Review paper 9 pages

Journal of Experimental Marine Biology and Ecology 535 (2021) 151489

Available online 13 November 2020 0022-0981/© 2020 Elsevier B.V. All rights reserved.

Irradiance, photosynthesis and elevated pCO2 effects on net calcification in tropical reef macroalgae

C. McNicholl , M.S. Koch *

Florida Atlantic University, Boca Raton, FL 33431, USA

A R T I C L E I N F O

Keywords: Coral reef Dissolution pH Climate change Ocean acidification

A B S T R A C T

Calcifying tropical macroalgae produce sediment, build three-dimensional habitats, and provide substrate for invertebrate larvae on reefs. Thus, lower calcification rates under declining pH and increasing ocean pCO2, or ocean acidification, is a concern. In the present study, calcification rates were examined experimentally under predicted end-of-the-century seawater pCO2 (1116 μatm) and pH (7.67) compared to ambient controls (pCO2 409 μatm; pH 8.04). Nine reef macroalgae with diverse calcification locations, calcium carbonate structure, photophysiology, and site-specific irradiance were examined under light and dark conditions. Species included five from a high light patch reef on the Florida Keys Reef Tract (FKRT) and four species from low light reef walls on Little Cayman Island (LCI). Experiments on FKRT and LCI species were conducted at 500 and 50 μmol photons m− 2 s− 1 in situ irradiance, respectively. Calcification rates independent of photosystem-II (PSII) were also investigated for FKRT species. The most consistent negative effect of elevated pCO2 on calcification rates in the tropical macroalgae examined occurred in the dark. Most species (89%) had net calcification rates of zero or net dissolution in the dark at low pH. Species from the FKRT that sustained positive net calcification rates in the light at low pH also maintained ~30% of their net calcification rates without PSII at ambient pH. However, calcifi- cation rates in the light independent of PSII were not sustained at low pH. Regardless of these low pH effects, most FKRT species daily net calcification rates, integrating light/dark rates over a 24h period, were not signif- icantly different between low and ambient pH. This was due to a 10-fold lower dark, compared to light, calci- fication rate, and a strong correspondence between calcification and photosynthetic rates. Interestingly, low-light species sustained calcification rates on par with high-light species without high rates of photosynthesis. Low-light species’ morphology and physiology that promote high calcification rates at ambient pH, may increase their vulnerability to low pH. Our data indicate that the negative effect of elevated pCO2 and low pH on tropical macroalgae at the organismal level is their impact on dark net calcification, probably enhanced dissolution. However, elevated pCO2 and low pH effects on macroalgae daily calcification rates are greatest in species with lower net calcification rates in the light. Thus, macroalgae able to maintain high calcification rates in the light (high and low irradiance) at low pH, and/or sustain strong biotic control with high [H+] in the bulk seawater, are expected to dominate under global change.

1. Introduction

Marine calcifier persistence and sustained calcification rates remain uncertain under future predictions of ocean acidification. Since the in- dustrial revolution, global ocean pH has decreased 0.1 pH units, and a further 0.3–0.4 reduction is predicted to occur by the year 2100 due to anthropogenic CO2 emissions (Gehlen et al. 2014; Hartin et al. 2016). A global decrease in ocean pH affects calcification rates in marine organ- isms, such as coral, shellfish, phytoplankton, and ecologically important

macroalgae (Andersson et al. 2009; Fabry et al. 2008; Hoegh-Guldberg et al. 2007; Koch et al. 2013; Orr et al. 2005; Ries et al. 2009). Dimin- ished calcification rates of tropical reef macroalgae is a concern because of their ecological role in carbonate sediment production, building of 3- dimensional reef habitat structure, and providing substrate for inverte- brate larval settlement (Adey 1998; Nelson 2009). Many studies have examined the effects of elevated partial pressure of CO2 (pCO2) that lowers seawater pH on macroalgal calcification, but often with con- flicting results (Hofmann et al. 2014; Koch et al. 2013; Nelson 2009;

* Corresponding author. E-mail addresses: [email protected] (C. McNicholl), [email protected] (M.S. Koch).

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https://doi.org/10.1016/j.jembe.2020.151489 Received 19 November 2019; Received in revised form 22 October 2020; Accepted 2 November 2020

Journal of Experimental Marine Biology and Ecology 535 (2021) 151489

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Porzio et al. 2011). Discrepancies in the literature may depend on species-specific calcification mechanisms, photophysiology, location of calcification site, and calcium carbonate (CaCO3) crystal form and mineral content (reviewed in Basso 2012; Hofmann and Bischof 2014; Koch et al. 2013).

In marine macroalgae, CaCO3 precipitation occurs in areas that are isolated or semi-isolated from bulk seawater where the saturation state (ΩCaCO3) can be elevated to promote calcification (Borowitzka and Larkum 1987). Calcification typically occurs in the cell walls of Rho- dophyta (red macroalgae), such as crustose coralline algae (CCA), and other Rhodophyta families (e.g., Peyssonneliacea) (Adey et al. 2013; Basso 2012). In the Chlorophyta (green macroalgae), calcification oc- curs in sheaths surounding filaments or specific compartments con- nected to external seawater by diffusive channels (Borowitzka and Larkum 1987, 1976). CCA are thought to be the most sensitive to declining pH and elevated pCO2 due to the proximity of their calcifying sites to overlying bulk seawater and the high magnesium-calcite content of their crystals. Magnesium concentrations in CaCO3 lattice have a positive relationship with temperature and higher concentrations of Mg result in a relatively more soluble polymorph of CaCO3 (Kamenos et al. 2009; Kamenos and Law 2010; Mccoy and Kamenos 2015). A number of studies have shown negative effects of elevated pCO2 and low pH on CCA calcification (Anthony et al. 2008; Basso 2012; Comeau et al. 2019; Diaz-Pulido et al. 2014; Gao et al. 1993; Kato et al. 2014; Noisette et al. 2013); however, other studies imply CCA resistance (Comeau et al. 2018, 2017, 2013; Cornwall et al. 2017; Dutra et al. 2015; Ries et al. 2009). Chlorophytes, and some rhodophytes, have an aragonite poly- morph of CaCO3, which is less soluble than the high magnesium poly- morph found in CCA (Borowitzka and Larkum 1987). Precipitating a less soluble CaCO3 polymorph within semi-isolated compartments may be advantageous to resist elevated pCO2 and low pH (Comeau et al. 2013; Peach et al. 2017b, 2016; Ries 2011; Vogel et al. 2015a), yet there are a number of studies showing lower net calcification under declining pH and elevated pCO2 conditions (Meyer et al. 2016; Price et al. 2011). In addition to the potential effects of morphology and polymorphs, photosynthesis affects calcification in marine macroalgae (Diaz-Pulido et al. 2007; Hofmann and Bischof 2014; Koch et al. 2013; Porzio et al. 2011; Raven and Hurd 2012).

