primer pair is used, only the 39-half of Sd-RanGAP is amplified. We used Exo-SAP to clean PCR products and then sequenced both strands of the PCR products using internal sequencing primers (Table S1), BigDye Terminator chemistry, and standard cycle sequencing protocols. All sequences were manually edited using Sequencher v. 4.5 (Gene Codes). We obtained outgroup sequences via BLAST searches of the D. simulans genome [62]. Sequencing Non-Coding Regions on Chromosome 2 In addition to Sd-RanGAP, we sequenced the parent gene and eight non-coding regions across chromosome 2 from a collection of SD chromosomes and from 10 wildtype chromosomes from Zimbabwe. As many SD chromosomes, and some wildtype ones, are homozygous lethal (see RESULTS), we could not make homozygous lines for sequencing for all stocks. Instead, for homozygous lethal lines, we used deficiencies to produce flies hemizygous for the focal chromosomal regions. The eight regions ranged in size from 567–874 bp long (Table 3). 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