<p>Outline of the hybridize, evolve, and re-sequence (HER) experiment illustrated for one strain pair (the experiment included two strain pairs, i.e., six heat-selection and six control lines altogether).</p>

The resulting F₁ females were divided into replicates and backcrossed with <i>D. flavomontana</i> males (replicate numbers shown in the upper left corner of the boxes). In the first backcross (BC₁) generation, replicate lines were split into heat-selection and control treatments.

Heat selection began with the BC₁ generation, where flies were subjected to 4 hours at 31 °C. Hybrids of the next two generations (BC₁F₂ and BC₁F₃) were exposed to 31.5 and 32 °C for 4 hours, respectively. The controls were not exposed to heat but were otherwise handled identically. In the BC₁F₄ generation, the heat-selection and control lines were sequenced and phenotyped.