PMC

Pairwise genetic divergence among P. xuthus, P. machaon and P. polytes was calculated in 50-kb windows. This yielded 70 highly divergent genomic regions, windows in the upper 95th percentile in all three comparisons, encompassing 915 genes. When overlaid with signatures of positive selection and differential expression throughout development, 11 genes (highlighted in yellow) emerged as strong candidates for a role in recent diversification, including frizzled. Degree of freedom (df) is the density of different single-nucleotide polymorphisms (SNPs) per bp of each 50-kb window.

(a) Phylogeny of butterfly families showing relationship of P. xuthus and P. machaon to D. plexippus, H. melpomene, M. cinxia, P. glaucus and P. polytes. (b) Chromosome mapping of P. xuthus (n=30) to B. mori (n=28). For P. machaon (n=31), we only plot chromosome 8 (chr8) and chr31, which were fused in P. xuthus. (c) Maximum-likelihood tree showing strong expansion of the scIPPS genes in the genomes of swallowtail butterflies. Included are all scIPPS genes identified in the genomes of ten holometabolous insects (P. xuthus, P. machaon, D. plexippus, H. melpomene, B. mori, P. xylostella, A. gambiae, D. melanogaster, T. castaneum and A. mellifera). The clades of P. xuthus and P. machaon are highlighted by light blue and deep blue, respectively. (d) Maximum-likelihood tree showing expansions of CYP6B genes in the genomes of P. xuthus and P. machaon, and of the CYP6AB genes in the genome of P. machaon, as compared with CYP6 genes of D. plexippus, H. melpomene, B. mori and D. melanogaster. In c,d, the clades of P. xuthus and P. machaon are highlighted by light blue and deep blue, respectively. (e,f) Expression profiles of FPPS and GGPPS genes at all development stage of P. xuthus (e) and P. machaon (f). (g,h) Expression profiles of CYP6 genes at all development stage of P. xuthus (g) and P. machaon (h). Expression measured in reads per kilobase of transcript per million reads mapped (RPKM).

(a–d) Mutations induced by the injection of Cas9 mRNA and Abdominal-B sgRNA with prolegs on segment A7–A10 (a), segments that do not normally have prolegs (c), or with a curled abdomen resulting from abnormal terga on segments A3 and after (b), compared with wild type (d). Green dots in a and c show normal prolegs of A6 and A10, while red stars in a denote the redundant prolegs on A7–A10. (e–j) Mutations induced by the injection of Cas9 mRNA and ebony sgRNA with enhanced melanic pigmentation (e) and an absence of orange colour in the false eyespot (f) in fifth instar larvae, compared with wild type (h,i), and with brown pigmentation (g) across the body in regions that were normally yellow and in wing patches that were normally orange in adult (j). (k–n) A mutant induced by the injection of Cas9 mRNA and frizzled sgRNA with smooth and colourless dorsal cuticle in right side (k), while normal in its left side (m), and its dorsal view (l) compared with wild type (n). Scale bars, 1 mm (a–d,f,i,k–n) and 10 mm (e,g,h,j).