Experimental Design (text extracted from Grottoli et al., 2014. See publication for figures and full methods description)

In July 2009, 10 coral fragments from nine healthy colonies of Porites divaricata (branching morphology), Porites astreoides (mounding/encrusting morphology), and Orbicella faveolata (formerly Montastraea faveolata (large, mounding morphology) were collected near Puerto Morelos, Mexico (20 deg 500 N, 86 deg 520 W region). Parent colonies were all at least 10m apart and thus assumed to be genotypically different. Fragments were mounted on labeled PVC tiles, and randomly placed in 10 shaded (600 umol photons m(-2) s(-1) ) outdoor flow-through seawater tanks, allowed to acclimate for 5 days, and then buoyantly weighed. The temperature in five tanks was gradually raised to 31.5C +- 0.20C (single bleaching treatment) over 7 days then maintained at the elevated temperature, while the other five tanks received ambient reef water (controls; 30.6 C +- 0.24C) . After 15 days, one treatment and one control fragment from each parent colony of each species were buoyantly weighed and then frozen at 80 C (0 weeks on the reef). The remaining fragments were transplanted back to the reef at 4.9 m depth (20 deg 52.8150 N, 86 deg 50.9890 W). After 6 weeks on the reef, one additional treatment and control fragment from each colony of each species were collected, buoyantly weighed, and frozen at 80C, while the remaining fragments stayed on the reef for a full year at ambient reef temperatures.

Chlorophyll a was determined according to Jeffrey & Humphrey (1975) and standardized to surface area (Marsh 1970). Chlorophyll a values at 0 and 1.5 months on the reef following repeat bleaching are from Schoepf et al. (2011).

Total soluble lipids, animal soluble protein and animal soluble carbohydrate were determined on ground, frozen coral fragments using established methods (Rodrigues & Grottoli 2007; Schoepf et al 2013) and then converted to Joules per gram ash-free dry weight (Gnaiger & Bitterlich 1984)).

Net calcification was determined using the buoyant weight technique (Jokiel et al 1978) and standardized to surface area. Calcification rates at 0 and 1.5 months on the reef following repeat bleaching are from Grottoli et al. (2014).

Tissue C and N isotopic analyses were performed on separated animal host and endosymbiont fractions using established methods (Rodrigues & Grottoli 2006; Hughes et al 2010). The difference between d13Ch and d13Ce (i.e. d13Ch2e) was calculated to determine the relative contribution of photoautotrophic versus heterotrophic carbon to the coral (Rodrigues & Grottoli 2006; Muscatine et al 1989). Repeated measurements of commercial standards (USGS-24, IAEA-N2) had a standard deviation of +- 0.04(permille) for d13C (n = 55) and +-0.11(permille) for d15N (n = 51).

References:

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Grottoli, Andréa G., et al. "The cumulative impact of annual coral bleaching can turn some coral species winners into losers." Global change biology 20.12 (2014): 3823-3833. http://dx.doi.org/10.1111/gcb.12658

Hughes, A. D., et al. "Acquisition and assimilation of carbon in non-bleached and bleached corals." Marine ecology progress series 420 (2010): 91-101. http://dx.doi.org/10.3354/meps08866

Jeffrey, SW T., and G. F. Humphrey. "New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton." Biochem Physiol Pflanz BPP (1975).

Jokiel, P. L., J. E. Maragos, and L. Franzisket. "Coral growth: buoyant weight technique." Coral reefs: research methods. UNESCO, Paris (1978): 529-541.

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Rodrigues, Lisa J., and Andréa G. Grottoli. "Calcification rate and the stable carbon, oxygen, and nitrogen isotopes in the skeleton, host tissue, and zooxanthellae of bleached and recovering Hawaiian corals." Geochimica et Cosmochimica Acta 70.11 (2006): 2781-2789. http://dx.doi.org/10.1016/j.gca.2006.02.014

Rodrigues, Lisa J., and Andréa G. Grottoli. "Energy reserves and metabolism as indicators of coral recovery from bleaching." Limnology and oceanography52.5 (2007): 1874-1882. http://doi:10.4319/lo.2007.52.5.1874

Schoepf, Verena, et al. "Short-term coral bleaching is not recorded by skeletal boron isotopes." PloS one 9.11 (2014): e112011. http://dx.doi.org/10.1371/journal.pone.0112011

Schoepf, V., et al. others (2013) "Coral energy reserves and calcification in a high-CO2 world at two temperatures." PLoS ONE 8: e75049. http://dx.doi.org/75010.71371/journal.pone.0075049

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