Artificial evolution experiments
We set up a 42-day-long artificial evolution experiment to assess how
strain-selection was affected by copper stress. We aimed for a period
that would be long enough to exhaust the strain selection but short
enough not to allow for novel mutations to play a role. De-novomutations are unlikely to affect the same strains across multiple
replicates (separate experimental bottles), which is how we later
validated the assumption of a low impact from new mutations. The two
original populations were re-assembled by pooling the 28-30 strains at
equal densities based on RFU and distributed at 0.0015 RFU density into
ten 100 mL cultures in tissue culture flasks (Polystyrene, Sarstedt).
This corresponded to ~10,000 S. marinoi chains
per replicate bottle (assuming five cells per chain), which are
sufficiently large populations to minimize genetic drift while allowing
for substantial effects of selection (Schlüter et al., 2014). Five
replicates were subjected to 8.65 µM copper exposure (strain RO5AC’s
EC50) and five to non-toxic control conditions. Exponential growth was
maintained through semi-continuous cultivation (Wood et al., 2005), with
dilutions every third day to maintain the density below 0.5 RFU
(Andersson et al., 2020). The two populations were processed six months
apart, with one replicate bottle in each treatment containing only
strain RO5AC as an internal reference to control for experimental
artifacts.
pH, Fv/Fm, and RFU were measured in connection with 3-day
re-inoculations and with daily resolution around days 0-3, 15-18, and
30-33. Measurements of pH were used as a control for inorganic carbon
limitation, which in our experimental setup occurs above
pH~8.2 (Andersson et al., 2020), and the Fv/Fm as a
control for development of chronic stress (Andersson et al., 2022).
Aliquot samples were preserved in Lugol’s solution and later screened
for signs of contamination, sexual reproduction, or spore formation, but
there were no such observations throughout the experiment. RFU was used
to calculate the growth rate across the three-day re-inoculation
periods.
Changes in copper tolerance of the populations was quantified using two
approaches. First, 72-h dose-response curves were collected as described
for individual strains after zero (N=1), 30 (N=3), and 42 (N=5) days.
For the copper selection treatment, copper toxicity was also relaxed for
three days at the end of the experimental selection phase, and
additional dose-response curves were determined (N=5) to assess if
increases in copper tolerance were reversible (plastic) or permanent
(evolved). Secondly, a rapid assay of copper tolerance was developed
that restricts the cells’ capacity to induce plastic responses during
the test period. For this test, cells were collected via gentle
centrifugation (10 min at 1500 g) and inoculated in lethal
concentrations of copper (15 µM), and loss of photosynthetic capacity
was quantified using a Phyto-PAM. Specifically, the yield of Photosystem
II was monitored under 166 µmol photons m-2s-1 actinic light throughout a three hour period. This
PAM assay was implemented in the artifical evolution experiments on days
three, 24 and 39.
The strain selection process was tracked using a recently developedS. marinoi barcoding locus (Pinder et al., 2023), as outlined in
the metabarcoding section below. Cell samples for this analysis were
collected from the artificial evolution experiment on day zero (N=4,
with two DNA extraction replicates × two PCR replicates), nine (N=5
bottle replicates), and 42 (N=5 bottle replicates), via centrifugation
(3000 g for 10 min), flash-freezing in liquid N2, and
storage at -80°C until subsequential DNA extraction.