Internal pH regulation facilitates in situ long-term acclimation of massive corals to end-of-century carbon dioxide conditions

The resilience of tropical corals to ocean acidification depends on their ability to regulate the pH within their calcifying fluid (pHcf). Recent work suggests pHcf homeostasis under short-term exposure to pCO2 conditions predicted for 2100, but it is still unclear if pHcf homeostasis can be maintained throughout a corals lifetime. At CO2 seeps in Papua New Guinea, massive Porites corals have grown along a natural seawater pH gradient for decades. This natural gradient, ranging from pH 8.1–7.4, provides an ideal platform to determine corals’ pHcf (using boron isotopes). Porites maintained a similar pHcf (~8.24) at both a control (pH 8.1) and seep-influenced site (pH 7.9). Internal pHcf was slightly reduced (8.12) at seawater pH 7.6, and decreased to 7.94 at a site with a seawater pH of 7.4. A growth response model based on pHcf mirrors the observed distribution patterns of this species in the field. We suggest Porites has the capacity to acclimate after long-time exposure to end-of-century reduced seawater pH conditions and that strong control over pHcf represents a key mechanism to persist in future oceans. Only beyond end-of-century pCO2 conditions do they face their current physiological limit of pH homeostasis and pHcf begins to decrease.

-Seawater pH T

characterization at the collection sites:
The seawater pH (pH T in total scale) was monitored at multiple locations within the seep site and at the control site by different instruments: a CTD (Seabird, SBE 19v2) equipped with a pH sensor (SBE 18), multi-channel loggers (XR-420, RBR Ltd.) with a pH sensor (AMT Analysenmesstechnik GmbH) and a SeaFET Ocean pH sensor (Satlantic). Prior to the deployment at the control site, the CTD was calibrated with NBS buffers (4.0, 7.0, 9.21; for slope evaluation of the calibration curve) and a TRIS buffer to derive seawater pH at total scale (pH T ) 2 . All instruments were placed at the control site side by side for cross-calibration. The CTD calibrated to total scale was used as reference, and all instruments were cross-referenced (deployed at the beginning at the control site) and corrected for their offset. The seawater pH T measurements were taken next to the cores collected and during the respective field campaign of core collection. The extreme site was only measured during the last trip, when 3 of the in total 4 cores were collected. Discrete water samples were repeatedly collected and analyzed for total alkalinity. Total alkalinity was determined by gran titration following Dickson et al. 2 .

-coral sample overview:
The sampled coral cores (1 to 5 cm diameter), their location and the date of sampling are listed in Table S1. The seawater pH T was monitored at the four sites (Fig. S1) during the three cruises. In Fig. S2 a overview of the different sample types are provided as well as the location of the measurements.  Fig.  S2: The small coral cores were collected as shown in (a). The diameter was approx.. 10 mm ((b), scale bar 10 mm) and a height of 4--10 mm (example in (c) scale bar 2 mm). The individual measurements were spread over the upper part (few mm) of the core ((d), scale bar 2 mm). The long core (e,f) had a diameter of 5 cm and the outer part was sent to . In the upper part was prepared for LA--ICP--MS (e) after x--ray of the part of the coral core (f).

-Boron isotopic signature:
The Thermo Fisher AXIOM multi collector inductive coupled plasma mass spectrometer (MC-ICP-MS) connected to a laser ablation system of New Wave Research UP193fx was used to measure boron isotopes following the method of Fietzke et al. 3 . Faraday cups were used to collect data simultaneously for 10 B (amu10) and 11 B (amu11) in the outer most cups (L4 and H4). The resulting instrumental settings are summarized in Table S2 and were valid throughout all measurements performed. The cones were cleaned on a regular basis (every 2-4 days). The tubes going from the ablation cell to the plasma torch were checked for material deposition and cleaned by high flow rates overnight and/or mobilization of the debris by increased flow rates transporting it out of the tubes. Prior to each measurement session, the standard and samples were pre-ablated to remove surface contaminations (spot size was used one size bigger than during analysis and 50shots per spot area = approx. 8-10 µm of surface material). A standard-sample-standard bracketing method was used. The data of one measurement session contained 5-6 brackets. Both 12 C and the variation of the standard for each session were used to check for instrument stability and contaminations. Sessions were excluded from further analysis when the standard drift was higher than the internal reproducibility of the standards (2SD of the session on the standards, 10 B/ 11 B of 0.00015 = 0.27‰). During each session approx. 2.5 µg of coral sample were ablated, corresponding to 0.13 ng of total B (using an average B concentration of 50 ppm for the coral carbonate). The individual boron isotopic signature area sampled for an individual δ 11 B data point was approx. 200-300 µm long with a spot diameter of 35-50µm. The spot were positioned at the edge of skeletal elements. Because the laser ablates in greater depth (~30µm) compared to SIMS (a few microns) we can not exclude that centres were not sampled in particular because in Porites centres are more discretely distributed and not always located in the central line e.g. 4,5 . However, we expect that we sampled for all individuals a similar portion of centres of calcification. The length encompasses approx. 10 days up to two weeks of growth considering the average linear extension of the corals measured by Farbricius et al. 1 was ~ 1.2 cm and this corresponds to ~ 32 µm day -1 . Hence, they are within the temporal scale of the seawater pH T monitoring.
The boron isotopic signature is reported in delta notation, normalizing the unknown sample relative to the known standard using the soda-lime glass NIST-SRM610 (δ 11 B=-0.55‰ ± 0.53) as bracketing standard during measurements: We compared individual spot measurements to investigate δ 11 B variations between the control site and the different seep site locations that differ in their intensity of seawater pH T variability 6 and also the relative changes between sites using average δ 11 B values.

