PLoS BIOLOGY A Conserved Supergene Locus Controls

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PLoS BIOLOGYA Conserved Supergene Locus Controls ColourPattern Diversity in Heliconius ButterfliesMathieu Joron1,2,3*, Riccardo Papa4, Margarita Beltrán1, Nicola Chamberlain5, Jesús Mavárez3,6, Simon Baxter1,Moisés Abanto7, Eldredge Bermingham6, Sean J. Humphray8, Jane Rogers8, Helen Beasley8, Karen Barlow8,Richard H. ffrench-Constant5, James Mallet3, W. Owen McMillan4, Chris D. Jiggins11 Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom, 2 Institute of Biology, Leiden University, Leiden,Netherlands, 3 The Galton Laboratory, Department of Biology, University College London, London, United Kingdom, 4 Department of Biology, University of Puerto Rico, SanJuan, Puerto Rico, 5 Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom, 6 Smithsonian Tropical Research Institute, Balboa, Panama, 7 JirónAlegrı́a Arias de Morey, Tarapoto, Peru, 8 The Wellcome Trust Sanger Institute, Cambridge, United KingdomWe studied whether similar developmental genetic mechanisms are involved in both convergent and divergentevolution. Mimetic insects are known for their diversity of patterns as well as their remarkable evolutionaryconvergence, and they have played an important role in controversies over the respective roles of selection andconstraints in adaptive evolution. Here we contrast three butterfly species, all classic examples of Müllerian mimicry.We used a genetic linkage map to show that a locus, Yb, which controls the presence of a yellow band in geographicraces of Heliconius melpomene, maps precisely to the same location as the locus Cr, which has very similar phenotypiceffects in its co-mimic H. erato. Furthermore, the same genomic location acts as a ‘‘supergene’’, determining multiplesympatric morphs in a third species, H. numata. H. numata is a species with a very different phenotypic appearance,whose many forms mimic different unrelated ithomiine butterflies in the genus Melinaea. Other unlinked colourpattern loci map to a homologous linkage group in the co-mimics H. melpomene and H. erato, but they are not involvedin mimetic polymorphism in H. numata. Hence, a single region from the multilocus colour pattern architecture of H.melpomene and H. erato appears to have gained control of the entire wing-pattern variability in H. numata, presumablyas a result of selection for mimetic ‘‘supergene’’ polymorphism without intermediates. Although we cannot at thisstage confirm the homology of the loci segregating in the three species, our results imply that a conserved yetrelatively unconstrained mechanism underlying pattern switching can affect mimicry in radically different ways. Wealso show that adaptive evolution, both convergent and diversifying, can occur by the repeated involvement of thesame genomic regions.Citation: Joron M, Papa R, Beltrán M, Chamberlain N, Mavárez J, et al. (2006) A conserved supergene locus controls colour pattern diversity in Heliconius butterflies. PLoS Biol4(10): e303. DOI: 10.1371/journal.pbio.0040303With strong divergence between geographic races of thesame species and near-perfect local mimetic convergencebetween species, the diverse wing patterns of Heliconiusbutterflies (Nymphalidae: Heliconiinae) provide an opportunity to link molecular genetics to adaptive evolution. A fewgenes of major effect are known to control patterns in theMüllerian co-mimics H. erato and H. melpomene [15]. This hasled to proposals that homologous genetic pathways [16] or alimited number of loci capable of controlling colour patternshifts [17] could play an important role in convergentmimicry. However, homology of genetic architecture inmimetic butterflies has never been directly tested, despitethe key role that mimicry has played in the history of thecontroversy [4,5].We investigated the genetic architecture of colour patternIntroductionRecent interest has focused on the genetic basis ofconvergent evolution [1,2]. Adaptive convergence betweenunrelated species, exemplified by colour pattern mimicry ininsects [3], has led to a long-standing controversy about therelative contribution of gradual evolution driven by naturalselection [4] versus occasional phenotypic leaps facilitated byconserved developmental pathways [5]. Recently, moleculargenetic studies have shed new light on this controversy andhave shown that regulation of the same genes [6,7], or evenrepeated recruitment of the same alleles [8], may explainconvergent phenotypes in nature.However, analysis of convergent phenotypes is only part ofthe story, because convergence and parallelism commonlyoccur in groups of organisms that have undergone recentadaptive radiations [9–11]. We are therefore interested in theevolution of phenotypic diversity and whether similardevelopmental genetic mechanisms are involved in convergent and divergent evolution. The repeated involvement ofhomologous loci in the evolution of convergent phenotypeswould appear to support a hypothesis of strong developmental constraints on adaptive evolution [11–13]. If the same lociare also recruited in divergent evolution, then they may begenerally important in phenotypic evolution rather thansolely playing a role in convergence [14].PLoS Biology www.plosbiology.orgAcademic Editor: Mohamed A. F. Noor, Duke University, United States of AmericaReceived April 12, 2006; Accepted July 14, 2006; Published September 26, 2006DOI: 10.1371/journal.pbio.0040303Copyright: Ó 2006 Joron et al. This is an open-access article distributed under theterms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original authorand source are credited.Abbreviations: BAC, bacterial artificial chromosome; cM, centimorgan* To whom correspondence should be addressed. E-mail: 2006 Volume 4 Issue 10 e303

