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

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_CF) and other carbonate chemistry parameters ([CO₃²⁻]_CF, DIC_CF, and Ω_CF) may be challenged by CO₂ driven acidification and temperature. Here, we examine the combined influence of changing temperature and CO₂ 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 δ¹¹B) to probe the pH_CF, [CO₃²⁻]_CF, and DIC_CF regulation in these corals, and δ¹³C to track changes in the sources of carbon for calcification. Temperature was found to not influence pH_CF regulation across all pCO₂ treatments in these corals, in contrast to recent studies on Indo-Pacific pocilloporid corals. We find that [DIC]_CF 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]_CF reductions may result from reduced input of respired CO₂ to the DIC pool for calcification. In addition, δ¹³C data suggest that under high temperature and CO₂ conditions, algal symbiont photosynthesis continues to influence the calcification pool and is associated with low [DIC]_CF 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.

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_SW) 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 [4,5,6,7].

Coral calcification responses to ocean acidification are known to be diverse, with observations of negative skeletal growth responses [8,9,10,11,12,13,14,15,16,17,18,19,20]. Coupled ocean acidification and temperature stress has revealed a variety of responses due to interactions with temperature stress, including negative synergistic [14,21,22,23,24].

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_CF and [CO₃²⁻]_CF 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 [27,28,29] have been reported in field-collected corals with different thermal exposure histories. As it is thought that biological upregulation of the pH_CF, [CO₃²⁻]_CF, [DIC]_CF, and the saturation state of aragonite (Ω_Ar)_CF is important in allowing corals to calcify [30,31,32,33,34,35], 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_CF, [DIC]_CF, and [CO₃²⁻]_CF. We applied a combined approach of boron geochemistry to constrain pH_CF, [DIC]_CF, and [CO₃²⁻]_CF and δ¹³C to trace changes in the source of carbon for calcification linked to coral and symbiont physiological responses to environmental changes.

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 (δ¹¹B_NEP) 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 [46,47,48]. 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 CamWuellestorfi, 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_CF

The pH of the calcifying fluid (pH_CF) 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_SW represents the boron isotopic composition of seawater (δ¹¹B_SW = 39.61‰) [52] and δ¹¹B_CARB 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_(B3-B4) = 1.0272. The dissociation constant of boron (K_B) was calculated from salinity, temperature and pressure [53], and the boron concentration was calculated from salinity after Lee et al., 2010 [54].

pH_CF = pK_B − log{−[δ¹¹B_SW − δ¹¹B_CARB]/[δ¹¹B_SW − α_(B3-B4)*δ¹¹B_CARB − ε_(B3-B4))]}

(1)

[H⁺]_SW and [H⁺]_CF were calculated based on pH_SW and pH_CF with [H⁺] = 10^−pH. The proton gradient [H⁺]_SW − [H⁺]_CF was evaluated to better visualize the scale of pH regulation in comparison to a logarithmic scale.

2.8. Modelled [CO₃²⁻]_CF, DIC_CF, and Ω_CF

DIC_CF and [CO₃²⁻]_CF were calculated using the method and Matlab© code from DeCarlo et al. [51]. This study uses combined B/Ca and δ¹¹B to calculate [CO₃²⁻]_CF, where:

$$\left[{\mathrm{CO}}_3^{2-}{]}_{\mathrm{CF}}\right]={\mathrm{K}}_{\mathrm{D}} \ast \frac{\left[\mathrm{B}\left(\mathrm{OH}{)}_4^{-}\right)\right]}{\mathrm{B}/\mathrm{Ca}}$$

(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_CF as calculated from δ¹¹B following Equation (1). Given pH_CF and [CO₃²⁻]_CF, DIC_CF can be calculated using the known dissociation constants for carbonate speciation at known temperatures and pressure. Ω_CF 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_CF) defined following Equation (3) is dependent on the accuracy of the [CO₃²⁻]_CF and is limited to the additional assumption on the [Ca²⁺] of the calcifying fluid.

$$\left[\mathsf{\Omega }{]}_{\mathrm{CF}}\right]=\frac{ \left[{\mathrm{CO}}_3^{2-}{]}_{\mathrm{CF}}\right]\times \left[{\mathrm{Ca}}_{}^{2+}{]}_{\mathrm{CF}}\right]}{\mathrm{Ksp}}$$

(3)

We calculated the saturation state of aragonite of the calcifying fluid using the [Ca²⁺]_sw determined using the salinity of the culture condition ([Ca²⁺]_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 CaCO₃ 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 δ¹³C and δ¹⁸O 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, δ¹¹B, δ¹³C, pH_CF, and Ω_CF across experimental treatment conditions relative to the control condition (28 °C, 400 μatm pCO₂). 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.

