# Physicochemical Control of Caribbean Coral Calcification Linked to Host and Symbiont Responses to Varying pCO2 and Temperature

Robert A Eagle, Maxence Guillermic, Illian de Corte, Blanca Alvarez

Caraveo, Colleen B Bove, Sambuddha Misra, Louise P Cameron, Karl D

Castillo, Justin B Ries

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Robert A Eagle, Maxence Guillermic, Illian de Corte, Blanca Alvarez Caraveo, Colleen B Bove, et al. Physicochemical Control of Caribbean Coral Calcification Linked to Host and Symbiont Responses to Varying pCO2 and Temperature. Journal of Marine Science and Engineering, 2022, 10 (8), pp.1075. 10.3390/jmse10081075. hal-04159173

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Article

# Physicochemical Control of Caribbean Coral Calcification Linked to Host and Symbiont Responses to Varying pCO₂ and Temperature

Robert A. Eagle<sup>1,2,*</sup>①, Maxence Guillermic<sup>1,2,3</sup>①, Illian De Corte<sup>1,2</sup>, Blanca Alvarez Caraveo<sup>1</sup>, Colleen B. Bove<sup>4,5</sup>, Sambuddha Misra<sup>6,7</sup>, Louise P. Cameron<sup>8</sup>, Karl D. Castillo<sup>4,5</sup> and Justin B. Ries<sup>8</sup>

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**Citation:** Eagle, R.A.; Guillermic, M.; De Corte, I.; Alvarez Caraveo, B.; Bove, C.B.; Misra, S.; Cameron, L.P.; Castillo, K.D.; Ries, J.B.
Physicochemical Control of Caribbean Coral Calcification Linked to Host and Symbiont Responses to Varying pCO₂ and Temperature. *J. Mar. Sci. Eng.* **2022**, *10*, 1075. https://doi.org/10.3390/jmse10081075

Academic Editor: Tom Spencer

Received: 13 January 2022

Accepted: 25 July 2022

Published: 5 August 2022

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**Abstract:** It is thought that the active physiological regulation of the chemistry of a parent fluid is an important process in the biomineralization of scleractinian corals. Biological regulation of calcification fluid pH (pH<sub>CF</sub>) and other carbonate chemistry parameters ([CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub>, DIC<sub>CF</sub>, and Ω<sub>CF</sub>) may be challenged by CO<sub>2</sub> driven acidification and temperature. Here, we examine the combined influence of changing temperature and CO<sub>2</sub> on calcifying fluid regulation in four common Caribbean coral species—*Porites astreoides*, *Pseudodiploria strigosa*, *Undaria tenuifolia*, and *Siderastrea siderea*. We utilize skeletal boron geochemistry (B/Ca and δ<sup>11</sup>B) to probe the pH<sub>CF</sub>, [CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub>, and DIC<sub>CF</sub> regulation in these corals, and δ<sup>13</sup>C to track changes in the sources of carbon for calcification. Temperature was found to not influence pH<sub>CF</sub> regulation across all pCO<sub>2</sub> treatments in these corals, in contrast to recent studies on Indo-Pacific pocilloporid corals. We find that [DIC]<sub>CF</sub> is significantly lower at higher temperatures in all the corals, and that the higher temperature was associated with depletion of host energy reserves, suggesting [DIC]<sub>CF</sub> reductions may result from reduced input of respired CO<sub>2</sub> to the DIC pool for calcification. In addition, δ<sup>13</sup>C data suggest that under high temperature and CO<sub>2</sub> conditions, algal symbiont photosynthesis continues to influence the calcification pool and is associated with low [DIC]<sub>CF</sub> in *P. strigosa* and *P. astreoides*. In *P. astreoides* this effect is also associated with an increase in chlorophyll a concentration in coral tissues at higher temperatures. These observations collectively support the assertion that physicochemical control over coral calcifying fluid chemistry is coupled to host and symbiont physiological responses to environmental change, and reveals interspecific differences in the extent and nature of this coupling.

**Keywords:** Caribbean; coral; calcification; pH regulation; boron isotopes; B/Ca; carbon isotopes; photosynthesis; bleaching; symbiont

<footnote n="1">¹ Department of Atmospheric and Oceanic Sciences, Institute of the Environment and Sustainability, Center for Diverse Leadership in Science, University of California, 520 Portola Plaza, Los Angeles, CA 90095, USA</footnote>

<footnote n="2">² Université de Brest Occidentale, Institut Universitaire Européen de la Mer, LGO, Rue Dumont d'Urville, 29280 Plouzané, France</footnote>

<footnote n="3">³ Department of Earth, Planetary, and Space Sciences, University of California, Los Angeles, 595 Charles Young Drive E., Los Angeles, CA 90095, USA</footnote>

<footnote n="4">⁴ Environment, Ecology, and Energy Program, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA</footnote>

<footnote n="5">⁵ Department of Earth, Marine, and Environmental Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA</footnote>

<footnote n="6">⁶ Indian Institute of Science, Centre for Earth Sciences, Malleshwaram, Bengaluru 560012, Karnataka, India</footnote>

<footnote n="7">⁷ The Godwin Laboratory for Palaeoclimate Research, Department of Earth Sciences, University of Cambridge, Cambridge CB2 3EQ, UK</footnote>

<footnote n="8">⁸ Department of Marine and Environmental Sciences, Marine Science Center, Northeastern University, 430 Nahant Rd, Nahant, MA 01908, USA</footnote>

<footnote>* Correspondence: robeagle@g.ucla.edu</footnote>
# 1. Introduction

Corals are foundational for some of earth's most biologically productive and diverse ecosystems, many of which are threatened by a combination of rising ocean temperatures, disease, ocean acidification (OA), and other pressures. Ocean warming can cause a breakdown in the symbiotic relationship between corals and their algal symbionts leading to coral "bleaching" and eventual mortality [1] and represents a significant threat to corals [2]. In addition, as atmospheric carbon dioxide partial pressure continues to rise, so does uptake of CO₂ into seawater, which reduces seawater pH (pH<sub>SW</sub>) and calcium carbonate saturation state (Ω), thereby making seawater less chemically favorable for organisms that produce CaCO₃ shells and skeletons [3]. However, it is understood that some species of marine calcifying organisms are resilient to external acidification or may even benefit by utilizing the additional dissolved inorganic carbon (DIC) available for shell building, and in some cases photosynthesis [4-7].

Coral calcification responses to ocean acidification are known to be diverse, with observations of negative skeletal growth responses [8-20]. Coupled ocean acidification and temperature stress has revealed a variety of responses due to interactions with temperature stress, including negative synergistic [14,21-24].

Whilst interactive effects of temperature and acidification on corals are known, the underlying physiological causes of those effects are less well understood. In two Indo-Pacific pocilloporid coral species, *Pocillopora damicornis* and *Stylophora pistillata*, we observed that corals cultured at 28 °C exhibit increased net calcification with increasing pCO₂, but a shift to negligible or negative net calcification at 31 °C [7]. At the 28 °C treatment, CO₂ fertilization of symbiont photosynthesis appeared to be occurring, which supplied the energy resources for corals to increase calcification [7,25]. At 31 °C, loss of symbiont density was observed from coral color analyses [7,25]. Using two independent approaches, pH microelectrodes and skeletal boron geochemistry, it was found that temperature compromised the corals' ability to maintain biological regulation of its internal parent fluids for calcification (calcifying fluids; CF), with pH<sub>CF</sub> and [CO₃²⁻]ₐ₆ reduced at 31 °C and more sensitive to changes in the carbonate chemistry of external seawater [7]. Similar observations using pH sensitive dyes [26] and geochemical proxies [27-29] have been reported in field-collected corals with different thermal exposure histories. As it is thought that biological upregulation of the pH<sub>CF</sub>, [CO₃²⁻]ₐ₆, [DIC]ₐ₆, and the saturation state of aragonite (Ω<sub>Ar</sub>)ₐ₆ is important in allowing corals to calcify [30-35], loss of this control on thermal stress may explain how temperature and pCO₂-induced ocean acidification could interact to negatively impact coral calcification [7]. Both laboratory experiments and observations on field collected corals have their limitations. In culture experiments, the question is how well are natural physiological responses to stressors simulated by the experiment and, in field collected corals, it can be difficult to constrain and disentangle complex and covarying relationships between the seawater carbonate system and their geochemical proxies [36]. Whilst coral calcifying chemistry regulation in response to acidification has been widely investigated in Indo-Pacific species, it has not to our knowledge been examined in depth in culture experiments on the Caribbean species studied here, with the exception of a study by [37] on *S. siderea* using a spatially resolved laser ablation approach.

Here, we further explore the independent and combined effect of temperature and pCO₂ on coral and algal symbiont physiology in a study on four species of common Caribbean scleractinian corals *Porites astreoides*, *Pseudodiploria strigosa*, *Undaria tenuifolia*, and *Siderastrea siderea* (Figure 1). These corals were recovered from the Belize Mesoamerican Barrier Reef System, cultured at 28 °C and 31 °C, and at ~280 to 3300 µatm pCO₂, and characterized for their structural and physiological responses to these conditions [38,39]. The coral species exhibited interspecific variations in host and symbiont responses to changing environmental conditions in the experiment, which allows us to investigate the link between host and symbiont physiology and the control of calcification and calcification fluid chemistry [38,39]. It was found that all four coral species exhibited nonlinear decreases in net calcification rate with increasing pCO₂ [38]. The 31 °C treatment reduced calcification
rate in only one species, *P. strigosa*, although *U. tenuifolia* exhibited too low survival rates at 31 °C to be assessed [38]. *Siderastrea siderea* was the most resilient coral maintaining positive rates of net calcification under all conditions, even when seawater was undersaturated with respect to aragonite [38]. *Siderastrea siderea* was found to maintain relatively unaltered symbiosis under experimental conditions, and relatively constant host energy reserves, consistent with its resilient calcification response [39]. *Pseudodiploria strigosa* did not maintain indicators of coral symbiont physiology under warming, such as chlorophyll content and symbiont cell density, and was the most bleached coral in the experiment [39]. Conversely, *P. astreoides* exhibited improved symbiont physiological indicators and chlorophyll content at 31 °C, which were reduced under the acidification conditions [39], although the 31 °C condition was characterized by lower survival rates [38]. *Pseudodiploria strigosa* and *S. siderea* showed stronger correlations between symbiont density and host energy reserves, consistent with a tighter coupling between these physiological indicators than were found in *P. astreoides* [39]. It was concluded in previous work that this may result from *P. astreoides* symbionts being more efficient in delivering autotrophically derived carbon to the host, whereas *P. strigosa* and *S. siderea* require relatively greater concentrations of their symbionts to support the host [39]. Therefore, this experiment represents an opportunity to expand on our previous work [7] to explore the underlying mechanisms behind the interaction of temperature and pCO₂ on corals and, in particular, how symbiont and host physiological responses could influence corals' ability to exert active biological control on physicochemical parameters, such as pH<sub>CF</sub>, [DIC]<sub>CF</sub>, and [CO₃²⁻]<sub>CF</sub>. We applied a combined approach of boron geochemistry to constrain pH<sub>CF</sub>, [DIC]<sub>CF</sub>, and [CO₃²⁻]<sub>CF</sub> and δ¹³C to trace changes in the source of carbon for calcification linked to coral and symbiont physiological responses to environmental changes.

