Amelioration of ocean acidification and warming effects through physiological buffering of a macroalgae

Abstract Concurrent anthropogenic global climate change and ocean acidification are expected to have a negative impact on calcifying marine organisms. While knowledge of biological responses of organisms to oceanic stress has emerged from single‐species experiments, these do not capture ecologically relevant scenarios where the potential for multi‐organism physiological interactions is assessed. Marine algae provide an interesting case study, as their photosynthetic activity elevates pH in the surrounding microenvironment, potentially buffering more acidic conditions for associated epiphytes. We present findings that indicate increased tolerance of an important epiphytic foraminifera, Marginopora vertebralis, to the effects of increased temperature (±3°C) and pCO2 (~1,000 µatm) when associated with its common algal host, Laurencia intricata. Specimens of M. vertebralis were incubated for 15 days in flow‐through aquaria simulating current and end‐of‐century temperature and pH conditions. Physiological measures of growth (change in wet weight), calcification (measured change in total alkalinity in closed bottles), photochemical efficiency (Fv/Fm), total chlorophyll, photosynthesis (oxygen flux), and respiration were determined. When incubated in isolation, M. vertebralis exhibited reduced growth in end‐of‐century projections of ocean acidification conditions, while calcification rates were lowest in the high‐temperature, low‐pH treatment. Interestingly, association with L. intricata ameliorated these stress effects with the growth and calcification rates of M. vertebralis being similar to those observed in ambient conditions. Total chlorophyll levels in M. vertebralis decreased when in association with L. intricata, while maximum photochemical efficiency increased in ambient conditions. Net production estimates remained similar between M. vertebralis in isolation and in association with L. intricata, although both production and respiration rates of M. vertebralis were significantly higher when associated with L. intricata. These results indicate that the association with L. intricata increases the resilience of M. vertebralis to climate change stress, providing one of the first examples of physiological buffering by a marine alga that can ameliorate the negative effects of changing ocean conditions.