Photosynthesis has been shown to increase calcification in marine macroalgae (Borowitzka and Larkum 1987; De Beer and Larkum 2001; Gao et al. 1993; Koch et al. 2013; Pentecost 1978; Semesi et al. 2009; Wizemann et al. 2014), but the influence of increasing pCO2 and [H

+] on the coupling of these two processes is only recently being disentangled (Brown et al. 2019; Comeau et al. 2018; Hofmann et al. 2016; McNicholl et al. 2020, 2019). A majority of marine macroalgae use carbon concentrating mechanisms (CCMs) to saturate RuBisCO with CO2 for photosynthesis (Raven and Hurd 2012). In the process of HCO3

− uptake, HCO3

− dehydrogenation to CO2 and OH − , catalyzed by external carbonic

anhydrase (CAext), can neutralize H + and raise the macroalgal surface

pH. Immediate (seconds) light-triggered pH increase in macro-and micro-algal surfaces detected with microsensors, combined with photosynthetic inhibitors, provides evidence that photosynthesis and light are major drivers of pH control at the seawater-cell surface inter- face (Chrachri et al. 2018; Cornwall et al. 2015, 2013; De Beer and Larkum 2001; Hofmann et al. 2016; McNicholl et al. 2019). Presence and maintenance of a high thalli surface pH may support calcification under declining pH and elevated pCO2 (Cornwall et al. 2014; Hofmann et al. 2016; McNicholl et al. 2019). In addition to photosynthesis, light- triggered H+ transport pumps independent of photosystem II (PSII) have been identified in several species of macroalgae and may facilitate calcification (De Beer and Larkum 2001; Hofmann et al. 2016; McNi- choll et al. 2019). Electron microscopy of epithallial cells show in- vaginations that have been postulated to promote proton pumping in coralline algae during decalcification/recalcification to support thalli growth (Pueschel et al., 2005). Thus, photosynthesis and active proton pumping will likely play an important role for continued calcification

(or dissolution) processes. While calcification continues to occur in some marine macroalgae in

the dark, rates are typically reduced or become net negative (Chisholm 2000; El Haïkali et al. 2004; Vogel et al. 2015b), and may rely on accumulated energy stored during periods of irradiance (Mccoy and Kamenos 2015). Dark dissolution is likely driven by lower pH at calci- fication sites (Borowitzka and Larkum 1976; Wizemann et al. 2014). Amplification of this effect may occur with lower seawater carbonate ion concentrations [CO3

2− ] and lower ΩCaCO3, as well as increased seawater [H+] and pCO2 in the bulk seawater (Comeau et al. 2012; McNicholl et al. 2020). A buildup of external CO2 or H

+ in the dark may prevent removing H+ from the calcifying space against a higher [H+] concen- tration in seawater (Cyronak et al. 2015; Jokiel 2011), although some biotic control and dissolution itself can buffer pH of the bulk seawater at the surface in the dark. Internal cellular acid-base regulation may also become difficult in the dark when the electrochemical gradient proton motive force reverses from passive diffusive efflux of H+ (pHsw:pHcell 8.2:7.2) to requiring active H+ transport (pHsw:pHcell 7.8:7.2), as exemplified in calcifying phytoplankton with proton channels (Taylor et al. 2012). Higher energetic demands for proton pumps or lack of H+

regulation could shift net calcification to net dissolution at night even for those groups, such as the CCA, with a known potential to biotically control calcification/decalcification as part of their life history (Pueschel et al., 2005).

The objective of this study was to determine the effects of a 0.4 pH decline (pH 7.7) from the current ambient pH (8.1), as predicted for 2100 (Gehlen et al. 2014; Hartin et al. 2016), on net calcification rates of nine tropical reef macroalgae. Species examined were characterized by diverse calcification locations, CaCO3 content, polymorph, CCMs, and site-specific irradiance levels. This included five species from a high- light patch reef on the Florida Keys Reef Tract (FKRT) and four spe- cies from under reef wall ledges with low light on Little Cayman Island (LCI). Calcification rates independent of photosystem-II (PSII) were also investigated for FKRT species. All calcification experiments were con- ducted in the light and dark. We hypothesized that algae from high-light patch reefs would continue to calcify at elevated pCO2 due to their high potential to raise surface pH through photosynthesis (McNicholl et al. 2019) and calcifying space pH (Cornwall et al. 2017). Low-light species were predicted to have low photosynthetic rates; therefore, calcification rates would be reduced at elevated pCO2. Further, we postulated that elevated pCO2 would not lower net calcification rates in FKRT species shown to exhibit light-triggered thalli H+ regulation independent of PSII based on microsensor studies (McNicholl et al. 2019). We proposed that net calcification rates would be lower in the dark and this decline would be amplified at low pH.

2. Materials & methods

2.1. Calcified macroalgae collection and site parameters

2.1.1. Florida keys reef tract (high light environment) Intact individuals of five dominant calcifying algae, including 3

Rhodophyta (Neogoniolithon strictum, Jania adhaerens, CCA) and 2 Chlorophyta (Halimeda scabra, Udotea luna) were collected (between March – June 2018) from a shallow Looe Key patch reef (~3–5 m) (24.62055◦ N, 81.37078◦ W) on the FKRT (Fig. 1a, Fig. 2a-e). Neo- goniolithon strictum and J. adhaerens were collected by hand from the benthos, while H. scabra and U. luna were collected by inserting a dive knife into the surrounding carbonate sediment and lifting out the thalli with rhizoids intact. CCA-covered carbonate rubble (Fig. 2c) was also collected.

Field light levels were measured during collections at midday (~11:00–13:00) just above the benthos with a 4π spherical PAR quan- tum sensor (LI-193, LI-COR Inc.). The shallow patch reef site had high- irradiance (~800 μmol photo m− 2 s− 1) based on average midday mea- surements during collections. Experiments with high-light species from

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the FKRT were conducted under a full-spectrum LED light (Kessil, A360W E-Series Tuna Sun) at 500 μmol photons m− 2 s− 1 (LI-COR Quantum 4 π Light Sensor). While this was lower than mid-day field irradiance at the benthos, 500 μmol photons m− 2 s− 1 PAR approximated light levels that saturated photosynthesis in these species across a range of pH (Zweng et al. 2018).

