(A) A nonsense variant in ACTN3 (rs1815739) is present at a higher frequency (left) in the Kalash than in their neighbors in Pakistan. Forward-time simulations (right) show that such a high frequency of the derived allele in the Kalash (dashed blue line) is only observed in a scenario that considers positive selection acting on the variant. The lower line represents the observed mean frequency of the derived allele in the Pakistani population, the orange lines represent the simulated allele frequency of the derived allele in each replicate in the scenario without selection, and the dark red lines represent each replicate in the scenario with positive selection. The observed frequency of the derived allele in Kalash population is reached only in the scenario with selection and only after 400 generations of drift (∼10,000 or 11,200 years ago if we assume a generation time of 25 or 28 years, respectively), suggesting that the observed pattern for this stop gain on ACTN3 can best be explained by selection acting in ancient times and not by any recent population split.