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 of Heliconius 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-mimics H. erato and H. 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 three Heliconius species that represent examples of both mimetic convergence and colour pattern diversification. H. melpomene and H. 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 to H. melpomene but has extremely divergent wing 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 of a different species of Melinaea (Nymphalidae: Ithomiinae); polymorphism in H. numata is thought to be maintained by strong selection for mimicry in a fine-scale spatial mosaic of ithomiine communities [19,20]. Figure 1 Colour Pattern Diversity of H. numata, H. melpomene, and their Respective Co-Mimics The 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 the figure 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, and co-mimics are known as postman forms. H. melpomene and H. erato are from divergent clades of Heliconius and are identified in the field using minor morphological 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 the arrangement of red versus white patches in H. m. plesseni and H. e. notabilis (first from right). Co-mimics H. numata and Melinaea 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). The differences in colour pattern between races of H. melpomene and H. erato are controlled by several Mendelian factors of large phenotypic effect [15,17]. In H. melpomene, a complex of at least three tightly linked loci (N, Yb, and Sb) control most of the variation in yellow and white pattern elements (Figures 1 and 2A), and recombination between these loci suggests that they lie just a few centimorgans (cM) apart [15,17,21]. Another pair of loci (B and D), situated on a different linkage group, controls most of the variation in the red pattern elements and interacts with N to control the colour of the forewing band [15,17] (Figure 1). Locus Ac controls the presence of a yellow patch in the discal cell of the forewing in some crosses [22]. Finally, locus K, unlinked to N–Yb–Sb or B–D, turns white patches to yellow in crosses between H. melpomene and H. cydno [21,23] (Table S1). Figure 2 Crosses Used for Mapping the Yb, P, and Cr Loci (A) Crossing scheme in H. melpomene showing segregation of tightly linked loci Yb and Sb (hindwing yellow bar and white margin, present in H. m. cythera, YbcYbc SbcSbc ) 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 linked N 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 in H. erato showing segregation of Cr alleles in brood CH-CH5; Cr controls the forewing yellow band (absent in H. e. cyrbia, CrcCrc ), and the hindwing yellow bar and white margin (present in H. e. cyrbia). The red-patterning gene D also segregates in this cross, but is unlinked to Cr; only progeny with a DhiDhi 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 in H. numata showing segregation of the P 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 same Pele and Psil alleles were sired by the same male or its full brother (Table S3). The radiation in H. erato has a similar genetic architecture, with a locus Cr that has similar phenotypic effects to the combined action of N, Yb, and Sb in H. melpomene. In crosses between H. 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 of H. himera (cf. N) [24] (Figure 2B). Nonetheless, there are differences between the species: in inter-racial H. erato crosses the forewing yellow band is controlled by an unlinked locus, D, rather than by Cr [17]. D also controls most of the variation in the red pattern elements in a way that is analogous to the B–D complex in H. melpomene. In contrast, mimicry polymorphism affecting yellow, brown/orange, and black colour patterns in H. numata is inherited entirely at a single Mendelian locus, P (Figure 2C). Populations are locally polymorphic, and nine distinctive alleles have been identified for the P locus in a narrow geographic area of Peru (Figures 1 and 2C) [19,20]. Alleles at the P 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 the P 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 different Heliconius 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 in H. melpomene; we used these markers to investigate synteny and homology of colour pattern genes between the three Heliconius species. Results We demonstrated homology of the genomic location of the P 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, lies within 1.1 cM of the H. melpomene pattern locus Yb 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 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 two recombinant individuals identified among 306 individuals derived from six mapping families (Table S2). The probability of finding Yb and P so tightly linked to a homologous