Conserved Mimicry Genes in HeliconiusFigure 1. Colour Pattern Diversity of H. numata, H. melpomene, and their Respective Co-MimicsThe upper half of the figure shows five sympatric forms of H. 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, and H. n. f. arcuella) with their distantly related comimetic Melinaea species (Nymphalidae: Ithomiinae) from the same area(first row: M. menophilus ssp. nov., M. ludovica ludovica, M. marsaeus rileyi, M. marsaeus mothone, and M. marsaeus phasiana) [20]. The lower half of thefigure shows five colour pattern races of H. melpomene, each from a different area of South America (third row: H. m. rosina, H. m. cythera, H. m. aglaope,H. m. melpomene, and H. m. plesseni) with their distantly related comimetic H. erato races from the same areas (fourth row: H. e. cf. petiveranus, H. e.cyrbia, H. e. emma, H. e. hydara, and H. e. notabilis). H. m. aglaope and H. e. emma are known as rayed forms, whereas H. m. rosina, H. m. melpomene, andco-mimics are known as postman forms. H. melpomene and H. erato are from divergent clades of Heliconius and are identified in the field using minormorphological characters, such as the different form of the red rays on the hindwing between H. m. aglaope and H. e. emma (third from left) or thearrangement of red versus white patches in H. m. plesseni and H. e. notabilis (first from right). Co-mimics H. numata and Melinaea spp. belong todifferent subfamilies of the Nymphalidae and have very different body morphology and wing venation. The phylogram on the left is a maximumlikelihood tree based on 1,541 bases of mitochondrial DNA (scale bar in substitutions per site, all bootstrap values over 99).DOI: 10.1371/journal.pbio.0040303.g001elements (Figures 1 and 2A), and recombination betweenthese loci suggests that they lie just a few centimorgans (cM)apart [15,17,21]. Another pair of loci (B and D), situated on adifferent linkage group, controls most of the variation in thered pattern elements and interacts with N to control thecolour of the forewing band [15,17] (Figure 1). Locus Accontrols the presence of a yellow patch in the discal cell of theforewing in some crosses [22]. Finally, locus K, unlinked to N–Yb–Sb or B–D, turns white patches to yellow in crossesbetween H. melpomene and H. cydno [21,23] (Table S1).The radiation in H. erato has a similar genetic architecture,with a locus Cr that has similar phenotypic effects to thecombined action of N, Yb, and Sb in H. melpomene. In crossesbetween H. e. cyrbia and a sister species, H. himera, Cr controls ahindwing yellow bar (cf. Yb), a white hindwing margin (cf. Sb)and the yellow forewing band of H. himera (cf. N) [24] (Figure2B). Nonetheless, there are differences between the species: ininter-racial H. erato crosses the forewing yellow band iscontrolled by an unlinked locus, D, rather than by Cr [17]. Dalso controls most of the variation in the red patternelements in a way that is analogous to the B–D complex inH. three Heliconius species that represent examples of bothmimetic convergence and colour pattern diversification. H.melpomene and H. erato are distantly related, yet are phenotypically identical and have undergone a parallel radiation intoover 30 named ‘‘rayed’’ or ‘‘postman’’ colour pattern racesacross the neotropics (Figure 1). H. erato is the probable modelfor this radiation [18], and local populations of the two comimics are monomorphic. The third species, H. numata, isclosely related to H. melpomene but has extremely divergentwing patterns. Unlike the patterns in H. melpomene or H. 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 ofa different species of Melinaea (Nymphalidae: Ithomiinae);polymorphism in H. numata is thought to be maintained bystrong selection for mimicry in a fine-scale spatial mosaic ofithomiine communities [19,20].The differences in colour pattern between races of H.melpomene and H. erato are controlled by several Mendelianfactors of large phenotypic effect [15,17]. In H. melpomene, acomplex of at least three tightly linked loci (N, Yb, and Sb)control most of the variation in yellow and white patternPLoS Biology www.plosbiology.org0002October 2006 Volume 4 Issue 10 e303