δ¹¹B, B/Ca, and δ¹³C 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 AVOVA 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 pCO₂ and temperature. The same approach was taken to analyze the calculated parameters pH_CF, [CO₃²⁻]_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 [7,17,32,34,35,47,57,58,59,60,61,62,63,64,65,66,67,68,69,70]. 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. Average Geochemical Data per Experimental Condition.

Species	T (°C)	CO2 (μatm)	δ11B (‰) 1	Error (SD)	N *	B/Ca (μmol/mol)	Error (SD)	N	δ13C (‰) 2	Error (SD)	N
P. astreoides
	28	311	23.2	0.9	6	396	54	6	−2.3	0.5	4
	28	405	23.6	0.9	6	437	31	6	−2.6	0.9	4
	28	702	22.7	1.4	5	432	53	4	−5.2	1.5	4
	28	3309	22.1	0.8	6	410	46	6	12.5	3.1	3
	31	288	24.1	0.1	1	389	17	1	−1.4	0.0	1
	31	442	23.4	1.3	5	424	32	5	−2.6	0.7	4
	31	674	23.1	0.4	5	443	82	5	−3.5	1.3	4
	31	3285	22.6	0.5	2	568	51	2	−5.0	1.2	2
P. strigosa
	28	312	24.2	0.6	6	496	43	6	−2.2	0.6	5
	28	406	24.5	1.5	5	495	70	4	−1.1	1.5	3
	28	702	23.3	0.7	6	481	35	6	−4.0	0.6	4
	28	3320	22.2	1.3	6	455	41	6	10.9	1.6	4
	31	288	23.9	0.9	4	527	44	4	−1.8	0.6	4
	31	443	23.9	0.8	5	495	50	5	−1.3	0.7	4
	31	674	23.7	0.5	4	528	33	4	−3.4	0.4	4
	31	3284	23.0	1.5	4	530	54	2	−6.4	2.9	4
S. siderea
	28	312	24.5	0.7	6	450	26	6	−3.3	0.2	4
	28	405	23.9	0.5	6	436	18	6	−3.9	0.6	4
	28	703	22.7	0.9	6	424	21	6	−6.2	1.1	4
	28	3317	21.8	1.2	6	415	27	6	12.5	1.7	3
	31	288	24.2	0.4	5	467	11	5	−2.6	0.2	3
	31	449	23.4	0.7	6	425	22	6	−2.6	1.4	4
	31	673	22.1	0.7	5	432	10	4	−5.5	1.4	3
	31	3285	21.3	1.4	6	428	21	6	14.3	2.0	3
U. tenuifolia
	28	312	24.59	0.68	4	490	51	4	−2.2	0.4	4
	28	404	24.25	0.56	4	518	32	4	−2.6	0.3	4
	28	698	23.54	1.25	4	464	76	4	−3.8	1.0	4
	28	3303	22.53	1.94	4	461	62	4	−7.2	2.8	4

* number of specimens analyzed; ¹ NIST; ² V-PDB.

, 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_SW) 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_SW that were best described by linear regressions seem best suited for this endeavor. P. strigosa showing the greatest sensitivity of δ¹¹B to pH_SW, 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_SW of 2.52 but was best fit by a nonlinear regression. At 31 °C, it is notable that the relationship between δ¹¹B and pH_SW 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).

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

Table 2. 2-way ANOVA output testing the influence of temperature and pCO₂ on measured geochemical parameters.

