Abstract

Citation:Joron M, Papa R, Beltrán M, Chamberlain N, Mavárez J, Baxter S, et al. (2006) A Conserved Supergene Locus Controls Colour Pattern Diversity inHeliconius Butterflies. PLoS Biol 4(10): e303. https://doi.org/10.1371/journal.pbio.0040303

Academic Editor:Mohamed A. F. Noor, Duke University, United States of America

Received:April 12, 2006;Accepted:July 14, 2006;Published: September 26, 2006

Copyright: © 2006 Joron et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding:This work was funded by 5th European Framework Programme Marie Curie Individual Fellowships to M. Joron and J. Mavárez; a European Molecular Biology Organization long-term fellowship to M. Joron; a Bogue Fellowship to M. Beltrán; several Natural Environment Research Council (NERC) grants to J. Mallet; a Smithsonian Institution grant to E. Bermingham; a National Science Foundation grant (IBN-0344705) to W. O. McMillan; a grant from the Sanger Institute to J. Rogers; and a Royal Society University Research Fellowship, Biotechnology and Biological Sciences Research Council grant 09074; and a NERC New Investigator grant to C. D. Jiggins.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations:BAC, bacterial artificial chromosome; cM, centimorgan

Introduction

Recent interest has focused on the genetic basis of convergent evolution [1,2]. Adaptive convergence between unrelated species, exemplified by colour pattern mimicry in insects [3], has led to a long-standing controversy about the relative contribution of gradual evolution driven by natural selection [4] versus occasional phenotypic leaps facilitated by conserved developmental pathways [5]. Recently, molecular genetic studies have shed new light on this controversy and have shown that regulation of the same genes [6,7], or even repeated recruitment of the same alleles [8], may explain convergent phenotypes in nature.

However, analysis of convergent phenotypes is only part of the story, because convergence and parallelism commonly occur in groups of organisms that have undergone recent adaptive radiations [9–11]. We are therefore interested in the evolution of phenotypic diversity and whether similar developmental genetic mechanisms are involved in convergent and divergent evolution. The repeated involvement of homologous loci in the evolution of convergent phenotypes would appear to support a hypothesis of strong developmental constraints on adaptive evolution [11–13]. If the same loci are also recruited in divergent evolution, then they may be generally important in phenotypic evolution rather than solely playing a role in convergence [14].

With strong divergence between geographic races of the same species and near-perfect local mimetic convergence between species, the diverse wing patterns ofHeliconius butterflies (Nymphalidae: Heliconiinae) provide an opportunity to link molecular genetics to adaptive evolution. A few genes of major effect are known to control patterns in the Müllerian co-mimicsH. erato andH. melpomene [15]. This has led to proposals that homologous genetic pathways [16] or a limited number of loci capable of controlling colour pattern shifts [17] could play an important role in convergent mimicry. However, homology of genetic architecture in mimetic butterflies has never been directly tested, despite the key role that mimicry has played in the history of the controversy [4,5].

We investigated the genetic architecture of colour pattern in threeHeliconius species that represent examples of both mimetic convergence and colour pattern diversification.H. melpomene andH. erato are distantly related, yet are phenotypically identical and have undergone a parallel radiation into over 30 named “rayed” or “postman” colour pattern races across the neotropics (Figure 1). H. erato is the probable model for this radiation [18], and local populations of the two co-mimics are monomorphic. The third species,H. numata, is closely related toH. melpomene but has extremely divergent wing patterns. Unlike the patterns inH. melpomene orH. erato, these patterns are highly polymorphic within populations, with up to seven “tiger”-patterned morphs in a single locality [20,21] (Figure 1). Each of these morphs is a precise mimic of a different species ofMelinaea (Nymphalidae: Ithomiinae); polymorphism inH. numata is thought to be maintained by strong selection for mimicry in a fine-scale spatial mosaic of ithomiine communities [19,20].