Seawater in situ chemistry was measured or calculated for each macroalgal collection (n = 3) (Table 1a). Site pH (Orion A211, 8302BNUMD; calibrated with NBS standards, Thermo Fisher Scienti- fic®), conductivity (salinity = 35.5) and temperature (28.5 ± 1.1 ◦C) (YSI 650 MDS) were determined in the field. Water samples (n = 3; 60 mL) were collected and total alkalinity determined within 48 h or fixed with HgCl2 (0.02%) and measured within eight weeks. For clarity and continuity, measurements for pH are reported on the NBS scale since correction to total scale using the TRIS buffer was not available for ex- periments conducted on LCI. Total alkalinity (TA) was measured by open-cell titration (Metrohm Titrando® 888) with 0.01 N HCl. Certified reference material (CRM Batch #156; Dickson Lab, Scripps Institute of

Oceanography) was also run for each batch of TA samples. The CRM offset was used to correct TA readings from each batch. This way the data had no systematic bias from the true value. A certified standard TRIS buffer was used to calibrate pH for total alkalinity analysis (TRIS buffer, Dickson Lab, Scripps Institute of Oceanography: pH = 8.21, mV = − 64.9, 25 ◦C and 35 salinity). TA measurements were performed in triplicate unless the initial two measurements were within ±5 μmol kg− 1 of each other. Total alkalinity, temperature, conductivity, and pHNBS data were used to calculate DIC speciation (CO2SYS, Pierrot et al. 2006 with; K1, K2 from Mehrbach et al., 1973 refit by Dickson and Millero, 1987) for each experiment.

Following collections, algal samples were immediately transported (4 h) in aerated coolers to Florida Atlantic University (FAU) in Boca Raton, FL. Samples were separated by species and acclimated for 48 h in 9 L aquaria held within a large mesocosm tank (~500 L) over which 2 (100 cm length x 10 cm width) full-irradiance spectrum fixtures (BuildMyLED Inc.) were hung. To provide all 9 aquaria with a similar light field, the fixtures were hung ~0.5 m above the tank and provided

Fig. 1. Collection sites at (a, top panel) a shallow (3–5 m) high irradiance patch reef of the Florida Keys Reef Tract (Looe Key Reef; 24.62055◦ N, 81.37078◦ W) and (b, middle panel) a deeper (20 m) low irradiance wall reef on Little Cayman Island (Rockbottom Reef; 19.7025◦N, 80.05694◦ W). The (c) experimental setup for total alkalinity anomaly incubations where macroalgae were raised above the bottom of the beakers on a perforated disk (see insert) to allow for continued, slow stirring below the macroalga. Irradiance on the reefs was measured using a 4 π sensor; on the deep wall reefs (d) an underwater data logger was deployed.

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Fig. 2. Species from the (a-e) shallow (3–5 m) high irradiance patch reef of the Florida Keys Reef Tract and (f-i) deeper (20 m) low irradiance wall reef on Little Cayman Island used to examine the effects of elevated pCO2 and low pH predicted for 2100 on net calcifica- tion rates in light and dark experiments. Species from the FKRT (a) Neogoniolithon strictum, (b) Jania adhae- rens, (c) CCA, (d) Halimeda scabra, (e) Udotea luna and LCI Peyssonneliaceae (f) Peypink, (g) Peyred, (h) Hal- imeda copiosa and (i) Halimeda goreauii.

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~250 μmol photons m− 2 s− 1 to each aquarium on a 12:12 light:dark cycle. All aquaria were semi-immersed (~80%) in the mesocosm tank with water maintained at 29 ◦C, the average temperature for the FKRT during summer (Kuffner et al. 2015). Water in the aquaria was aerated and circulated via small submersible pumps and replenished (75%) every 2 d with seawater from the flow-through seawater system at the FAU marine lab (coastal Atlantic Ocean). Experiments were completed within 5 d of collections.

2.1.2. Little Cayman Island reef (low light environment) Calcified macroalgae were collected from a low-light reef wall

(Fig. 1b) on LCI and experiments conducted at the Little Cayman Research Center (LCRC) (July 2018). Four species, including 2 Rhodo- phyta prostrate lobed crustose algae (Family Peyssonneliaceae; Peyred and Peypink) and 2 Chlorophyta (Halimeda goreauii and Halimeda copiosa) were collected under ledges and crevices along the upper reaches of a reef wall (Rockbottom ~20 m; 19.7025◦N, 80.05694◦ W; Fig. 2 f-i). The Halimeda species hung under ledges (Fig. 1b, Fig. 2h-i) and were collected from the holdfast removing the loosely attached filaments. Peyssonneliaceae samples (Fig. 2 f-g) were carefully removed with a small chisel (20 cm) where the crustose lobes were attached to the substrate. While macroalgae were being collected, light was measured (LI-COR Quantum 4 π Light Sensor) in situ under ledges (Fig. 1d), col- lecting data during midday (~11:00–13:00) for ~5 min with s− 1 interval (RBRsolo3 single channel data logger). The range of light under ledges at the collection site was ~5 to 50 μmol photons m− 2 s− 1. Macroalgae were kept in aerated seawater from the reef at low light and immediately transportation to LCRC (< 2 h). At LCRC, algae were separated into 4 L aquaria, kept aerated in the shade (~ 50 μmol photons m− 2 s− 1) with natural sunlight and experiments run within 24 h. Seawater was collected and immediately analyzed for pH and salinity upon returning to the lab, and samples were fixed with HgCl2 (0.02%) for total alkalinity analysis within 8 weeks.

2.2. Elevated pCO2 experiments

Net calcification rates were determined for macroalgae in seawater

adjusted to pH (7.7) predicted for 2100 (Gehlen et al. 2014; Hartin et al. 2016) and ambient controls (8.1). Final pH and pCO2 treatment levels attained (see results) were determined from the initial and end mea- surement of each light (between 0800 and 1500) and dark (between 1900 and 0300) experiment. Calcification rates of FKRT and LCI species were determined using the TAA technique. The TAA technique for determining calcification rates in marine calcifiers is based on the changes in seawater TA over time. The TAA target was 3–10 times the accuracy of the method (~10 μmol kg− 1) and within 10% of TA (Lang- don et al. 2010). Based on this protocol, experimental incubation time for high-light species from the FKRT (n = 3–5) was 1–3 h in the light, depending on species, and 4 h in the dark. Incubation time for low-light species from LCI (n = 4) was 4.5 h in the light and 5.5 h in the dark. Blank runs with seawater were also conducted. Experiments were con- ducted in glass beakers (150–250 mL) covered with parafilm and secured with rubber bands to reduce atmospheric gas exchange (Fig. 1c) according to Chisholm and Gattuso (1991). Individual thalli were sus- pended approximately 2 cm above the bottom of the beaker on a perforated disk with a stirbar underneath and flow created with a stir- plate (Fig. 1c insert).