| INTRODUC TI ON
Increased anthropogenic CO 2 has caused physical and chemical changes to oceans worldwide causing global climate change (GCC; warming; IPCC, 2013) and ocean acidification (OA; Caldeira & Wickett, 2003). These physical and chemical changes are expected to have severe impacts on marine biota and in particular organisms that produce calcium carbonate shells or tests (Byrne & Fitzer, 2019;Kroeker et al., 2013;Kroeker, Kordas, & Harley, 2017). Although most studies to date have focused on single-species responses to climate change stressors (reviewed in Byrne & Fitzer, 2019;Hofmann et al., 2010;Przeslawski, Byrne, & Mellin, 2015), recent work highlights the importance of considering multispecies interactions in future multistressor ocean conditions (Kroeker et al., 2017). These interactions, framed in the context of ecological theory, provide relevant and more accurate predictions of how organisms will respond to climate change stress (Gaylord et al., 2015), and nuanced aspects of responses to climate change are overlooked in single-species studies. Examples of compensatory mechanisms whereby interactions between multiple organisms modify organism physiology have been shown to buffer against stress to maintain ecological equilibria (Doo, Carpenter, & Edmunds, 2018;Ghedini, Russell, & Connell, 2015). In particular, metabolic processes, such as photosynthesis in primary producers, have the potential to ameliorate the negative effects of projected climate change conditions (e.g., OA) through physiological interactions between multiple species (Connell et al., 2017).
On coral reefs, ecological interactions in the form of symbiosis (mutualism, parasitism, commensalism) between organisms drive high diversity (Hughes et al., 2003). Endosymbiotic marine relationships (and the transfer of energy from the endosymbiont to the host) have been relatively well studied in corals (e.g., Little, van Oppen, & Willis, 2004), but the effects of climate change on epibiont and ectosymbiont relationships are largely unknown. Macroalgae present an interesting case study, as they produce a diffusive boundary layer (DBL) of increased pH due to photosynthesis (Cornwall, Hepburn, Pilditch, & Hurd, 2013;Hurd et al., 2011). These layers have been observed on the micro-scale and have been hypothesized to buffer against the effects of ocean acidification on macroalgae and in seagrass beds (Bergstrom, Silva, Martins, & Horta, 2019;Cornwall et al., 2013;Hurd et al., 2011), although the corresponding increase in respiration can potentially negate these positive effects (Kapsenberg & Cyronak, 2019). Although macroalgal DBLs should influence the physiology of their calcifying epiphytes, observations at CO 2 seeps in Papua New Guinea and Mexico have shown negligible effects of buffering by seagrasses on calcifying epibionts in response to decreasing pH (Fabricius et al., 2011;Pettit, Smart, Hart, Milazzo, & Hall-Spencer, 2015). In contrast, a recent study identified increased abundance of mollusk species in turf algae at a temperate CO 2 seep site compared with adjacent ambient sites, suggesting that turf algae provide large DBLs, which benefit calcifying organisms residing on that substrata (Connell et al., 2017).
Foraminifera are single-celled organisms that reside in oceans worldwide, serving as a major carbon sink in both pelagic and benthic marine habitats (Langer, 2008). Shell geochemistry of planktic foraminifera has been extensively used as paleoindicators of past ocean conditions and climactic change (Hönisch et al., 2012;Spero, Bijma, Lea, & Bemis, 1997). Recently, anthropogenic-driven alterations in ocean chemistry have been identified in planktic foraminifera through changes in population density and shell thickness (Moy, Howard, Bray, & Trull, 2009;Osborne, Thunell, Gruber, Feely, & Benitez-Nelson, 2020). Benthic species, specifically large benthic foraminifera (LBFs), often cohabit with macroalgal substrata in shallow-water coral reef flats where they benefit from this symbiosis (Doo, Hamylton, & Byrne, 2012). Many LBFs such as including Marginopora vertebralis form symbioses with marine microalgae ( Figure 1a). These protists are especially important in terms K E Y W O R D S large benthic foraminifera, macroalgae, ocean acidification, ocean warming, physiological buffering, species interaction F I G U R E 1 Photograph of (a) Marginopora vertebralis taken using light microscopy. The dark green color indicates the presence of Symbiodinium sp. microsymbionts. Scale bar is 1 mm. (b) Postmortem, M. vertebralis is important in beach sand production, as seen in the foreground of the photograph, where white tests are seen. This image was taken at Coconut Beach, Lizard Island Reef, Australia of biogeochemical processes of carbon sequestration and generation of biogenic calcium carbonate (Langer, 2008;Langer, Silk, & Lipps, 1997). LBFs generate >95% of carbonate sands in some areas of coral reefs (Figure 1b; Baccaert, 1987;Davies & West, 1981) and up to 4%-6% of total carbonate storage of certain coral reef cays (Doo, Hamylton, Finfer, & Byrne, 2016). Recent studies on LBFs have showed varied but generally negative results of calcification and growth when exposed to increasing OA and GCC (reviewed in Doo, Fujita, Byrne, & Uthicke, 2014). Previous work on M. vertebralis has found increased productivity in associated dinoflagellate symbionts in response to near-future OA conditions, but this did not compensate for increased metabolic demands, and ultimately resulted in lower calcification rates for this species when compared to ambient conditions (Naidu, Hallock, Erez, & Maata, 2017;Uthicke & Fabricius, 2012).
To date, nearly all climate change studies of LBFs have involved experiments with these organisms in isolation, often not representative of natural systems. Most tropical LBFs occur in high densities associated with corticated and calcareous marine macroalgae such as Laurencia spp. and Halimeda spp. (Langer, 1993). In this study, we explore the potential for the noncalcifying macroalgae L. intricata

| Collection and acclimation
Specimens of M. vertebralis (as identified by Renema, 2018) and