Conserved Mimicry Genes in HeliconiusFigure 2. Crosses Used for Mapping the Yb, P, and Cr Loci(A) Crossing scheme in H. melpomene showing segregation of tightlylinked loci Yb and Sb (hindwing yellow bar and white margin, present inH. m. cythera, YbcYbc SbcSbc) in brood B033. Genotypes are shown on thefigure. The hindwing image in the box has been manipulated tohighlight the shadow hindwing bar characteristic of heterozygotegenotypes. Segregation of the linked N locus controlling the yellowforewing band was followed in a different set of crosses not shown here(Table S1; Materials and Methods).(B) Crossing scheme in H. erato showing segregation of Cr alleles inbrood CH-CH5; Cr controls the forewing yellow band (absent in H. e.cyrbia, CrcCrc), and the hindwing yellow bar and white margin (present inH. e. cyrbia). The red-patterning gene D also segregates in this cross, butis unlinked to Cr; only progeny with a DhiDhi genotype are shown on thefigure (Table S2; see also [24] for a figure of a similar cross showing allnine possible genotypes).(C) Crossing scheme in H. numata showing segregation of the P alleles inintercross B502. F1 parents are heterozygous for different alleles, thusproducing four different genotypes in the progeny. P switches the entirecolour pattern, with strong dominance between sympatric alleles. Otherbroods (not shown) segregating for the very same Pele and Psil alleleswere sired by the same male or its full brother (Table S3).DOI: 10.1371/journal.pbio.0040303.g002In contrast, mimicry polymorphism affecting yellow,brown/orange, and black colour patterns in H. numata isinherited entirely at a single Mendelian locus, P (Figure 2C).Populations are locally polymorphic, and nine distinctivealleles have been identified for the P locus in a narrowgeographic area of Peru (Figures 1 and 2C) [19,20]. Alleles atthe P locus are nearly all completely dominant, with a linearhierarchy of dominance relationships [19,20], as might beexpected in order to prevent the segregation of intermediateand nonmimetic phenotypes in wild populations. Occasionalrecombinant phenotypes occur, suggesting that the P locusmay be a tight cluster of genes, or ‘‘supergene’’ [19,25].Despite suggestions in the literature that there might begenetic homology between some of these mimicry genes indifferent Heliconius species [16,26,27], such homology has notbeen directly tested. Here we describe the development ofmolecular markers that are tightly linked to a colour patternlocus in H. melpomene; we used these markers to investigatesynteny and homology of colour pattern genes between thethree Heliconius species.ResultsWe demonstrated homology of the genomic location of theP locus in H. numata, the N–Yb–Sb complex in H. melpomene,and the Cr locus in H. erato (Figure 3). A noncoding region(a41), cloned from an amplified fragment length polymorphism marker in a linkage mapping study of H. melpomene, lieswithin 1.1 cM of the H. melpomene pattern locus Yb on linkagegroup 15 (out of a total map length of 1,616 cM) [22] (FigureS1). Among 413 individuals with both genotype and phenotype information from four mapping families, there were justfive individuals recombinant between a41 and Yb (Table S1).This same marker is located within 0.7 cM of the P locus,which controls polymorphism in H. numata, with only tworecombinant individuals identified among 306 individualsderived from six mapping families (Table S2). The probabilityof finding Yb and P so tightly linked to a homologous markerin the two species by chance is p , 0.002 (see Materials andMethods).The primers for the noncoding a41 marker did not amplifya product in H. erato. However, we used a PCR amplicon ofPLoS Biology www.plosbiology.org0003October 2006 Volume 4 Issue 10 e303