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Figure 1. Colour Pattern Diversity ofH. numata, H. melpomene, and their Respective Co-Mimics

The upper half of the figure shows five sympatric forms ofH. numata from northern Peru (second row, left to right:H. n. f.tarapotensis, H. n. f.silvana, H. n. f.aurora, H. n. f.bicoloratus, andH. n. f.arcuella) with their distantly related comimeticMelinaea species (Nymphalidae: Ithomiinae) from the same area (first row:M. menophilus ssp. nov.,M. ludovica ludovica, M. marsaeus rileyi, M. marsaeus mothone, andM. marsaeus phasiana) [20]. The lower half of the figure shows five colour pattern races ofH. melpomene, each from a different area of South America (third row:H. m. rosina, H. m. cythera, H. m. aglaope, H. m. melpomene, andH. m. plesseni) with their distantly related comimeticH. erato races from the same areas (fourth row:H. e. cf.petiveranus, H. e. cyrbia, H. e. emma, H. e. hydara, andH. e. notabilis).H. m. aglaope andH. e. emma are known as rayed forms, whereasH. m. rosina, H. m. melpomene, and co-mimics are known as postman forms.H. melpomene andH. erato are from divergent clades ofHeliconius and are identified in the field using minor morphological characters, such as the different form of the red rays on the hindwing betweenH. m. aglaope andH. e. emma (third from left) or the arrangement of red versus white patches inH. m. plesseni andH. e. notabilis (first from right). Co-mimicsH. numata andMelinaea spp. belong to different subfamilies of the Nymphalidae and have very different body morphology and wing venation. The phylogram on the left is a maximum-likelihood tree based on 1,541 bases of mitochondrial DNA (scale bar in substitutions per site, all bootstrap values over 99).

https://doi.org/10.1371/journal.pbio.0040303.g001

The differences in colour pattern between races ofH. melpomene andH. erato are controlled by several Mendelian factors of large phenotypic effect [15,17]. InH. melpomene, a complex of at least three tightly linked loci(N, Yb, andSb) control most of the variation in yellow and white pattern elements (Figures 1 and2A), and recombination between these loci suggests that they lie just a few centimorgans (cM) apart [15,17,21]. Another pair of loci(B andD), situated on a different linkage group, controls most of the variation in the red pattern elements and interacts withN to control the colour of the forewing band [15,17] (Figure 1). LocusAc controls the presence of a yellow patch in the discal cell of the forewing in some crosses [22]. Finally, locusK, unlinked toN–Yb–Sb orB–D, turns white patches to yellow in crosses betweenH. melpomene andH. cydno [21,23] (Table S1).

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Figure 2. Crosses Used for Mapping theYb, P, andCr Loci

(A) Crossing scheme inH. melpomene showing segregation of tightly linked lociYb andSb (hindwing yellow bar and white margin, present inH. m. cythera, Yb^cYb^c Sb^cSb^c) in brood B033. Genotypes are shown on the figure. The hindwing image in the box has been manipulated to highlight the shadow hindwing bar characteristic of heterozygote genotypes. Segregation of the linkedN locus controlling the yellow forewing band was followed in a different set of crosses not shown here (Table S1; Materials and Methods).

(B) Crossing scheme inH. erato showing segregation ofCr alleles in brood CH-CH5;Cr controls the forewing yellow band (absent inH. e. cyrbia, Cr^cCr^c), and the hindwing yellow bar and white margin (present inH. e. cyrbia). The red-patterning geneD also segregates in this cross, but is unlinked toCr; only progeny with aD^hiD^hi genotype are shown on the figure (Table S2; see also [24] for a figure of a similar cross showing all nine possible genotypes).

(C) Crossing scheme inH. numata showing segregation of theP alleles in intercross B502. F1 parents are heterozygous for different alleles, thus producing four different genotypes in the progeny.P switches the entire colour pattern, with strong dominance between sympatric alleles. Other broods (not shown) segregating for the very sameP^ele andP^sil alleles were sired by the same male or its full brother (Table S3).

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The radiation inH. erato has a similar genetic architecture, with a locusCr that has similar phenotypic effects to the combined action ofN, Yb, andSb inH. melpomene. In crosses betweenH. e. cyrbia and a sister species,H. himera, Cr controls a hindwing yellow bar (cf.Yb), a white hindwing margin (cf.Sb) and the yellow forewing band ofH. himera (cf.N) [24] (Figure 2B). Nonetheless, there are differences between the species: in inter-racialH. erato crosses the forewing yellow band is controlled by an unlinked locus,D, rather than byCr [17].D also controls most of the variation in the red pattern elements in a way that is analogous to theB–D complex inH. melpomene.

