Corals from Rebotel Reef on the western barrier reef of Palau were collected for offshore samples, while nearshore corals were collected in Nikko Bay approximately 28 km away. The corals Acropora muricata and Coelastrea aspera were sampled in March of 2014 from both locations and used in the initial thermal experiments. Two additional coral species, Pachyseris rugosa, and Cyphastrea chalcidicum, were sampled from the same locations and treated the same way in March of 2015. A total of 8 colonies of each species were collected using a hammer and chisel at a depth of 5–10 meters (offshore) or 1–5 meters (nearshore) to ensure similar irradiance conditions, and each colony was sampled a minimum distance of 10 meters from surrounding colonies to better ensure sampling of unique coral genets. Despite the thermal experiments being conducted in multiple years (2014 and 2015), the thermal histories and light levels indicate similar conditions during this time period and allowed physiological comparisons across host species and population origin (see Hoadley et al. 2021). Colonies were transported to the Palau International Coral Research Center (PICRC) and fragmented into replicate pieces (clone ramets) and placed into 1200 L flow-through aquariums supplied with natural seawater and held at 27.5°C. Corals were allowed to heal for a minimum of 2 days and were then placed on individual 5 cm square PVC tiles with marine epoxy (splash zone compound A-788) and returned to the holding aquariums for 12 – 16 days to recover before the start of the experiment.

For each treatment, two replicate fragments from each coral colony were placed in separate treatment bins. In the heated treatment, the temperature was gradually increased from 27.5°C to 32°C over 4 days, and then maintained at 32°C for an additional 10 days, totaling 14 days of heating. The control treatment was kept at a constant temperature of 27.5°C throughout the 14-day experiment. All the experimental coral fragments were kept outdoors, and covered by non-UV filtering clear plastic film (Sun Selector, Ginegar Plastic Products) to protect them from periodic rainfall. Additionally, a 60% shade cloth was used to provide a peak midday light intensity of 800 μmol quanta m^-2s^-1, measured with a PAR sensor (LiCor LI-192), similar to the maximum light levels of natal colony habitats at collection depth. 

At the beginning of the experiment (day 0), one fragment from each coral colony (if available; n=4-8) was removed, and ¹³C and ¹⁵N isotope measurements of unlabeled colonies were made for enrichment comparison. On day 14 (4 days of temperature ramping and 10 days at 32°C), coral fragments were removed from treatments and processed the same as day 0.

A pulse amplitude modulation fluorometer (Diving PAM, Waltz, Germany) was used to measure the maximum quantum yield of photosystem II (PSII, Fv/Fm) one hour after sunset in three separate locations using a 0.6 second saturation pulse (saturation intensity > 1000 mmol quanta m-2s-1). Three intracolony Fv/Fm measurements were averaged together to calculate the mean Fv/Fm for each fragment.

Coral tissue was removed using an airbrush (100 psi) and filtered (0.22 µm) seawater. The resulting slurry, containing coral tissue and symbiotic dinoflagellates, was homogenized for approximately 10 seconds using a Tissue Tearor (BioSpec Products, Bartlesville, OK, USA). Aliquots (1 ml) were taken from the homogenized slurry and preserved with 1% glutaraldehyde for symbiotic algal enumeration. Algal densities were quantified using an EVOS digital fluorescent microscope from 4–6 replicate haemocytometer counts (AO Spencer Bright Line Improved Neubauer haemocytometer) and normalized to coral surface area using the aluminum foil method (Marsh 1970) for C. aspera, C. chalcidicum, and P. rugosa, and the hot wax method (Stimson and Kinzie 1991) for the branching coral A. muricata. The influence of thermal treatments (32°C) on areal symbiotic dinoflagellate densities were compared to clone fragments at the control temperature (28°C).

On day 14, control and treatment fragments were placed into glass beakers containing 400 ml of freshly filtered seawater (0.45 µm) that was enriched with 0.633 mM of NaH¹³CO₃ (99 atom % 13C, Cambridge Isotope Lab Inc., Andover, MA, USA), and 1.5 µM of Na¹⁵NO₃^– (98 atom % ¹⁵N, Cambridge Isotope Lab Inc., Andover, MA, USA).  The beakers were fitted with false bottoms and continually stirred with magnetic stir bars. All beakers were held constant at the experimental temperatures for 5 h (28°C or 32°C) and illuminated by LED lights (Cree Cool White XP-G R5) set to a light intensity of 500 μmol quanta m^-2s^-1. Preliminary measurements determined this irradiance level was sufficient to maximize photosynthesis (Pmax) and the H¹³CO₃ and ¹⁵NO₃^– concentrations were sufficient to be used for elemental tracing across the biological compartments.  After isotopic labeling, the fragments were removed, rinsed in filtered seawater, and immediately frozen at -60°C. The impact of symbiotic dinoflagellates on the uptake and assimilation of ¹³C and ¹⁵N across biological compartments (symbiotic dinoflagellates, coral tissue, and coral skeleton) was assessed by comparing colonies containing D. trenchii with colonies containing Cladocopium spp. at a temperature of 28°C  and the influence of thermal treatments (32°C) on ¹³C and ¹⁵N uptake and assimilation was compared.

Coral tissue was removed with an airbrush as previously described, followed by the addition of 0.02% (w/v) sodium dodecyl sulfate (SDS) and homogenization for 10 s with a Tissue-Tearor (Biospec Products, Inc). Symbiotic dinoflagellates and coral tissue were separated by 2–3 centrifugation washes (550 g for 5 min) with 10 second homogenization between each wash (Lesser and Shick 1989). Algal fractions were microscopically verified to ensure the efficiency of the separation technique and to confirm the homogeneity and removal of the bulk animal material (Tremblay et al. 2012). Clean algal cells were pelleted via centrifugation (5,000 g for 5 min) and frozen at -20°C until analyzed. Accumulated supernatants (animal portion) were microscopically verified to not contain symbiotic dinoflagellates or skeletal material and were filtered onto pre-combusted (450° C for 5h) glass 0.7 mm filters (Whatman GF/F) until clogged and frozen at -20°C.

Due to the relatively high concentration of ¹³C assimilation by the symbiotic dinoflagellates during incubations, coral skeletons were placed in 100% bleach for 24 hours to remove any remnant organic material from host-algal tissue, rinsed in freshwater for 24 hours, and dried under low heat. Approximately 20 mg of CaCO₃ was sampled from the corallite and coenosarc regions of the coral skeleton using a Dremel tool with a diamond bit. Skeletal samples were stored at -20°C until analyzed. Elemental ¹³C and ¹⁵N analyses were performed on a Carlo Erba CHN Elemental Analyzer (Model NA1500) coupled to Thermo Finnigan Delta V Isotope Ratio Mass Spectrometer via a Thermo Finnigan Conflo III Interface at the University of Georgia, Center for Applied Isotope Studies.