Experimental seawater was filtered (0.45 μM) and brought to tem- perature (29 ◦C) in a waterbath before assigning treatment. Low pH treatment was obtained by bubbling seawater with pure CO2 prior to incubations. Initial and post-incubation pH, O2 (Orion A329), and temperature (Orion A211, 8302BNUMD) were recorded. Algae were acclimated to experimental seawater for 15 min. A relatively short acclimation time was needed to keep carbonate chemistry and △pH to a minimum before incubations. Low-light experiments with LCI species were conducted using the same experimental setup and protocols with the exception that these species were incubated at 50 μmol photons m− 2 s− 1.

Ambient and low pH treatment runs were randomized so ~50% of the runs were low pH treatments, followed by ambient controls, and the inverse for the other 50%. In this experiment, repeated measures were used (i.e., treatments were applied sequentially to the same individual thalli). When similar TAA experiments were run during 45Ca experi- ments (McNicholl et al. 2020), similar results were found for TAA in

Table 1 Summary of (a) average seawater carbonate chemistry during incubations averaged across experiments in light/dark and ambient (A) or low (L) pH. In situ seawater conditions (n = 3) from collection sites Florida Keys Reef Tract (FKRT) and Little Cayman Island (LCI) shown. Carbonate chemistry parameters were calculated using CO2SYS (Pierrot et al. 2006) applying experimental seawater temperature (29 ◦C) and salinity (35.5), and (b) change (△) in seawater pH, total alkalinity (TA) and oxygen (O2) during incubations in ambient and low pH experiments in the light and dark averaged across all experiments (n = 38). Means ± SD. Details of carbonate chemistry and △pH, △TA and △O2 are presented for all experiments by species and treatments in supplemental tables (Table S1, S2). Summary of (a) average seawater carbonate chemistry during incubations averaged across experiments in light/dark and ambient (A) or low (L) pH. In situ seawater conditions (n = 3) from collection sites on the Florida Keys Reef Tract (FKRT) and Little Cayman Island (LCI) reefs are shown. Carbonate chemistry parameters were calculated using CO2SYS (Pierrot et al. 2006) applying experimental seawater temperature (29 ◦C) and salinity (35.5).The (b) change (△) in seawater pH, total alkalinity (TA) and oxygen (O2) in the light and dark are averaged across all experiments (n = 38). Means ± SD. Details of carbonate chemistry and △pH, △TA and △O2 are presented for all experiments by species and treatments in supplemental tables (Table S1, S2).

(a) pH pCO2 (μatm)

HCO3 −

(μmol kg− 1) CO3

2−

(μmol kg− 1) TA

(μmol kg− 1) ΩCa ΩArag

Experimental Light - A

Light - L 8.07 ± 0.06 7.71 ± 0.05

367 ± 64 992 ± 140

1665 ± 76 1981 ± 58

248 ± 23 130 ± 12

2283 ± 29 2302 ± 39

6.0 ± 0.6 3.1 ± 0.3

4.0 ± 0.4 2.1 ± 0.2

Dark - A Dark - L

8.00 ± 0.03 7.62 ± 0.03

451 ± 34 1239 ± 112

1760 ± 31 2062 ± 29

222 ± 11 111 ± 8

2305 ± 31 2329 ± 31

5.4 ± 0.3 2.7 ± 0.2

3.6 ± 0.2 1.8 ± 0.1

In Situ FKRT

LCI 8.10 ± 0.02 8.05 ± 0.03

343 ± 11 402 ± 37

1662 ± 17 1752 ± 39

259 ± 4 243 ± 13

2292 ± 31 2348 ± 24

6.2 ± 0.1 5.9 ± 0.3

4.2 ± 0.1 3.9 ± 0.2

(b) Ambient pH Experiments Low pH Experiments

△ pH △ TA

(μmol kg− 1) △ O2

(mg L− 1) △ pH

△ TA (μmol kg− 1)

△ O2 (mg L− 1)

Experimental Light <0.01 ± 0.09 − 70 ± 41 1.60 ± 0.93 0.09 ± 0.09 − 43 ± 50 1.82 ± 0.90 Dark − 0.11 ± 0.03 − 17 ± 12 − 0.42 ± 0.33 − 0.08 ± 0.03 22 ± 16 − 0.51 ± 0.31

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FKRT species using completely independent thalli for each run.

2.3. PSII inhibitor experiments

To examine light-dark PSII-independent calcification rates, an in- hibitor, 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU), was added to the seawater according to Hofmann et al. (2016), after De Beer and Larkum (2001) and Borowitzka and Larkum (1976). DCMU was also highly effective at arresting photosynthesis in microsensor studies, eliminating O2 flux from the diffusive boundary layer (100 μm) at the thalli surface (McNicholl et al. 2019). A stock solution of 0.05 M DCMU was added to reach a concentration of 4 μM. Algae were left in DCMU- amended seawater until no change in O2 production was detected upon illumination (~30 min). Incubations were conducted (as above) at both pH levels and light/dark conditions with DCMU. DCMU experi- ments were only performed on species from the FKRT species due to time constraints on LCI.

2.4. Calculation of net calcification and chemistry analysis

Net calcification (Gnet) rates in μmol CaCO3 g dwt− 1 h− 1 were calculated from TA changes (△TA) during the experiments based on the following equation (Eq. 1):

Gnet = − 0.5ρw ∆TA*v Wa*t

(1)

where: ρw is seawater density (kg L− 1) and ∆TA (μmol kg− 1) is the final TA minus the initial TA, v is the volume of seawater (L), Wa is the algal dry weight (g) and t is the incubation time in hours. Calcification rates were normalized to dry weight (60 ◦C) for FKRT species with the exception of CCA that had a complex 3-D structure. CCA was normalized to g of a flexible 2-D surface (foil) according to Marsh (1970). Calcifi- cation rates of Halimeda spp. from LCI were normalized to dry weight as above and to cm− 2 of thalli surface for LCI Peyssonneliaceae. Daily calcification rates were calculated by combining light (LGnet) and dark (DGnet) net calcification rates using a 12:12 light:dark cycle, as a first- order estimate of rates over 24 h (Eq. 2).

Daily Gnet = (LGnet*12) + (DGnet*12) (2)

2.5. Photosynthesis and respiration

Photosynthesis and respiration rates were determined from initial and final dissolved O2 measurements (optical probe) using data from incubation runs in the light and dark, respectively. The O2 flux rates were normalized to dry mass (g) or surface area, as described above, and adjusted to seawater volume and time.