In contrast, mimicry polymorphism affecting yellow, brown/orange, and black colour patterns inH. numata is inherited entirely at a single Mendelian locus,P (Figure 2C). Populations are locally polymorphic, and nine distinctive alleles have been identified for theP locus in a narrow geographic area of Peru (Figures 1 and2C) [19,20]. Alleles at theP locus are nearly all completely dominant, with a linear hierarchy of dominance relationships [19,20], as might be expected in order to prevent the segregation of intermediate and nonmimetic phenotypes in wild populations. Occasional recombinant phenotypes occur, suggesting that theP locus may be a tight cluster of genes, or “supergene” [19,25].

Despite suggestions in the literature that there might be genetic homology between some of these mimicry genes in differentHeliconius species [16,26,27], such homology has not been directly tested. Here we describe the development of molecular markers that are tightly linked to a colour pattern locus inH. melpomene; we used these markers to investigate synteny and homology of colour pattern genes between the threeHeliconius species.

Results

We demonstrated homology of the genomic location of theP locus inH. numata, theN–Yb–Sb complex inH. melpomene, and theCr locus inH. erato (Figure 3). A noncoding region(a41), cloned from an amplified fragment length polymorphism marker in a linkage mapping study ofH. melpomene, lies within 1.1 cM of theH. melpomene pattern locusYb on linkage group 15 (out of a total map length of 1,616 cM) [22] (Figure S1). Among 413 individuals with both genotype and phenotype information from four mapping families, there were just five individuals recombinant betweena41 andYb (Table S1). This same marker is located within 0.7 cM of theP locus, which controls polymorphism inH. numata, with only two recombinant individuals identified among 306 individuals derived from six mapping families (Table S2). The probability of findingYb andP so tightly linked to a homologous marker in the two species by chance isp < 0.002 (seeMaterials and Methods).

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Figure 3. Chromosomal Maps for Linkage Group Homologues inH. melpomene (LG15),H. numata (LG15), andH. erato (LG02)

Distances are in Haldane centimorgans. The alternative orders forP anda41 relative toHm01 inH. numata are not significantly different (ΔLnL = −1.40). Similarly, most orders ofN, Sb, Yb, anda41 inH. melpomene are not significantly different (from ΔLnL = −0.15 for the ordera41–Yb–N–Sb– to ΔLnL = −0.77 fora41–N–Yb–Sb–). Finally, the two orders forCr andGerTra inH. erato are also equally significant. Therefore, we here show the most likely gene orders but cannot exclude that the colour loci are on the other side of the anchor locia41, Fox, orGerTra. In contrast, anchor loci orderGerTra–RpP40–Hm01–Hm08 is robust, with alternative orders significantly worse (ΔLnL < −2), although the relative placement ofRpL22 andeIF3-S9 is uncertain inH. melpomene andH. erato (ΔLnL > −2).

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The primers for the noncodinga41 marker did not amplify a product inH. erato. However, we used a PCR amplicon of this marker to probe a whole-genome bacterial artificial chromosomal (BAC) library ofH. melpomene. A 118-kb BAC clone was identified and its genomic location confirmed by the following: (a) alignment with sequences of thea41 locus generated fromH. melpomene genomic DNA and (b) recombination mapping of at least one marker derived from the end sequences of this clone in bothH. melpomene andH. numata. In both species, these end sequences showed complete linkage toa41 in at least 100 individuals. This clone was then sequenced and annotated by BLAST comparison with nucleotide and protein sequence databases (seeMaterials and Methods;Figure 4). In addition to identifying thea41 locus, we identified nine genes and three retrotransposon-associated coding regions (Figure 4).

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Figure 4. Annotation of Clone AEHM-41C10 from theHeliconius melpomene BAC Library

The region is situated on LG15 in theH. melpomene genome [22]. The sequence contains open reading frames of strong homology to 12 reported genes, three of which appear to be retrotransposon-associated coding regions (dotted boxes). Also highlighted in double frames are thea41 marker, which was used inH. numata andH. melpomene crosses and to isolate the clone from the library, and theRabgeranylgeranyl transferase gene, used as a marker inH. erato crosses.