<figure><img src="image_5.png" /><figcaption>**Figure 1.** Sample photographs of the coral species cultured in the experiment of Bove et al. (2019) [38] and investigated here: (A) *Porites astreoides*, (B) *Pseudodiploria strigosa*, (C) *Undaria tenuifolia*, and (D) *Siderastrea siderea*.</figcaption></figure>

**2. Materials and Methods**

**2.1. Sample Collection**

Samples from the tropical Caribbean Scleractinian coral species *Siderastrea siderea*, *Pseudodiploria strigosa*, *Porites astreoides*, and *Undaria tenuifolia* (Figure 1) were collected from inshore and offshore reef environments along the southern portion of the Mesoamerican
Barrier Reef System (MBRS) off the coast of Belize in June 2015, at depths of 3–5 m [38]. For each species, six colonies were collected from both an inshore and offshore reef environment, totaling 48 colonies (4 species × 6 colonies × 2 reef environments), as described in Supplementary Table S3. Samples were then transported to a natural seawater flow-through aquarium system at Northeastern University’s Marine Science Center in Nahant, MA, USA, where each colony was sectioned into eight fragments to be acclimated and subsequently undergo the 93-day experiment [38]. New data from a second experiment on Indo-Pacific corals are also presented here, in this experiment specimens of *Stylophora pistillata*, were obtained from DeJong MarineLife (Lingewaal, The Netherlands) and cultured as described previously [7,25].

## 2.2. *Experimental Culturing*

Following a ~70 day conditioning period, fragmented coral colonies from *S. siderea*, *P. strigosa*, *P. astreoides*, and *U. tenuifolia* were reared for 93 days under four *pCO₂* conditions corresponding to preindustrial (311 µatm), present-day control (405 µatm), end-of-century (701 µatm), and extreme (3309 µatm) carbon dioxide partial pressures, and under two temperature conditions corresponding to control (28 °C, as determined by in situ reef temperature records near the collection site) and elevated temperature treatments (31 °C), as predicted for end-of-century mean sea-surface temperature near the collection site [40]. The extreme *pCO₂* condition allows for an exploration of the physiological limits of the species, as well as simulates conditions in past greenhouse periods in earth history which are relevant to understand coral evolution. Coral fragments within each tank were fed every other day with a mixture of ca. 6 g frozen adult *Artemia* sp. and 250 mL concentrated newly hatched live *Artemia* sp. (500 mL⁻¹) to satisfy any heterotrophic feeding by each species. Supplementary Tables S1 and S2 reproduce the measured and calculated experimental parameters from the original study [38].

## 2.3. *Net Calcification Rate, Coral Symbiont and Host Physiological Measurements*

Net coral calcification rates from this experiment were estimated from surviving coral fragments using the buoyant weight method, as described in [38], and reported in units of mg cm⁻² d⁻¹. Algal symbiont cell density was assessed using Lugol's iodine staining, with cell counts standardized to total coral tissue volume to yield values in cells per cm² as described in [39]. Chlorophyll a (Chl a) was measured on a Turner Design 10-AU fluorometer and expressed in units of g of pigment per cm² of coral tissue surface area [39]. Coral host protein, host lipids, and host carbohydrate were determined by spectrophotometry and recorded in units of mg per cm² of coral tissue surface area. Whilst these data have been reported in previous publications [38,39], they are reproduced here in Table S3 to provide easy reference for interpreting the geochemical data.

## 2.4. *Coral Sample Preparation for Geochemical Analyses*

New skeletal growth was identified relative to a calcein marker emplaced in the coral skeleton at the start of the experiment, which was visualized using fluorescent light microscopy [38]. Powdered and homogenized samples underwent clay-removal and oxidative cleaning following the method from Barker et al. [41]. Sample material was then dissolved in 1 M HCl (80–100 µL).

## 2.5. *Boron Isotopic Analysis*

Boron was purified via microdistillation [42,43] following similar methods described in our previous work [7,44]. Measurements were carried out on a Thermo Scientific® Neptune Plus MC-ICP-MS at the University of Cambridge equipped with 10¹³ Ω resistors [45]. Boron isotopic composition is reported in standard δ¹¹B per mil (‰) notation with respect to the NIST SRM 951a boric acid standard (Catanzaro et al., 1970). A laboratory coral standard (NEP; *Porites* sp.) from the University of Western Australia and the Australian National
University was analyzed for assessing internal reproducibility between analytical sessions as well as external reproducibility with other labs.

Sample blanks typically contained less than 0.6 ng-B (<5 ppb B). The δ¹¹B composition of the NEP (δ¹¹BNEP) was measured at 25.6 ± 1.0‰ (2 SD, n = 20) across 13 analytical sessions (Table S4), with each number representing an ab initio processed sample from the present study, which are within analytical uncertainty of published values for the same in-house standard of 25.96 ± 0.30‰, 26.2 ± 0.88‰, n = 27, (25.8 ± 0.89‰, n = 6, 25.71 ± 0.79‰, n = 27, from [46–48]). The proposed international standard JCP-1 [49] was not analyzed across our analytical sessions due to lack of availability, nevertheless, previous work [7,44] was performed over the same period and report values within analytical uncertainty of published data, thereby validating the method utilized in the present study [7,49].

## 2.6. B/Ca Analysis

Elemental ratios were analyzed on a Thermo Scientific® Element HR ICP-MS at Cambridge University, UK and at European Institute for Marine Studies (IUEM), France, after calcium concentration checks on the Agilent® ICP-OES at Cambridge University, UK and at IUEM, France. Isotope concentrations of the acid blanks relative to a typical Ca concentration session (10 ppm) were: Li⁷ < 7%, B¹¹ < 0.75%, Mg²⁵ < 0.5%, Sr⁸⁷ < 0.01%, Ca⁴³ < 0.1%. External reproducibility is reported relative to the consistency standard CamWuellestorf, as published by [43] and measurements of the NEP coral. Our data were within one standard error of published values (Table S5). The analytical uncertainties (2sd) on the B/Ca ratios were 15 µmol/mol.

## 2.7. Modelled pH<sub>CF</sub>

The pH of the calcifying fluid (pH<sub>CF</sub>) was calculated from the boron isotopic composition of the coral skeleton following rationale from Hemming and Hanson [50] and using the MATLAB code provided in De Carlo et al. [51], where δ¹¹B<sub>SW</sub> represents the boron isotopic composition of seawater (δ¹¹B<sub>SW</sub> = 39.61‰) [52] and δ¹¹B<sub>CARB</sub> represents the boron isotopic composition of the coral skeleton. The isotopic fractionation factor between the boron forms from Klochko et al. (2006) was used, α<sub>(B3-B4)</sub> = 1.0272. The dissociation constant of boron (K<sub>B</sub>) was calculated from salinity, temperature and pressure [53], and the boron concentration was calculated from salinity after Lee et al., 2010 [54].

$$ \text{pH}_{\text{CF}} = \text{pK}_{\text{B}} - \log\left\{-\frac{[\delta^{11}\text{B}_{\text{SW}} - \delta^{11}\text{B}_{\text{CARB}}]}{[\delta^{11}\text{B}_{\text{SW}} - \alpha_{(\text{B3-B4})} * \delta^{11}\text{B}_{\text{CARB}} - \varepsilon_{(\text{B3-B4})}]}\right\} \quad (1) $$

[H⁺]<sub>SW</sub> and [H⁺]<sub>CF</sub> were calculated based on pH<sub>SW</sub> and pH<sub>CF</sub> with [H⁺] = 10⁻⁵pH. The proton gradient [H⁺]<sub>SW</sub> - [H⁺]<sub>CF</sub> was evaluated to better visualize the scale of pH regulation in comparison to a logarithmic scale.

## 2.8. Modelled [CO₃²⁻]<sub>CF</sub>, DIC<sub>CF</sub>, and Ω<sub>CF</sub>

DIC<sub>CF</sub> and [CO₃²⁻]<sub>CF</sub> were calculated using the method and Matlab® code from DeCarlo et al. [51]. This study uses combined B/Ca and δ¹¹B to calculate [CO₃²⁻]<sub>CF</sub>, where:

$$ [\text{CO}_3^{2-}]_{\text{CF}} = K_D * \frac{[\text{B(OH)}_4^-]}{\text{B/Ca}} \quad (2) $$

We use the KD defined in McCulloch et al. [33], KD = 0.00297*exp(-0.0202*[H⁺]), where [H⁺] is determined based on pH<sub>CF</sub> as calculated from δ¹¹B following Equation (1). Given pH<sub>CF</sub> and [CO₃²⁻]<sub>CF</sub>, DIC<sub>CF</sub> can be calculated using the known dissociation constants for carbonate speciation at known temperatures and pressure. Ω<sub>CF</sub> can also be calculated but requires and additional assumption on the [Ca²⁺] of the calcifying fluid. The saturation state of aragonite in the calcifying medium (Ω<sub>CF</sub>) defined following Equation (3)
is dependent on the accuracy of the $[CO_3^{2-}]_{CF}$ and is limited to the additional assumption on the $[Ca^{2+}]$ of the calcifying fluid.

$$[\Omega]_{CF} = \frac{[CO_3^{2-}]_{CF} \times [Ca^{2+}]_{CF}}{K_{sp}} \quad (3)$$

We calculated the saturation state of aragonite of the calcifying fluid using the $[Ca^{2+}]_{sw}$ determined using the salinity of the culture condition ($[Ca^{2+}]_{sw}$ (mol/kg sw) = 0.02128/40.1* ((Salinity - 0.03)/1.805)).

## 2.9. Carbon Stable Isotope Analyses

Analyses on Caribbean corals were conducted on two Nu Perspective isotope ratio mass spectrometers (IRMS) at UCLA [55]. Most samples were analyzed using a Nu Carb specific sample preparation system. This system reacts 0.48 mg (±0.03 mg) of pure calcium carbonate material, for 20 min at 70 °C in individual reaction vials, thus eliminating any potential memory effects that are associated with analyses using a common acid bath system. Gases released by acid digestion of $CaCO_3$ are purified in a series of liquid N2-cooled, temperature controlled cold-fingers, an Adsorption Trap (AdTrap), an in-line, short Gas Chromatograph (GC) column packed with Porapak Type-QTM50/80 and silver wool, before introduction to the mass-spectrometer dual inlet for isotope analysis. The second digestion system is the common acid bath (CAB) system [55]. Analyses of *Stylophera pistillata* skeletal $\delta^{13}$C and $\delta^{18}$O was carried out on a GasBench II coupled to a Delta V mass spectrometer at the stable isotope facility of Pôle spectrometrie Océan (PSO), Plouzané, France. Results were calibrated to the Vienna Pee Dee Belemnite (V-PDB) scale and referenced to the international standard NBS19.

