Coral Reefs

, Volume 35, Issue 1, pp 357–368

Increased temperature mitigates the effects of ocean acidification in calcified green algae (Halimeda spp.)

  • Justin E. Campbell
  • Jay Fisch
  • Chris Langdon
  • Valerie J. Paul

DOI: 10.1007/s00338-015-1377-9

Cite this article as:
Campbell, J.E., Fisch, J., Langdon, C. et al. Coral Reefs (2016) 35: 357. doi:10.1007/s00338-015-1377-9


The singular and interactive effects of ocean acidification and temperature on the physiology of calcified green algae (Halimeda incrassata, H. opuntia, and H. simulans) were investigated in a fully factorial, 4-week mesocosm experiment. Individual aquaria replicated treatment combinations of two pH levels (7.6 and 8.0) and two temperatures (28 and 31 °C). Rates of photosynthesis, respiration, and calcification were measured for all species both prior to and after treatment exposure. Pre-treatment measurements revealed that H. incrassata displayed higher biomass-normalized rates of photosynthesis and calcification (by 55 and 81 %, respectively) relative to H. simulans and H. opuntia. Furthermore, prior to treatment exposure, photosynthesis was positively correlated to calcification, suggesting that the latter process may be controlled by photosynthetic activity in this group. After treatment exposure, net photosynthesis was unaltered by pH, yet significantly increased with elevated temperature by 58, 38, and 37 % for H. incrassata, H. simulans, and H. opuntia, respectively. Both pH and temperature influenced calcification, but in opposing directions. On average, calcification declined by 41 % in response to pH reduction, but increased by 49 % in response to elevated temperature. Within each pH treatment, elevated temperature increased calcification by 23 % (at pH 8.0) and 74 % (at pH 7.6). Interactions between pH, temperature, and/or species were not observed. This work demonstrates that, in contrast to prior studies, increased temperature may serve to enhance the metabolic performance (photosynthesis and calcification) of some marine calcifiers, despite elevated carbon dioxide concentrations. Thus, in certain cases, ocean warming may mitigate the negative effects of acidification.


Increases in oceanic carbon dioxide (CO2) concentrations may serve to alter the physiology and functioning of many marine organisms (Orr et al. 2005). As CO2 concentrations rise from anthropogenic activities (e.g., fossil fuel combustion), gradual shifts in seawater carbonate chemistry occur, whereby both pH and carbonate ion (CO32−) concentrations decline. As a result, saturation states of the various polymorphs of calcium carbonate (aragonite and calcite) decrease, which alters mineral solubility and can ultimately inhibit organismal calcification. Currently, oceanic pH has declined by 0.1 units since the industrial era, and projections forecast an additional 0.3–0.5 unit reduction as we approach the year 2100 (Caldeira and Wickett 2003, 2005). While marine organisms that produce calcium carbonate (CaCO3) shells and skeletons may be adversely influenced (Orr et al. 2005; Hall-Spencer et al. 2008), recent research has documented a wide variety of responses, some of which may be attributable to either taxonomic distinctions (Ries et al. 2009; Kroeker et al. 2010), or the interactive effects of other environmental parameters such as temperature, irradiance, water flow, and/or nutrient regimes (Atkinson et al. 1995; Reynaud et al. 2003; Suggett et al. 2013; Comeau et al. 2014; Hofmann et al. 2014, 2015; Vogel et al. 2015a). While CO2, as a solitary factor, can induce physiological responses, it remains difficult to ascertain the ultimate consequences of ocean acidification (OA) without the consideration of other abiotic parameters that vary across spatial and temporal dimensions.