Conserved Mimicry Genes in Heliconiussequences of this clone in both H. melpomene and H. numata. Inboth species, these end sequences showed complete linkage toa41 in at least 100 individuals. This clone was then sequencedand annotated by BLAST comparison with nucleotide andprotein sequence databases (see Materials and Methods;Figure 4). In addition to identifying the a41 locus, weidentified nine genes and three retrotransposon-associatedcoding regions (Figure 4).None of the genes identified in the 118-kb BAC clone is acandidate for the Yb locus itself, because recombinants wereidentified between markers derived from the BAC endsequences and Yb in H. melpomene (unpublished data).However, coding sequences were used to design conservedPCR primers for gene-based markers that cross-amplifybroadly across Heliconius. One of these markers, GerTra,amplifies using primers anchored in two putative exons ofthe Rab geranylgeranyl transferase beta subunit (bggt-II) gene andspans an intron showing substantial allelic size variation in H.erato (Figure S3). This region was 14 kb from the a41 markerin H. melpomene (Figure 4), and variation at this locussegregated nearly perfectly with the colour locus Cr in H.erato. Only one recombinant between Cr and GerTra alleleswas identified among 197 individuals from two mappingfamilies of H. erato (Table S2), thus locating GerTra within 0.3cM of the Cr locus (Figure 3; total map length in H. erato wasestimated at 1,430 cM [27,28]). The probability of the H.melpomene gene Yb and H. erato gene Cr being tightly linked tohomologous markers by chance is p , 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 as Yb in H. melpomene(Figure 2). In H. numata, Hm01, Hm08, and RpP40 show aconserved pattern of linkage with H. melpomene both in termsof gene order and estimated distances between loci (Figure 2;eIF3-S9 and RpL22 were not variable in mapped broods of H.numata). The two microsatellite loci unfortunately do notcross-amplify in H. erato, but RpL22 and eIF3-S9 both map tothe linkage group containing the Cr locus (Figure 2). Thesedata reinforce our observation that linkage order is preservedbetween distantly related Heliconius species [27] and suggestthat the chromosomes bearing colour genes P, Yb, and Cr havenot undergone large-scale rearrangement between the threespecies.In addition to genotyping a41 and the markers derivedfrom the BAC sequence, we have genotyped and assigned tolinkage groups a total of 48 codominant molecular markersfrom across the genome, including 12 markers for genesknown to be involved in the development of wings andpatterns in other butterflies or in Drosophila (so-calledcandidate genes) [29–31], and 37 other conserved single-copynuclear genes and microsatellites used as anchor loci incomparative mapping [22,27,28] (see Materials and Methods).We found no conflicting linkage relationship between thethree species on the 16 linkage groups anchored with sharedmarkers (Table 1) out of a total of 21 in each species [22,27],suggesting a widely conserved pattern of synteny at thegenome scale. In H. melpomene, we have also mapped thefollowing: (a) patterning loci B and D, which lie 66.7 cM fromthe gene Cubitus-interruptus on linkage group 18, (b) locus Ac,which is assigned to LG10, and (c) a locus we here term Khw,which lies 10 cM from the gene wingless on linkage group 1(Table 1). Khw controls the white/yellow switch of theFigure 3. Chromosomal Maps for Linkage Group Homologues in H.melpomene (LG15), H. numata (LG15), and H. erato (LG02)Distances are in Haldane centimorgans. The alternative orders for P anda41 relative to Hm01 in H. numata are not significantly different (DLnL ¼ 1.40). Similarly, most orders of N, Sb, Yb, and a41 in H. melpomene arenot significantly different (from DLnL ¼ 0.15 for the order a41–Yb–N–Sb–to DLnL ¼ 0.77 for a41–N–Yb–Sb–). Finally, the two orders for Cr andGerTra in H. erato are also equally significant. Therefore, we here showthe most likely gene orders but cannot exclude that the colour loci are onthe other side of the anchor loci a41, Fox, or GerTra. In contrast, anchorloci order GerTra–RpP40–Hm01–Hm08 is robust, with alternative orderssignificantly worse (DLnL , 2), although the relative placement of RpL22and eIF3-S9 is uncertain in H. melpomene and H. erato (DLnL . 2).DOI: 10.1371/journal.pbio.0040303.g003this marker to probe a whole-genome bacterial artificialchromosomal (BAC) library of H. melpomene. A 118-kb BACclone was identified and its genomic location confirmed bythe following: (a) alignment with sequences of the a41 locusgenerated from H. melpomene genomic DNA and (b) recombination mapping of at least one marker derived from the endPLoS Biology www.plosbiology.org0004October 2006 Volume 4 Issue 10 e303

Conserved Mimicry Genes in HeliconiusFigure 4. Annotation of Clone AEHM-41C10 from the Heliconius melpomene BAC LibraryThe region is situated on LG15 in the H. melpomene genome [22]. The sequence contains open reading frames of strong homology to 12 reportedgenes, three of which appear to be retrotransposon-associated coding regions (dotted boxes). Also highlighted in double frames are the a41 marker,which was used in H. numata and H. melpomene crosses and to isolate the clone from the library, and the Rabgeranylgeranyl transferase gene, used as amarker in H. erato crosses.DOI: 10.1371/journal.pbio.0040303.g004homologous colour pattern loci in closely related species(Figure 1). Our results in H. numata argue strongly against theidea that shared genetic architecture [8,32] constrainsmorphological diversification [7,33]. Instead, the data implythat natural selection has shaped a developmental switchingmechanism capable of responding to a wide variety ofmimetic pressures and producing loc

A Conserved Supergene Locus Controls Colour Pattern Diversity in Heliconius Butterflies Mathieu Joron1,2,3*, Riccardo Papa4, Margarita Beltra n1, Nicola Chamberlain5, Jesu s Mava rez3,6, Simon Baxter1, Moise s Abanto7, Eldredge Bermingham6, Sean J. Humphray8, Jane Rogers8, Helen Beasley8, Karen Barlow8, Richard H. ffrench-Constant5, Ja

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