A genetic switch for male UV iridescence in an incipient species pair of sulphur butterflies

Abstract

Mating cues evolve rapidly and can contribute to species formation and maintenance. However, little is known about how sexual signals diverge and how this variation integrates with other barrier loci to shape the genomic landscape of reproductive isolation. Here, we elucidate the genetic basis of ultraviolet (UV) iridescence, a courtship signal that differentiates the males of Colias eurytheme butterflies from a sister species, allowing females to avoid costly heterospecific matings. Anthropogenic range expansion of the two incipient species established a large zone of secondary contact across the eastern United States with strong signatures of genomic admixtures spanning all autosomes. In contrast, Z chromosomes are highly differentiated between the two species, supporting a disproportionate role of sex chromosomes in speciation known as the large-X (or large-Z) effect. Within this chromosome-wide reproductive barrier, linkage mapping indicates that cis-regulatory variation of bric a brac (bab) underlies the male UV-iridescence polymorphism between the two species. Bab is expressed in all non-UV scales, and butterflies of either species or sex acquire widespread ectopic iridescence following its CRISPR knockout, demonstrating that Bab functions as a suppressor of UV-scale differentiation that potentiates mating cue divergence. These results highlight how a genetic switch can regulate a premating signal and integrate with other reproductive barriers during intermediate phases of speciation.

Keywords: UV iridescence; evo-devo; genetic coupling; large-Z effect; speciation.

Fig. 1.

Large-Z architecture of species differentiation includes the U-locus candidate gene bab. (A) UV iridescence differentiates males from two incipient species. (B and B′) PCA (B) and distance-based phylogenetic network (B′) of 22 male whole-genome SNPs from the admixed Maryland population. (C and D) F_ST values for C. philodice vs. C. eurytheme plotted against recombination rate (C), and Manhattan plot (D). Red indicates windows with above-median recombination rate and in the 95^th percentile of F_ST, including on the Z chromosome (asterisk). (E) Quantitative trait locus (QTL) analysis of the presence/absence of UV in 252 male offspring from F₂ and BCs. (F) Genotype plot for the whole Z chromosome with resequencing data from 23 individuals. Each row is an individual, and each column is a color-coded SNP. The red bracket indicates a 2.5-Mb interval with high F_ST and above-median recombination rate. (G) Annotation of the U-locus zero-recombinant interval (box) and surrounding region.

Fig. 2.

Bab negatively correlates with UV-scale precursors in C. eurytheme male wings. (A) Pseudocolored SEM images highlighting the ultrastructural differentiation of the UV-iridescent dorsal cover scales (dcs; magenta), relative to non-UV ground scales (gs; orange) and non-UV ventral cover scales (vcs; green). (B) Microphotographs of adult C. eurytheme male wing surfaces in the visible and UV ranges. Line: damaged areas exposing non-UV ground scales. (C) Immunofluorescence detection of Bab (green) in all UV precursors at 46% pupal development. Magenta: DAPI (nuclei); orange: Dve; circles: cover-scale nuclei. (Scale bars, 2 μm [A], 100 μm [B], and 10 μm [C].)

Fig. 3.

Bab represses UV-scale fate. (A) G₀ phenotypes resulting from CRISPR mosaic KOs (mKO) targeting the first exon of bab. Gain of UV iridescence was observed across both species and both sexes, including ventral wing surfaces. (B and C) Magnified views of male C. philodice wings featuring extensive ectopic iridescence following bab mKO. WT, wild type. (D and D′) False-colored SEM views of a mosaic region (box in C), with complete transformation of wild-type cover (cs; orange) and ground scales (green) into UV-iridescent scales with dense longitudinal ridges. Arrowheads indicate examples of ground scales in a wild-type state (white) and in a bab mutant clone. (E and E′) Correlative light and electron microscopy images featuring a superimposed view of UV-reflected light (magenta) on an SEM view. Both ground and cover scales from bab mutant clones are UV-iridescent. (Scale bars, 10 μm.)