Differential expression analysis of HLAU genes. (A) Principal component analysis of HLAU genes’ expression in head and larval tissues. Both tissues had HLAU genes’ variance representing the phylogenetic distance between species: D. buzzatii and D. koepferae, compared with the mojavensis cluster corresponds to the PC1 (45%), and 26% between them in PC2. Species from the mojavensis cluster had strong similarity, with D. arizonae slightly separated from D. mojavensis. (B) HLAU genes’ expression in the Opuntia sp. dweller D. m. wrigleyi versus its sister species D. arizonae, which does not have preference between Opuntia sp. and columnar cacti. The left plot represents expression in the head; the right plot, in larval tissue. (C) HLAU expression in head: differential expression analysis of HLAU genes from heads in the Opuntia sp. dweller D. m. wrigleyi versus the three columnar dwellers D. m. mojavensis, and D. buzzatii versus D. koepferae. (D) HLAU expression in larvae: the same pairwise species as in C with Opuntia sp. dweller versus columnar dwellers, but from larvae tissue. (B–D) Filled dots represent HLAU genes with log₂ fold-change > |1| and FDR < 0.05; transparent dots represent nonsignificant differential expression.