2.6. Statistical analysis

A repeated measures two-way ANOVA was performed (SigmaPlot v13.0, Systat Software Inc.) to compare calcification rates across light/ dark at ambient and low pH treatments for experiments with and without DCMU. The assumptions of normality and homogeneity of variance were tested using the Shapiro-Wilkes and Brown-Forsythe tests, respectively. The assumption of sphericity in repeated measures was tested using the Mauchly’s test. Differences amongst means were established using the Holm-Sidak post-hoc test. The effect of pH treat- ments on the calculated daily calcification rates were determined using a t-test. Regression analysis was used to establish the relationship between net photosynthesis and calcification (SigmaPlot v13.0, Systat Software Inc.). Significance levels were established at p < 0.05 unless otherwise stated.

3. Results

3.1. Carbonate chemistry and △pH, △TA and △O2

Carbonate chemistry, treatment and in situ pH and pCO2, △pH, TA and △O2 are summarized in Table 1. All data by species and

treatments are presented in supplemental tables (Tables S1, S2). The resulting average pH and pCO2 across experiments and treatments was 7.67 and 1116 μatm, respectively (Table 1). The average ambient pH and pCO2 for controls were 8.04 and 409 μatm, respectively (Table 1). The averages of the initial and end pCO2 during all experiments was approximately 3-fold higher in the elevated pCO2 treatments compared to controls, resulting in a pH of 7.71 for the low pH treatment compared to 8.07 for ambient pH (Table 1a). The dark experiments were ~ 0.08 pH lower than in the light due to metabolic differences, but the values were within the pH variance found in the light experiments (Table 1a). The concentrations of CO3

2− and saturation state of CaCO3 declined ~50% in the high pCO2 treatments but remained above CaCO3 saturation (Ω > 1) for all experiments (Table 1a). The △pH and △O2 across experiments indicate only modest changes in chemistry occurred throughout the experiments (Table 1b). The △pH was on average ± 0.03 to ±0.09 showing that our treatments were close to those applied at the initiation of the experiment. This is also indicated by a small change in metabolic O2, which was similar in the light for both high and low pH treatments (Table 1b). Average TA changes in the light were at least 3 times our accuracy for CRM TA (± 10 μmol kg− 1) and remained significantly lower than 10% of seawater TA. Further, the TA data for all the experiments highlight our general result that in the light △ TA was negative (indi- cating net calcification occurred) (Table 1b).

3.2. High light species (FKRT) calcification

Greater net calcification rates were found in the light compared to the dark for all species (Fig. 3) based on 2-way ANOVA main effects. Net calcification rates were similar at low and ambient pH for N. strictum (Holm-Sidak, t = 1.38 p = 0.214) and J. adhaerens (Holm-Sidak, t = 1.99 p = 0.085) in the light (Fig. 3a,b). Halimeda scabra net calcification rates increased 16% at low pH in the light (Fig. 3d; Holm-Sidak, t = 2.54, p = 0.035), indicating a positive response to elevated pCO2. In contrast, U. luna (Holm-Sidak, t = 3.07 p = 0.018) and CCA (Holm-Sidak, t = 4.56 p = 0.028) net calcification rates significantly declined at low pH rela- tive to controls in the light with CCA eliciting the strongest negative effect (Fig. 3c,e).

Only N. strictum and J. adhaerens showed significant positive net calcification rates in the dark and only at ambient pH (Fig. 3a,b). Dark calcification rates for these two species were only a tenth the rates observed in the light. Even at ambient pH, U. luna exhibited net disso- lution in the dark (Fig. 3c), while H. scabra and CCA had net calcification rates approximating zero (Fig. 3d,e). Neogoniolithon strictum, J. adhaerens, and H. scabra (Holm-Sidak, t = 2.73 p = 0.032, t = 2.34 p = 0.050, t = 2.71 p = 0.027, respectively) had significantly lower net calcification rates in the dark at low pH compared to ambient pH (Fig. 3a,b,d). CCA had 87% less net calcification on average at low pH compared to ambient pH in the dark, although not significant (Holm- Sidak, t = 1.59 p = 0.226).

The two species that were negatively affected by low pH in the light, U. luna and CCA, had lower daily calcification rates at low pH relative to ambient controls (Fig. 3c,e; t-test, t = 2.65 p = 0.038, t = 5.27 p = 0.006, respectively). In contrast, the three species with no significant negative low pH effect on net calcification in the light, N. strictum (t-test, t = 1.21 p = 0.262), J. adhaerens (t-test, t = 1.16 p = 0.278), and H. scabra (t-test, t = 0.03 p = 0.977), had similar daily calcification rates in low and ambient pH treatments (Fig. 3a,b,d). Halimeda scabra’s significantly lower net calcification rate in the dark at low pH was offset by increased calcification rates in the light at low pH, resulting in no difference in daily calcification rates (Fig. 3d).

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3.2.1. Photosynthesis and calcification Net photosynthesis and respiration were not different between the

low and ambient pH for any species from the FKRT or LCI (Table 2). Species from the FKRT that exhibited a broad range in photosynthetic rates amongst individuals exhibited a strong correlation between net photosynthesis and net calcification rates (Fig. 4). Linear relationships between net photosynthesis and calcification rates for N. strictum and H. scabra were stronger at low pH (R2 = 0.96; R2 = 0.94) compared to ambient pH (R2 = 0.68; R2 = 0.64), respectively (Fig. 4a,c), while for J. adhaerens the relationships were relatively similar (R2 = 0.87, R2 = 0.98; Fig. 4b). The other two species did not have a broad range in photosynthetic and calcification rates, thus no relationship could be established.

3.2.2. Inhibitor experiments (FKRT) The photosynthesis inhibitor (DCMU) arrested O2 flux in the light for

all species, providing confidence that calcification in the presence of DCMU was non-PSII light-dependent calcification (Table 2). Calcifica- tion was stimulated in the light without PSII in three FKRT species,

including N. strictum, J. adhaerens, and H. scabra (Fig. 5a,b,d). These species maintained a relatively high percentage (22 to 34%) of the calcification rates attained in the light without PSII inhibition (Fig. 3a,b, d). This light-triggered non-PSII net calcification was significantly greater than calcification rates measured in the dark at ambient pH (Holm-Sidak, t = 3.58 p = 0.012, t = 3.98 p = 0.004, t = 3.00 p = 0.032, N. strictum, J. adhaerens, and H. scabra, respectively). However, non-PSII light-dependent calcification rates were not sustained at low pH. Calcification rates at low pH without PSII were similar in the light and dark (Holm-Sidak, t = 0.43 p = 0.681, t = 1.84 p = 0.104, t = 1.01 p = 0.351, N. strictum, J. adhaerens, and H. scabra, respectively). No significant light-triggered calcification with DCMU was observed for U. luna (Two-way ANOVA, F1,4 = 1.55 p = 0.281) or CCA (Two-way ANOVA, F1,4 = 0.065 p = 0.812). For all species examined, no net posi- tive calcification rates were measured in the presence of DCMU at low pH in the dark (Fig. 5).