| Incubation parameters and seawater chemistry
The seawater pH and temperature conditions were controlled using a Neptune Apex system dosing pure CO 2 to regulate pH. In the experiment, a total of 4 sumps were used, one for each manipulated seawater condition (see above). This water was pumped into individual jars (see above), maintaining independence between replicates. Total alkalinity, pH, and temperature of the header tanks were measured on a daily basis, from randomly selected drippers. Total alkalinity samples were filtered with a 0.22-µm filter prior to analysis to eliminate possible contamination of calcium carbonate in the sample and measured using open-cell potentiometric titrations (Dickson, Sabine, & Christian, 2007). Seawater pH was monitored using mcresol spectrophotometric measurements on an Ocean Optics USB4000+ spectrophotometer, and pH Total calculated based on standard protocols (Dickson, Sabine, & Christian, 2007). These were referenced to seawater Certified Reference Material (CRM), Batch 161, prepared by A. Dickson in the Scripps School of Oceanography.
Temperature and salinity measurements were collected with Vernier TMP-BTA and CON-BTA probes, respectively. Seawater parameters remained stable throughout the experimental incubation (Table 1).

| Growth measurements
The

| Instantaneous calcification measurements
After a 2-week incubation, alkalinity anomaly measurements were made using close bottle experiments as a proxy of instantaneous calcification. Organisms were carefully sealed in ~20 ml glass scintillation vials with their chosen treatment group water, and in the case of the algal associated groups, with the algal hosts. The sealed vials were immersed in the appropriate flow-through water system to maintain treatment temperature. Treatment groups were incubated for 8 hr in light conditions.
Analyses of water samples for total alkalinity were as above, and calcification (G) was calculated using Equation (1)

| Oxygen flux measurements
Oxygen flux measurements were made with a PreSens Oxy-10 mini 10-channel optical sensor. At the end of the 15-day incubation period, oxygen flux measurements were taken in 30 ml glass scintillation vials that were gently stirred. Replicate samples (including L. intricata in association treatments) were gently placed in the glass jars with corresponding pH and temperature conditions, and allowed to acclimate for at least 5 min before measurements were recorded.

For oxygen production measurements, light conditions in replicates
were ~100 µmol photons m −2 s −1 during measurements (similar to incubation levels) and measured for a total of ~30 min in light conditions first. Subsequently, respiration was measured in dark conditions for ~30 min, allowing for 5 min of acclimation, and rate of oxygen consumption measured after the acclimation period. All analyses were performed using standard protocols for LBFs outlined

| Statistical analyses
For growth rate, instantaneous calcification, maximum photochemical efficiency (Fv/Fm), total chlorophyll, and oxygen flux measurement data, a three-way ANOVA was performed using pH (amb, −0.3 pH units), temperature (amb, and +3°C), and association (no association-treatments of M. vertebralis only, and with association-treatments of M. vertebralis and L. intricata) as fixed factors. Assumptions of ANOVA (homogeneity of variance and normality) were tested and met. All analyses were performed in R Tukey HSD test analyses conducted with the agricolae package.

| Growth and calcification parameters
A 250% decrease in growth (wet weight change) was observed in M. vertebralis in low-pH conditions when they were not associated with L. intricata. In the presence of the algae, growth of M. vertebralis was not affected by acidification conditions (F 1,72 = 7.94, p < 0.001; Figure 2a; Figure S1; Table S1a). There was no effect of increased  Figure S2; Table S1b).

| Physiology of Laurencia intricata in GCC and OA conditions
Photosynthesis rates of L. intricata were not significantly impacted by

| D ISCUSS I ON
Our study highlights the importance of algae-calcifier relationships as a key component in the resilience of benthic assemblages Note: These data were used to provide a baseline metabolic rate and subtracted from Marginopora vertebralis and L. intricata oxygen flux measurements to infer rates of metabolism for M. vertebralis in associated treatment groups (see Methods). Values are mean ± SE (n = 10).

