Sample Collection

The experiment was performed during the austral summer from mid-January to late March 2021 at Heron Island Research Station (HIRS), southern Great Barrier Reef (23 27°S, 151 55°E). Heron Reef is composed of five distinct geomorphological habitats characterized by diverse benthic communities and biogeochemical conditions. Fragments of the coral P. damicornis were collected from the reef flat and slope locations within the same depth range (1–3 m) on 14 and 15 January 2021. Four fragments were collected from each individual colony (genetic clones), totalling 96 fragments from 24 colonies (n = 12 per habitat). For more information about sample collection methods and treatment, see Brown et al., 2022.

Skeletal micromorphological analysis

The limited amount of new CaCO3 deposition observed during the 8-week exposure (~15–30% for each fragment; (Brown et al., 2022)) precluded our resolution to detect changes in net calcification or CaCO3 density of newly formed skeleton that were attributable to experimental pCO2 treatment conditions. To better resolve changes in biomineralization resulting from the seawater pCO2 variability treatments, a total of 16 coral fragments (n=4 per origin per treatment) were selected for skeletal micromorphological analyses. All tissue was removed from the skeletons by soaking the fragments in 10% sodium hypochlorite for 24 hr, rinsing with deionized (DI) water, and drying. Areas of CaCO3 deposition that occurred during the experiment were identified by comparing images at the start and end of the 8-week experiment. These deposits of new CaCO3 were carefully chipped off of the experimental fragments using a razor blade and imaged using a scanning electron microscope (SEM; Quanta 600 FEG Mark II Environmental Scanning Electron Microscope, Field Electron and Ion Company). Using the SEM, fragments were imaged across scales with magnification maintained between samples: an overall view of the skeleton (56x), individual whole calyxes (124x), spine structures between (141x) and inside (164x) the calyxes, and the rapid accretion deposits (RADs) on the spines (1013x). Several features of interest previously used to investigate coral biomineralization (Scucchia et al., 2023; Scucchia, Malik, Zaslansky, et al., 2021) were quantified using ImageJ (v1.53c) (Schneider et al., 2012), including: number of corallites, distance between corallites (i.e., coenosteum width), corallite diameter, circularity of the corallite, number of spines within calyx, spine length and maximum spine width (on spines both between and inside the calyx), number of RADs, and size of RADs.

The significant interaction between treatment and origin was explored on all micromorphological features using linear mixed effects models, with colony as a random effect. The significance of fixed effects and their interactions was determined using an analysis of variance with a type III error structure using the Anova function in car package (Fox et al., 2012). Significant interactive effects were followed by pairwise comparison of estimate marginal means using the emmeans package with Tukey HSD adjusted p values (Lenth et al., 2018). Data were tested for homogeneity of variance and normality of distribution through graphical analyses of residual plots for all models. All statistical analyses were done using R version 4.0.3 software (R Core Team, 2020), and graphical representations were produced using the package ggplot2 (Wickham, 2016).