Salinity experiments

We performed experiments at three salinities–a ‘low salinity’ treatment of 25, an ‘ambient salinity’ control of 35, and a ‘high salinity’ treatment of 45. These three salinity conditions encompass the global range of open ocean sea surface salinities, which range from a minimum of ~29 to a maximum of ~38 [29]. These salinity treatments also span the very low salinities present in coastal regions with large freshwater inputs as well as the very high salinities recorded in restricted, evaporation-dominated basins (for example salinities of ~41 observed in the northern Red Sea [55]). For strain RCC1210 from an isolation salinity of ~20 (Table 1), the ‘low salinity’ treatment of 25 is not a true low salinity condition in regard to its isolation from the much lower marine salinities present in the Baltic Sea, which is a marginal sea heavily influenced by freshwater inputs [56]. All six strains were maintained in stock culture under salinity 35 for several months prior to the start of the experiments.

Each salinity medium was prepared by diluting sterilized salt solution described above with sterile distilled water (prior to the addition of f/2 media enrichment solution) until the desired salinity was reached, as determined by a portable conductivity meter (WTW Multi 3400i, Xylem Analytics, Germany). Experiment cultures for each salinity treatment were grown in triplicate flasks under continuous (24 hour) light at an irradiance of ~70–100 μmol photons m^-2 s^-1 in 70 mL sterile polycarbonate flasks with ventilated lids to aid gas equilibration. Continuous light was used to desynchronize the timing of cell division to ensure that coccosphere size data were not influenced by cell cycle phase [57]. Each flask contained 60 mL of salinity 25, 35, or 45 media that was directly inoculated (no period of pre-experiment acclimation) with exponentially growing cells from salinity 35 stock cultures that had been maintained in semi-continuous batch culture prior to the start of the experiments. The absence of a pre-experimental acclimation phase allowed the observation of growth and morphological responses to an abrupt onset of low or high salinity conditions, which may occur naturally in the event of severe hydrological conditions or glacial/icesheet meltwater events for example (see Discussion). After inoculation each experimental flask had an initial concentration of approximately 2x10⁵ cells mL^-1. Cultures were gently mixed daily to keep cells in suspension and flask headspace was able to re-equilibrate daily when flasks were opened under a laminar flow hood (sterile environment) to enable cell counts to be performed.

The pH and total alkalinity (TA) were measured at the start and end of the experiment for strains PLYB11 and RCC1232 (on which morphological analyses were perform). The pH was measured using a portable pH meter (WTW Multi 3400i, Xylem Analytics, Germany) and TA was measured using a titration method (MQuant Alkalinity Test, Merck). Dissolved inorganic carbon (DIC) was calculated approximately as the difference between the two acid capacity values determined through titration (KS4.3 –K_S8.2). The measured and calculated carbonate system data are reported in S4 Table.