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Fig. S1 Thoracic segments of some inbred strains from Iriomote reared at 25 and 20 °C.

Fig. S2 Polymorphism table of nucleotide sites in the 0.9-kb ECR.

Fig. S3 Polymorphism table of indels in the 0.9-kb ECR.

Fig. S4 Nucleotide differences in the 0.9-kb ECR of TW1(G23) and Mel6(G59).

Fig. S5 Indels in the 0.9-kb ECR of TW1(G23) and Mel6(G59).

Fig. S6 Enhancer activity of the e_ECR0.9Light.

Fig. S7 Polymorphism status of the 5 nucleotide sites that have caused expression level changes of ebony in abdomens of African flies.

Fig. S8 Sliding window analyses of the nucleotide diversity (π) among standard (n = 5) and In(3R)Payne (n = 8) sequences in the ebony gene region.

Table S1 RT-qPCR quantities of ebony in each strain for Fig. 1C

Table S2 Pyrosequencing results for Table 2

Table S3 Pyrosequencing results for Fig. 5

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