Fly collection, DNA isolation and sequencing
We collected wild Drosophila at the four mountainous locations across Arizona between the 22nd of August and the 11th of September 2017: the Southwest research station in the Chiricahua mountains (CH, ~5,400 feet elevation, 31.871 latitude -109.237 longitude, 96 flies), in Prescott National Forest (PR, ~7,900 feet elevation, 34.586 latitude -112.559 longitude, 96 flies), Madera Canyon in the Santa Rita mountains (SR, ~4,900 feet elevation, 31.729 latitude -110.881 longitude, 96 flies) and Miller Peak in the Huachuca mountains (HU, ~5,900 feet elevation, 31.632 latitude -110.340 longitude, 53 flies) (Coe et al. 2012). Baits consisted of store-bought white button mushrooms (Agaricus bisporus ) placed in large piles about 30cm in diameter, with at least 5 baits per location. We used a sweep net to collect flies over the baits in either the early morning or late afternoon between one and three days after the bait was set. We sorted flies by sex and species at the University of Arizona in Tucson, AZ and flash frozen at -80°C before shipping on dry ice to the University of Kansas in Lawrence KS.
We sorted 343 flies (172 females and 171 males) which phenotypically matched D. innubila. We then homogenized and extracted DNA using the Qiagen Gentra Puregene Tissue kit (USA Qiagen Inc., Germantown, MD, USA). We also prepared the DNA of 40 D. innubila collected in 2001 from CH. We prepared a genomic DNA library of these 383 DNA samples using a modified version of the Nextera DNA library prep kit (~ 350bp insert size) meant to conserve reagents. We sequenced the libraries on four lanes of an Illumina HiSeq 4000 (150bp paired end) (Supplementary Table 1, Data to be deposited in the NCBI SRA).