## 2.10. Statistical Methods

For elemental data, the most restrictive false discovery rate (Q = 0.1%) was used to thin data using the ROUT method set, meaning that <0.1% of identified outliers would have been falsely identified as such. To test for statistically significant changes in net calcification, we performed a one-way ANOVA followed by Dunnett's multiple comparisons tests on skeletal B/Ca composition, $\delta^{11}$B, $\delta^{13}$C, $pH_{CF}$, and $\Omega_{CF}$ across experimental treatment conditions relative to the control condition (28 °C, 400 µatm $pCO_2$). To determine the best fit by linear or second-order polynomial (quadratic) regressions, we used a combined linear and quadratic fit test, using the Akaike's Information Criteria (AIC). We compared the best-fit regressions across temperature conditions by first determining if the slopes of the two regressions are statistically different, and if the slopes are not significantly different ($p$-value > 0.05), we tested to see if the intercepts were significantly different ($p$-value < 0.05). This was done using an Analysis of Covariance (ANCOVA) method. The statistical analyses above were carried out GraphPad Prism version 9.0.0 for macOS, GraphPad Software, San Diego, CA, USA.

$\delta^{11}$B, B/Ca, and $\delta^{13}$C data were tested to see if it met assumptions of normality using the Shapiro-Wilks test ($p$ > 0.05). At the level $p$ < 0.05 we found that most of the data were not normally distributed. In order to perform a two-way ANOVA test, we transformed the data using the Tukey ladder of powers method to reduce negative and positive skew of non-normal data methodologically [56]. Method until it met the test of normality. Table S6 gives information about whether data were transformed and how they were transformed. Following confirmation that the data were normal, we analyzed isotope and elemental data using a two-way ANOVA to test the influence of the individual and combined effects of $pCO_2$ and temperature. The same approach was taken to analyze the calculated parameters $pH_{CF}$, $[CO_3^{2-}]_{CF}$, and DIC$_{CF}$ and the coral and symbiont physiological data; calcification rate, symbiont cell density, chlorophyll a, and total host energy data from [38,39]. All statistical analysis was conducted using the 'MASS' package in R version 4.0.2 (R Core Development Team 2016, Vienna, Austria).
As coral species were collected from inshore and offshore reef environments, so we also tested whether there were significant effects of temperature, pCO₂, and reef environment on geochemical and physiological parameters in a three-way ANOVA. As this analysis did not resolve a significant effect of reef environments, it justified the pooling of data across reef environments to conduct the two-way ANOVA tests described above.

## 2.11. Data Compilation

Data from previous publications was compiled to construct synthesis figures on coral calcifying fluid chemistry derived from geochemistry and other approaches such as electrodes and pH sensitive dyes. Literature data used for this metanalysis is summarized in Table S12 and was derived from the following publications [7,17,32,34,35,47,57–70]. In order to include the data from Allison et al., 2014 [32] on the carbonate chemistry of the calcification fluid from *P. damicornis* cultured under different concentrations of the ruthenium red (RR, 3.7 and 5.3 µM) inhibitor of Ca-ATPase activity, new calculations of the original data were performed using the framework described in this paper. Carbonate chemistry of the cultured experiments were calculated from alkalinity and pH provided in a previous publication [71] and using the CO₂sys program [72].

# 3. Results

## 3.1. *Analyses of (δ¹¹B), B/Ca, and δ¹³C*

Mean boron geochemistry (δ¹¹B and B/Ca) and δ¹³C for each experimental condition of the Caribbean coral experiment are reported in Table 1, individual coral specimen data are reported in Table S7. B/Ca (±SD) measurements for the control culturing conditions were averaged for each species under the experimental control conditions of 28 °C and 405 µatm pCO₂: *P. astreoides* (437 ± 31 µmol/mol), *P. strigosa* (495 ± 70 µmol/mol), *S. siderea* (436 ± 18 µmol/mol), *U. tenuifolia* (518 ± 32 µmol/mol). Under the experimental control culturing conditions, coral skeletal aragonite δ¹¹B compositions (±SD) were averaged for each species: *P. astreoides* (24.54 ± 0.74‰), *P. strigosa* (24.40 ± 1.40‰), *U. tenuifolia* (24.63 ± 0.68‰), and *S. siderea* (23.87 ± 0.47‰). Mean δ¹³C (±SD) for each species under the control temperature and pCO₂ experimental condition (28 °C, 405 µatm pCO₂) was: *P. astreoides* (–2.58 ± 0.92‰), *P. strigosa* (–1.11 ± 1.51‰), *S. siderea* (–3.94 ± 0.61‰), and *U. tenuifolia* (–2.65 ± 0.34‰) (Table 1).

Boron geochemical data for *S. pistillata* have previously been reported in Guillermic et al. Here, we report δ¹³C and δ¹⁸O data from *S. pistillata* skeletal aragonite from this additional experiment on Indo-Pacific corals, given in Table S8. The principal reason for presenting these data in this study is that they provide context for the interpretation of data from the Caribbean corals, as described in more detail below.

Trends in measured geochemical parameters as a function of culture seawater pH (pH<sub>SW</sub>) were explored using Akaike's Information Criteria (AIC) to determine whether a linear or second-order polynomial (quadratic) regression best fit the data (Figure 2; Table S9). In all cases, a linear regression was found to fit the data better, with the exception of δ¹¹B and B/Ca data for *S. siderea*, where a nonlinear fit was preferred (Figure 2, Table S9). In terms of geochemical proxy development for historical seawater pH reconstruction, the three species that exhibited relationships between δ¹¹B and pH<sub>SW</sub> that were best described by linear regressions seem best suited for this endeavor. *P. strigosa* showing the greatest sensitivity of δ¹¹B to pH<sub>SW</sub>, with a regression slope of 2.15 compared to 1.46 in *P. astreoides* and *U. tenuifolia* at the 28 °C temperature condition. However, *S. siderea* showed the greatest sensitivity with a slope of the linear regression between δ¹¹B and pH<sub>SW</sub> of 2.52 but was best fit by a nonlinear regression. At 31 °C, it is notable that the relationship between δ¹¹B and pH<sub>SW</sub> within *P. strigosa* was no longer statistically significant, indicating a potential influence of temperature on δ¹¹B in this species, although this was not significant in the ANOVA tests described below (Figure 2, Tables S9 and S11).
<table><caption>**Table 1.** Average Geochemical Data per Experimental Condition.</caption><thead><tr><th>Species</th><th>T (°C)</th><th>CO<sub>2</sub> (μatm)</th><th>δ<sup>11</sup>B (‰)<sup>1</sup></th><th>Error (SD)</th><th>N*</th><th>B/Ca (μmol/mol)</th><th>Error (SD)</th><th>N</th><th>δ<sup>13</sup>C (‰)<sup>2</sup></th><th>Error (SD)</th><th>N</th></tr></thead><tbody><tr><td rowspan="8">P. astreoides</td><td>28</td><td>311</td><td>23.2</td><td>0.9</td><td>6</td><td>396</td><td>54</td><td>6</td><td>-2.3</td><td>0.5</td><td>4</td></tr><tr><td>28</td><td>405</td><td>23.6</td><td>0.9</td><td>6</td><td>437</td><td>31</td><td>6</td><td>-2.6</td><td>0.9</td><td>4</td></tr><tr><td>28</td><td>702</td><td>22.7</td><td>1.4</td><td>5</td><td>432</td><td>53</td><td>4</td><td>-5.2</td><td>1.5</td><td>4</td></tr><tr><td>28</td><td>3309</td><td>22.1</td><td>0.8</td><td>6</td><td>410</td><td>46</td><td>6</td><td>12.5</td><td>3.1</td><td>3</td></tr><tr><td>31</td><td>288</td><td>24.1</td><td>0.1</td><td>1</td><td>389</td><td>17</td><td>1</td><td>-1.4</td><td>0.0</td><td>1</td></tr><tr><td>31</td><td>442</td><td>23.4</td><td>1.3</td><td>5</td><td>424</td><td>32</td><td>5</td><td>-2.6</td><td>0.7</td><td>4</td></tr><tr><td>31</td><td>674</td><td>23.1</td><td>0.4</td><td>5</td><td>443</td><td>82</td><td>5</td><td>-3.5</td><td>1.3</td><td>4</td></tr><tr><td>31</td><td>3285</td><td>22.6</td><td>0.5</td><td>2</td><td>568</td><td>51</td><td>2</td><td>-5.0</td><td>1.2</td><td>2</td></tr><tr><td rowspan="8">P. strigosa</td><td>28</td><td>312</td><td>24.2</td><td>0.6</td><td>6</td><td>496</td><td>43</td><td>6</td><td>-2.2</td><td>0.6</td><td>5</td></tr><tr><td>28</td><td>406</td><td>24.5</td><td>1.5</td><td>5</td><td>495</td><td>70</td><td>4</td><td>-1.1</td><td>1.5</td><td>3</td></tr><tr><td>28</td><td>702</td><td>23.3</td><td>0.7</td><td>6</td><td>481</td><td>35</td><td>6</td><td>-4.0</td><td>0.6</td><td>4</td></tr><tr><td>28</td><td>3320</td><td>22.2</td><td>1.3</td><td>6</td><td>455</td><td>41</td><td>6</td><td>10.9</td><td>1.6</td><td>4</td></tr><tr><td>31</td><td>288</td><td>23.9</td><td>0.9</td><td>4</td><td>527</td><td>44</td><td>4</td><td>-1.8</td><td>0.6</td><td>4</td></tr><tr><td>31</td><td>443</td><td>23.9</td><td>0.8</td><td>5</td><td>495</td><td>50</td><td>5</td><td>-1.3</td><td>0.7</td><td>4</td></tr><tr><td>31</td><td>674</td><td>23.7</td><td>0.5</td><td>4</td><td>528</td><td>33</td><td>4</td><td>-3.4</td><td>0.4</td><td>4</td></tr><tr><td>31</td><td>3284</td><td>23.0</td><td>1.5</td><td>4</td><td>530</td><td>54</td><td>2</td><td>-6.4</td><td>2.9</td><td>4</td></tr><tr><td rowspan="8">S. siderea</td><td>28</td><td>312</td><td>24.5</td><td>0.7</td><td>6</td><td>450</td><td>26</td><td>6</td><td>-3.3</td><td>0.2</td><td>4</td></tr><tr><td>28</td><td>405</td><td>23.9</td><td>0.5</td><td>6</td><td>436</td><td>18</td><td>6</td><td>-3.9</td><td>0.6</td><td>4</td></tr><tr><td>28</td><td>703</td><td>22.7</td><td>0.9</td><td>6</td><td>424</td><td>21</td><td>6</td><td>-6.2</td><td>1.1</td><td>4</td></tr><tr><td>28</td><td>3317</td><td>21.8</td><td>1.2</td><td>6</td><td>415</td><td>27</td><td>6</td><td>12.5</td><td>1.7</td><td>3</td></tr><tr><td>31</td><td>288</td><td>24.2</td><td>0.4</td><td>5</td><td>467</td><td>11</td><td>5</td><td>-2.6</td><td>0.2</td><td>3</td></tr><tr><td>31</td><td>449</td><td>23.4</td><td>0.7</td><td>6</td><td>425</td><td>22</td><td>6</td><td>-2.6</td><td>1.4</td><td>4</td></tr><tr><td>31</td><td>673</td><td>22.1</td><td>0.7</td><td>5</td><td>432</td><td>10</td><td>4</td><td>-5.5</td><td>1.4</td><td>3</td></tr><tr><td>31</td><td>3285</td><td>21.3</td><td>1.4</td><td>6</td><td>428</td><td>21</td><td>6</td><td>14.3</td><td>2.0</td><td>3</td></tr><tr><td rowspan="4">U. tenuifolia</td><td>28</td><td>312</td><td>24.59</td><td>0.68</td><td>4</td><td>490</td><td>51</td><td>4</td><td>-2.2</td><td>0.4</td><td>4</td></tr><tr><td>28</td><td>404</td><td>24.25</td><td>0.56</td><td>4</td><td>518</td><td>32</td><td>4</td><td>-2.6</td><td>0.3</td><td>4</td></tr><tr><td>28</td><td>698</td><td>23.54</td><td>1.25</td><td>4</td><td>464</td><td>76</td><td>4</td><td>-3.8</td><td>1.0</td><td>4</td></tr><tr><td>28</td><td>3303</td><td>22.53</td><td>1.94</td><td>4</td><td>461</td><td>62</td><td>4</td><td>-7.2</td><td>2.8</td><td>4</td></tr></tbody></table>

