Convergent evolution in the genetic basis of Müllerian mimicry in heliconius butterflies

Abstract

The neotropical butterflies Heliconius melpomene and H. erato are Müllerian mimics that display the same warningly colored wing patterns in local populations, yet pattern diversity between geographic regions. Linkage mapping has previously shown convergent red wing phenotypes in these species are controlled by loci on homologous chromosomes. Here, AFLP bulk segregant analysis using H. melpomene crosses identified genetic markers tightly linked to two red wing-patterning loci. These markers were used to screen a H. melpomene BAC library and a tile path was assembled spanning one locus completely and part of the second. Concurrently, a similar strategy was used to identify a BAC clone tightly linked to the locus controlling the mimetic red wing phenotypes in H. erato. A methionine rich storage protein (MRSP) gene was identified within this BAC clone, and comparative genetic mapping shows red wing color loci are in homologous regions of the genome of H. erato and H. melpomene. Subtle differences in these convergent phenotypes imply they evolved independently using somewhat different developmental routes, but are nonetheless regulated by the same switch locus. Genetic mapping of MRSP in a third related species, the "tiger" patterned H. numata, has no association with wing patterning and shows no evidence for genomic translocation of wing-patterning loci.

Figure 1.—

Heliconius melpomene and H. erato comimics. D phenotype: Dorsal and ventral wings from H. melpomene ecuadorensis and H. erato etylus, displaying the D phenotype. Specimens were collected near Zamora, Ecuador. The yellow forewing patch is controlled by HeD in H. erato and by a second locus (called N) in H. melpomene. B phenotype: H. melpomene melpomene and H. erato hydara were collected near Cayenne, French Guiana (4°91′ N, 52°36′ W). All individuals are males.

Figure 2.—

HmB and HmD phenotypes are inherited in repulsion. Schematic of LG18 chromosomes from an F₁ cross between the heterozygous Bb/Dd female (dark gray) and male (light gray). Crossing over of sister chromatids does not occur during oogenesis, so F₂ progeny inherit either bD or Bd genotypes from the F₁ female. As all LG18 chromosomal markers are completely linked when inherited from the F₁ mother, molecular markers are used to assign chromosome prints. Recombination between HmD and HmB or Hmd and Hmb may occur in the F₁ male during spermatogenesis. Consequently, phenotypes are only informative for one of the B/b alleles or one of the D/d alleles and are therefore in repulsion. For example in the case of a bD/b- F₂ individual, the F₁ female-derived chromosome contributed b/D and F₁ male-derived chromosome b/-. Here, it is unclear whether one or two HmD alleles are present, as the F₁ female HmD phenotype masks any contribution by the F₁ male. Male informative genotypes from heterozygous B-/D- progeny were deduced using chromosome prints. A B-/D- F₂ heterozygote that inherited a b/D chromosome from the F₁ female must have inherited a dominant HmB allele from the F₁ father to produce the observed phenotype. A hyphen “-” signifies unknown male-derived alleles.

Figure 3.—

Linkage map of H. melpomene LG18 (77.3 cM, log likelihood = −92.61). Three nuclear genes (Ci, RpS30, and Bm44), one microsatellite marker (Hm14), and six AFLP sequence-tagged sites were genetically mapped in 164 F₂ individuals from brood 44. The linkage map was constructed using MapMaker 2.0. Four AFLP markers identified by an asterisk were positioned manually on the basis of AFLP banding patterns for up to 35 individuals. All AFLP markers are within a 6.5-cM region.

Figure 4.—

Analysis of recombinants spanning the HmB/D region on LG18. A H. melpomene BAC library was probed with three AFLP markers (open circles) linked to the HmB and HmD loci. Hm_eACTmGTA195 hybridized with 1 BAC clone, bHM40C14, within contig 1729. Hm_eCCmCTA386 hybridized to 19 clones from contig 196, (containing 32 clones). Hm_eCGmATG213 hybridized to 6 clones from contig 446 (containing 6 clones) plus 5 clones from contig 936 (containing 10 clones). PCR amplification using BAC end primers designed from contig 446 successfully amplified in BAC clones from contig 936, and vice versa, demonstrating these two contigs overlap (supplemental Figure 1). This implies contigs 446 and 936 may represent very divergent haplotypes for the B/D region, as fingerprinting did not group these clones into a single contig. The BAC ends of some clones were sequenced and used to design genotyping assays for the 372 progeny (solid circles). As HmB and HmD are inherited in repulsion, recombinants from BAC ends to these loci are listed independently. Recombinants between contig ends were observed, enabling orientation of the HmB and HmD loci. The H. melpomene BAC library was screened using additional BAC end probes (solid squares), bHM40A21_sp6 (contig 936), bHM28L23_sp6 (contig 1305), and bHM36K2_sp6 (contig 196), which created a single tile path spanning the HmB locus and part of the HmD locus. Finally, a gene near the H. erato HeD locus (MRSP) was used as a probe. For full details of BAC screening results see supplemental Table 1. Accession nos. for BAC end sequences are FI107283–FI107295 and FI136209–FI136223. The figure scale is based on relative sizes of fingerprinted contigs.

Figure 5.—

Linkage maps comparing homologous chromosomes from H. erato, H. melpomene, and H. numata. H. erato linkage map adapted from Kapan et al. (2006), showing HeD in the most likely position (70% probability density). BAC library screening using H. erato AFLP marker He_CCAC491 identified clone BBAM-25K4. Sequence analysis identified a MRSP gene, which was genetically mapped along with Ci in all three species. Two H. melpomene AFLP markers (Hm_eACTmGTA195 and Hm_eCTmCTC156) and one microsatellite (Hm14) cross amplified in H. numata and were genetically mapped to LG18. Numbers shown are centimorgans.