Strain isolation
Sediment samples were collected using a Gemax corer and processed as described in the Supplemental Material and Methods. We have previously shown that S. marinoi is the most abundant resting stage species in the sediments, with up to a million viable cells per gram sediment (Andersson, Rengefors, et al., 2023). In this study, individual strains of S. marinoi were germinated and isolated from resting stages in the sediment one to one-and-a-half years after sampling using the sediment immediately below the surface (1-2 cm depth for the mining and 2-4 cm depth for the reference inlet). Resting stages were germinated and cultured in standard f/2 media (Guillard 1975) amended with 106 µM SiO2. All media was mixed from the same batch of sterile-filtered (Sarstedt’s [Helsingborg, Sweden] 0.2 mm polyethersulfone membrane filter) water sourced from Askö Marine Laboratory (station B1: www.smhi.se) with a salinity of 7. Sediment (0.1 mg) was diluted in 1 mL medium in replicates on 24-well plates (Polystyrene, FalconTM), incubated at 16°C and 10 µmol photons m-2 s-1 of photosynthetically active radiation (PAR) at a 12:12h light:dark cycle. After 3-6 days, the light intensity was increased to 180 µmol photons m-2 s-1, and only one individual chain of S. marinoi was isolated from each well using standard micro-pipetting techniques (Härnström et al. 2011).
Sixty-nine and 55 strains were isolated from the mining and reference inlet with 88% and 94% survival, respectively. We did not attempt to make cultures axenic as this stresses cells and could impose unwanted selection. Strains were re-inoculated from one to 100 mL of medium, divided and processed in parallel for DNA samples, phenotyping, and the artificial evolution experiments. To minimize laboratory adaptation, all strains were processed within one month after germination, corresponding to <37 generations. During each sub-culture step, the culture was screened for contamination of other phytoplankton species and auxospore formation or bimodal cell sizes indicating sexual inbreeding in S. marinoi (Ferrante et al. 2019), and such cultures were discarded (ca. 30%). This left 30 mining and 28 reference strains available for experiments.