To further constrain trends in the data, two-way ANOVA tests were used to explore the individual and combined effects of pCO₂ and temperature on geochemical parameters. For *P. astreoides*, pCO₂ alone had a significant individual effect on δ¹¹B and δ¹³C, while temperature had no effect on geochemical parameters. However, the combined effect of temperature and pCO₂ was found to significantly impact B/Ca. For *P. strigosa*, pCO₂ had a significant effect on δ¹¹B and δ¹³C, and temperature only influenced B/Ca. In *S. siderea*, pCO₂ had a significant effect on δ¹¹B, δ¹³C, and B/Ca, whereas temperature had an effect on δ¹³C alone. Lastly, in *U. tenuifolia*, only pCO₂ had a significant effect on δ¹³C, and no temperature effects were observed. Results of these analyses are presented in Tables 2 and S10.

Coral specimens were collected from both the inshore and offshore reef environments (Supplementary Table S3). Although previous work showed that reef environment had an effect on the physiological response of the corals [39], we found no significant effect of reef environment on the geochemical data or on the carbonate system parameters calculated for the coral calcifying fluid (Table S10). A lack of effect of local reef environment in skeleton boron and carbon geochemistry is a positive feature in terms of using the species as palaeoceanographic archives.

<footnote>* number of specimens analyzed; ¹ NIST; ² V-PDB.</footnote>
<figure><img src="image_6.png" /><figcaption>**Figure 2.** Measured δ¹¹B (A), δ¹³C (B), and B/C (C) composition of coral aragonite following the 93-day culturing experiment. Large triangular symbols represent the mean value (±1SD) for each treatment condition. Blue symbols represent the control temperature (28 °C) treatment condition and red represents the high temperature (31 °C) treatment condition. Linear versus centered quadratic fit was determined using the Akaike's Information Criteria test (Table S8), with shaded areas representing the 95% confidence interval. Dashed blue and red curves represent the expected δ¹¹B composition of borate ion (B(OH)⁴⁻) in solution at 28 °C and 31 °C, respectively. * indicates a significant difference between 28 °C and 31 °C data for a given pCO₂ treatment as demonstrated by a *p*-value < 0.05 in a Welch's T test.</figcaption></figure>

It is important to note that the principal driver of trends in δ¹³C data across variable pCO₂ and pH<sub>SW</sub> treatments is the isotopic composition of the CO₂ gas used to manipulate the pCO₂ of the treatment seawater. This gas is combustion sourced and therefore has a relatively negative δ¹³C composition that influences the δ¹³C of DIC more in cultures with higher pCO₂. Despite these trends being influenced by the δ¹³C of the source gas, there is potential to meaningfully interpret the difference between the δ¹³C values between the two temperature treatments for a given pCO₂. For example, Welch's T-Tests indicate a statistically significant difference in δ¹³C data between the 28 °C and 31 °C treatments at the highest pCO₂ treatment for *P. astreoides* and *P. strigosa*, but not at the lowest pCO₂ condition (Figure 3). Additionally, ANCOVA tests reveal the linear regressions through δ¹³C data as a function of pCO₂ for both *P. astreoides* and *P. strigosa* have significantly different slopes at 28 °C versus 31 °C (*p* < 0.0001 and *p* = 0.0021, respectively), while *S. siderea* did not exhibit a significant change in slope across temperature conditions (*p* = 0.102). These trends in δ¹³C are likely to reflect changes in the sources of DIC for calcification linked to the coral host and symbiont physiological responses to temperature and pCO₂, as further explored in the following sections.
<figure><img src="image_7.png" /><figcaption>**Figure 3.** (A) Measured net calcification rate (mg cm⁻² d⁻¹), replotted from Bove et al. [38]. (B) δ¹¹B-derived pH<sub>CF</sub>. Triangle symbols represent the mean value (±1 SD) for each treatment condition, blue and red symbols represent the 28 °C and 31 °C treatments, respectively. Linear versus centered quadratic fit was determined using the Akaike's Information Criteria test (Table S8), with shading representing the 95% confidence interval. * indicates a significant difference between 28 °C and 31 °C data for a given pCO₂ treatment, as demonstrated by a *p*-value < 0.05 in a Welch's T-test.</figcaption></figure>
<table><caption>**Table 2.** 2-way ANOVA output testing the influence of temperature and pCO₂ on measured geochemical parameters.</caption><thead><tr><th>Geochemical Parameter</th><th>Species</th><th>Experimental Variable</th><th>ANOVA p-Value</th></tr></thead><tbody><tr><td rowspan="6">δ<sup>11</sup>B</td><td rowspan="2">P. astreoides</td><td>Temperature</td><td>0.1171</td></tr><tr><td>CO<sub>2</sub></td><td><strong>0.0152</strong></td></tr><tr><td rowspan="2">P. strigosa</td><td>Temperature + CO<sub>2</sub></td><td>0.6094</td></tr><tr><td>CO<sub>2</sub></td><td>0.5521</td></tr><tr><td rowspan="2">S. siderea</td><td>Temperature + CO<sub>2</sub></td><td><strong>0.00532</strong></td></tr><tr><td>CO<sub>2</sub></td><td>0.59757</td></tr><tr><td rowspan="12">δ<sup>13</sup>C</td><td rowspan="2">U. tenuifolia</td><td>Temperature</td><td>0.36</td></tr><tr><td>CO<sub>2</sub></td><td><strong>7.58 × 10<sup>-10</sup></strong></td></tr><tr><td rowspan="2">P. astreoides</td><td>Temperature + CO<sub>2</sub></td><td>0.441</td></tr><tr><td>CO<sub>2</sub></td><td>0.322</td></tr><tr><td rowspan="2">P. strigosa</td><td>Temperature</td><td>0.152</td></tr><tr><td>CO<sub>2</sub></td><td><strong>4.12 × 10<sup>-5</sup></strong></td></tr><tr><td rowspan="2">S. siderea</td><td>Temperature + CO<sub>2</sub></td><td>0.193</td></tr><tr><td>CO<sub>2</sub></td><td>0.5521</td></tr><tr><td rowspan="2">U. tenuifolia</td><td>Temperature + CO<sub>2</sub></td><td><strong>0.00532</strong></td></tr><tr><td>CO<sub>2</sub></td><td>0.59757</td></tr><tr><td rowspan="12">B/Ca</td><td rowspan="2">P. strigosa</td><td>Temperature</td><td>0.36</td></tr><tr><td>CO<sub>2</sub></td><td><strong>7.58 × 10<sup>-10</sup></strong></td></tr><tr><td rowspan="2">S. siderea</td><td>Temperature + CO<sub>2</sub></td><td>0.441</td></tr><tr><td>CO<sub>2</sub></td><td>0.322</td></tr><tr><td rowspan="2">P. astreoides</td><td>Temperature</td><td>0.22201</td></tr><tr><td>CO<sub>2</sub></td><td>0.78951</td></tr><tr><td rowspan="2">P. strigosa</td><td>Temperature + CO<sub>2</sub></td><td><strong>0.00939</strong></td></tr><tr><td>CO<sub>2</sub></td><td><strong>0.00911</strong></td></tr><tr><td rowspan="2">S. siderea</td><td>Temperature</td><td>0.74462</td></tr><tr><td>CO<sub>2</sub></td><td>0.16759</td></tr><tr><td rowspan="2">U. tenuifolia</td><td>Temperature + CO<sub>2</sub></td><td><strong>0.00103</strong></td></tr><tr><td>CO<sub>2</sub></td><td>0.84438</td></tr></tbody></table>

### 3.2. Net Calcification, pH<sub>CF</sub>, [CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub>, and DIC<sub>CF</sub>

Net calcification rates for the Caribbean corals were reported in Bove et al. [38], which found that all four coral species showed non-linear reductions in net calcification with increasing pCO₂. Corals showed interspecific differences, with *S. siderea* maintaining positive net calcification under all experimental conditions, *P. strigosa* exhibited a shift to negative net calcification at 31 °C only, and *P. astreoides* exhibited an increase in calcification with elevated temperature at the lower pCO₂ condition, but a shift toward negative calcification (i.e., net dissolution) under the higher pCO₂ treatments (Figure 3).

The pH of the coral calcifying fluid (pH<sub>CF</sub>) was calculated from δ<sup>11</sup>B data using canonical methods from the literature, as described in the methods section. Although the δ<sup>11</sup>B approach to calculating pH<sub>CF</sub> in corals has been independently verified based on comparison with pH sensitive dye and pH microelectrode approaches [7,30,47,61,73], significant differences in sensitivities in those approaches appear to exist, and different hypotheses have been advanced to explain these differences [7,47]. At the control temperature and pCO₂ experimental condition (28 °C, 405 µatm pCO₂), mean δ<sup>11</sup>B-derived pH<sub>CF</sub> (total scale, ±1 SD) for each species was as follows: *P. astreoides* (8.45 ± 0.06), *P. strigosa* (8.50 ± 0.11), *S. siderea* (8.47 ± 0.03), *U. tenuifolia* (8.49 ± 0.04) (Table 3).

