Acute and Long-Term Consequences of Co-opted doublesex on the Development of Mimetic Butterfly Color Patterns

Abstract

Novel phenotypes are increasingly recognized to have evolved by co-option of conserved genes into new developmental contexts, yet the process by which co-opted genes modify existing developmental programs remains obscure. Here, we provide insight into this process by characterizing the role of co-opted doublesex in butterfly wing color pattern development. dsx is the master regulator of insect sex differentiation but has been co-opted to control the switch between discrete nonmimetic and mimetic patterns in Papilio alphenor and its relatives through the evolution of novel mimetic alleles. We found dynamic spatial and temporal expression pattern differences between mimetic and nonmimetic butterflies throughout wing development. A mimetic color pattern program is switched on by a pulse of dsx expression in early pupal development that causes acute and long-term differential gene expression, particularly in Wnt and Hedgehog signaling pathways. RNAi suggested opposing, novel roles for these pathways in mimetic pattern development. Importantly, Dsx co-option caused Engrailed, a primary target of Hedgehog signaling, to gain a novel expression domain early in pupal wing development that is propagated through mid-pupal development to specify novel mimetic patterns despite becoming decoupled from Dsx expression itself. Altogether, our findings provide multiple views into how co-opted genes can both cause and elicit changes to conserved networks and pathways to result in development of novel, adaptive phenotypes.

Keywords: co-option; development; evolution; gene regulation; mimicry; polymorphism; sexual dimorphism.

Fig. 1.

Dynamic doublesex expression patterns across Papilio alphenor hindwing development. (A) Papilio alphenor color patterns and dsx genotypes. dsx^H and dsx^h are the mimetic and nonmimetic alleles, respectively. (B) dsx expression in bulk hindwing RNA-seq data in relation to phases of hindwing development (see fig. 2; supplementary fig. S2, Supplementary Material online). (C) Anti-Dsx antibody staining at select stages of hindwing development. Scale bars: 2 mm for full wings, 50 μm in zooms. Replicate stains and additional timepoints can be found in supplementary figure S1, Supplementary Material online. (D) Schematic of select stages of wing development and observed Dsx expression patterns. Scale cells produce the mature scales that comprise the adult wing color pattern; socket cells secure scale cells in the wing. These two cells derive from division of a single precursor cell ∼40% PD. Epidermal cells comprise the bulk of the wing structure but do not contribute to color pattern.

Fig. 2.

Differential expression underlying the mimicry switch. (A) Genes differentially expressed between mimetic and nonmimetic females at each stage, identified using DESeq2 (overall FDR < 0.01). Up/down is mimetic relative to nonmimetic females. (B) Euler diagram of genes with significantly different expression profiles in mimetic dsx^H females relative to the indicated groups, identified using maSigPro (overall FDR < 0.01). (C) Euler diagram of overlap between DESeq2 and maSigPro results. (D) Median expression profiles of three largest clusters for each set in (C). (E) Relationships, DE gene enrichment, and Gene Ontology BP term enrichment of co-expressed gene modules identified using WGCNA (full results in supplementary fig. S5 and table S3, Supplementary Material online). Module numbers are arbitrary. Dashed red line indicates cuts to define metamodules. *Benjamini–Hochberg corrected Fisher Exact Test P value < 0.05.

Fig. 3.

RNAi shows DE genes primarily affect mimetic color pattern development. (A) siRNAs are injected and electroporated into the ventral left hindwing at pupation. Landmarks, orientations, and keys applicable to all panels. (B) dsx RNAi phenotypes in mimetic and nonmimetic females. The area typically affected by RNAi injections is circled in the mimetic female. (C–H) Expression patterns from bulk RNA-seq data and phenotypes in mimetic and nonmimetic females for six target genes. Note that bulk RNA-seq data cannot distinguish between changes in gene expression level and pattern. All images are of the ventral surface. The effect of RNAi should only be judged by the color patterns relative to wing veins between the right wing (wild-type, wt) and left wing (RNAi) of the same individual. Full phenotypes and over 70 additional RNAi individuals, including males of both genotypes, are found in supplementary data set S1, Supplementary Material online.

Fig. 4.

The Doublesex mimicry switch acts through Engrailed. (A) Temporal en expression patterns. (B) en RNAi phenotypes. Red dashed lines indicate the discal cell. (C) inv RNAi phenotypes. (D) En and Dsx antibody staining patterns at 15–20% PD. Dashed lines in zoom images mark the edges of dsx RNAi clones. (E) En and Dsx antibody staining patterns at 40–45% PD. Brightness levels between pairs of zoom images in (D) are comparable because they were taken with the same settings and are not adjusted. Note that En is still present in the pale patch in E3 (left side of the image), just at relatively low levels compared with the adjacent melanic patch. Scale bars: 2 mm for full wings; 25 μm for D1, 2, 5, 6; and 50 μm for remaining for zooms. (F) Schematic of En expression across early- to mid-pupal hindwing development. See additional stains in supplementary figure S6, Supplementary Material online.