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None of the genes identified in the 118-kb BAC clone is a candidate for theYb locus itself, because recombinants were identified between markers derived from the BAC end sequences andYb inH. melpomene (unpublished data). However, coding sequences were used to design conserved PCR primers for gene-based markers that cross-amplify broadly acrossHeliconius. One of these markers,GerTra, amplifies using primers anchored in two putative exons of theRab geranylgeranyl transferase beta subunit (βggt-II) gene and spans an intron showing substantial allelic size variation inH. erato (Figure S3). This region was 14 kb from thea41 marker inH. melpomene (Figure 4), and variation at this locus segregated nearly perfectly with the colour locusCr inH. erato. Only one recombinant betweenCr andGerTra alleles was identified among 197 individuals from two mapping families ofH. erato (Table S2), thus locatingGerTra within 0.3 cM of theCr locus (Figure 3; total map length inH. erato was estimated at 1,430 cM [27,28]). The probability of theH. melpomene geneYb andH. erato geneCr being tightly linked to homologous markers by chance isp < 0.003.

At a broader scale, two microsatellite markers(Hm01 andHm08) and three conserved gene regions(eIF3-S9, RpL22, andRpP40) map to the same linkage group asYb inH. melpomene (Figure 2). InH. numata, Hm01, Hm08, andRpP40 show a conserved pattern of linkage withH. melpomene both in terms of gene order and estimated distances between loci (Figure 2;eIF3-S9 andRpL22 were not variable in mapped broods ofH. numata). The two microsatellite loci unfortunately do not cross-amplify inH. erato, butRpL22 andeIF3-S9 both map to the linkage group containing theCr locus (Figure 2). These data reinforce our observation that linkage order is preserved between distantly relatedHeliconius species [27] and suggest that the chromosomes bearing colour genesP, Yb, andCr have not undergone large-scale rearrangement between the three species.

In addition to genotypinga41 and the markers derived from the BAC sequence, we have genotyped and assigned to linkage groups a total of 48 codominant molecular markers from across the genome, including 12 markers for genes known to be involved in the development of wings and patterns in other butterflies or inDrosophila (so-called candidate genes) [29–31], and 37 other conserved single-copy nuclear genes and microsatellites used as anchor loci in comparative mapping [22,27,28] (seeMaterials and Methods). We found no conflicting linkage relationship between the three species on the 16 linkage groups anchored with shared markers (Table 1) out of a total of 21 in each species [22,27], suggesting a widely conserved pattern of synteny at the genome scale. InH. melpomene, we have also mapped the following: (a) patterning lociB andD, which lie 66.7 cM from the geneCubitus-interruptus on linkage group 18, (b) locusAc, which is assigned to LG10, and (c) a locus we here termKhw, which lies 10 cM from the genewingless on linkage group 1 (Table 1).Khw controls the white/yellow switch of the hindwing margins inH. melpomene, and it is putatively distinct fromK, which controls the yellow/white switch of the forewing patch inH. cydno [23]. In both cases, the allele for white is dominant to that for yellow.

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Table 1.

Linkage Group Associations inH. melpomene, H. numata, andH. erato

https://doi.org/10.1371/journal.pbio.0040303.t001

Discussion

The data provide strong support for the hypothesis that a homologous gene or complex of genes regulates pattern diversity inH. numata, H. melpomene, andH. erato. The hypothesis of genetic homology of mimetic patterns in the geographic radiation of the Müllerian co-mimicsH. erato andH. melpomene is a long-standing question, and our data provide the first explicit test, to our knowledge, of this hypothesis. It was initially suggested that shared developmental pathways might facilitate the convergence seen between mimetic species [5]. Subsequently, a more extreme hypothesis was proposed, which states that the actual genes (rather than merely pathways) might be homologous between species [16]. Here we have confirmed the hypothesis of homology, to under 10⁻³ of theHeliconius genome [22,27], of at least one of the major loci controlling convergent patterns betweenH. erato andH. melpomene (Figure 1).

Taken on its own, this result would apparently support the hypothesis that strong developmental constraints are important in mimicry evolution. Nonetheless, the positional genetic homology we demonstrate stands in striking contrast to the lack of colour pattern similarity [16] between theH. erato–H. melpomene pair and the patterns ofH. numata, which are controlled by the same genomic region but involved in totally different mimicry rings (Figure 1). Rather than a constraint, this implies an extraordinary “jack-of-all-trades” flexibility of homologous colour pattern loci in closely related species (Figure 1). Our results inH. numata argue strongly against the idea that shared genetic architecture [8,32] constrains morphological diversification [7,33]. Instead, the data imply that natural selection has shaped a developmental switching mechanism capable of responding to a wide variety of mimetic pressures and producing locally adapted but highly divergent colour patterns.

