Chaetoceros simplex cultures, were obtained from population strain CCMP 200 (National Center for Marine Algae and Microbiota, NCMA).

Thermal performance curve (TPC) assays:
We assayed the TPCs of our populations twice during the evolution experiment. This involved pre‐acclimating sub‐cultures from each population to 28 °C (in‐between the 25 °C control and 31 °C experimental treatment) in N‐replete medium for 20 days (20–25 generations) to remove any effects of acclimation to previous temperatures (31 or 25 °C) and N levels. Subsequently, separate flasks containing N‐replete medium were placed at each assay temperature, inoculated with pre‐acclimated populations, and allowed to acclimate for six more days.

After ~ 200 generations of evolution (165–186 days: 194–232 generations), we characterised the TPCs of two randomly selected N‐replete evolved populations that were 34 °C‐tolerant (see the 34 °C challenge section below) and the control and an ancestral population. Assay temperatures were 10, 20, 25, 29, 31, 32, 34 and 35 °C, and three replicate populations were grown in 50‐mL culture flasks (instead of well plates) (96 growth rate estimates). We measured in vivo chlorophyll‐a fluorescence (excitation wavelength: 436 nm, emission wavelength: 680 nm) daily using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA) to estimate the growth rate.

Growth rate calculations:
From the daily biomass estimations (in vivo chlorophyll‐a fluorescence ), we calculated population growth rates (day−1), as the slope of the linear regression of ln(biomass) vs. time (days).
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Also see data for 100 generations: https://www.bco-dmo.org/dataset/778749
More details in Aranguren-Gassis et al. 2019, Ecology Letters.