Symbiodinium culture preparation: Cultures were obtained from the LaJeunesse algal collection at Penn State University. Phylogenetic relationships of these strains are described in Parkinson et al., (2015). Symbiodinium cultures were grown and maintained in liquid media (ASPA-8A, Blank, 1987) at 26 ˚C with fluorescent lights delivering 80-100 μmol quanta m-2 s-1 (measured using a DIVING-PAM equipped with a flat cosine-corrected Fiber Quantum Sensor, Walz, Germany) on a 12:12 hours Light:Dark photoperiod in Innovator 44 incubators (New Brunswick, USA). Symbiodinium cultures of equal starting concentration (3 x 104 cells 126 mL-1) for each strain were grown for 7.5 days at 26 ˚C at logarithmic growth phase prior to experimentation. After 7.5 days of incubation at 26 ˚C, one culture of each strain was subjected to a high temperature treatment of 31 ˚C for two light cycles and one dark cycle (i.e., 1.5 d), whilst another was maintained at 26 ˚C.

Data collection: 100 μL aliquots (8.33 x 104 cells mL-1, calculated as the average of twelve independent hemocytometer measurements) of dark-adapted cultures were then subjected to 5 minutes of elevated temperatures in a thermocycler (Eppendorf Mastercycler Pro S).

Chlorophyll fluorescence parameters were measured immediately after the 5 min temperature exposure using a Fluorometer (see below). Measurements were performed at 25 temperatures (i.e. 26, 30.3, 30.5, 31, 31.7, 32.6, 33.7, 34.8, 35.9, 36.8, 37.6, 38.1, 38.3, 38.8, 38.9, 39.4, 40.1, 41,42, 43, 44, 44.9, 45.6, 46.1, 46.2 ˚C) and replicated three times per Symbiodinium strain per treatment. A modified protocol by Díaz-Almeyda et al., 2011 was used.

Fv/Fm was automatically calculated/determined by fitting each fluorescence transient to the bio-physical model of Kolber et al. (1998) using the FIREPRO software (Satlantic, Version 1.4.3) integrated with the FIRe system.

Reference excitation profile used by FIReView to normalize the variable fluorescence profile: EXCFOP.TXT
Column 1: incremental sample counter
Column 2: elapsed time measured in microSeconds (uS)
Column 3: reference excitation profile
Column 4: relative fluorescence profile normalized to the reference excitation, or the fluorescence yield
Calibration date: 1/ 1/2002
Time (in seconds) of day reading was taken = 79931

Methodology Reference:
Díaz-Almeyda, E., Thomé, P. E., Hafidi, M. El, and Iglesias-Prieto, R. (2011). Differential stability of photosynthetic membranes and fatty acid composition at elevated temperature in Symbiodinium. Coral Reefs, 30(1), 217–225. https://doi.org/10.1007/s00338-010-0691-5.