a, Schematic alignment of carbonaria and typica haplotypes showing the position of the three primers (A, B and C, not to scale) used in the same PCR to detect the presence and absence of the 22 kb carb-TE. In the presence of the carb-TE, primers A and C are too far apart to generate a product; the repeat structure of the carb-TE presents three annealing sites for primer B but only the shortest primer B–C combination is amplified when using 45-s extension (primer sequences are listed in Supplementary Table 1). b, carb-TE genotypes for father (lane 2), mother (lane 3) and 15 offspring (lanes 4–18); the two brightest bands in the size ladder are 300 bp and 1 kb (lane 1). The parents were full siblings and known to be heterozygous (c/t), and therefore expected to generate c/c, c/t and t/t offspring. The larger band (primers B–C) indicates the presence of the carb-TE and the smaller band (primers A–C) its absence (typica allele in this family); heterozygotes have both bands. The individual in lane 15 (135F1-12) is the homozygous male used for whole genome sequencing. c, Presence or absence of the carb-TE in a carbonaria haplotype fosmid clone (lane 2), three different typica haplotype clones (lanes 3–5; one fosmid, two BACs), wild carbonaria homozygotes (lanes 6 and 7), wild carbonaria heterozygotes (lanes 8–10), typica with a flanking haplotype similar to the carbonaria haplotype but lacking the carb-TE (lanes 11–13), light insularia (lanes 14–16), intermediate insularia (lanes 17–19), dark insularia (lanes 20–22) and carbonaria-like insularia (lanes 23–25).