Geochemical Parameter	Species	Experimental Variable	ANOVA p-Value
δ11B	P. astreoides	Temperature	0.1171
		CO2	0.0152
		Temperature + CO2	0.6094
	P. strigosa	Temperature	0.5521
		CO2	0.00532
		Temperature + CO2	0.59757
	S. siderea	Temperature	0.36
		CO2	7.58 × 10−10
		Temperature + CO2	0.441
	U. tenuifolia	CO2	0.322
δ13C	P. astreoides	Temperature	0.152
		CO2	4.12 × 10−5
		Temperature + CO2	0.193
	P. strigosa	Temperature	0.5521
		CO2	0.00532
		Temperature + CO2	0.59757
	S. siderea	Temperature	0.36
		CO2	7.58 × 10−10
		Temperature + CO2	0.441
	U. tenuifolia	CO2	0.322
B/Ca	P. astreoides	Temperature	0.22201
		CO2	0.78951
		Temperature + CO2	0.00939
	P. strigosa	Temperature	0.00911
		CO2	0.74462
		Temperature + CO2	0.16759
	S. siderea	Temperature	0.17474
		CO2	0.00103
		Temperature + CO2	0.84438
	U. tenuifolia	CO2	0.484

and Table 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.

It is important to note that the principal driver of trends in δ¹³C data across variable pCO₂ and pH_SW 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.

3.2. Net Calcification, pH_CF, [CO₃²⁻]_CF, and DIC_CF

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_CF) was calculated from δ¹¹B data using canonical methods from the literature, as described in the methods section. Although the δ¹¹B approach to calculating pH_CF 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 δ¹¹B-derived pH_CF (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. Net calcification data and calculated calcifying fluid pH and carbonate system parameters.

Species	T (°C)	CO2 (μatm)	Net Calcification (mg cm−2 d−1)	Error (SD)	N 1	pHCF	Error (SD)	DICCF (mmol/mol)	Error (SD)	[CO32−]CF (mmol/mol)	Error (SD)	ΩCF 2	Error (SD)	N
P. astreoides
	28	311	−0.08	0.26	11	8.42	0.06	4983	863	1131	181	20.9	3.4	6
	28	405	0.07	0.52	12	8.45	0.06	4421	352	1053	81	19.5	1.5	6
	28	702	−0.17	0.4	10	8.39	0.10	4561	618	957	180	17.8	3.3	4
	28	3309	−0.65	0.5	12	8.35	0.05	4841	512	969	102	17.9	1.9	6
	31	288	0.5	0.5	6	8.45	0	4777	0	1242	0	23	0	1
	31	442	0.04	0.29	8	8.40	0.09	4434	394	1055	107	19.5	2	5
	31	674	−0.02	0.24	9	8.38	0.02	4365	703	1004	133	18.6	2.5	5
	31	3285	−0.54	0.27	3	8.35	0.03	3356	286	736	106	13.6	2	2
P. strigosa
	28	312	0.92	1.28	13	8.49	0.04	3862	362	989	59	18.3	1.1	6
	28	406	1	1.12	6	8.50	0.10	3881	590	1013	85	18.7	1.6	4
	28	702	0.66	0.79	14	8.43	0.04	4025	311	933	47	17.3	0.9	6
	28	3320	0.03	0.71	16	8.36	0.08	4339	452	876	68	16.2	1.3	6
	31	288	0.41	0.3	9	8.43	0.06	3269	9	889	12	16.4	0.2	2
	31	443	−0.32	0.74	6	8.43	0.05	3782	399	959	134	17.8	2.5	5
	31	674	−0.23	0.55	7	8.42	0.03	3450	109	850	78	15.7	1.4	3
	31	3284	−0.5	0.39	8	8.37	0.10	3609	423	759	35	14	0.6	2
S. siderea
	28	312	1.30	0.57	10	8.51	0.05	4216	261	1115	76	20.6	1.4	6
	28	405	1.37	0.70	12	8.47	0.03	4391	192	1087	63	20.1	1.2	6
	28	703	1.08	0.45	11	8.39	0.06	4596	194	993	129	18.4	2.4	6
	28	3317	0.13	0.52	11	8.33	0.08	4765	333	926	133	17.1	2.5	6
	31	288	1.00	0.54	8	8.46	0.03	3969	117	1033	34	19.1	0.6	4
	31	449	1.16	0.70	11	8.41	0.05	4407	252	1060	62	19.6	1.1	6
	31	673	1.06	0.51	11	8.32	0.05	4449	120	909	47	16.8	0.9	3
	31	3285	0.43	0.53	12	8.26	0.10	4480	284	851	119	15.7	2.2	5
U. tenuifolia
	28	312	0.16	0.10	11	8.52	0.04	3886	449	1037	85	19.2	1.6	4
	28	404	0.24	0.17	7	8.49	0.04	3676	188	950	102	17.6	1.9	4
	28	698	0.05	0.08	4	8.44	0.08	4230	767	996	99	18.5	1.8	4
	28	3303	−0.23	0.05	5	8.38	0.13	4286	657	908	198	16.8	3.7	4