Fig. 3. Net calcification rates (n = 3–5) of Florida Keys Reef Tract macroalgal species (a = Neogoniolithon strictum, b = Jania adhaerens, c = Udotea luna, d = Halimeda scabra, e = CCA) in the light (500 μmol photons m− 2 s− 1) and dark at ambient and low pH. Totals are daily net calcification rates calculated by combining light and dark calcification rates at the respective treatment pH and normalized to 24 h. Means ± standard errors are shown. Different lowercase letters represent significant differences from a two-way ANOVA with Light x pCO2 treatments and a post-hoc Holm-Sidak to compare between means (P < 0.05). *CCA calcification rates are normalized to g of flexible 2-D surface due to high 3-D complexity. Differences in daily calcification rates and CCA within light treatments were determined by t-tests; daily differences shown with capital letters (P < 0.05).

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3.3. Low light species (LCI) calcification

Although the low-irradiance Halimeda species from LCI reefs were incubated at 10-fold lower irradiance and had 10 times lower photo- synthetic rates compared to FKRT Halimeda species (Table 2), their calcification rates in the light (Fig. 6a,b) were similar (Fig. 3d). At ambient pH, net calcification rates were significantly higher in the light relative to the dark (Fig. 6; Holm-Sidak, t = 4.92 p = 0.003, t = 3.28 p = 0.038, t = 3.37 p = 0.023, H. goreauii, H. copiosa, Peyr, respectively) with the exception of Peyp, that approached significance (Fig. 6d; Holm- Sidak, t = 2.59 p = 0.073). Lower dark calcification rates compared to the light were consistent with results from the FKRT species (Fig. 3). However, in contrast to Halimeda from the FKRT, relatively high positive net calcification rates were maintained in the dark by Halimeda species from LCI reefs, H. goreauii (39%) and H. copiosa (22%) (Fig. 6a,b). These dark calcification rates are more than twice those of N. strictum and J. adhaerens from the FKRT (Fig. 3a,b). Calcification rates at low pH in the light decreased by 49% and 28% for H. goreauii and H. copiosa, respec- tively (Fig. 6a,b), but was only significant for H. goreauii (Holm-Sidak, t = 3.19 p = 0.021), likely due to high variance in H. copiosa (Holm- Sidak, t = 1.78 p = 0.149). Although both H. goreauii and H. copiosa had on average ~ 50% lower daily calcification rates at low pH in the light, the differences only approached significance for H. goreauii (t-test, t = 2.15 p = 0.0748) and was not significant for H. copiosa (t-test, t = 1.18 p = 0.281). Peyred from LCI only exhibited positive net calcifi- cation rates in the light at ambient pH and showed net dissolution at low pH in the light and dark (Fig. 6c). Although similar trends were observed, low pH only significantly reduced Peypink (Holm-Sidak, t = 3.17 p = 0.047) net calcification rates in the dark, but not Peyred (Holm-Sidak, t = 0.800 p = 0.460). Daily calcification rates were

significantly lower at low compared ambient pH for Peypink (t-test, t = 2.93 p = 0.026) and approached significance for Peyred (t-test, t = 2.30 p = 0.061) (Fig. 6c,d).

4. Discussion

The most consistent negative effect of low pH and elevated pCO2 on calcification rates in tropical macroalgae examined from FKRT and LCI occurred in the dark, albeit effects in the light controlled daily calcifi- cation rates. Most of the species examined (89%) had calcification rates of zero or net dissolution in the dark under 2100 predictions for pH and ocean carbonate chemistry. Other experimental and field studies also indicate negative effects of elevated pCO2 and low pH on dark calcifi- cation rates. Dark calcification rates of Halimeda opuntia decreased 167% at a low pH (~7.8) tropical CO2 seep site compared to non-seep adjacent control areas (Vogel et al. 2015a). These data are comparable to the 171% decrease in dark calcification rates observed at low pH for the three Halimeda species in this study. A temperate coralline alga (Lithothamnion glaciale) with net positive calcification rates in the dark at ambient pH, also exhibited net dissolution when exposed to low pH in

Table 2 Net photosynthesis and respiration rates during the light and dark calcification incubations at ambient and low pH and with and without a photosystem II in- hibitor (DCMU). All data are normalized to gram dry weight per hour with the exception of CCA from the Florida Reef Tract and Peyssonneliaceae from Little Cayman Island (see below). Mean +/− SE (n = 5–3).

Oxygen (μmol O2 g dwt− 1 or cm− 2 h− 1)*

Net Photosynthesis Respiration

Amb pH Low pH Amb pH Low pH

Florida Reef Tract (No DCMU)

N. strictum 8.66 ± 1.66 8.83 ± 1.42 -0.76 ± 0.08 -0.96 ± 0.17 J. adhaerens 15.47 ± 4.13 19.29 ± 4.09 -2.39 ± 0.49 -2.97 ± 0.70 U. luna 12.85 ± 1.51 12.54 ± 0.95 -2.08 ± 0.34 -2.10 ± 0.48 H. scabra 8.12 ± 1.95 8.40 ± 2.12 -1.30 ± 0.22 -1.36 ± 0.45 *CCA 37.24 ± 13.08 48.30 ± 13.59 -6.28 ± 1.84 -8.62 ± 2.79

Florida Reef Tract (+ DCMU)

N. strictum -1.09 ± 0.42 -1.75 ± 0.44 -1.29 ± 0.21 -1.37 ± 0.26 J. adhaerens -2.63 ± 1.08 -3.81 ± 0.76 -2.92 ± 0.50 -4.13 ± 0.70 U. luna -2.70 ± 0.88 -3.56 ± 0.24 -3.31 ± 0.71 -3.39 ± 0.99 H. scabra -2.51 ± 0.73 -2.94 ± 0.52 -2.45 ± 0.71 -2.31 ± 0.53 *CCA -11.76 ± 2.64 0.02 ± 0.35 -14.40 ± 4.62 -11.19 ± 1.27

Little Cayman Island (No DCMU)

H. goreauii 1.37 ± 0.98 2.55 ± 0.84 -0.49 ± 0.19 -0.33 ± 0.31 H. copiosa 3.34 ± 0.94 3.26 ± 1.00 -0.53 ± 0.32 -0.92 ± 0.32 *Peyred 0.19 ± 0.01 0.19 ± 0.04 -0.05 ± 0.01 -0.05 ± 0.03 *Peypink 0.12 ± 0.03 0.17 ± 0.03 -0.01 ± 0.01 -0.06 ± 0.02

* CCA from the Florida Reef Tract normalized to g of flexible 2-D surface due to high 3-D complexity and Peyssonneliaceae; Peyred and Peypink from Little Cayman Island to cm− 2 of thalli surface.