The [CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub> and DIC<sub>CF</sub> were calculated from δ<sup>11</sup>B and B/Ca data using the K<sub>D</sub> of McCulloch et al. [74] and the approach of DeCarlo et al. [51] (Table 3), as described in the methods section. The B/Ca proxy is still prone to uncertainties [70,75] and would benefit from additional validation. One critique is that the proxy is based on limited inorganic precipitation datasets [76,77] which have been difficult to reconcile due to different experimental conditions and from varying [Ca<sup>2+</sup>] in the experiment leading in turn
to varying saturation states of aragonite (Ω) within the precipitation experiments [70]. This
leads to potential difficulties when translating to highly biologically controlled fluids (such
as the calcifying fluid in corals). Additionally, it is possible that coral biomineralization
involves transformation of amorphous precursor phases, as evidenced by the detection of
amorphous calcium carbonate (ACC) in corals [78], although it is unclear whether this is
the universal pathway by which corals calcify. Amorphous or metastable phases of calcium
carbonate may have different K<sub>D</sub>, which could contribute to differences in biomineral
chemistry [79,80], although given that ACC transformation seems to occur via dissolution
and reprecipitation into crystalline phases [81], precursor phase K<sub>D</sub> may not be relevant for
the final mineral. Despite these uncertainties, results of Guillermic et al. [7] showed that
the K<sub>D</sub> formulations used by DeCarlo et al. [51] (Termed D18); McCulloch et al. [74] (M17);
Holcomb et al. [77] (H16) resulted in similar reconstructed [CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub> among biological
replicates and that the outcome was within error of independent measurements from
[CO<sub>3</sub><sup>2-</sup>] microelectrodes as reported by Sevilgen et al. [35] (S19) for the same coral species
(S. pistillata) (M17: 909 ± 313 (SD, n = 8), H16: 757 ± 342 (SD, n = 8), D18: 848 ± 392 (SD,
n = 7), S19: 679 ± 183 (SD); at 311 ppm pCO<sub>2</sub>, 28 °C) [7].

Regression analysis of net calcification, calculated pH<sub>CF</sub>, [CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub>, and DIC<sub>CF</sub> as a function of pH<sub>SW</sub> were explored using an AIC approach (Figures 3 and 4; Table S9). pH<sub>CF</sub> as a function of pH<sub>SW</sub> follows a similar pattern to the originating δ<sup>11</sup>B data (Figure 3; Table S9). Patterns in [CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub> as a function of pH<sub>SW</sub> differ from patterns in pH<sub>CF</sub> as a function of pH<sub>SW</sub>, with only *P. strigosa* and *S. siderea* showing statistically significant regressions under the 28 °C treatment (Figure 4; Table S9).

In addition, regression analyses of net calcification as a function of pH<sub>CF</sub>, [CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub>, and [DIC]<sub>CF</sub> were explored using the AIC approach. Here, linear regressions were typically favored but rarely reached statistical significance (Table S9). This result is not surprising given the previous findings of Liu et al. [69] that pH<sub>CF</sub> is not necessarily tightly coupled to net calcification responses to seawater acidification in a wide range of marine calcifiers, including the temperate coral *Oculina arbuscula*. Net calcification represents a balance of dissolution and calcification and will be influenced by both physicochemical and other controls over calcification, all of which may confound a clear relationship with pH<sub>CF</sub>.

Two-way ANOVA tests were again used to explore the individual and combined effects of *pCO*₂ and temperature on calculated calcifying fluid parameters of pH<sub>CF</sub>, [CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub>, and DIC<sub>CF</sub>. In *P. astreoides*, *pCO*₂ had a significant effect on pH<sub>CF</sub> and [CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub>. Temperature and the combined effect of temperature and *pCO*₂ had significant effects on DIC<sub>CF</sub>. Similarly, in *P. strigosa*, *pCO*₂ had a significant effect on pH<sub>CF</sub> and [CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub>, but temperature only had an individual effect on DIC<sub>CF</sub>. In *S. siderea*, the calculated pH<sub>CF</sub> data did not meet assumptions of normality after transformation and, consequently, ANOVA results are not presented. There was a significant effect of *pCO*₂ on [CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub> of *S. siderea* and significant individual effects of both *pCO*₂ and temperature on DIC<sub>CF</sub>. Lastly, U. tenuifolia displayed no significant effects of either *pCO*₂ or temperature on the calculated calcifying fluid parameters. Results of these analyses are presented in Table 4.
<figure><img src="image_8.png" /><figcaption>**Figure 4.** (A) Measured net calcification rate (mg cm⁻² d⁻¹) replotted from Bove et al. [38] (B) B/Ca-derived carbonate ion concentration of the coral calcifying fluid ([CO₃²⁻]CF, µmol/mol), and (C) combined δ¹¹B and B/Ca-derived dissolved inorganic carbon concentration of the coral calcifying fluid (DICCF, µmol/mol). Triangle symbols (±1 SD) represent the mean value for each treatment condition, blue and red symbols represent the 28 °C and 31 °C treatment conditions, respectively. Linear versus centered quadratic fit was determined using the Akaike Information Criteria test (Table S8), with shading representing the 95% confidence interval. * indicates a significant difference between 28 °C and 31 °C data for a given pCO₂ treatment, as demonstrated by a *p*-value < 0.05 in a Welch's-T test.</figcaption></figure>

<table><caption>**Table 3.** Net calcification data and calculated calcifying fluid pH and carbonate system parameters.</caption><thead><tr><th>Species</th><th>T (°C)</th><th>CO<sub>2</sub> (µatm)</th><th>Net Calcification (mg cm<sup>-2</sup> d<sup>-1</sup>)</th><th>Error (SD)</th><th>N<sup>1</sup></th><th>pH<sub>CF</sub></th><th>Error (SD)</th><th>DIC<sub>CF</sub> (mmol/mol)</th><th>Error (SD)</th><th>[CO<sub>3</sub>²⁻]CF (mmol/mol)</th><th>Error (SD)</th><th>Ω<sub>CF</sub><sup>2</sup></th><th>Error (SD)</th><th>N</th></tr></thead><tbody><tr><td rowspan="6">P. astreoides</td><td>28</td><td>311</td><td>-0.08</td><td>0.26</td><td>11</td><td>8.42</td><td>0.06</td><td>4983</td><td>863</td><td>1131</td><td>181</td><td>20.9</td><td>3.4</td><td>6</td></tr><tr><td>28</td><td>405</td><td>0.07</td><td>0.52</td><td>12</td><td>8.45</td><td>0.06</td><td>4421</td><td>352</td><td>1053</td><td>81</td><td>19.5</td><td>1.5</td><td>6</td></tr><tr><td>28</td><td>702</td><td>-0.17</td><td>0.4</td><td>10</td><td>8.39</td><td>0.10</td><td>4561</td><td>618</td><td>957</td><td>180</td><td>17.8</td><td>3.3</td><td>4</td></tr><tr><td>28</td><td>3309</td><td>-0.65</td><td>0.5</td><td>12</td><td>8.35</td><td>0.05</td><td>4841</td><td>512</td><td>969</td><td>102</td><td>17.9</td><td>1.9</td><td>6</td></tr><tr><td>31</td><td>288</td><td>0.5</td><td>0.5</td><td>6</td><td>8.45</td><td>0</td><td>4777</td><td>0</td><td>1242</td><td>0</td><td>23</td><td>0</td><td>1</td></tr><tr><td>31</td><td>442</td><td>0.04</td><td>0.29</td><td>8</td><td>8.40</td><td>0.09</td><td>4434</td><td>394</td><td>1055</td><td>107</td><td>19.5</td><td>2</td><td>5</td></tr><tr><td rowspan="6">P. strigosa</td><td>31</td><td>674</td><td>-0.02</td><td>0.24</td><td>9</td><td>8.38</td><td>0.02</td><td>4365</td><td>703</td><td>1004</td><td>133</td><td>18.6</td><td>2.5</td><td>5</td></tr><tr><td>31</td><td>3285</td><td>-0.54</td><td>0.27</td><td>3</td><td>8.35</td><td>0.03</td><td>3356</td><td>286</td><td>736</td><td>106</td><td>13.6</td><td>2</td><td>2</td></tr><tr><td>28</td><td>312</td><td>0.92</td><td>1.28</td><td>13</td><td>8.49</td><td>0.04</td><td>3862</td><td>362</td><td>989</td><td>59</td><td>18.3</td><td>1.1</td><td>6</td></tr><tr><td>28</td><td>406</td><td>1</td><td>1.12</td><td>6</td><td>8.50</td><td>0.10</td><td>3881</td><td>590</td><td>1013</td><td>85</td><td>18.7</td><td>1.6</td><td>4</td></tr><tr><td>28</td><td>702</td><td>0.66</td><td>0.79</td><td>14</td><td>8.43</td><td>0.04</td><td>4025</td><td>311</td><td>933</td><td>47</td><td>17.3</td><td>0.9</td><td>6</td></tr><tr><td>28</td><td>3320</td><td>0.03</td><td>0.71</td><td>16</td><td>8.36</td><td>0.08</td><td>4339</td><td>452</td><td>876</td><td>68</td><td>16.2</td><td>1.3</td><td>6</td></tr><tr><td>31</td><td>288</td><td>0.41</td><td>0.3</td><td>9</td><td>8.43</td><td>0.06</td><td>3269</td><td>9</td><td>889</td><td>12</td><td>16.4</td><td>0.2</td><td>2</td></tr><tr><td>31</td><td>443</td><td>-0.32</td><td>0.74</td><td>6</td><td>8.43</td><td>0.05</td><td>3782</td><td>399</td><td>959</td><td>134</td><td>17.8</td><td>2.5</td><td>5</td></tr><tr><td>31</td><td>674</td><td>-0.23</td><td>0.55</td><td>7</td><td>8.42</td><td>0.03</td><td>3450</td><td>109</td><td>850</td><td>78</td><td>15.7</td><td>1.4</td><td>3</td></tr><tr><td>31</td><td>3284</td><td>-0.5</td><td>0.39</td><td>8</td><td>8.37</td><td>0.10</td><td>3609</td><td>423</td><td>759</td><td>35</td><td>14</td><td>0.6</td><td>2</td></tr></tbody></table>
<table><caption>**Table 3. Cont.**</caption><thead><tr><th>Species</th><th>T (°C)</th><th>CO<sub>2</sub> (µatm)</th><th>Net Calcification (mg cm<sup>-2</sup> d<sup>-1</sup>)</th><th>Error (SD)</th><th>N<sup>1</sup></th><th>pH<sub>CF</sub></th><th>Error (SD)</th><th>DIC<sub>CF</sub> (mmol/mol)</th><th>Error (SD)</th><th>[CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub> (mmol/mol)</th><th>Error (SD)</th><th>Ω<sub>CF</sub><sup>2</sup></th><th>Error (SD)</th><th>N</th></tr></thead><tbody><tr><td rowspan="6">S. siderea</td><td>28</td><td>312</td><td>1.30</td><td>0.57</td><td>10</td><td>8.51</td><td>0.05</td><td>4216</td><td>261</td><td>1115</td><td>76</td><td>20.6</td><td>1.4</td><td>6</td></tr><tr><td>28</td><td>405</td><td>1.37</td><td>0.70</td><td>12</td><td>8.47</td><td>0.03</td><td>4391</td><td>192</td><td>1087</td><td>63</td><td>20.1</td><td>1.2</td><td>6</td></tr><tr><td>28</td><td>703</td><td>1.08</td><td>0.45</td><td>11</td><td>8.39</td><td>0.06</td><td>4596</td><td>194</td><td>993</td><td>129</td><td>18.4</td><td>2.4</td><td>6</td></tr><tr><td>28</td><td>3317</td><td>0.13</td><td>0.52</td><td>11</td><td>8.33</td><td>0.08</td><td>4765</td><td>333</td><td>926</td><td>133</td><td>17.1</td><td>2.5</td><td>6</td></tr><tr><td>31</td><td>288</td><td>1.00</td><td>0.54</td><td>8</td><td>8.46</td><td>0.03</td><td>3969</td><td>117</td><td>1033</td><td>34</td><td>19.1</td><td>0.6</td><td>4</td></tr><tr><td>31</td><td>449</td><td>1.16</td><td>0.70</td><td>11</td><td>8.41</td><td>0.05</td><td>4407</td><td>252</td><td>1060</td><td>62</td><td>19.6</td><td>1.1</td><td>6</td></tr><tr><td rowspan="3">U. tenuifolia</td><td>31</td><td>673</td><td>1.06</td><td>0.51</td><td>11</td><td>8.32</td><td>0.05</td><td>4449</td><td>120</td><td>909</td><td>47</td><td>16.8</td><td>0.9</td><td>3</td></tr><tr><td>31</td><td>3285</td><td>0.43</td><td>0.53</td><td>12</td><td>8.26</td><td>0.10</td><td>4480</td><td>284</td><td>851</td><td>119</td><td>15.7</td><td>2.2</td><td>5</td></tr><tr><td>28</td><td>312</td><td>0.16</td><td>0.10</td><td>11</td><td>8.52</td><td>0.04</td><td>3886</td><td>449</td><td>1037</td><td>85</td><td>19.2</td><td>1.6</td><td>4</td></tr><tr><td rowspan="2">S. siderea</td><td>28</td><td>404</td><td>0.24</td><td>0.17</td><td>7</td><td>8.49</td><td>0.04</td><td>3676</td><td>188</td><td>950</td><td>102</td><td>17.6</td><td>1.9</td><td>4</td></tr><tr><td>28</td><td>698</td><td>0.05</td><td>0.08</td><td>4</td><td>8.44</td><td>0.08</td><td>4230</td><td>767</td><td>996</td><td>99</td><td>18.5</td><td>1.8</td><td>4</td></tr><tr><td>28</td><td>3303</td><td>-0.23</td><td>0.05</td><td>5</td><td>8.38</td><td>0.13</td><td>4286</td><td>657</td><td>908</td><td>198</td><td>16.8</td><td>3.7</td><td>4</td></tr></tbody></table>