¹ Refers to number of specimens analyzed; ² Ω_CF values calculated using the assumption of [Ca]_CF = [Ca]_SW.

).

The [CO₃²⁻]_CF and DIC_CF were calculated from δ¹¹B and B/Ca data using the K_D of McCulloch et al. [74] and the approach of DeCarlo et al. [51] (Table 3), as described in 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 [Ca²⁺] 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_D, 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_D may not be relevant for the final mineral. Despite these uncertainties, results of Guillermic et al. [7] showed that the K_D formulations used by DeCarlo et al. [51] (Termed D18); McCulloch et al. [74] (M17); Holcomb et al. [77] (H16) resulted in similar reconstructed [CO₃²⁻]_CF among biological replicates and that the outcome was within error of independent measurements from [CO₃²⁻] 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 pCO₂, 28 °C) [7].

Regression analysis of net calcification, calculated pH_CF, [CO₃²⁻]_CF, and DIC_CF as a function of pH_SW were explored using an AIC approach (Figure 3 and Figure 4; Table S9). pH_CF as a function of pH_SW follows a similar pattern to the originating δ¹¹B data (Figure 3; Table S9). Patterns in [CO₃²⁻]_CF as a function of pH_SW differ from patterns in pH_CF as a function of pH_SW, 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_CF, [CO₃²⁻]_CF, and [DIC]_CF 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_CF 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_CF.

Two-way ANOVA tests were again used to explore the individual and combined effects of pCO₂ and temperature on calculated calcifying fluid parameters of pH_CF, [CO₃²⁻]_CF, and DIC_CF. In P. astreoides, pCO₂ had a significant effect on pH_CF and [CO₃²⁻]_CF. Temperature and the combined effect of temperature and pCO₂ had significant effects on DIC_CF. Similarly, in P. strigosa, pCO₂ had a significant effect on pH_CF and [CO₃²⁻]_CF, but temperature only had an individual effect on DIC_CF. In S. siderea, the calculated pH_CF data did not meet assumptions of normality after transformation and, consequently, ANOVA results are not presented. There was a significant effect of pCO₂ on [CO₃²⁻]_CF of S. siderea and significant individual effects of both pCO₂ and temperature on DIC_CF. Lastly, U. tenuifolia displayed no significant effects of either pCO₂ or temperature on the calculated calcifying fluid parameters. Results of these analyses are presented in

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.

