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.