The nature and mode of action for this developmental hot spot [34] of wing-pattern evolution remains to be determined. The tightly linked loci known to segregate in both theH. melpomene N–Yb–Sb complex and theH. erato Cr locus might represent a number ofcis regulatory elements of a single switch gene [2,35], a cluster of duplicated genes with divergent function [35,36], or a cluster of nonparalogous but functionally related genes [37]. One or probably more of these distinct elements could be involved in the switch supergene of theH. numata polymorphic mimicry. The three tightly linked colour pattern lociYb, Sb, andN clearly segregate on LG15 inH. melpomene, whereasP (H. numata) andCr (H. erato) show only extremely rare recombinant phenotypes, which could reflect higher crossing-over rates in this genomic region inH. melpomene and/or the involvement of more genetic elements (Figure 3).

Colour patterns develop by the maturation and spatial arrangement of different types of scales on the surface of the developing wing, each characterised by specific pigments and cuticular ultrastructure [16,38]. Our data show that genes on many different chromosomes are involved in the development of the colour pattern. The yellow, red, and orange pigments inHeliconius are ommochromes, and the ommochrome pathway genes —vermilion, white, andscarlet— are all unlinked to patterning genes segregating in our crosses in at least one of the species (Table 1). Furthermore, signalling-pathway genes known to be involved in establishing spatial information in developing butterfly wings —such asengrailed, Distal-less, anddecapentaplegic [29,30,39,40]—are also unlinked to switch genes. Two candidate genes were found to be linked to patterning loci(Cubitus-interruptus withB andD, andwingless withKhw), but in both cases, recombination mapping ruled out a direct role for these loci (see below). We have shown that genes involved in wing development and pigment formation are distributed across the genome and not tightly linked to the patterning loci that we have mapped.

A number of observations from these and previous crosses, combined with the results obtained here, offer some clues as to the nature of theN–Yb–Sb/Cr/P complex. (a) The same pigment types are found in different genotypes at this locus inH. erato andH. melpomene (Figure 2B), demonstrating that these loci control placement of pigments but do not switch particular pigment pathways on or off constitutively. (b) InH. melpomene, several tightly linked loci control distinct pattern elements that can be separated by rare recombination. These loci have a similar function in that they all control placement of white or yellow pattern elements (Figure 2A), suggesting that they are either linked paralogous copies of the same gene, or clusters ofcis regulatory elements of a single gene. (c) The locus controls patterns across both fore- and hindwings in all three species, but most strikingly inH. numata (Figure 2C). (d) The same allele can both increase and decrease the extent of the same pigment in different areas of the wing surface. In general, alleles adding yellow elements are recessive to those for black, but in the recessivesilvana form ofH. numata (alleleP^sil,Figure 2C), dominance of melanic elements is reversed relative to other forms such astarapotensis (P^tar). Items (c) and (d) imply that the gene product(s) are not directly involved in determining spatial positioning across the wing but are more likely transcribed in response to spatial information. Therefore, this complex locus most likely acts by communicating between spatial coordinate pathways and pigment pathways to create colour pattern elements. We hypothesise that the switch gene is most likely a transcription factor with a number ofcis regulatory elements that respond to the spatial information present in different parts of the wing. This transcription factor then triggers a response in sequentially acting downstream pathways to affect pigment deposition and scale morphology that are characteristic of each pattern element. Such regulatory elements would segregate in our crosses between wild forms and might vary in numbers and/or distance across species.