-Seawater carbonate chemistry
The seawater pH T is reduced where the almost pure volcanic CO 2 gas seeps through the seafloor. Persistent seeping has been confirmed for at least 70 years, but it has likely persisted for much longer 1 . At the control site, the seawater pH T displayed only slight variations throughout the day, with an average seawater pH T of 8.1 (SD = 0.10; Table S3). The pH T fluctuations increase and the seawater pH T become lower the closer to the major seeps. The intermediate site and low pH T site showed average seawater pH T values of 7.9 (SD = 0.20) and 7.6 (SD = 0.17), respectively. Seawater pH T at the seep sites revealed strong variations in seawater pH T at time scales of hours ( Fig. S3-S4). During increased tidal or wind driven currents and waves, the seawater pH T differences were damped, while stagnant conditions resulted in a stronger seawater pH T reduction. The seawater pH T value for the extreme site ranged from 6.80 to 7.91 with mean values of 7.4 (SD = 0.26).   2.6.13 0:00 3.6.13 0:00 4.6.13 0:00 5.6.13 0:00 6.6.13 0:00 7.6.13 0:00 8.6.13 0:00 9.6.13 0:00 pH T Date previous measurements e.g. 1,7 , and were used in combination with the measured pH T to calculate dissolved inorganic carbon (DIC), pCO 2 and the aragonite saturation state (Ω arag ). Table S3 summarizes the carbonate chemistry for the four different regions where the coral cores were collected and seawater pH T was monitored. This unique field setting allowed us to verify for the first time the application of δ 11 B as a seawater pH T proxy for tropical corals growing naturally in situ under a wide range of seawater pH T levels. The corals were exposed to the full range of natural environmental heterogeneity in light, currents, temperature and food supply, which distinguishes them from specimens taken from controlled laboratory conditions.