<table><caption>**Table 4.** 2-way ANOVA output testing the influence of temperature and pCO₂ on calculated calcifying fluid carbonate system parameters and coral and symbiont physiological parameters.</caption><thead><tr><th colspan="2">Parameter/Species/Experimental Variable</th><th>p Value</th><th></th><th>p Value</th></tr></thead><tbody><tr><td colspan="2"><strong>Symbiont cell density</strong></td><td></td><td><strong>pH<sub>CF</sub></strong></td><td></td></tr><tr><td rowspan="3">P. astreoides</td><td>Temperature</td><td>0.417</td><td rowspan="3">P. astreoides</td><td>Temperature</td><td>0.676</td></tr><tr><td>CO<sub>2</sub></td><td>0.11</td><td>CO<sub>2</sub></td><td><strong>6.54 × 10<sup>-5</sup></strong></td></tr><tr><td>Temperature + CO<sub>2</sub></td><td>0.466</td><td>Temperature + CO<sub>2</sub></td><td>0.286</td></tr><tr><td rowspan="3">P. strigosa</td><td>Temperature</td><td><strong>2.59 × 10<sup>-11</sup></strong></td><td rowspan="3">P. strigosa</td><td>Temperature</td><td>0.42275</td></tr><tr><td>CO<sub>2</sub></td><td>0.648</td><td>CO<sub>2</sub></td><td><strong>0.00536</strong></td></tr><tr><td>Temperature: CO<sub>2</sub></td><td>0.15</td><td>Temperature + CO<sub>2</sub></td><td>0.62005</td></tr><tr><td rowspan="3">S. siderea</td><td>Temperature</td><td><strong>9.09 × 10<sup>-5</sup></strong></td><td rowspan="3">S. siderea</td><td>Temperature</td><td>Not Normal *</td></tr><tr><td>CO<sub>2</sub></td><td><strong>0.000251</strong></td><td>CO<sub>2</sub></td><td>Not Normal *</td></tr><tr><td>Temperature + CO<sub>2</sub></td><td>0.858348</td><td>Temperature + CO<sub>2</sub></td><td>Not Normal *</td></tr><tr><td>U. tenuifolia</td><td>CO<sub>2</sub></td><td>0.727</td><td>U. tenuifolia</td><td>CO<sub>2</sub></td><td>0.317</td></tr><tr><td colspan="2"><strong>Chl a</strong></td><td></td><td><strong>[CO<sub>3</sub><sup>2-</sup>]</strong></td><td></td></tr><tr><td rowspan="3">P. astreoides</td><td>Temperature</td><td><strong>9.89 × 10<sup>-7</sup></strong></td><td rowspan="3">P. astreoides</td><td>Temperature</td><td>0.41309</td></tr><tr><td>CO<sub>2</sub></td><td><strong>6.10 × 10<sup>-11</sup></strong></td><td>CO<sub>2</sub></td><td><strong>0.00333</strong></td></tr><tr><td>Temperature + CO<sub>2</sub></td><td><strong>2.80 × 10<sup>-9</sup></strong></td><td>Temperature + CO<sub>2</sub></td><td>0.22932</td></tr><tr><td rowspan="3">P. strigosa</td><td>Temperature</td><td><strong>6.15 × 10<sup>-8</sup></strong></td><td rowspan="3">P. strigosa</td><td>Temperature</td><td>0.0988</td></tr><tr><td>CO<sub>2</sub></td><td><strong>0.000217</strong></td><td>CO<sub>2</sub></td><td><strong>0.0224</strong></td></tr><tr><td>Temperature: CO<sub>2</sub></td><td>0.070441</td><td>Temperature: CO<sub>2</sub></td><td>0.855</td></tr><tr><td rowspan="3">S. siderea</td><td>Temperature</td><td><strong>0.00394</strong></td><td rowspan="3">S. siderea</td><td>Temperature</td><td>0.679</td></tr><tr><td>CO<sub>2</sub></td><td><strong>1.37 × 10<sup>-7</sup></strong></td><td>CO<sub>2</sub></td><td><strong>5.98 × 10<sup>-5</sup></strong></td></tr><tr><td>Temperature + CO<sub>2</sub></td><td>0.25756</td><td>Temperature + CO<sub>2</sub></td><td>0.996</td></tr><tr><td>U. tenuifolia</td><td>CO<sub>2</sub></td><td><strong>0.033</strong></td><td>U. tenuifolia</td><td>CO<sub>2</sub></td><td>0.181</td></tr><tr><td colspan="2"><strong>Total host energy</strong></td><td></td><td><strong>[DIC]<sub>CF</sub></strong></td><td></td></tr><tr><td rowspan="3">P. astreoides</td><td>Temperature</td><td><strong>0.02755</strong></td><td rowspan="3">P. astreoides</td><td>Temperature</td><td><strong>0.00163</strong></td></tr><tr><td>CO<sub>2</sub></td><td><strong>0.00659</strong></td><td>CO<sub>2</sub></td><td>0.69095</td></tr><tr><td>Temperature: CO<sub>2</sub></td><td>0.06197</td><td>Temperature + CO<sub>2</sub></td><td><strong>0.04313</strong></td></tr><tr><td rowspan="3">P. strigosa</td><td>Temperature</td><td><strong>8.35 × 10<sup>-5</sup></strong></td><td rowspan="3">P. strigosa</td><td>Temperature</td><td><strong>0.00395</strong></td></tr><tr><td>CO<sub>2</sub></td><td>0.103</td><td>CO<sub>2</sub></td><td>0.26074</td></tr><tr><td>Temperature + CO<sub>2</sub></td><td>0.857</td><td>Temperature + CO<sub>2</sub></td><td>0.3616</td></tr></tbody></table>

<footnote n="1">¹ Refers to number of specimens analyzed; ² Ω<sub>CF</sub> values calculated using the assumption of [Ca]<sub>CF</sub> = [Ca]<sub>SW</sub>.</footnote>
<table><caption>**Table 4. Cont.**</caption><thead><tr><th colspan="2">Parameter/Species/Experimental Variable</th><th>p Value</th><th></th><th>p Value</th></tr></thead><tbody><tr><td rowspan="2"><i>S. siderea</i></td><td>Temperature</td><td><strong>0.000522</strong></td><td rowspan="2"><i>S. siderea</i></td><td>Temperature</td><td><strong>0.00119</strong></td></tr><tr><td>CO<sub>2</sub></td><td>0.118746</td><td>CO<sub>2</sub></td><td><strong>2.62 × 10<sup>-5</sup></strong></td></tr><tr><td rowspan="2"><i>U. tenuifolia</i></td><td>Temperature + CO<sub>2</sub></td><td>0.710086</td><td rowspan="2"><i>U. tenuifolia</i></td><td>Temperature + CO<sub>2</sub></td><td>0.3765</td></tr><tr><td>CO<sub>2</sub></td><td>0.168</td><td>CO<sub>2</sub></td><td>0.463</td></tr><tr><td colspan="6"><strong>Calcification rate</strong></td></tr><tr><td rowspan="2"><i>P. astreoides</i></td><td>Temperature</td><td><strong>0.0178</strong></td><td rowspan="2"></td><td>Temperature</td><td><strong>0.000142</strong></td></tr><tr><td>CO<sub>2</sub></td><td><strong>5.11 × 10<sup>-6</sup></strong></td><td>CO<sub>2</sub></td><td><strong>0.000364</strong></td></tr><tr><td rowspan="2"><i>P. strigosa</i></td><td>Temperature + CO<sub>2</sub></td><td>0.1779</td><td rowspan="2"></td><td>Temperature + CO<sub>2</sub></td><td>0.678234</td></tr><tr><td>CO<sub>2</sub></td><td><strong>0.000142</strong></td><td>CO<sub>2</sub></td><td><strong>0.000364</strong></td></tr><tr><td rowspan="2"><i>S. siderea</i></td><td>Temperature</td><td>0.52</td><td rowspan="2"></td><td>Temperature</td><td><strong>0.000142</strong></td></tr><tr><td>CO<sub>2</sub></td><td><strong>3.27 × 10<sup>-8</sup></strong></td><td>CO<sub>2</sub></td><td><strong>0.000364</strong></td></tr><tr><td></td><td>Temperature + CO<sub>2</sub></td><td>0.209</td><td></td><td>Temperature + CO<sub>2</sub></td><td>0.209</td></tr></tbody></table>

We combined our data with a compilation of coral data from the literature using different approaches (geochemistry, microelectrode, pH-sensitive dye; Table S11). It is clear from almost all published studies that pH<sub>CF</sub>, [CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub>, and DIC<sub>CF</sub> are differentiated from seawater values and that this is an active compensatory process as pH<sub>CF</sub>, and [CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub> values are increasingly elevated relative to seawater values under acidified conditions (Figures 5 and 6).

