Conceptual illustration of the ‘during-winter’ and ‘after-winter’ hypotheses for apple versus hawthorn fly race divergence in gene expression and the experimental design. (A,B) Transcripts regulating earlier diapause termination in apple relative to hawthorn flies could be differentially regulated between populations during winter (‘during-winter’ hypothesis; A), or only after exposure to warmer temperatures necessary to begin post-diapause development (‘after-winter’ hypothesis; B). Green and pink represent up- and down-regulation of transcripts in apple relative to hawthorn flies and not absolute values, so the key feature is when the shifts in transcription levels occur between the host races, i.e. the fork in up- and down-regulation can be seen prior to 0 h in the during-winter hypothesis and extends afterward, whereas it is initiated only after the 0 h time point in the after-winter hypothesis. Note that both positive and negative values of this relative measure may reflect up- or down-regulation of transcripts in one population compared with the other. Solid and dashed lines in the upper panel denote accelerating and constant differences between populations following the temperature shift. (C) Experimental design: upon pupariation, apple and haw fly pupae were exposed to common, simulated overwinter conditions, then sampled for RNAseq at three time points – one at the end of winter (0 h), one 24 h after the end of winter, and one 48 h after transfer to warmer conditions.