## 2 Materials and Methods
### Experimental Setup  
#### Experiment 1: Iron and B\({}_{12}\) Limitation  
To study the growth rate and photophysiological responses of _Proorecentrum_ sp. and _Phaeocystis antarctica_ under iron and vitamin B\({}_{12}\) limitation in monospecific and mixed cultures, we performed an experiment including three different growth conditions, i.e., 100% L1 medium without iron (i.e., with added desferoxamine B (DFB), Sigma-Aldrich), 100% L1 medium without vitamin B\({}_{12}\) (-B12) Sigma-Aldrich, and 100% L1 medium as the control (CTRL) as follows:  
Figure 2: Chromatograms in absorbance (mAU) of the two species.  
1. For the DFB treatment, we prepared L1 medium without iron (II). As natural oligotrophic seawater was used as a base for the medium, we added DFB as a ligand to chelate all dissolved iron, to achieve a final DFB/iron (II) ratio of 100:1, assuming an iron concentration of 5 nM.
2. For the -B12 treatment, modified 100% L1 medium was prepared by excluding vitamin B\({}_{12}\) from the vitamin stock solution. The amount of vitamin B\({}_{12}\) present in the prefiltered (0.2 \(\upmu\)m mesh size) seawater used as the culture medium before the addition of L1 medium would be negligible as seawater samples were sterilized by autoclaving at 1.06 kg cm\({}^{-2}\) for 20 min.
3. For the CTRL treatment (i.e., with both iron and B\({}_{12}\)) 100% L1 medium was used.  
The flasks used for incubating the samples were soaked overnight in 1% citranox (Alconox) to remove trace metals, rinsed six times with Milli-Q water, soaked in 10% HC1 (Baker Instra-analyzed) for at least 3 days, and then rinsed three times with Milli-Q water (Ph = 2) prior to use. Flasks were prepared in a positive-pressure trace metal clean area using established trace metal clean techniques for incubation.  
The two species were grown in flasks under continuous light at 40 \(\upmu\)mol photons m\({}^{-2}\) s\({}^{-1}\), in 100% L1 medium at 1.5 \({}^{\circ}\)C, until the maximum growth rate was achieved. In the case of _Prorocentrum_ sp., 20 mL of the isolates were inoculated in 250 mL flasks containing the appropriate medium for one of the three treatments. All treatments were performed in triplicate, yielding a total of 27 flasks for the entire experiment, i.e., 9 flasks for each culture and 3 cultures. _P. antarctica_ were cultured following the same procedure. For the mixed cultures, flasks were inoculated with 10 mL each of _Prorocentrum_ sp. and _P. antarctica_. At the beginning of the experiment, the pigment spectral composition, photosynthetic efficiency, total biomass, and total cell count of the cultures were determined using HPLC, Phyto-PAM, spectrofluorometric analysis of the chlorophyll-a (Chl-a) content, and Burker counting chamber, respectively.  
Single time point sampling was performed daily at the same time for 16 days, for evaluating the maximum PSII photochemical efficiency (Fv/Fm), the maximum PSII effective absorption cross-section (\(\sigma\)PSII), and Chl-a content. At the end of the experiment, i.e., after 16 days, the pigment spectral composition of each treatment was obtained using HPLC, and the total cell count was determined to calculate the Chl-a/cell ratio for _Prorocentrum_ sp. and _P. antarctica_.