Fig. 4. The linear relationship between net photosynthetic and calcification rates (n = 5) in three Florida Reef Tract species (a = Neogoniolithon strictum, b = Jania adhaerens, c = Halimeda scabra). Equations and R2 for linear re- gressions are shown for ambient pH (circles with solid line) and low pH (tri- angles with dashed line) treatments.`

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the dark (Kamenos et al. 2013). Reduced net calcification by 164% in macroalgae (H. opuntia) from the Great Barrier Reef at low pH in the dark was interpreted as a negative amplifying effect of low pH (Vogel et al. 2015b). The impact of low pH and elevated pCO2 on preferentially nighttime net calcification (Kamenos et al. 2013; Venn et al. 2019; Vogel et al. 2015b; this study) necessitates a greater understanding of these mechanisms.

The constraints on net calcification rates in the dark at low pH is likely attributable to greater rates of dissolution. This conjecture is reasonable given McNicholl et al. (2020) found in 45Ca experiments either no significant difference between gross calcification rates in the dark between low (7.7) and ambient (8.1) pH, or a shift from positive gross calcification rates to net dissolution at low pH in the dark, in a majority of the FKRT species examined herein (N. strictum, J. adhaerens, H. scabra, U. luna). This was the case, even though net calcification rates significantly declined at low pH in the dark in all four of the FKRT species examined (McNicholl et al. 2020). McNicholl et al. (2020) also established a strong relationship (R2 = 0.82) between increasing TA and

loss of 45Ca from pre-labelled 45CaCO3 thalli only in the low pH treat- ment, suggesting dissolution. These data indicate that the ability to form new CaCO3 is not the primary factor constraining net calcification rates in the dark at low pH for the majority of FKRT species examined herein. Further, respiration rates cannot account for the increased dissolution at low pH in darkness. None of the species examined exhibited greater respiration rates in the dark at low pH. Comeau et al. (2016) also found respiration rates to be insensitive to elevated pCO2 in 6 coral and 6 macroalgal species from reefs in Moorea (French Polynesia). Other macroalgal studies support the conclusion that respiration rates do not increase in response to low pH conditions in the dark (Kamenos et al. 2013; Martin et al. 2013a; Semesi et al. 2009; Zou et al. 2011; Zweng et al. 2018). A general amplifying effect of low pH on dissolution in the dark was observed in both high- and low-light macroalgae in the present study, regardless of species-specific photophysiology, calcification location, taxonomy, or thalli CaCO3 polymorph, suggesting a funda- mental relationship that necessitates further research.

In contrast to results in the dark, low pH had no significant negative

Fig. 5. Net calcification rates (n = 3–5) of Florida Reef Tract species (a = Neogoniolithon strictum, b = Jania adhaerens, c = Udotea luna, d = Halimeda scabra, e = CCA) with the photosynthesis inhibitor DCMU in the light (500 μmol photons m− 2 s− 1) and dark at ambient and low pH. Means ± standard errors are shown. Different lowercase letters represent significant differences from a two-way ANOVA with Light x pCO2 treatments (P < 0.05). *CCA calcification rates are normalized to g of flexible 2-D surface due to high 3-D complexity.

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effect on FKRT species net calcification rates under high irradiance (500 μmol photons m− 2 s− 1) except for U. luna. Neogoniolithon strictum and J. adhaerens maintained 87% of their calcification rates and H. scabra increased net calcification rates 16% at high light and low pH. Two other Halimeda species (H. digitate and H. opuntia) from CO2 seep sites in Indonesia at low pH (7.7) were also found to increase calcifi- cation rates in the light (131% and 41%, respectively) relative to adja- cent ambient pH control sites (Vogel et al. 2015a). The FKRT species that maintained calcification rates under low pH and elevated pCO2 in the light had a strong positive relationship between net photosynthesis and calcification at ambient (R2 0.64 to 0.98) and low pH (R2 0.87 to 0.96). While the importance of photosynthesis in macroalgal calcification has long been appreciated (Borowitzka 1981; Koch et al. 2013; Pentecost 1978), how this relationship is sustained under low pH has not been resolved. Further, in some species, as was shown for H. heteromorpha, net photosynthetic rates do not always positively correspond to rates of calcification at low pH (Brown et al. 2019). Photosynthesis has been shown to elevate pH at the macroalgal thalli surface under low pH and elevated pCO2 (Cornwall et al. 2015; Hofmann et al. 2016; McNicholl et al. 2019). This photosynthetically-driven increase in surface pH can mitigate the negative effects of bulk seawater acidification on net calcification (Cornwall et al. 2014). Photosynthesis also elevates pH within the calcifying space of corals and macroalgae, even under low external bulk seawater pH (Comeau et al. 2018; Cornwall et al. 2017; Venn et al. 2019).

In addition to photosynthesis, light-dependent proton pumps inde- pendent of PSII may be important for macroalgal calcification. Three species from the FKRT (H. scabra, N. strictum, and J. adhaerens) main- tained 22% to 34% of their calcification rates in the light independent of PSII. These same three species were observed to control thalli surface H+

light/dark dynamics independent of PSII (McNicholl et al. 2019). Thus, active pH regulation independent of PSII may be linked to calcification

in macroalgae. Calcification rates in the light without PSII were not sustained at low pH in the present study, even though H+ dynamics seemingly continue at low pH (McNicholl et al. 2020). This was possibly due to unsustainable H+ transport requirements, acid-base disfunction, and/or changes in the electrochemical gradients of H+ across the plas- malemma. Thus, we suggest photosynthesis and light-triggered proton pumps may promote calcification in the light at ambient pH, but proton pumps become overwhelmed under elevated [H+] at low pH. Under this scenario, calcification would be more dependent on high rates of photosynthesis as ocean pH declines.