In conjunction with CO2, surface seawater temperatures are similarly on the rise, with increases of up to 1.0 °C over the past century in tropical regions (Deser et al. 2010), and forecasts of an additional 1–2 °C increase by the year 2100 (Collins et al. 2013). Temperature influences a variety of metabolic processes in marine organisms, such as photosynthesis, respiration, and calcification. For corals, elevated temperature can increase calcification up to a thermal optimum (Lough and Barnes 2000). However, beyond this point, bleaching can occur, whereby coral hosts expel photosynthetic symbionts and exhibit significant declines in calcification (Cooper et al. 2008; De’ath et al. 2009; Cantin et al. 2010). Substantial temperature effects have also been documented for calcified algae, primarily in temperate coralline species. For instance, seasonal increases in photosynthesis and calcification have been reported during the warmer, summer months for several species in the Mediterranean (Potin et al. 1990; Martin et al. 2006, 2007). Furthermore, experimental work has shown that elevated temperature (+3 °C) can enhance the productivity and calcification of the encrusting coralline alga, Lithophyllum cabiochae (Martin and Gattuso 2009; Martin et al. 2013). However, it is important to note that these benefits were only evident during the cooler months, as elevated temperature increased algal necrosis during the summer, again highlighting the consideration of thermal optima. While corals and calcified algae clearly exhibit fundamental distinctions in calcification mechanisms (Comeau et al. 2012), the relatively broad influence of temperature suggests that organismal responses to OA may be partially driven by thermally induced shifts in metabolic functioning, as studies have demonstrated that increased temperature can either (1) exacerbate OA effects by initiating bleaching and declines in productivity (Reynaud et al. 2003; Anthony et al. 2008; Martin and Gattuso 2009; Rodolfo-Metalpa et al. 2011; Diaz-Pulido et al. 2012; Dove et al. 2013), or (2) mitigate OA by serving to increase basal rates of calcification and photosynthesis (Muehllehner and Edmunds 2008; Edmunds et al. 2012; Johnson and Carpenter 2012; Castillo et al. 2014). These reports suggest that variation in OA responses may be linked to experimental temperatures and the thermal optima of the species under consideration. While the concurrent effects of OA and temperature have been documented for calcified invertebrates and some algal groups (Martin and Gattuso 2009; Martin et al. 2013; Noisette et al. 2013), we know comparatively little in regard to the responses from other marine organisms such as calcified green algae.

Calcified algae within the genus Halimeda (order Bryopsidales) are widely distributed across tropical and subtropical environments and play important ecological roles in both habitat provisioning and biogenic sediment production. Production rates in certain locations can approach 1.4 kg CaCO3 m−2 yr−1, positioning Halimeda as a major contributor to carbonate budgets within shallow waters around the globe (Payri 1988). This group further occupies a diverse range of environments (mangroves, seagrass beds, and coral reefs) and can produce structurally complex mounds which serve as critical habitat for a diversity of marine life (Rees et al. 2007). Given the importance of this algal group within coastal habitats, examining OA responses is critical to providing an increasingly comprehensive perspective on climate change effects within marine systems. While some studies document the singular effects of OA on Halimeda spp. (Ries et al. 2009; Price et al. 2011; Comeau et al. 2013; Campbell et al. 2014; Vogel et al. 2015b), there is an increasing need to examine how other parameters may ultimately modify these responses.

Work by Sinutok et al. (2011, 2012) has suggested that warming increases the negative effects of acidification in H. macroloba and H. cylindracea, with both factors eliciting declines in photosynthesis and calcification. While these findings have been attributed to the effects of high temperature on enzyme activity and CO2 fixation, many of these effects occurred at high temperatures (34 °C), presumably far beyond the thermal optima for these species (Sinutok et al. 2011). Subsequent experiments conducted at lower temperatures (32 °C) revealed that calcification was solely dependent on pH and uninhibited by high temperature (Sinutok et al. 2012). Moreover, it was documented that short-term exposure to elevated temperature increased photosynthesis (O2 flux) at the segment surface. Hence, while extreme temperatures can negatively impact Halimeda spp., moderate increases may raise photosynthesis in the face of OA.

