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.