Although the FKRT species were reliant on photosynthesis to facili- tate high calcification rates, Halimeda species growing on reef walls maintained similarly high calcification rates at 10-fold lower irradiance. The distinct morphology and physiology of the three Halimeda species examined in this study may explain the divergent responses to low pH. H. scabra from the FKRT that sustained calcification in the light at low pH has relatively high organic:inorganic carbon ratios (Peach et al. 2017b; Vroom et al. 2003) indicating a high photosynthetic capacity. In contrast, species with lower organic:inorganic carbon ratios, H. goreauii and H. copiosa, did not compensate for low pH via photosynthesis while growing in low light (50 μmol photons m− 2 s− 1). Low-light adapted Halimeda species likely have an alternative strategy to promote high rates of calcification. One hypothesis is that short diffusive pathways (<5 μm, Peach et al. 2017a) that connect their calcifying space to external bulk seawater allow for efficient export of H+, a byproduct of calcification that can lower internal pH and limit further calcification. The path-length may also facilitate diffusive uptake of Ca2+ and CO3

2−

into the calcifying space in support of calcification. It has been shown by Peach et al. (2017a) that the shorter the diffusive path length the greater the %CaCO3 in Halimeda species from LCI, including the species exam- ined herein. A short path-length morphology in H. goreauii and H. copiosa, combined with a low respiration rate due to a low organic:

Fig. 6. Net calcification rates (n = 4) of Little Cayman Island species (two chlorophyte Halimeda species: a = Halimeda goreauii, b = Halimeda copiosa, and two rhodophyte species from the Peyssonneliaceae family: c = Peyr, d = Peyp) in low light (50 μmol photons m− 2 s− 1) and dark at ambient and low pH. Totals are daily net calcification rates calculated by combining light and dark calcification rates at respective pH and normalized to 24 h. Means ± standard errors are shown. Different lowercase letters represent significant differences from a two-way ANOVA with Light x pCO2 treatments and post-hoc analysis with Holm-Sidak (P < 0.05). Peys- sonneliaceae calcification rates were normalized to surface area of thalli lobes (cm2). Significant differences in daily calcification rates were determined by t-tests and shown with capital letters (P < 0.05, unless otherwise shown).

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inorganic ratio, likely accounts for high calcification rates measured for LCI Halimeda at ambient pH, regardless of low photosynthetic rates. Low light adaptation, and potentially a greater dependence on proton pumps associated with biotic control, was shown by H. goreauii’s and H. copiosa’s ability to maintain 39% and 22% of their calcification rates in the dark at ambient pH. However, low-light adapted morphology and physiology that allow Halimeda species to be effective and dominant calcifiers on deep reefs (Littler et al. 1985; Vroom et al. 2003), may also enhance their vulnerability to low pH. For example, low-irradiance Halimeda species are likely more dependent on proton pumps for calci- fication. We observed a significant loss of non-PSII light-induced calci- fication in FKRT species at low pH, potentially an indicator of a decline in biotic control of calcification. While we (McNicholl et al. 2019) and others (De Beer and Larkum 2001) observed Halimeda species to possess proton pumps, the loss of proton pump function in regards to calcifi- cation has not been examined in low-light species at low pH.

Two species from the high light FKRT site (U. luna and CCA) also had significant declines in net calcification in the light at low pH. Net dissolution occurred in these two species in the dark under both ambient and low pH. The apparent lower resistance to dissolution and lower pH in the light may correspond to the proximity of these species’ calcifi- cation sites to bulk seawater. Udotea luna calcification occurs within external sheaths along thalli filaments that are directly exposed to bulk seawater (Bohm 1978). Udotea luna dark dissolution rates were also found to be high in both ambient and low pH treatments in 45Ca ex- periments (McNicholl et al. 2020). Further, microsensor experiments (McNicholl et al. 2019) singled out U. luna amongst FKRT species as having the least ability to raise pH at the thalli surface in response to light at low pH, leading to the suggestion that it has weak biotic control. Meyer et al. (2016) showed Udotea flabellum to also exhibit net disso- lution in the dark under ambient pH. Further, U. flabellum had a 36% lower net calcification rate under low pH in the light, relative to con- trols. CCA calcification sites are also proximate to seawater because of its prostrate form. This group is often recognized as being highly vulnerable to declining pH and elevated pCO2 due to a high‑magnesium CaCO3 structure (Ries 2011). Secondary calcification also occurs be- tween filaments in coralline macroalgae which are more exposed to bulk seawater than cell wall calcification, and thus less resistant to increased [H+] (Cornwall et al. 2017; Hofmann et al. 2012). While we observed a negative low pH effect on calcification rates of prostrate encrusting rhodophytes in these short-term incubations, Peyssonneliaceae (Dutra et al. 2015) and CCA (Kamenos et al. 2016; Martin et al. 2013b) have exhibited more robust responses to elevated pCO2 in longer-term studies. Kamenos et al. (2013) also detected molecular-level changes in CCA carbonate minerals that were exposed to abrupt, but not slow, treatments of low pH (7.77). These data correspond to recent results demonstrating the ability of CCA to acclimate to lower pH over several generations (Cornwall et al. 2020). Thus, mechanisms and species- specific resistance/vulnerabilities to future changes in pH and carbon- ate chemistry need further examination in both short and long-term studies.

Based on our research, we propose that negative responses to elevated pCO2 and low pH at the organismal level for calcifying tropical macroalgae are primarily associated with effects on net calcification in the dark for high-light species. This is likely attributable to greater dissolution in the dark at low pH. Even with greater dissolution in the dark at low pH, daily net calcification rates can be unaffected by low pH because of high net calcification rates in the light and low overall calcification rates in the dark. Low pH and elevated pCO2 effects on daily calcification rates appear to be greatest in species that exhibit declines in net calcification rates in the light. Further, our inhibition experiments lead us to suggest that PSII-independent calcification mechanisms may become overwhelmed at low pH with greater [H+] in the bulk seawater. Thus, low-light species’ morphology and strategy that evolved to sustain calcification without high rates of photosynthesis might make them more vulnerable to greater [H+] in the bulk seawater. Light-driven

processes, including photosynthesis and/or H+ control, will be essen- tial to sustain or enhance daytime calcification to offset nighttime dissolution and maintain a net positive daily calcification rate. Thus, macroalgae able to maintain high calcification rates in the light (high and low irradiance) at low pH, and/or sustain strong biotic control with high [H+] in the bulk seawater, are expected to dominate under global change.

Supplementary data to this article can be found online at https://doi. org/10.1016/j.jembe.2020.151489.

CRediT authorship contribution statement

C. McNicholl: Investigation, Formal analysis, Writing - original draft, Visualization, Writing - review & editing, Validation, Software, Methodology, Conceptualization. M.S. Koch: Writing - original draft, Visualization, Writing - review & editing, Project administration, Su- pervision, Resources, Validation, Methodology, Conceptualization.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research was funded by the National Science Foundation Ocean Acidification Program-CRI-OA Grant #1416376. The authors would like to thank Chris Johnson, Kimberly McFarlane, and the undergraduate students that assisted in the field and lab. Dr. Carrie Manfrino is recognized for her support in the field, and the Cayman Island Marine Conservation Board and Department of the Environment for permitting our LCI research. We also appreciate the anonymous reviewers and editors that significantly improved the manuscript.

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