Diversification of complex butterfly wing patterns by repeated regulatory evolution of a Wnt ligand

Abstract

Although animals display a rich variety of shapes and patterns, the genetic changes that explain how complex forms arise are still unclear. Here we take advantage of the extensive diversity of Heliconius butterflies to identify a gene that causes adaptive variation of black wing patterns within and between species. Linkage mapping in two species groups, gene-expression analysis in seven species, and pharmacological treatments all indicate that cis-regulatory evolution of the WntA ligand underpins discrete changes in color pattern features across the Heliconius genus. These results illustrate how the direct modulation of morphogen sources can generate a wide array of unique morphologies, thus providing a link between natural genetic variation, pattern formation, and adaptation.

Fig. 1.

Linkage mapping of forewing pattern shape variation to WntA. (A) Examples of forewing pattern shapes found in Heliconius shown with their associated Sd and Ac genotypes. Mapped Ac polymorphisms in the H. melpomene/H. cydno clade consist of presence or absence of a proximal melanic patch (arrows). (B) Linkage-mapping of Sd and Ac. Crossing-over events between genetic variation and individual phenotypes out of N individuals are featured at each marker. The depicted region spans the halves of two scaffolds separated by an assembly gap; however, the reference H. melpomene genetic map (27) and the colinearity of the Impact-Dpr1 syntenic block with the silkworm genome assembly (46) provide independent evidence of contiguity between these two scaffolds (SI Appendix, Fig. S2).

Fig. 2.

Variable expression of WntA explains pattern shape diversity in Heliconius. (A) Larval wing disk in situ hybridizations showing WntA expression in presumptive black territories, and delineating pattern boundaries. Vein landmarks define homologous positions between larval and adult wings. In the intermediate panels, dashed lines corresponding to the presumptive position of the light-color patterns (hashed on adult wings) were added to facilitate the comparison of larval and adult wing topologies. (B) WntA marks presumptive optix-negative territories in H. melpomene plesseni. Expression of optix was reproduced from ref. . (C) Heparin injections result in dose-dependent expansions of black patterns that phenocopy the effects of WntA heterozygosity (D, dashed lines). The absence of effect on basal iridescent (H. sara sara) and red (H. erato erato and H. erato lativitta) patterns rules out a generic effect of heparin on wing scale phenotypes. H. erato lativitta resembles H. erato etylus (used in mapping crosses) and both originate from Eastern Ecuador low-lands.

Fig. 3.

Summary of phylogenetic replication for linkage mapping of forewing pattern variation (Sd and Ac loci), WntA in situ hybridizations, and heparin injections presented in this study. Multiple lines of evidence suggest that forewing pattern shapes are adaptive across the genus (, , , –53). NA, not applicable; NPC, narrow phenotypic cline; MüMi: Müllerian mimicry; —, not assessed because of stock limitations or unavailability. Note that H. erato lativitta/H. melpomene malleti, and H. erato notabilis/H. melpomene plesseni are convergent comimics that occur in Eastern Ecuador low- and highlands, respectively (dashed lines). Phylogenetic relationships between species are derived from a recent molecular phylogeny (47) after retaining nodes with bootstrap support ≥0.98. Estimated age of Heliconius radiation is derived from a recent molecular clock study (48). Only a subset of Heliconius wing pattern diversity is represented here.