintenance of mimetic signals, the relative frequencies of models and mimics necessary to establish and maintain Batesian mimicry in natural populations remain understudied. Here we investigate the frequency-dependent dynamics of imperfect Batesian mimicry, using predation experiments involving artificial butterfly models. We use two geographically distinct populations of Adelpha butterflies that vary in their relative frequencies of a putatively defended model (Adelpha iphiclus) and Batesian mimic (Adelpha serpa). We found that in Costa Rica, where both species share similar abundances, Batesian mimicry breaks down, and predators more readily attack artificial butterfly models of the presumed mimic, A. serpa By contrast, in Ecuador, where A. iphiclus (model) is significantly more abundant than A. serpa (mimic), both species are equally protected from predation. Our results provide compelling experimental evidence that imperfect Batesian mimicry is frequency-dependent on the relative abundance of models and mimics in natural populations, and contribute to the growing body of evidence that complex dynamics, such as seasonality or the availability of alternative prey, influence the evolution of mimetic traits.

Phylogeography of Heliconius cydno and its closest relatives: disentangling their origin and diversification.

The origins of the extraordinary diversity within the Neotropics have long fascinated biologists and naturalists. Yet, the underlying factors that have given rise to this diversity remain controversial. To test the relative importance of Quaternary climatic change and Neogene tectonic and paleogeographic reorganizations in the generation of biodiversity, we examine intraspecific variation across the Heliconius cydno radiation and compare this variation to that within the closely related Heliconius melpomene and Heliconius timareta radiations. Our data, which consist of both mtDNA and genome-scan data from nearly 2250 amplified fragment length polymorphism (AFLP) loci, reveal a complex history of differentiation and admixture at different geographic scales. Both mtDNA and AFLP phylogenies suggest that H. timareta and H. cydno are probably geographic extremes of the same radiation that probably diverged from H. melpomene prior to the Pliocene-Pleistocene boundary, consistent with hypotheses of diversification that rely on geological events in the Pliocene. The mtDNA suggests that this radiation originated in Central America or the northwestern region of South America, with a subsequent colonization of the eastern and western slopes of the Andes. Our genome-scan data indicate significant admixture among sympatric H. cydno/H. timareta and H. melpomene populations across the extensive geographic ranges of the two radiations. Within H. cydno, both mtDNA and AFLP data indicate significant population structure at local scales, with strong genetic differences even among adjacent H. cydno colour pattern races. These genetic patterns highlight the importance of past geoclimatic events, intraspecific gene flow, and local population differentiation in the origin and establishment of new adaptive forms.

Wing patterning gene redefines the mimetic history of Heliconius butterflies.

The mimetic butterflies Heliconius erato and Heliconius melpomene have undergone parallel radiations to form a near-identical patchwork of over 20 different wing-pattern races across the Neotropics. Previous molecular phylogenetic work on these radiations has suggested that similar but geographically disjunct color patterns arose multiple times independently in each species. The neutral markers used in these studies, however, can move freely across color pattern boundaries, and therefore might not represent the history of the adaptive traits as accurately as markers linked to color pattern genes. To assess the evolutionary histories across different loci, we compared relationships among races within H. erato and within H. melpomene using a series of unlinked genes, genes linked to color pattern loci, and optix, a gene recently shown to control red color-pattern variation. We found that although unlinked genes partition populations by geographic region, optix had a different history, structuring lineages by red color patterns and supporting a single origin of red-rayed patterns within each species. Genes closely linked (80-250 kb) to optix exhibited only weak associations with color pattern. This study empirically demonstrates the necessity of examining phenotype-determining genomic regions to understand the history of adaptive change in rapidly radiating lineages. With these refined relationships, we resolve a long-standing debate about the origins of the races within each species, supporting the hypothesis that the red-rayed Amazonian pattern evolved recently and expanded, causing disjunctions of more ancestral patterns.

Mate preference across the speciation continuum in a clade of mimetic butterflies.

Premating behavioral isolation is increasingly recognized as an important part of ecological speciation, where divergent natural selection causes the evolution of reproductive barriers. A number of studies have now demonstrated that traits under divergent natural selection also affect mate preferences. However, studies of single species pairs only capture a snapshot of the speciation process, making it difficult to assess the role of mate preferences throughout the entire process. Heliconius butterflies are well known for their brightly colored mimetic warning patterns, and previous studies have shown that these patterns are also used as mate recognition cues. Here, we present mate preference data for four pairs of sister taxa, representing different stages of divergence, which together allow us to compare diverging mate preferences across the continuum of Heliconius speciation. Using a novel Bayesian approach, our results support a model of ecological speciation in which strong premating isolation arises early, but continues to increase throughout the continuum from polymorphic populations through to "good," sympatric ecologically divergent species.