<figure><img src="image_9.png" /><figcaption>**Figure 5.** Compilation of δ<sup>11</sup>B-derived calcification fluid pH<sub>CF</sub> data from shallow-water zooxanthellate coral culturing experiments. We include data from cold-water azooxanthellate corals as a point of reference, as well as ranges of pH<sub>CF</sub> data as determined by SNARF pH-sensitive dyes and pH microelectrode analyses. 'RR' label denotes corals treated with the Ca-ATPase inhibitor ruthenium red [32]. Previously published data used in the figure are available in Table S12 alongside citations for data sources.</figcaption></figure>

<footnote>* Data did not reach normality even after transformation steps, so ANOVA test was not performed.</footnote>
<figure><img src="image_10.png" /><figcaption>**Figure 6.** Compilation of combined δ¹¹B and B/C-derived DIC<sub>CF</sub> and [CO₃²⁻]₄F measurements from available published data, as well as published microelectrode data. We include cultured shallow-water zooxanthellate corals, cold-water azooxanthellate corals, as well as a range of DIC<sub>CF</sub> values as determined by SNARF-1 pH-sensitive dyes and pH microelectrode analyses. (A) DIC<sub>CF</sub> as a function of pH<sub>SW</sub> (total scale), (B) DIC<sub>CF</sub> as a function of DIC<sub>SW</sub>, and (C) [CO₃²⁻]₄F as a function of [CO₃²⁻]₅W. 'RR' label denotes corals treated with the Ca-ATPase inhibitor ruthenium red [32]. Previously published data used in the figure are available in Table S12 alongside citations for data sources.</figcaption></figure>

### 3.3. Biological Compensation for External pH Changes, and the Importance of Coral and Symbiont Physiology

Changes in pH<sub>CF</sub> across experimental conditions are relatively small, with values changing, for example, from 8.52 to 8.26 in the most sensitive coral species *S. siderea*. However, it is important to note that this represents a substantial compensation or buffering by the coral with respect to the much larger changes in pH<sub>SW</sub>, which range from 8.3 to 7.3 in the experiment.

To illustrate the chemical effects of this compensation, we provide calculations of the proton differential ([H⁺]<sub>SW</sub> – [H⁺]₄F), maintained by the coral with respect to seawater under all experimental conditions (Figure 7, Table S9), for which larger values represent a greater difference in proton concentration in the calcification fluid relative to seawater. For example, for *P. astreoides* at 28 °C, the comparison between the 311 µatm *pCO₂* and the ~33,309 µatm *pCO₂* treatments show a proton differential of 0.5 × 10⁻⁸ mol/L at 400 µatm *pCO₂* and 6.3 × 10⁻⁸ mol/L at 3000 µatm, which represents a differential 12.4 times higher at the elevated *pCO₂* condition. At 31 °C for *P. astreoides*, the differential is 13.4 times higher between the ~400 µatm and ~3000 µatm *pCO₂* conditions. The same comparison shows a proton differential 11.4 and 13.4 times higher for *P. strigosa* at 28 °C and 31 °C, 11.9 and 13.9 times higher for *S. siderea* at 28 °C and 31 °C and 11.5 times higher for *U. tenuifolia* at 28 °C.
<figure><img src="image_11.png" /><figcaption>**Figure 7.** Proton differentials maintained by the coral species between seawater and their calcifying fluid ([H⁺]SW – [H⁺]CF). Values are given for preindustrial, present day, mid-century, and extreme (~3300 ppm) pCO₂ conditions, with precise pCO₂ values given in Table S1. Calculated values are provided in Table S9.</figcaption></figure>

Using the compilation of pH<sub>CF</sub> data from the literature and from the present study, we compare the slopes of the relationships between pH<sub>CF</sub> and pH<sub>SW</sub> (Figure 8), with a slope of 1 indicating that changes in pH<sub>CF</sub> perfectly track changes in pH<sub>SW</sub>, and a slope of 0 indicating that the coral is perfectly compensating for external changes in pH<sub>SW</sub> and that pH<sub>CF</sub> is invariant across the experimental conditions. This compilation demonstrates that although there are cases where corals perfectly compensate for external pH<sub>SW</sub> changes, in most cases corals exhibit a modest decrease in pH<sub>CF</sub> with decreasing pH<sub>SW</sub> (Figure 8). Furthermore, δ<sup>11</sup>B-based estimates of pH<sub>CF</sub> were generally lower than that found by the microelectrode approach (Figure 8). We hypothesized in previous work that this could reflect different time intervals being recorded by these approaches [7], but it cannot be ruled out that there are systematic differences in the techniques, such as recording different microenvironments.

<figure><img src="image_12.png" /><figcaption>**Figure 8.** The sensitivity of pH<sub>CF</sub> to pH<sub>SW</sub> within the coral species studied here and reported in the literature. Y-Axis is the slope of the relationship between pH<sub>CF</sub> and pH<sub>SW</sub>, with a value of 1 showing that change in pH<sub>CF</sub> directly tracks the magnitude of the change in pH<sub>SW</sub>, and a value of 0 indicating perfect control over coral pH<sub>CF</sub> amidst variable pH<sub>SW</sub>. On X axis, numbers in parenthesis are the temperature of the experiment. Previously published data used in the figure are available in Table S12 alongside citations for data sources.</figcaption></figure>
It should ultimately be ΩCF that is the most influential factor in coral calcification. If pH<sub>CF</sub> modestly decreases, but ΩCF remains high enough to support efficient coral calcification, then this may explain how corals continue calcifying despite acidification of their calcifying fluid. Calculating ΩCF requires an additional assumption due to the lack of constraints on [Ca<sup>2+</sup>]<sub>CF</sub>. We conducted a set of calculations where [Ca<sup>2+</sup>]<sub>CF</sub> is assumed to be equivalent to [Ca<sup>2+</sup>]<sub>SW</sub> (Table 2 and Table S7). The calculated saturation states were then plotted against coral calcification data (Figure 9). Our compilation of ΩCF exhibits inter-species variation, but none of the four species (*S. siderea*, *P. strigosa*, *P. astreoides* and *U. tenuifolia*) exhibit significantly different values from each other at under control temperature and pCO<sub>2</sub> conditions (ANOVA test, *p* = 0.2), but were significantly higher compared to that calculated for *S. pistillata* [7], *A. yongei* (*p* < 0.05) and *P. damicornis* (*p* < 0.05) [7,67] using the same assumptions. Species-specific threshold values of ΩCF, where calcification is outweighed by dissolution, can be estimated from Figure 9, but care should be taken interpreting those absolute values due to the lack of constraints on [Ca<sup>2+</sup>]<sub>CF</sub>. There is a possibility that [Ca<sup>2+</sup>]<sub>CF</sub> could differ between species, as highlighted by previous work on *A. yongei* and *P. damicornis* [51]. In addition, the highest calculated ΩCF values do not translate to highest calcification rates for the corals, which may reflect prior observations that coral calcification is complex process beyond simple physicochemical manipulation of the calcifying fluid [78,82].

<figure><img src="image_13.png" /><figcaption>**Figure 9.** Relationships between boron isotope derived ΩCF and net calcification rates. ΩCF is calculated assuming that [Ca²⁺] of seawater is equivalent to [Ca²⁺] of the calcifying fluid. Published data used in this figure is given in Table S12 with citations for data sources.</figcaption></figure>

Statistical analyses of coral and symbiont physiological data from this experiment have been explored elsewhere [39]. Here, we perform additional 2-way ANOVA tests on the same data to produce statistical tests that are handled identically between the geochemical and physiological data. In *P. astreoides*, we found significant individual and combined effects of pCO₂ and temperature on Chl a, and total host energy reserves, but not on symbiont cell density. Significantly, and as noted in previous work of Bove et al. [39], the significant effect of temperature on Chl a in *P. astreoides* is actually to increase levels, whereas for all other significant effects reported from our ANOVA test they are characterizing decreases. However, it is important to note that temperature actually improved symbiont physiological parameters in this species, rather than repressing them as reported previously for this species [39]. In *P. strigosa*, only temperature had an effect on both symbiont cell density and total host energy reserves. Significant individual effects of both temperature and pCO₂ on both symbiont cell density and Chl a were observed for *S. siderea*, whilst only temperature had an effect on total host energy for this species. For *U. tenuifolia*, only pCO₂
had an effect on Chl a. Results of these analyses are presented in Table 4. Specimens of *S. siderea* were collected from both the inshore and offshore environments and previous work showed that reef environment affected physiological response; however, our results show a nonsignificant effect of reef environment on physiological data (Table S10), which likely reflects the difference in statistical approaches, with ANOVA tests employed here on single parameters versus the principal component analyses conducted by Bove et al. [39].

These physiological data are relevant for understanding how host and symbionts
responses to environmental change may influence the chemistry of the calcifying fluid. It is
notable that in the three coral species examined (excluding *U. tenuifolia*), temperature was
found to influence total host energy reserves, or the total of the lipid, carbohydrate, and
protein concentrations for a given area of coral host tissue. Temperature was not found to
influence pH<sub>CF</sub> and [CO<sub>3</sub><sup>2-</sup>]<sub>CF</sub>, but in all three corals was found to reduce [DIC]<sub>CF</sub> (Table 4).
Whilst this observation does not prove cause and effect, it is consistent with temperature
stress causing depletion of host energy reserves, and reduced host input of respiration
produced CO<sub>2</sub> into the DIC pool for calcification. As temperature increase was associated
with reduced symbiont cell density and Chl a in some of the corals, this is consistent with a
scenario where the symbiont was less efficiently supplying energy to the host, leading to
the observed decrease in energy reserves.