Halimeda spp. are siphonalean coenocytic algae, commonly attached to either hard substrates (via rhizoid structures) or buried in soft sediments (via a bulbous holdfast). The algal architecture is structured around a series of connected, calcified segments which are joined together at flexible, uncalcified nodes. Segments are comprised of a series of central filaments (siphons) which extensively branch into smaller utricles and fuse within the peripheral cortex (Verbruggen and Kooistra 2004). Calcification occurs in semi-enclosed spaces between medullary filaments that comprise the segment cortex, and as this inter-utricular space is partially isolated from the external environment, photosynthetic and respiratory processes likely play a strong role in regulating aragonite deposition via control of seawater pH and aragonite saturation (Ωar) (Borowitzka and Larkum 1977; Jensen et al. 1985; Wizemann et al. 2014). The link between photosynthesis and calcification has further been supported by the observation that new segments generally do not calcify until chloroplast maturity (Borowitzka and Larkum 1977; Hay et al. 1988; Larkum et al. 2011). Given the close association between photosynthesis and calcification for this group, this study examines whether thermally induced shifts in photosynthesis influence the OA responses of three common species of Halimeda. Using a series of short- and long-term incubations, we demonstrate that for thermally tolerant species, increases in temperature may elevate photosynthesis and calcification, even under high CO2 concentrations.


Sample collection

Algal specimens were sourced from the nearshore marine environment (1–3 m depth) in south Florida and the Florida Keys. Halimeda opuntia was collected in Summerland Key, Florida USA (25.02°N, 80.49°W) on June 8, 2014. Halimeda incrassata and H. simulans were similarly collected along Key Biscayne (25.71°N, 80.15°W) on June 15, 2014. Large vegetative fragments of H. opuntia were carefully detached from the substrate, and individual thalli of H. simulans and H. incrassata were carefully uprooted with the holdfast intact. All samples were transported in aerated coolers to the Smithsonian Marine Station (SMS) within 24 h, gently rinsed with clean seawater, and allowed to acclimate to ambient tank conditions (salinity 36, temperature 28 °C, pH 8.1). Irradiance within the tanks was provided by a series of full spectrum, T5 fluorescent bulbs, which delivered 200 μmol photons m−2 s−1 to the bottom of the tank. While light levels at the site of collection were comparatively higher (400–500 μmol photons m−2 s−1), prior work has demonstrated that saturation irradiances (Ik) for field populations of H. opuntia and H. tuna typically vary from 70 to 240 μmol photons m−2 s−1 at a reef site in the Florida Keys (Beach et al. 2003). All species were maintained in an upright orientation using shallow acrylic containers. For psammophytic species, the holdfasts remained attached and were associated with natural sediments from the site of collection. During the acclimation and experimental periods, all individuals displayed healthy green segments, and instances of mortality were not detected.

Initial incubations (photosynthesis, respiration, instantaneous calcification)

Prior to treatment exposure, preliminary incubations were conducted to determine baseline, species-specific distinctions in net photosynthesis, respiration and instantaneous calcification. On June 25, replicate light and dark incubations (n = 5–6) were conducted on a subsample of haphazardly selected individuals of each species. Incubations were conducted in fully sealed, clear 2 L chambers that were filled with ambient seawater, and submerged in temperature-controlled aquaria (28 °C). Battery-powered magnetic stirrers within each chamber provided circulation, and all incubations were conducted for 60 min. For the light incubations, each chamber received a single algal specimen (approximately 3.0 g dry weight with holdfast intact) and was exposed to 200 µmol photons m−2 s−1 of irradiance. Subsequent dark incubations to measure respiration were conducted using fresh seawater with the same algal specimens. Blank incubations without algae (both light and dark) were conducted to account for background shifts in water chemistry, which were negligible. Water samples for total alkalinity (TA) and dissolved oxygen (DO) were taken at the beginning and end of each incubation. TA samples were fixed with saturated HgCl2 to halt biological activity, and the DO samples were fixed with Winkler reagents according to a modified Winkler protocol (Langdon 2010). TA was determined using open-cell, potentiometric titration with a Mettler Toledo DL15. Seawater-certified reference materials (CRM, obtained from A. Dickson, Scripps Institution of Oceanography) were analyzed every 10 samples to ensure the accuracy of the TA measurements. On average, measured values were within ±0.5 % of the CRM. DO was determined by Winkler titration using an automated titrator with amperometric endpoint detection. Salinity and temperature was measured with a calibrated handheld YSI meter. After the incubations, all algal samples were dried to a constant weight for 72 h in a 60 °C convection oven.