-Average boron isotopic signature, variability and corresponding internal calcifying conditions:
The boron isotopic signature of the coral skeletons differed significantly between the four sites (non-parametric Kruskal Wallis Test, p<0.001). Post-hoc pairwise comparisons showed that the corals from the extreme site were significantly different from all other sites. Those from the intermediate site and low pH T site were not significantly different from each other, nor were the intermediate site and control site, while the low pH T site was significantly different from the control site (Table S4).  Boron isotopic signatures were converted into internal calcifying pH (pH cf ), used to calculate the extent of pH cf up-regulation (∆pH = pH cf -pH T ). As seawater pH T declined 0.5 units from the mean control site value of 8.1 to 7.6 at the low pH T site, the δ 11 B decreased by 1.45‰. This suggested a reduction in internal pH cf of only 0.12 (Table S3). The 0.2 unit decrease in seawater pH T between the low pH T site and the extreme site resulted in a 1.7‰ decline of δ 11 B, suggesting a change in the internal pH cf of 0.18. Hence, this resulted in a relationship between δ 11 B and seawater pH T that deviates from the boron fractionation curve, rather reflecting polynomial response (AIC criteria were used to test for the best fit: linear vs polynomial of 2 nd and 3 rd rank: 1071 > 1067 < 1068, respectively).
Laboratory studies derived empirical δ 11 B-pH calibration equations 9,10 for tropical corals that differ between species and suggest different vital effects on δ 11 B incorporation. Both studies used different species (in each case two different coral species were tested) to derive such relationship. The shape of the calibrations within a study is similar but off-set between the two species. Comparing the two studies, the calibration relationships deviate from each other ( Fig. 1; main manuscript). Likely vital effects on boron incorporation can play a role (which were also shown to differ with skeletal region within the same coral species analyzed 11 ). Also different sites of origin of the corals used in the culturing experiment could be essential and contribute to differences in vital effects (in case of Hönisch et al. 9 Ruykyus Island, Japan and in case of Krief et al. 10 the Red Sea). A recent study showed no effect of seawater pH on δ 11 B in massive Porites grown at the Heron Island reef flat 12 . This highlights that the response (in particular the shape of the relationship) can be very different. In this latter case it was argued to derive from strong environmental fluctuations on the reef flat at Heron Island and supports the idea that origin can make a difference and determine vital effects.   (Table S1). Skeletal δ 11 B values were converted into internal calcifying fluid pH (pH cf ) and pH up-regulation intensity (∆pH = pH cf -pH T ). Other internal conditions at the site of calcification were adopted from McCulloch et al. 13  Based on the assumption that the dissolved inorganic carbon (DIC) concentration is doubled at the site of calcification, the internal aragonite saturation state Ω arag cf , the derived mean Ω cf for the extreme site is around 9.41 (s.e.m. 0.53). A recent study argues that corals can concentrate DIC cf within the internal calcifying fluid to foster calcification 14 , which is supported by an up-regulation of HCO 3 transporter genes 15 , allowing them to maintain a high Ω cf even at reduced pH cf . This would furthermore enable corals to continue calcifying even at lower pH cf values.
High spatial resolution studies of boron isotopes in corals have revealed pronounced variation in the same genus 16,17 and also other coral species 18 . In this study, δ 11 B values were highly variable even at the control site ( Fig. S5; average range 7.3 for the control site and of 8.1 for the extreme site) where the seawater pH T was more stable compared to the other sites ( Fig. S3-4). Seep site corals, however, showed few values > 23‰ (corresponding to an internal pH cf of 8.39), in contrast to control site corals with δ 11 B of up to 25.7‰ or a pH cf of 8.56. Such high variations have been previously described for tropical corals, and it was argued that these variations reflect the effect of biological processes on skeletal isotopic composition rather than external seawater pH T variations 16,17 . Several factors are thought to contribute to these internal variations in pH cf , but they are not yet fully understood. The general assumption is based on passive diffusion of seawater to the site of calcification and an active elevation of pH cf by Ca-ATPase e.g. [19][20][21][22] . However, at the site of calcification, the DIC might be different from seawater (potentially elevated), and this affects the carbonate saturation state. The type, source and transportation of DIC to the site of calcification is still not fully understood 23 . Elevation of pH cf requires energy. This demand needs to be met, but investment might vary temporally depending on energy availability and energetic trade-offs between other energy-requiring processes. Light is known to enhance the calcification rate 24 and calcification rate is suggested to be linked to internal pH cf up-regulation intensity 13 , suggesting that differences in light level can affect the internal pH cf . Hence, days with cloudy conditions might be reflected at the δ 11 B level. However, laboratory δ 11 B experiments appear to contradict such assumptions, showing no change of δ 11 B with differing light levels 25,9 , suggesting that the internal pH cf is the same at different light conditions. This raises the question of what other mechanisms could enhance light calcification without affecting internal pH cf ? In addition, we also observed boron derived pH cf below seawater values. This can be related to two essential processes: First corals also grow at night. In case of Porites from the Papua New Guinea vent sites (subjected to pH of 7.8) these corals still have a not negligible growth at night (almost 40% of day growth 7 ) that will contribute to the boron signature. Second the pH surrounding the corals (within their diffusive boundary layer (DBL)) are also lower than seawater pH during the night and internal pH cf values were shown to become even lower within the coral tissue close to the skeleton 21,26-28 . The pH value strongly depends on the flow rate and during rather stagnant conditions pH within the DBL in the night can be really low. Together this can contribute to lower boron isotopic signature in the skeleton. Apart from that we still do not know what factors contribute to the high variability observed in boron isotopic signature. However, this goes beyond the scope of this manuscript. Here we followed the current mainly applied interpretation and application of boron isotopes in coral skeletons.

-Calcification rates:
Relative calcification rates (Table S6) of the individual corals did not change between the control and intermediate sites, based on the corals apparent ability to maintain a similar high internal pH cf (Table S5). Internal pH cf and calcification rate are reduced at the extreme site (Table S6).