Urbanization and its urban‐heat‐island effect (UHI) have expanding footprints worldwide. The UHI means that urban habitats experience a higher mean and more frequent extreme high temperatures than rural habitats, impacting the ontogeny and resilience of urban biodiversity. However, many organisms occupy different microhabitats during different life stages and thus may experience the UHI differently across their development. While evolutionary changes in heat tolerance in line with the UHI have been demonstrated, it is unknown whether such evolutionary responses can vary across development. Here, using common‐garden‐reared Chiasmia clathrata moths from urban and rural populations from three European countries, we tested for urban evolution of heat shock tolerance in two life stages: larvae and adults. Our results indicate widespread urban evolution of increased heat tolerance in the adult stage only, suggesting that the UHI may be a stronger selective agent in adults. We also found that the difference in heat tolerance between urban and rural populations was similar to the difference between Mid‐ and North‐European regions, suggesting similarity between adaptation to the UHI and natural, latitudinal temperature variation. Our observations incentivize further research to quantify the impact of these UHI adaptations on fitness during urbanization and climate change, and to check whether life‐stage‐specific adaptations in heat tolerance are typical of other ectothermic species that manage to survive in urbanized settings.

From a total of 50 different F2 and F3 full-sib moth families, 688 individuals were tested. Of these, 82 were tested at the larval stage only, 81 were tested both as larvae and adults, and 525 individuals were tested at the adult stage only, which resulted overall in 769 heat knock-down measurements. All larvae from the subset of individuals assessed at the larval stage (N=163) were tested two days after entering the final (fifth) larval instar. For increased precision, each individual was tested separately. Each of them was weighed (Precisa 202A; precision: 0.1 mg) immediately prior to the heat knock-down time (HKDT) measurement. After weighing, each individual was placed in a plastic, conical centrifuge tube (Falcon, 50 ml) without lid. This tube was then fully immersed, except for the top of the tube, in a programmable water bath (Grant Instruments Inc., Cambridge, UK: GD100) kept at 50.0°C for the duration of the experiment. The tube was held in place vertically by manually pressing it down to the bottom. The HKDT was recorded with a timer as the time in seconds from tube insertion into the bath until the main part of the larval body (i.e., the abdomen) stopped moving for a full 20 seconds (these 20 seconds were not included into the HKDT). During the heat exposure, larvae initially either remained curled on the bottom for some seconds or immediately started crawling around, but all showed energetic crawling movements for some time before typically and suddenly being knocked down by falling motionless on their back. Immediately afterwards, larvae were placed back in their individual rearing cups and climate room, with identical rearing conditions for tested and non-tested larvae. Tested larvae that successfully completed pupation were retested as adults (N=81; i.e., 50% survival).

Adults (N=606) followed a similar test procedure, except that they were not weighed, and that after placing them in the test tube now with cotton pushed into the conical tip to form a flat surface for the adult to stand on a transparent lid covered the tube to prevent escape. HKDT was recorded as the time in seconds from tube insertion into the bath until the individual fell on its side, without being able to recover a stable position for at least 20 s (small, trembling movements of appendages were allowed). After insertion in the tube, individuals typically waited around 20 to 50 s before walking and flying around until knock-down.