Heliconius wing patterns: an evo-devo model for understanding phenotypic diversity

Evolutionary Developmental Biology aims for a mechanistic understanding of phenotypic diversity, and present knowledge is largely based on gene expression and interaction patterns from a small number of well-known model organisms. However, our understanding of biological diversification depends on our ability to pinpoint the causes of natural variation at a micro-evolutionary level, and therefore requires the isolation of genetic and developmental variation in a controlled genetic background. The colour patterns of Heliconius butterflies (Nymphalidae: Heliconiinae) provide a rich suite of naturally occurring variants with striking phenotypic diversity and multiple taxonomic levels of variation. Diversification in the genus is well known for its dramatic colour-pattern divergence between races or closely related species, and for Müllerian mimicry convergence between distantly related species, providing a unique system to study the development basis of colour-pattern evolution. A long history of genetic studies has showed that pattern variation is based on allelic combinations at a surprisingly small number of loci, and recent developmental evidence suggests that pattern development in Heliconius is different from the eyespot determination of other butterflies. Fine-scale genetic mapping studies have shown that a shared toolkit of genes is used to produce both convergent and divergent phenotypes. These exciting results and the development of new genomic resources make Heliconius a very promising evo-devo model for the study of adaptive change.     

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.     

Genomic tools and cDNA derived markers for butterflies

The Lepidoptera have long been used as examples in the study of evolution, but some questions remain difficult to resolve due to a lack of molecular genetic data. However, as technology improves, genomic tools are becoming increasingly available to tackle unanswered evolutionary questions. Here we have used expressed sequence tags (ESTs) to develop genetic markers for two Müllerian mimic species, Heliconius melpomene and Heliconius erato. In total 1363 ESTs were generated, representing 330 gene objects in H. melpomene and 431 in H. erato. User-friendly bioinformatic tools were used to construct a nonredundant database of these putative genes (available at http://www.heliconius.org), and annotate them with blast similarity searches, InterPro matches and Gene Ontology terms. This database will be continually updated with EST sequences for the Papilionideae as they become publicly available, providing a tool for gene finding in the butterflies. Alignments of the Heliconius sequences with putative homologues derived from Bombyx mori or other public data sets were used to identify conserved PCR priming sites, and develop 55 markers that can be amplified from genomic DNA in both H. erato and H. melpomene. These markers will be used for comparative linkage mapping in Heliconius and will have applications in other phylogenetic and genomic studies in the Lepidoptera.     

A Gene-Based Linkage Map for Bicyclus anynana Butterflies Allows for a Comprehensive Analysis of Synteny with the Lepidopteran Reference Genome

Lepidopterans (butterflies and moths) are a rich and diverse order of insects, which, despite their economic impact and unusual biological properties, are relatively underrepresented in terms of genomic resources. The genome of the silkworm Bombyx mori has been fully sequenced, but comparative lepidopteran genomics has been hampered by the scarcity of information for other species. This is especially striking for butterflies, even though they have diverse and derived phenotypes (such as color vision and wing color patterns) and are considered prime models for the evolutionary and developmental analysis of ecologically relevant, complex traits. We focus on Bicyclus anynana butterflies, a laboratory system for studying the diversification of novelties and serially repeated traits. With a panel of 12 small families and a biphasic mapping approach, we first assigned 508 expressed genes to segregation groups and then ordered 297 of them within individual linkage groups. We also coarsely mapped seven color pattern loci. This is the richest gene-based map available for any butterfly species and allowed for a broad-coverage analysis of synteny with the lepidopteran reference genome. Based on 462 pairs of mapped orthologous markers in Bi. anynana and Bo. mori, we observed strong conservation of gene assignment to chromosomes, but also evidence for numerous large- and small-scale chromosomal rearrangements. With gene collections growing for a variety of target organisms, the ability to place those genes in their proper genomic context is paramount. Methods to map expressed genes and to compare maps with relevant model systems are crucial to extend genomic-level analysis outside classical model species. Maps with gene-based markers are useful for comparative genomics and to resolve mapped genomic regions to a tractable number of candidate genes, especially if there is synteny with related model species. This is discussed in relation to the identification of the loci contributing to color pattern evolution in butterflies.     

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.     