We have shown that another major mimicry locus lies on a homologous chromosome in the two co-mimics (Table 1).Cubitus-interruptus is 75 cM fromD inH. erato [27] and 66.7 cM fromB andD inH. melpomene (unpublished data). Given the loose linkage, a more precise positional comparison of these loci awaits fine-scale mapping of this linkage group, but the similarity of phenotypic effects of those loci and their location on homologous chromosomes hint at possible genetic homology ofB–D andD, and, together with colour pattern loci on other linkage groups (Table 1), hint at a largely shared multilocus colour pattern architecture between the distantly related co-mimicsH. erato andH. melpomene. Taken together, these findings in turn reveal a probable route for the evolution of the unusual “supergene” pattern control ofH. numata. Local mimicry polymorphism inH. numata is stable and is associated with selection favouring single-locus control of the entire pattern(P) with hierarchical dominance and avoiding nonmimetic intermediates [19,20,41]. However, the evolution of such supergene architecture, where the cosegregation of wing characters can be broken up by recombination [19], and which is most widely known from polymorphic Batesian mimics such asPapilio dardanus orP. memnon [42–44], is a puzzle. Theory predicts that selection against nonmimetic recombinants will rarely lead to the evolution of closer linkage between unlinked elements [41]. Genes must be rather tightly linked in the first place [41,45–47]—for instance via local gene duplications or regulatory element expansion [35,36]—to provide a useful starting point for the evolution of tighter linkage. In contrast to that ofH. numata, geographic radiation inH. melpomene is controlled by several unlinked regulatory loci of large effect(N–Yb–Sb, B–D, Ac, andK), and nonadaptive recombinants are probably not a focus of selection because they occur only in narrow hybrid zones [15,17,21]. More distantly relatedHeliconius, such asH. erato, also have a similar and probably largely homologous multichromosomal mimicry architecture [17,27,38] (Table 1), so that the single-locus inheritance inH. numata is a derived state (Figure 1). Our results thus suggest that part or all of the existingN–Yb–Sb complex ofH. melpomene has evolved intoP inH. numata, by taking control of regulation of the entire wing pattern [43,47], whereas the remaining unlinked colour pattern loci(B, D, Ac, andK inH. melpomene; D andSd inH. erato) do not cosegregate with major colour pattern variation inH. numata (Table 1). This result provides the first empirical evidence against the hypothesis of a “supergene” evolving via a gradual tightening of linkage between previously loosely linked or unlinked genes; this hypothesis has previously been challenged only on theoretical grounds [41,45–47]. The elucidation of the mechanism by whichP may have gained control of the entire regulation of wing pattern inH. numata will require the precise identification of the regulatory regions involved at this locus and the developmental pathways in which they take part [35].

To this end, the markers we developed provide a decisive step towards positional cloning of loci underlying colour pattern shifts. Our markers on LG15 are situated within a fraction of a centimorgan of the actual loci under selection, which may represent as little as 150 kb, given the estimated physical-to-map distance of ~165–180 kb/cM [22,28]. The genomic resources now available for positional cloning and large-scale sequencing in the threeHeliconius species mean that we are now close to identifying the genes involved in this adaptive radiation [48]. Fine-scale mapping using densely distributed markers will locate the recombination breakpoints in our crosses and narrow the segregating locus to a region of a few kilobases. Furthermore, the phenotypes studied occur in the wild and segregate across natural hybrid zones or in polymorphic populations [17,18,20], which will facilitate the use of association studies to test candidate loci [8]. On a broader phylogenetic scale, the identification of the colour pattern alleles segregating in different forms, races, and species in the wild will allow insights into the history of variation at these major loci and lead to testable hypotheses regarding the historical, developmental, or genetic constraints underlying the repeated recruitment of alleles at specific genes in mimetic lineages. Unravelling the molecular structure and developmental role of this locus inHeliconius will therefore provide important insights into the evolutionary basis of adaptive novelty.

Materials and Methods

Supporting Information

Acknowledgments

We gratefully acknowledge C. Estrada, F. Simpson, S. Gallusser, and G. Lamas for their help and collaboration with field and insectary work, and R. D. Reed for his help with the development of theGerTra marker. The authors also thank ANAM, Panama; the Ministerio de Agricultura, Peru; and the Ministerio del Medio Ambiente, Ecuador, for permission to carry out this project.

Author Contributions

M. Joron, W. O. McMillan and C. D. Jiggins developed the project. M. Joron, M. Abanto, W. O. McMillan, and C. D. Jiggins performed the insectary crosses. M. Beltrán, N. Chamberlain, J. Mavárez, S. Baxter, and C. D. Jiggins developed the markers in the laboratories of E. Bermingham, R. H. Ffrench-Constant, and C. D. Jiggins. M. Joron, R. Papa, S. Baxter, and C. D. Jiggins carried out the genotyping. N. Chamberlain performed the BAC library screen. S. J. Humphray performed the BAC sequencing, and H. Beasley and K. Barlow did the finishing in the laboratory of J. Rogers. M. Joron, E. Bermingham, J. Mallet, W. O. McMillan, and C. D. Jiggins wrote the paper.

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