Parameter/Species/Experimental Variable
	p Value		p Value
Symbiont cell density		pHCF
P. astreoides		P. astreoides
Temperature	0.417	Temperature	0.676
CO2	0.11	CO2	6.54 × 10−5
Temperature + CO2	0.466	Temperature + CO2	0.286
P. strigosa		P. strigosa
Temperature	2.59 × 10−11	Temperature	0.42275
CO2	0.648	CO2	0.00536
Temperature: CO2	0.15	Temperature + CO2	0.62005
S. siderea		S. siderea
Temperature	9.09 × 10−5	Temperature	Not Normal *
CO2	0.000251	CO2	Not Normal *
Temperature + CO2	0.858348	Temperature + CO2	Not Normal *
U. tenuifolia		U. tenuifolia
CO2	0.727	CO2	0.317
Chl a		[CO32−]
P. astreoides		P. astreoides
Temperature	9.89 × 10−7	Temperature	0.41309
CO2	6.10 × 10−11	CO2	0.00333
Temperature + CO2	2.80 × 10−9	Temperature + CO2	0.22932
P. strigosa		P. strigosa
Temperature	6.15 × 10−8	Temperature	0.0988
CO2	0.000217	CO2	0.0224
Temperature: CO2	0.070441	Temperature: CO2	0.855
S. siderea		S. siderea
Temperature	0.00394	Temperature	0.679
CO2	1.37 × 10−7	CO2	5.98 × 10−5
Temperature + CO2	0.25756	Temperature + CO2	0.996
U. tenuifolia		U. tenuifolia
CO2	0.033	CO2	0.181
Total host energy		[DIC]CF
P. astreoides		P. astreoides
Temperature	0.02755	Temperature	0.00163
CO2	0.00659	CO2	0.69095
Temperature: CO2	0.06197	Temperature + CO2	0.04313
P. strigosa		P. strigosa
Temperature	8.35 × 10−5	Temperature	0.00395
CO2	0.103	CO2	0.26074
Temperature + CO2	0.857	Temperature + CO2	0.3616
S. siderea		S. siderea
Temperature	0.000522	Temperature	0.00119
CO2	0.118746	CO2	2.62 × 10−5
Temperature + CO2	0.710086	Temperature + CO2	0.3765
U. tenuifolia		U. tenuifolia
CO2	0.168	CO2	0.463
Calcification rate
P. astreoides
Temperature	0.0178
CO2	5.11 × 10−6
Temperature + CO2	0.1779
P. strigosa
Temperature	0.000142
CO2	0.000364
Temperature + CO2	0.678234
S. siderea
Temperature	0.52
CO2	3.27 × 10−8
Temperature + CO2	0.209

* Data did not reach normality even after transformation steps, so ANOVA test was not performed.

.

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_CF, [CO₃²⁻]_CF, and DIC_CF are differentiated from seawater values and that this is an active compensatory process as pH_CF, and [CO₃²⁻]_CF values are increasingly elevated relative to seawater values under acidified conditions (Figure 5 and Figure 6).

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

Changes in pH_CF 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_SW, 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⁺]_SW − [H⁺]_CF), 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.

Using the compilation of pH_CF data from the literature and from the present study, we compare the slopes of the relationships between pH_CF and pH_SW (Figure 8), with a slope of 1 indicating that changes in pH_CF perfectly track changes in pH_SW, and a slope of 0 indicating that the coral is perfectly compensating for external changes in pH_SW and that pH_CF is invariant across the experimental conditions. This compilation demonstrates that although there are cases where corals perfectly compensate for external pH_SW changes, in most cases corals exhibit a modest decrease in pH_CF with decreasing pH_SW (Figure 8). Furthermore, δ¹¹B-based estimates of pH_CF 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.

It should ultimately be Ω_CF that is the most influential factor in coral calcification. If pH_CF modestly decreases, but Ω_CF remains high enough to support efficient coral calcification, then this may explain how corals continue calcifying despite acidification of their calcifying fluid. Calculating Ω_CF requires an additional assumption due to the lack of constraints on [Ca²⁺]_CF. We conducted a set of calculations where [Ca²⁺]_CF is assumed to be equivalent to [Ca²⁺]_SW (Table 2 and Table S7). The calculated saturation states were then plotted against coral calcification data (Figure 9). Our compilation of Ω_CF 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 pCO₂ 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 Ω_CF, 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 [Ca²⁺]_CF. There is a possibility that [Ca²⁺]_CF could differ between species, as highlighted by previous work on A. yongei and P. damicornis [51]. In addition, the highest calculated Ω_CF 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].

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_CF and [CO₃²⁻]_CF, but in all three corals was found to reduce [DIC]_CF (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 CO₂ 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 ∆δ¹³C (δ¹³C_31°C − δ¹³C_28°C) for each pCO₂ treatment. In Figure 10, ∆δ¹³C is plotted against ∆[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, ∆δ¹³C of P. astreoides and P. strigosa (Figure 10) shifted to more positive values and that ∆[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.

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_CF 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_CF 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_CF 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_CF relative to seawater, as shown in Figure 5.

Furthermore, we are able to examine associations between coral and symbiont physiological 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. Photosynthesis 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 ∆δ¹³C and ∆[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 ∆δ¹³C. 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 increased 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 physiological 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 resilient 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.