Dataset: Acropora hyacinthus isotope data from coral samples collected at a north shore site in Mo’orea in May 2019

In May 2019, during the height of the bleaching event, SCUBA divers observed bleached Acropora hyacinthus (urn:lsid:marinespecies.org:taxname:207044) coral colonies in both the shallow fore reef (~5 m depth) and deep fore reef (~14 m depth) habitats at a north shore site in Mo’orea (fore reef: 17.4731°S, 149.8176°W). Bleaching was variable across habitats. On the shallow fore reef some A. hyacinthus colonies were bleached and others were not bleached. On the deep fore reef all A. hyacinthus colonies were bleached. Despite extensive searching at the site, it was not possible to locate any unbleached colonies in May 2019. Colonies from the shallow and deep fore reef habitats were tagged for future sampling.

In August 2019, after the period of accumulated thermal stress subsided, the previously tagged colonies on the deep and shallow fore reef were relocated. By August, all the previously tagged colonies on the deep fore reef (~14 m depth) had died. Despite this high mortality, August surveys on the deep fore reef identified previously untagged colonies that were visibly recovering from bleaching (Leinbach et al. 2021), and therefore are assumed to have bleached. These colonies were photographed with an Olympus Tough TG-5 camera with the underwater macro mode on and the auto fill in flash setting, tagged, and sampled. Corals were photographed with a scale bar that included black and white markings and a Coral Watch Coral Health Chart. Due to the high prevalence of bleaching at this site in May (100% of colonies had some level of bleaching, including 53.2% that were severely bleached and 46.8% that were partially bleached), it is maintained that these previously untagged colonies were bleached during the marine heatwave (MHW) (Leinbach et al. 2021). In October 2019, 30 and 28 previously tagged colonies at ~5 and 14 m, respectively, were again located, photographed, and sampled via SCUBA for physiological metrics and/or reproductive histology (Leinbach et al. 2021). Water column particulate organic matter (POM) was also sampled by collecting 4 L of seawater at ~2 m depth and filtering through pre-combusted 0.7 µm Whatman glass fiber filters using vacuum filtration. Samples were collected at the fore reef site where corals were sampled.

For all corals sampled, bleaching severity was determined visually from photographs with a color standard. Each colony was assigned a score from 1 to 5 according to their bleaching severity (Siebeck et al. 2006), with 1 indicating stark white bleaching and 5 indicating no visible bleaching (not observed for any colonies in this study, for more details on color-scoring see Leinbach et al., 2021). Using the same photographs, planar colony area was measured by tracing the outline of each colony and calculating the planar surface area using ImageJ (Schneider et al. 2012). Coral fragments of approximately 2 cm were collected from each colony and frozen at - 40°C. Using filtered seawater (FSW), coral fragments were airbrushed to remove coral tissue and endosymbiont algal cells (blastate) from the calcium carbonate skeleton. The blastate was then homogenized and centrifuged (2,000 x g for 2 min) to separate coral host tissue from endosymbiont cells. After supernatant (coral host) removal, the endosymbiont pellet was then resuspended in 2 mL of FSW and centrifuged an additional time to separate out any remaining coral host tissue. The supernatant from this centrifugation was combined with the coral host fraction. The coral host fraction was centrifuged an additional time to pellet any endosymbiont cells remaining in solution. The endosymbiont fraction was cleaned (resuspended in FSW and centrifuged) six times to ensure the removal of animal tissue from the pellet. Following separation of coral host tissue and endosymbiont cells, each fraction (and POM samples) was filtered through a 0.7 µm Whatman glass fiber filter. Samples were rinsed with 1mL of 1N HCl to remove any residual calcium carbonate from the coral skeleton, then rinsed once more with 1mL of deionized water. Filters were placed in a drying oven overnight set at 60 °C and kept dry until transportation to the University of California - Santa Barbara where they were immediately placed into a -80 °C freezer until analysis. Of the colonies sampled in Leinbach et al. (2021), we sampled 27 of these for fatty acid analysis and 20 for isotope analysis.

Coral host, symbiont and POM samples were extracted using a modified Folch method (Folch et al. 1957) following Taipale et al. (2013) and Radice et al. (2019). Nonadecenoic acid (C19:1) was used as an internal standard. Fatty acids were analyzed with a Gas Chromatograph equipped with a Flame Ionization Detector (GC-FID, Hewlett Packard HP5890) at University of California Santa Barbara using a Supelco Omegawax 250 Column (30 m x 0.25 mm ID x 0.25 um film thickness) with a 1 uL injection and a 30 second splitless hold time. Fatty acids were identified by a mixture of techniques, including: comparison of retention times and peak area to a certified reference material (Supelco 37 component FAME mix, FAME-37), spiking experiments with known analytes, and comparison with previously identified peaks from our in-house tissue reference material of Red Sea Stylophora pistillata (urn:lsid:marinespecies.org:taxname:206982) from Love (2023). Mass of fatty acid per sample was calculated by dividing peak area by the daily calibrated response factor for that compound in a standard mix (Supelco FAME-37). If the fatty acid of interest was not in the FAME-37 mix, a response factor was generated by the next closest fatty acid with the same carbon length tail and the same or similar number of double bonds since response factor follows a linear trend, decreasing systematically throughout the chromatographic run time as carbon number and degree of unsaturation increases. Analytical precision for relative abundance data (calculated from FAME-37) was ± 0.04%. Note, samples were oven dried as an unavoidable step due to covid-19 related sampling challenges. As such, the proportions of FA measured here should not be compared across studies. However, since all samples were of the same species and received the same handling, treatment, and storage, making relative difference comparisons among samples and tissue types in this study is valid (Ingemansson et al. 1995; Nazemroaya et al. 2011; Rudy et al. 2016).

Coral host, symbiont, and POM samples were analyzed for δ13C and δ15N values using a Thermo Finnigan Delta-Plus Advantage isotope mass spectrometer coupled with a Costech EAS elemental analyzer in the University of California - Santa Barbara Marine Science Institute Analytical Laboratory. Instrument calibration and linearity were conducted using acetanilide reference standards. Instrument precision, determined using replicate analyses of L-glutamic acid USGS40 (δ13CVPDB-LSVEC = –26.39 ± 0.04‰, δ15NAIR = –4.52 ± 0.06‰), was ± 0.12‰ for δ13C and ± 0.06‰ for δ15N. Isotope ratios are expressed in standard δ notation, expressed as per mil (‰) relative to Pee Dee Belemnite (PDB) for carbon and atmospheric Air (N2) for nitrogen.