Comparative population genetics of mimetic Heliconius butterflies in an endangered habitat; Brazil's Atlantic Forest.

BACKGROUND: Brazil's Atlantic Forest is a biodiversity hotspot endangered by severe habitat degradation and fragmentation. Habitat fragmentation is expected to reduce dispersal among habitat patches resulting in increased genetic differentiation among populations. Here we examined genetic diversity and differentiation among populations of two Heliconius butterfly species in the northern portion of Brazil's Atlantic Forest to estimate the potential impact of habitat fragmentation on population connectivity in butterflies with home-range behavior. RESULTS: We generated microsatellite, AFLP and mtDNA sequence data for 136 Heliconius erato specimens from eight collecting locations and 146 H. melpomene specimens from seven locations. Population genetic analyses of the data revealed high levels of genetic diversity in H. erato relative to H. melpomene, widespread genetic differentiation among populations of both species, and no evidence for isolation-by-distance. CONCLUSIONS: These results are consistent with the hypothesis that the extensive habitat fragmentation along Brazil's Atlantic Forest has reduced dispersal of Heliconius butterflies among neighboring habitat patches. The results also lend support to the observation that fine-scale population genetic structure may be common in Heliconius. If such population structure also exists independent of human activity, and has been common over the evolutionary history of Heliconius butterflies, it may have contributed to the evolution of wing pattern diversity in the genus.

Gene flow and the genealogical history of Heliconius heurippa.

BACKGROUND: The neotropical butterfly Heliconius heurippa has a hybrid colour pattern, which also contributes to reproductive isolation, making it a likely example of hybrid speciation. Here we used phylogenetic and coalescent-based analyses of multilocus sequence data to investigate the origin of H. heurippa. RESULTS: We sequenced a mitochondrial region (CoI and CoII), a sex-linked locus (Tpi) and two autosomal loci (w and sd) from H. heurippa and the putative parental species, H. cydno and H. melpomene. These were analysed in combination with data from two previously sequenced autosomal loci, Dll and Inv. H. heurippa was monophyletic at mtDNA and Tpi, but showed a shared distribution of alleles derived from both parental lineages at all four autosomal loci. Estimates of genetic differentiation showed that H. heurippa is closer to H. cydno at mtDNA and three autosomal loci, intermediate at Tpi, and closer to H. melpomene at Dll. Using coalescent simulations with the Isolation-Migration model (IM), we attempted to establish the incidence of gene flow in the origin of H. heurippa. This analysis suggested that ongoing introgression is frequent between all three species and variable in extent between loci. CONCLUSION: Introgression, which is a necessary precursor of hybrid speciation, seems to have also blurred the coalescent history of these species. The origin of Heliconius heurippa may have been restricted to introgression of few colour pattern genes from H. melpomene into the H. cydno genome, with little evidence of genomic mosaicism.

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.

No genomic mosaicism in a putative hybrid butterfly species.

Recent descriptions of hybrid animal species have spurred interest in this phenomenon, but little genomic data exist to support it. Here, we use frequency variation for 657 amplified fragment length polymorphism (AFLP) markers and DNA sequence variation from 16 genes to determine whether the genome of Heliconius pachinus, a suspected hybrid butterfly species, is a mixture of the putative parental species, Heliconius cydno and Heliconius melpomene. Despite substantial shared genetic variation among all three species, we show that the genome of H. pachinus is not a mosaic; both AFLP and DNA sequence data overwhelmingly associate H. pachinus with just one of the potential parents, H. cydno. This pattern also applies to the gene wingless, which is tightly linked to the locus that determines forewing colour-one specific H. pachinus trait that has been hypothesized to have originated from H. melpomene. As a whole, the data support a traditional, bifurcating model of speciation in which H. pachinus split from a common ancestor with H. cydno without a genetic contribution from H. melpomene. However, comparison of our data to DNA sequence data for another putative hybrid Heliconius species, Heliconius heurippa, suggests that the H. heurippa genome may be a mosaic.

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.