Patterns on the insect wing

The evolution of wings and the adaptive advantage they provide have allowed insects to become one of the most evolutionarily successful groups on earth. The incredible diversity of their shape, size, and color patterns is a direct reflection of the important role wings have played in the radiation of insects. In this review, we highlight recent studies on both butterflies and Drosophila that have begun to uncover the types of genetic variations and developmental mechanisms that control diversity in wing color patterns. In butterflies, these analyses are now possible because of the recent development of a suite of genomic and functional tools, such as detailed linkage maps and transgenesis. In one such study, extensive linkage mapping in Heliconius butterflies has shown that surprisingly few, and potentially homologous, loci are responsible for several major pattern variations on the wings of these butterflies. Parallel work on a clade of Drosophila has uncovered how cis-regulatory changes of the same gene correlate with the repeated gain and loss of pigmented wing spots. Collectively, our understanding of formation and evolution of color pattern in insect wings is rapidly advancing because of these recent breakthroughs in several different fields.     

Genomic studies on the nature of species: adaptation and speciation in Mimulus

Evolutionary biology is in an exciting era, in which powerful genomic tools make the answers accessible to long‐standing questions about variation, adaptation and speciation. The availability of a suite of genomic resources, a shared knowledge base and a long history of study have made the phenotypically diverse plant genus Mimulus an important system for understanding ecological and evolutionary processes. An international Mimulus Research Meeting was held at Duke University in June 2014 to discuss developments in ecological and evolutionary genetic studies in Mimulus. Here, we report major recent discoveries presented at the meeting that use genomic approaches to advance our understanding of three major themes: the parallel genetic basis of adaptation; the ecological genomics of speciation; and the evolutionary significance of structural genetic variation. We also suggest future research directions for studies of Mimulus and highlight challenges faced when developing new ecological and evolutionary model systems.     

First-generation linkage map of the warningly colored butterfly Heliconius erato

We report the first genetic linkage map of Heliconius erato, a species that shows remarkable variation in its warningly colored wing patterns. We use crosses between H. erato and its sister species, H. himera, to place two major color pattern genes, D and Cr, on a linkage map containing AFLP, allozyme, microsatellite and single-copy nuclear loci. We identified all 21 linkage groups in an initial genetic screen of 22 progeny from an F1 female x male H. himera family. Of the 229 markers, 87 used to identify linkage groups were also informative in 35 progeny from a sibling backcross (H. himera female x F1 male). With these, and an additional 33 markers informative in the second family, we constructed recombinational maps for 19 of the 21 linkage groups. These maps varied in length from 18.1 to 431.1 centimorgans (cM) and yielded an estimated total length of 2400 cM. The average distance between markers was 23 cM, and eight of the 19 linkage groups, including the sex chromosome (Z) and the chromosome containing the Cr locus, contained two or more codominant anchor loci. Of the three potential candidate genes mapped here, Cubitus interruptus (Ci), Decapentaplegic (Dpp) and Wingless (Wg), only Ci was linked, although loosely, to a known Heliconius color pattern locus. This work is an important first step for constructing a denser genetic map of the H. erato color pattern radiation and for a comparative genomic study of the architecture of mimicry in Heliconius butterflies.     

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.     

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.     

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.     

Ecologically relevant genetic variation from a non-Arabidopsis perspective

Ecologically relevant genetic variation occurs in genes harbouring alleles that are adaptive in some environments but not in others. Analysis of this type of genetic variation in model organisms has made substantial progress, and is now being expanded to other species in order to better cover the diversity of plant life. Recent advances in connecting ecological and molecular studies in non-model species have been made with regard to edaphic and climatic adaptation, plant reproduction, life-history parameters and biotic interactions. New research avenues that increase biological complexity and ecological relevance by integrating ecological experiments with population genetic and functional genomic approaches provide new insights into the genetic basis of ecologically relevant variation.     

Biodiversity assessment using markers for ecologically important traits

Most studies of genetic variation within species to date are based on random markers. However, how well this correlates with quantitative variation is contentious. Yet, functional, or'ecotypic' variation in quantitative traits determines the ecological niche of a species, its future evolutionary potential, and, for livestock, crops and their wild relatives, their usefulness as a genetic resource for breeding. But nowadays we can also assess genetic diversity using markers directly targeted at specific genes or gene families. Such gene-targeted, multilocus profiles of markers can contribute to ex-situ management of genetic resources, ecological studies of diversity, and conservation of endangered species.     

