## 2 Materials and Methods  
### Phytoplankton Collection, Isolation, and Culture  
Searwater samples were collected during the austral summer of 2017 onboard the R/V Italica, using a rosette sampler equipped with Niskin bottles, under the P-ROSE Project (Plankton biodiversity and functioning of the Ross Sea ecosystems in a changing Southern Ocean-XXXII Italian Antarctic Expedition). At each station, 30 mL of sea water was withdrawn from the Niskin bottles, prefiltered through a 300 um mesh (Nitexs, Wildlife Supply Company, Buffalo, NY, USA) to remove most metazoan plankton, and inoculated in 60 mL flasks filled with sterilized Ross Sea water enriched with 20% L1 medium. After inoculation, the flasks were incubated at 3 \({}^{\circ}\)C under continuous light at an intensityof \(\sim\)40 \(\upmu\)mol photons m\({}^{-2}\) s\({}^{-1}\). All samples were checked periodically using a light microscope and refreshed every 2 weeks until the end of the cruise. On reaching Italy, the incubator with the live mixed samples was transported from the R/V Italica to the laboratory of Marine Ecology at the University of Naples Federico II. All flasks were refrigerated at 1.5 \({}^{\circ}\)C under continuous light at \(\sim\)40 \(\upmu\)mol photons m\({}^{-2}\) s\({}^{-1}\) and refreshed using 50% L1 medium with a salinity of 34, generating a series of replicates for each sample.  
After acclimatization for 2 weeks, individual phytoplankton species were isolated to prepare monospecific cultures. Phytoplankton were isolated by [PERSON], following the procedure suggested by [PERSON], of the Center for Marine Science, University of North Carolina, Wilmington, USA (personal communication), and involved both serial dilution and single cell collection using handheld micropipettes and an inverted light microscope (Leica DMIL led).  
Considering the isolation of _P. antarctica_, single balloon-like colonies were collected using a modified micropipette and transferred to a 96-well rack in sterile conditions under a laminar hood (ASALAIR 700), until one colony per well was obtained. Each well was filled with 0.3 mL of seawater enriched with 50% L1 medium. Once cells started to replicate, we transferred three colonies to a 24-well rack, and after two weeks, when cells achieved the maximum growth rate, \(\sim\)2 mL of the sample from each well were inoculated in 60 mL flasks, and new medium was added at 50% volume of the sample. Flasks were periodically checked using a Phyto-PAM Compact Unit (Walz) to evaluate photosynthetic quantum efficiency, and samples were subjected to high performance liquid chromatography (HPLC) to determine the pigment spectral composition and to identify the marker pigment of each isolated species [34; 35; 36]. By doing this, a signal corresponding to Peridinin (Perid), the marker pigment of dinoflagellates, was detected in colonial _P. antarctica_ cultures, especially under nutrient starvation. Thorough microscopic examination of the culture yielded a previously unknown species of dinoflagellate, resembling _Prorocentrum minimum_ (Dinophyceae), associated with the mucilaginous sheath of _P. antarctica_ colonies. This unknown dinoflagellate species was isolated using a modified micropipette, breaking colonies mechanically, and the first monospecific culture was obtained after culturing for eight weeks, and then subjected to HPLC to obtain the pigment spectra. The isolates were cultured under standard growth conditions of 1.5 \({}^{\circ}\)C, 100% L1 medium, and continuous light at 40 \(\upmu\)mol photons m\({}^{-2}\) s\({}^{-1}\) (Figure 1). The pigment spectra of the two species are reported in Figure 2.  
Figure 1: **(a)**_Phaeocystis antarctica_; **(b)**_Prorocentrum_ sp.