A long noncoding RNA at the cortex locus controls adaptive coloration in butterflies

Abstract

Evolutionary variation in the wing pigmentation of butterflies and moths offers striking examples of adaptation by crypsis and mimicry. The cortex locus has been independently mapped as the locus controlling color polymorphisms in 15 lepidopteran species, suggesting that it acts as a genomic hotspot for the diversification of wing patterns, but functional validation through protein-coding knockouts has proven difficult to obtain. Our study unveils the role of a long noncoding RNA (lncRNA) which we name ivory, transcribed from the cortex locus, in modulating color patterning in butterflies. Strikingly, ivory expression prefigures most melanic patterns during pupal development, suggesting an early developmental role in specifying scale identity. To test this, we generated CRISPR mosaic knock-outs in five nymphalid butterfly species and show that ivory mutagenesis yields transformations of dark pigmented scales into white or light-colored scales. Genotyping of Vanessa cardui germline mutants associates these phenotypes to small on-target deletions at the conserved first exon of ivory. In contrast, cortex germline mutant butterflies with confirmed null alleles lack any wing phenotype and exclude a color patterning role for this adjacent gene. Overall, these results show that a lncRNA gene acts as a master switch of color pattern specification and played key roles in the adaptive diversification of wing patterns in butterflies.

Keywords: butterfly; evo-devo; lncRNA; pigmentation.

Fig. 1.

Expression and CRISPR mKOs of the ivory lncRNA reveal color patterning roles. (A) Examples of H. melpomene and H. erato morphs exhibiting color pattern polymorphisms associated with the cortex locus, alongside representative phenotypes of the ivory^Δ78k deletion (17). (B) Summary of the association intervals previously mapped at the cortex locus for H. melpomene and H. erato (7, 15, 17, 30, 31). For H. melpomene: Yb(d) dorsal yellow bar (Hmel215003o: 1,403,500 to 1,405,500), Yb(v) ventral yellow bar (1,429,500 to 1,430,500), and ivory^Δ78k deletion (1,494,903 to 1,573,176). For H. erato: Cr1 Peruvian (West of Andes) yellow bar (Hera1505: 2,053,037 to 2,171,230), Cr2 Panamanian (East of Andes) yellow bar (2,211,881 to 2,315,926), and FBs forewing base spot (2110,000 to 2113,800). Current mapping intervals for H. melpomene loci N, Sb, and Vf, and the H. erato Cr locus are too large to indicate on the figure (–36). Combined RNA-seq read junction events are shown for both ivory (red, positive strand) and cortex (blue, negative strand), with annotations for the adjacent miRNAs193, 2788 (37) and for the gene parn. ATAC-seq tracks from normalized average read depth from 36 h pupal hindwings are shown in green (38). Gray lines indicate aligned regions >250 bp between H. melpomene and H. erato.

Fig. 2.

Expression of ivory associates with melanic scales in nymphalid butterflies. (A–C) Expression of ivory in 30% pupal wing tissue of H. erato prefigures adult melanic scales, and mKO results in a complete loss of black scales. Unbanded morphs of H. erato express ivory in the presumptive yellow bar region (white arrowheads). Expression is absent in presumptive red (D–D”) and yellow (E–E”) regions in the banded morph. (F–J”) Expression and function are conserved in H. charithonia, strongly marking future black scales, resulting in yellow/white states in the mKOs. (K–M) Function is conserved outside Heliconius, with V. cardui displaying similar loss of melanic scales in the mKOs and a strong correlation between ivory expression and adult black patterns. (N–N”) Expression of ivory is absent from ventral forewing orange-red ommochrome scales, as well as from (O–O”) white margin patterns and yellow eyespot rings.

Fig. 3.

Phenotypes are explained by ivory-specific mutations. (A–C) Loss of melanic scales in ivory germline mutants. (D) Radiolabeling of Tyrosine highlighting melanin containing scales and (E) Tryptophan radiolabeling revealing the distribution of ommochrome-containing patterns. Ommochromes are not affected by ivory KOs in V. cardui (arrowheads). (F) Strong ivory phenotype shown in (B) is explained by a compound heterozygous deletion of 82/97 bp at its first exon. (G and H) Exon 2 G₀ cortex crispants display no wing phenotypes, but a large fraction of cells carrying mutations at the expected cut site. These crispants were crossed to produce F₁ offspring (I and I’), again displaying no wing phenotypes. (J and K) Genotyped alleles recovered in the F₁ lead to premature protein truncation due to an 8 nt and 10 nt deletion at cortex exon 2. Panel J, yellow: predicted Beta-sheet structures; magenta: predicted alpha-helices; orange: frame-shifted amino acid residues. Panel K, blue: sgRNA sequence; red: protospacer adjacent motif; dotted line: predicted CRISPR cutting site.

Fig. 4.

Conserved and pattern-specific effects of ivory mKOs on nymphalid butterflies. Wing pattern effects of ivory perturbation in representative A. incarnata and D. plexippus G₀ crispants. (A–E”) Ventral wing views of A. incarnata comparing wild-type to full (nonmosaic) ivory crispant and a clonal ivory crispant (i.e., with fragmented mosaic of WT and mutant clones). Phenotypes include melanic (black/brown)-to-silver transformations and lighter orange (dashed lines). Insets feature magnified views of the Cu₁–Cu₂ silver spots in forewings (D and D”), and hindwings (E and E”). (F–J”) Dorsal views, featuring ivory crispant clones with lightened orange and black coloration (I and I”, Discalis II spots), and melanic-to-white conversions (J and J”, marginal M₃-Cu₂ region). (K–O”) Ventral wing views of D. plexippus comparing wild-type to full and clonal ivory crispants. Insets feature the forewing M₁-M₃ white spot region (N and N”) and the hindwing discal crossvein (O and O”), with conversions of both melanic and orange to white states, as well as a residual orange coloration in some of the affected areas. (P–T”) Dorsal views, with insets featuring the forewing Cu₁–Cu₂ vein junction (S and S”) and the hindwing M₁–M₂ vein junctions (T and T”).