Genome size variation in butterflies (Insecta,Lepidotera, Papilionoidea): a thorough phylogenetic comparison

Butterflies have been of great interest to naturalists for centuries, and the study of butterflies has been an integral part of ecology and evolution ever since Darwin proposed his theory of natural selection in 1859. There are > 18 000 butterfly species worldwide, showing great diversity in morphological traits and ecological niches. Compared with butterfly diversity, however, patterns of genome size variation in butterflies remain poorly understood, especially in a phylogenetic context. Here, we sequenced and assembled the mitogenomes of 68 butterflies and measured the genome sizes (C-values) of 67 of them. We also assembled 10 mitogenomes using reads from GenBank. Among the assembled 78 mitogenomes, those from 59 species, 23 genera and one subfamily are reported for the first time. Combining with published data of mitogenomes and genome size, we explored the patterns in genome size variation for 106 butterfly species in a phylogenetic context based on analyses of mitogenomes from 264 species covering six families. Our results show that the genome size of butterflies has a 6.4-fold variation ranging from 0.203 pg (199 Mb) (Nymphalidae: Heliconius xanthocles) to 1.287 pg (1253 Mb) (Papilionidae: Parnassius orleans). Within families, the largest variation was found in Papilionidae (5.9-fold: 0.22–1.29 pg), followed by Nymphalidae (4.8-fold: 0.2–0.95 pg), Pieridae (4.4-fold: 0.22–0.97 pg), Hesperiidae (2.2-fold: 0.3–0.66 pg), Lycaenidae (2.6-fold: 0.39–1.02 pg) and Rioidinidae (1.8-fold: 0.48–0.87 pg). Our data also suggest that butterflies have an ancestral genome size of c. 0.5 pg, and some ancestral genome size increase or decrease events along different subfamilies or tribes produce the diversity of genome size variation in diverse butterflies. Our data provide novel insights into patterns of genome size variation in butterflies and are an important reference for future genome sequencing programmes.     

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.     

Decoupling of rapid and adaptive evolution among seminal fluid proteins in heliconius butterflies with divergent mating systems

Reproductive proteins often diverge rapidly between species. This pattern is frequently attributed to postmating sexual selection. Heliconius butterflies offer a good opportunity to examine this hypothesis by contrasting patterns of reproductive protein evolution between clades with divergent mating systems. Pupal-mating Heliconius females typically mate only once, limiting opportunity for postmating sexual selection. In contrast, adult-mating females remate throughout life. Reproductive protein evolution is therefore predicted to be slower and show little evidence of positive selection in the pupal-mating clade. We examined this prediction by sequencing 18 seminal fluid protein genes from a dozen Heliconius species and a related outgroup. Two proteins exhibited dN/dS > 1, implicating positive selection in the rapid evolution of at least a few Heliconius seminal fluid proteins. However, contrary to predictions, the average evolutionary rate of seminal fluid proteins was greater among pupal-mating Heliconius. Based on these results, we suggest that positive selection and relaxed constraint can generate conflicting patterns of reproductive protein evolution between mating systems. As predicted, some loci may show elevated evolutionary rates in promiscuous taxa relative to monandrous taxa resulting from adaptations to postmating sexual selection. However, when monandry is derived (as in Heliconius), the opposite pattern may result from relaxed selective constraints.     

Correlations between scale structure and pigmentation in butterfly wings

SUMMARY We examined the correlation between color and structure of wing scales in the nymphalid butterflies Bicyclus anynana and Heliconius melpomene . All scales in B. anynana are rather similar in comparison to the clear structural differences of differently pigmented scales in H. melpomene . Where scale structural differences in H. melpomene are qualitative, they seem to be quantitative in B. anynana . There is a "gradient" in the density of some structural elements, the cross ribs, in the scales of B. anynana : black, gold, and brown scales show progressively lower cross rib density within an individual. There is, however, high individual variation in the absolute cross rib densities (i.e., scales with a particular color and cross rib density in one individual may have a different color but similar density in another individual). By ectopically inducing color pattern during early pupal development, we examined whether a scale's color and its microstructure could be uncoupled. The effect of these manipulations appears to be different in B. anynana and H. melpomene . In Bicyclus , "black" scales induced by wing damage at an ectopic location normally containing brown scales acquire both an intermediate structure and color between that of brown and normal black scales. In Heliconius , however, intermediate colors or scale structure were never observed, and scales with an altered color (due to damage) always have the same structure as normal scales with that color. The results are discussed on the basis of gene expression patterns, variability in rates of scale development and pigment, and scale sclerotization pathways.