TA B L E 2 Respiration rates for Laurencia intricata in isolation
in the future ocean conditions. In particular, we show the potential for physiological modulation of calcifying organisms to GCC and OA through species interactions with a macroalgae (e.g., Bergstrom et al., 2019;Doo et al., 2018). Consistent with previous studies, we observed decreased growth (wet weight) in response to OA when M. vertebralis was incubated in isolation (Doo et al., 2014;Sinutok, Hill, Doblin, Wuhrer, & Ralph, 2011). As seen in previous culturing experiments, this decrease in wet weight is likely an integrated signal of longer-term (weeks) net dissolution associated with unfavorable carbonate chemistry conditions; however, other effects such as metabolic narcosis could also contribute to decreases in growth observed (Christensen, Nguyen, & Byrne, 2011). fish and algal growth rates (Ghedini et al., 2015), and ectosymbiotic crabs ameliorating the impacts of OA on host scleractinian coral calcification (Doo et al., 2018). At a temperate CO 2 seep site (analog of projected OA conditions), increased thickness of turf algae is directly linked to increases in calcifier (grazing gastropods) abundance through increased provisioning of suitable habitat and food (Connell et al., 2017). However, studies on LBFs in tropical CO 2 seep sites F I G U R E 2 (a) Growth, wet weight change per day with, and (b) calcification (alkalinity anomaly) of Marginopora vertebralis incubated in temperature (ambient, +3°C) and pH (ambient, −0.3 pH units) treatments for 2 weeks in isolation and in association with Laurencia intricata.
Fully factorial mean ± SE, n = 10 are expressed, and significance is shown with differing letters from Tukey HSD post hoc analyses show association with seagrass did not ameliorate the negative effects of OA on LBFs (Fabricius et al., 2011;Pettit et al., 2015). As such, a further understanding of biological and ecological interactions between species is a key to understanding climate change impacts in the context of ecological theory (Gaylord et al., 2015).
In studies that have investigated the role of alga-calcifier interactions, photosynthesis of the marine alga is hypothesized to ameliorate the negative effects of OA by increasing pH and saturation state. Although these increases in oxygen production from photosynthesis are demonstrated, DBLs have elevated pH in the micro-scale due to photosynthesis Hurd et al., 2011), potentially only benefiting organisms in close association with the algal basiphyte (Semesi, Beer, & Björk, 2009). Although we did not directly test for DBLs in this study, previous results in addition to other photophysiological parameters of total chlorophyll indicate that L. intricata may provide a stable refugium for associated LBFs due to production of a large DBL (Borowitzka, Larkum, & Borowitzka, 1978). Marginopora vertebralis also produces a large DBL (Glas & Fabricius, 2012) suggesting a shift in nutrient acquisition strategy from actively acquiring organic carbon resources through photosynthesis to passive environmental acquisition of similar resources maintaining homeostasis (calcification rate) in association with L. intricata. Marginopora vertebralis is also known to exhibit flexibility in biochemical responses to OA, allowing for acclimatization to changing ocean conditions (Prazeres, Uthicke, & Pandolfi, 2015).
Further, a decrease in total chlorophyll levels of M. vertebralis was observed when in association with L. intricata, while increasing maximum photochemical efficiency at ambient temperatures. These results suggest that photo-oxidative stress from endosymbiont photosynthesis for the LBF holobiont could be limited through adaptation mechanisms of decrease in total chlorophyll in conjunction with increased maximum photochemical efficiency (Prazeres, Uthicke, & Pandolfi, 2016). The increased stability that is gained through living on algae may lead to more favorable conditions, in which decreased total chlorophyll levels of M. vertebralis in association with L. intricata are able to maintain similar calcification rates (Prazeres et al., 2016). These shifts in physiological responses of M. vertebralis acquired through interaction with L. intricata highlight that LBFs may be adapted to algal substrata, and have the potential to use this interaction to buffer against changing ocean conditions. drastically in calcifiers incubated in isolation versus in ecologically relevant scenarios in association with their natural substrata, due to biological buffering from the algal DBL. In addition, our study indicates that the substratum choice is likely a key factor in the survival of M. vertebralis in a changing ocean, as the status of calcifiers that live in isolation from algal substrata will be impaired compared to those living in association with certain algal habitats. Species interactions, particularly multispecies symbioses that provide biological buffering services, are an important ecological process that needs further investigation to more accurately estimate the mechanistic changes that may occur under future climate change scenarios (Doo et al., 2020). International Research Scholarship. Renata Ferrari is thanked for assistance with fieldwork.

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data included in this manuscript have been included in the supporting information file and have been collated and deposited on the Dryad repository https://doi.org/10.5061/dryad.qv9s4 mwbw.