marker in the two species by chance is p −2). The primers for the noncoding a41 marker did not amplify a product in H. erato. However, we used a PCR amplicon of this marker to probe a whole-genome bacterial artificial chromosomal (BAC) library of H. melpomene. A 118-kb BAC clone was identified and its genomic location confirmed by the following: (a) alignment with sequences of the a41 locus generated from H. melpomene genomic DNA and (b) recombination mapping of at least one marker derived from the end sequences of this clone in both H. melpomene and H. numata. In both species, these end sequences showed complete linkage to a41 in at least 100 individuals. This clone was then sequenced and annotated by BLAST comparison with nucleotide and protein sequence databases (see Materials and Methods; Figure 4). In addition to identifying the a41 locus, we identified nine genes and three retrotransposon-associated coding regions (Figure 4). Figure 4 Annotation of Clone AEHM-41C10 from the Heliconius melpomene BAC Library The region is situated on LG15 in the H. 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 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 a marker in H. erato crosses. None of the genes identified in the 118-kb BAC clone is a candidate for the Yb locus itself, because recombinants were identified between markers derived from the BAC end sequences and Yb in H. melpomene (unpublished data). However, coding sequences were used to design conserved PCR primers for gene-based markers that cross-amplify broadly across Heliconius. One of these markers, GerTra, amplifies using primers anchored in two putative exons of the Rab geranylgeranyl transferase beta subunit (βggt-II) gene and spans an intron showing substantial allelic size variation in H. erato (Figure S3). This region was 14 kb from the a41 marker in H. melpomene (Figure 4), and variation at this locus segregated nearly perfectly with the colour locus Cr in H. erato. Only one recombinant between Cr and GerTra alleles was identified among 197 individuals from two mapping families of H. erato (Table S2), thus locating GerTra within 0.3 cM of the Cr locus (Figure 3; total map length in H. erato was estimated at 1,430 cM [27,28]). The probability of the H. melpomene gene Yb and H. erato gene Cr being tightly linked to homologous markers by chance is p 84) confirms that the fragments represent orthologous markers in both species. The large insertions and deletions in the middle of the sequence allowed easy genotyping (Beltrán M, Mavárez J, González M, Bermingham E, Jiggins C, unpublished data). (30 KB DOC) Click here for additional data file. Figure S2 Alignment of a H. melpomene a41 Sequence with BAC Clone AEHM-41C10 The marker corresponds to positions 5,829–6,170 on the BAC sequence. (27 KB DOC) Click here for additional data file. Figure S3 Alignment of H. erato GerTra Sequences with H. melpomene BAC Clone AEHM-41C10 Because the PCR amplicons in H. erato are too large for complete sequencing, we provide here the alignment of both end sequences with the respective H. melpomene Rab geranylgeranyl transferase exons from which the primers were designed. Exon 1 lies at position 19,970:20,290 and exon 2 at 21,535:21,846, with a 1,245-bp intron in between. (38 KB DOC) Click here for additional data file. Table S1 Mapping Families and Colour Pattern Genotypes in H. melpomene Details of the H. m. cythera × H. m. melpomene F2 crosses segregating for Ybc/Yb, Sbc/Sb, and Khww/Khw, and H. m. malleti × H. m. melpomene F2 crosses segregating for NN/NB, B/b, and D/d (full pedigree information available upon request; codes in brackets identify the brood from which each parent originates). Khw is only expressed in a Sbc/Sbc background. See Figure 2A for details of wing patterns. (98 KB DOC) Click here for additional data file. Table S2 Mapping Families and Colour Pattern Genotypes in H. erato Details of the F2 cross (CH-CH5) and the backcross (CH-Cy6) of H. e. cyrbia × H. himera segregating for Crcyr/Crhim . Cr alleles do not segregate in the NOTF2–9 reference F2 cross H. e. notabilis × H. himera, which was used to map gene markers GerTra and RpL22. Segregation at unlinked colour pattern loci D and Sd is given for reference. See Figure 2B for details of wing patterns. (45 KB DOC) Click here for additional data file. Table S3 Mapping Families and Colour Pattern Genotypes in H. numata Coloured superscript numbers identify chromosomes identical by descent in different broods (full pedigree information available upon request; codes in brackets give the brood from which each parent originates). See Figure 2C for details of wing patterns. (70 KB DOC) Click here for additional data file. Accession Numbers The Genbank (http://www.ncbi.nlm.nih.gov) accession number for the H. melpomene BAC clone AEHM-41C10 is CR974474.