To further explore the relationship between these host and symbiont physiological
changes and the source of carbon in the calcification fluid, we examined Δδ¹³C (δ¹³C₃₁°C –
δ¹³C₂₈°C) for each *pCO₂* treatment. In Figure 10, Δδ¹³C is plotted against Δ[DIC] ([DIC]₃₁°C – [DIC]₂₈°C). This analysis shows that, on one hand, there is a significant difference in the
source of carbon for calcification between *S. siderea* and *S. pistillata*, for which temperature
did not influence δ¹³C at the higher *pCO₂* conditions. On the other hand, it shows that at
higher *pCO₂* conditions, Δδ¹³C of *P. astreoides* and *P. strigosa* (Figure 10) shifted to more
positive values and that Δ[DIC] shifted towards more negative values compared to the other
corals, with the overall effect most pronounced in *P. astreoides*. The potential mechanisms
driving these trends are discussed below.
<figure><img src="image_14.png" /><figcaption>**Figure 10.** Combined influence of temperature and pCO₂ on the δ¹³C and [DIC] of the coral calcifying fluid. Data are expressed as Δδ¹³C (δ¹³C₃₁°C – Δδ¹³C₂₈°C) and Δ[DIC] ([DIC]₃₁°C – [DIC]₂₈°C) for each pCO₂ treatment.</figcaption></figure>

**4. Discussion**

Collectively, the data from this study and the literature suggest that most corals experience a degree of acidification of their internal calcification fluid media in response to external seawater acidification. Nonetheless, considerable biological compensation is observed for the studied species, which maintain a larger proton differential between the calcifying fluid and seawater under elevated pCO₂. Therefore, the slight decreases in pH<sub>CF</sub> under elevated pCO₂ may represent either a practical tradeoff by the coral, or a biological limit to controlling calcifying fluid chemistry that still allows the coral to maintain [CO₃²⁻]�CF and Ω<sub>CF</sub> at a level that supports efficient calcification. The hypothesis that this is an active and energy-consuming adaptation by corals is supported by the finding that the ability of corals to regulate pH and carbonate chemistry of their calcifying fluid can be impaired by thermal stress and symbiont loss [7,25]. In contrast to our studies of *S. pistillata* and *P. damicornis* [7,25], we found that temperature did not result in a statistically significant change in pH<sub>CF</sub> regulation across all conditions in the Caribbean species investigated here, although it did under a subset of the experimental conditions in *S. siderea*. This may reflect that the highest temperature treatment in the present experiment [38,39], did not elicit as severe an effect on the corals and their symbionts as in the prior experiment [7,25], with the exception of *U. tenuifolia* that had too high a mortality at elevated temperature to be examined. Notably, the Ca-ATPase inhibitor experiments of Allison et al. [32], and the heat stress experiments of Guillermic et al. [7], on *Pocillopora damicornis* are the only experiments
where corals appeared to almost completely lose the ability to elevate pH<sub>CF</sub> relative to seawater, as shown in Figure 5.

Furthermore, we are able to examine associations between coral and symbiont physio-
logical responses and calcifying fluid regulation. This revealed a significant depletion in
total host energy reserves at elevated temperature, as well as a reduction in [DIC]CF in all
three corals examined. The reduction in calculated [DIC]CF with temperature increase was
also seen in the *S. pistillata* and *P. damicornis* experiment described in Guillermic et al. [7].
This result is consistent with a reduction in the energy transfer between the symbiont and
host leading to the host depleting its energy reserves and/or the host consuming more
energy in response to temperature stress. Although we cannot prove cause and effect,
changes in the energy balance between host and symbiont could then lead to a reduction in
the production of metabolic CO2 that is contributed to the DIC pool for calcification, which
is consistent with the reduction we see in calculated [DIC]CF in these corals. The story is
also more complex, however, with additional interactions apparent from the δ13C data.

Although the δ¹³C data did not indicate major differences in host and symbiont physiological influence on [DIC]CF for the different coral species under the control CO₂ conditions, significant differences in δ¹³C emerged under elevated temperature and CO₂ amongst the species, with shifts towards positive δ¹³C associated with amplified reductions in [DIC]CF in two of the coral species studied.

It is thought that changes in the flux of carbon to and from photosynthesis and
respiration, respectively, may change the δ¹³C of the DIC pool used for calcification. Phos-
tosynthesis preferentially consumes ¹²C, which may then result in ¹³C enrichment in the
DIC pool for calcification if this pool acts as a source of CO₂ for photosynthesis [83]. As
the rate of photosynthesis increases and/or the rate of respiration decreases, the DIC pool
should have more positive δ¹³C resulting in more positive coral skeletal δ¹³C [83,84]. In
the present experiment, we can see that host respiration input is likely decreasing as the
host consumes energy reserves at elevated temperature. There is also an overall reduction
in [DIC]CF, which is likely due to a reduction in CO₂-input from respiration. One way to
explain the relationship between the Δδ¹³C and Δ[DIC] data in Figure 10 is that, in addition
to the reduction in respiration-sourced CO₂ to the DIC pool for calcification, photosynthetic
drawdown of ¹²CO₂ by the symbiont is significantly enriching the δ¹³C of the DIC pool
in *P. astreoides* and *P. strigosa* at highest temperature and *pCO₂* conditions, which could
further deplete [DIC]CF and drive more positive Δδ¹³C. This effect was not seen in a similar
experiment on *S. pistillata* [7] where symbiont loss and bleaching of the coral occurred [25],
which suggests that photosynthetic influence over the DIC pool was minimal at the higher
temperature and *pCO₂* conditions. This interpretation is also congruent with observations
from this experiment that the symbiont in *P. astreoides* is not negatively impacted by in-
creased temperature [39] and is evidence for a divergent host and symbiont physiological
response to environmental parameters in this coral species. Conversely, the effects of host
respiration and symbiont photosynthesis remained relatively balanced in terms of their
influence on the source of DIC for calcification in *S. siderea*. It has been previously noted
that *P. astreoides*, *S. siderea*, and *P. strigosa* host differing algal symbiont communities which
may result in differing carbon allocation between the host and symbiont [39]. For example,
*S. siderea* mainly hosts *Cladocopium* and *Breviolum* whereas *P. astreoides* hosts *Breviolum* and
*Symbiodinium* [39]. The potential changes in carbon flux between the host and symbiont
is interesting to consider in the context of the debate on the extent of mutualism between
the coral host and symbiont, and whether the symbiont develops features that are more
parasitic in nature under environmental change [39,85,86].

Overall, the results presented here and in previous work on these corals [7,38,39,87] highlight the considerable complexity in host and symbiont responses to ocean warming and acidification, demonstrating that these responses are closely coupled to a coral's physicochemical control of their calcification process. The combined physiological data from this study, preceding studies [38,39,87], as well as the work of other groups (e.g., [88]), converge on the conclusion that *S. siderea* is a relatively resilient species to environmental
change and so may be amongst the least impacted by future warming and acidification, compared to a number of more sensitive Caribbean species studied such as *O. faveolata*, *M. cavernosa*, and *U. tenuifolia* [38,88]. Some differences in observations on *P. astreoides* between studies exist in its temperature sensitivity [38,88] which may reflect intraspecific differences in host or symbiont physiology, with the evidence here suggesting thermal sensitivity can be relatively low for the symbiont of at least some Caribbean *P. astreoides* but that reductions in net calcification can still occur in non-bleached corals in acidified seawater.

The type of data presented here provides useful insights into the underlying physi-
logical mechanisms of coral adaptation, and the variation in responses between species in
an experiment. Whilst difficult to make regional or global predictions from single studies, a
recent metanalyses of experimental data suggested that at the levels predicted over the next
century acidification is less of a threat to Caribbean corals compared to warming but that
there is evidence for variation across the region and considerable data gaps [89]. Despite
this finding, the evidence presented in this paper suggest acidification resilient Caribbean
corals maintain increasingly large proton differentials with seawater under acidification,
highlighting that there may be an energetic cost to this adaption that corals would have to
maintain indefinitely in the future. The work here also highlights that warming can have
complex influences on the calcification processes even outside a severe bleaching event
through changes to the partitioning of carbon between the host, symbiont, and the pool
of ions used for calcification. The sustainability of these adaptations to acidification and
warming are largely unexplored in Caribbean corals, but is highly relevant given findings
from this experiment that even if positive net calcification is maintained in environmental
challenge experiments corals could be depleting energy reserves [39]. Therefore, it will
be important for future research to attempt to determine whether even the relatively re-
silient species such as *S. siderea* can maintain their short-term adaptive responses over the
longer term.

Overall, the results presented here and in previous work on these corals [7,38,39,87] highlight the considerable complexity in host and symbiont responses to ocean warming and acidification, and demonstrate that these responses are closely coupled to a coral's physicochemical control of their calcification process.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jmse10081075/s1, Table S1: Measured Culture Conditions in the 8 Experimental Setups from Bove et al., 2019; Table S2: Calculated Culture Conditions in the 8 Experimental Setups from Bove et al., 2019; Table S3: Physiological data of the host and the symbionts originally published in Bove et al. (2021) from the same corals used in this study; Table S4: Boron isotope standard reproducibility; Table S5: Reproducibility of B/Ca analyses of CamWuellestorf and NEP standards; Table S6: Functions used for data transformations according the method of Tukey et al., 1977; Table S7: Geochemical data and calculated parameters from individual Caribbean coral specimens, of which the average per experimental condition are given in main text Tables 1 and 2; Table S8: Average and individual coral specimen geochemical data and calculations from *Stylophera pistillata*; Table S9: AIC results for Caribbean coral geochemical data as a function of the pH of seawater in the experiment and net calcification as a function of calculated CF parameter data; Table S10: Calculations of pH gradient between seawater and the calcifying fluid; Table S11: ANOVA results for Caribbean coral geochemical and physiological data; Table S12: Compilation of published data used in the main text.

**Author Contributions:** Conceptualization, R.A.E., K.D.C. and J.B.R.; methodology, R.A.E., S.M., K.D.C. and J.B.R.; formal analysis, C.B.B., L.P.C., S.M., B.A.C., M.G., I.D.C. and R.A.E.; investigation, C.B.B., L.P.C., M.G., I.D.C., K.D.C., R.A.E. and J.B.R.; resources, R.A.E., K.D.C. and J.B.R.; data curation, M.G. and R.A.E.; writing—original draft preparation, R.A.E. and I.D.C.; writing—review and editing, all authors; visualization, M.G., I.D.C. and R.A.E.; supervision, R.A.E., K.D.C. and J.B.R.; project administration, R.A.E., K.D.C. and J.B.R.; funding acquisition, R.A.E., K.D.C. and J.B.R. All authors have read and agreed to the published version of the manuscript.
**Funding:** RAE and JBR acknowledge support from National Science Foundation grants OCE-1437166 and OCE-1437371. KDC acknowledges support from NSF grant OCE-1459522. RAE, MG, ID, and BA were also supported by the "Laboratoire d'Excellence" LabexMER (ANR-10-LABX-19) Chair to RAE, co-funded by a grant from the French government under the program "Investissements d'Avenir" and MG was supported by an IAGC student grant 2017. RAE acknowledges support from the Pritzker Endowment to UCLA IoES, and JBR acknowledges support from the ZMT and the Hanse-Wissenschaftskolleg Fellowship Program. BA was supported by an NSF GRFP. M.G., I.D., and BA were supported by the Center for Diverse Leadership in Science, funded by NSF, the Packard Foundation, Sloan Foundation, and Silicon Valley Community Foundation.

**Data Availability Statement:** Data will be archived in the BCO-DMO archive on publication of this article (https://www.bco-dmo.org/, accessed on 24 July 2022).

**Acknowledgments:** We thank Belize Fisheries Department for all associated permits, the Toledo Institute for Development and Environment (TIDE), and the Southern Environmental Association (SEA) for their continued support, as well as Garbutt's Marine for assistance in the field.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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