Experimental design

Four experiments were set up in this study (Supplementary Figure S2). Firstly, to assess the effect of elevated pCO₂ on the physiological performance of T. pseudonana, a selection experiment was set up in laboratory, where algae were cultured under ambient and elevated pCO₂ for 720 days. Secondly, to assess the evolutionary response of T. pseudonana to elevated pCO₂, shift experiments were set up in the laboratory, where the selected lineages at ambient and elevated pCO₂ were transferred into five different concentrations of pCO₂. Thirdly, to compare the differences in response between the indoor and outdoor experiments, an outdoor culture system was set up, where T. pseudonana was cultured under ambient and elevated pCO₂ using natural temperature and light. Fourthly, we conducted transcriptome sequencing of T. pseudonana using the long-term selected population (after 720 days’ selection in laboratory), to gain a mechanistic understanding of how elevated pCO₂ influences copper metabolism of T. pseudonana. For all four experiments, algae were acclimated under ambient or elevated pCO₂ for more than 30 generations, and then exposed to Cu without acclimation. In all experiments, the respective pCO₂ levels of 420, 1,000, and 2,000 μatm were established by bubbling the liquid medium with air or air/CO₂ premixed gas using a CO₂ chamber (HP1000G-D, China, for the laboratory experiment) or CO₂ Enricher (CE-100B; Wuhan Ruihua Instrument & 25 Equipment Ltd., for the outdoor culture system).