Shifts in TA and dissolved oxygen (DO) were used to compute instantaneous rates of calcification (G), net photosynthesis (NP), and respiration (R). All calculations were normalized to the dry weights of the algae (excluding the holdfasts) used in the incubations. Calcification was determined by the following equation (Schneider and Erez 2006)
$$ {\text{G}}\left(\upmu{{\text{mol CaCO}}_{3} {\text{g}}^{ - 1} {\text{h}}^{ - 1} } \right) = - 0.5 \cdot \Delta {\text{TA}} \cdot \uprho \cdot {\text{V}}/{\text{DW}} \cdot {\text{T}}, $$
where G is calcification, ΔTA is the change in total alkalinity (μmol kg−1), ρ is seawater density, V is the chamber volume (L), DW is algal dry weight (g), and T is the incubation time (h). NP and R were calculated based on shifts in DO using the following equation
$$ {\text{NP}}\,{\text{or}}\,{\text{R}} = \Delta {\text{DO}} \cdot {\text{V/DW}} \cdot {\text{T}}, $$
where ΔDO represents the change in dissolved oxygen over the course of either the light or dark incubations.

Experimental design

Individuals that were not used in the preliminary incubations were subjected to experimental manipulations of temperature and pH within aquaria for 4 weeks (July 16, 2014–August 13, 2014). After 30 d of acclimation, all algae were weighed using the buoyant weight technique (Davies 1989) with an analytical balance (±0.01 g). Three individuals of each species were placed within 12 independent, temperature-controlled tanks (37 L) at SMS. Mean initial buoyant weights were 5.07 ± 0.4, 1.04 ± 0.1, and 2.14 ± 0.1 g for H. incrassata, H. simulans, and H. opuntia, respectively. Individuals were evenly spaced across the tank area to avoid shading. Each tank consisted of an enclosed recirculating system, and water flow was provided by a 473 LPH powerhead. All tanks were filled with filtered seawater (<10 µm) collected from an offshore location near Fort Pierce, FL. Four treatments were established in triplicate tanks representing a factorial combination of both pH (8.0, 7.6) and temperature (28, 31 °C). Reductions in pH and increases in temperature approximate ‘worst-case’ future scenarios for climate change over the next century (Caldeira and Wickett 2003; Moss et al. 2010; Collins et al. 2013). A computer (Aqua Medic) continuously monitored pH within each tank, and treatment adjustments were achieved by periodically dosing 100 % gaseous CO2 at a slow rate (25 mL min−1). Temperature control was achieved by independent dual-stage digital controllers attached to water-jacketed heat exchangers. Salinity was maintained near 36 by replenishing evaporative losses with deionized (DI) water. Water changes (50 % volume) were conducted twice a week to minimize biologically induced shifts in alkalinity, and lighting was similar to that of the preliminary incubations, and set on a 12/12 cycle.

Seawater chemistry

Water chemistry in each tank was monitored during the experiment via periodic measurements of pH, temperature, and salinity (3–4 times per week) using a calibrated Thermo Scientific Orion Ross combination pH electrode (relative accuracy ± 0.01 units) and an YSI meter. Weekly water samples were collected at midday to measure TA via open-cell potentiometric titration. Carbonate parameters within each tank were calculated with the program CO2SYS (Lewis and Wallace 1998), using the measured parameters of pH, TA, temperature, and salinity with the carbonate dissociation constants of Mehrbach et al. (1973), as refit by Dickson and Millero (1987). Measured carbonate parameters (Table 1) reveal depressed alkalinities within our tanks (mean of 1779 μmol kg−1) relative to typical values documented across the Florida Keys, which range 2162–2480 μmol kg−1 (Manzello et al. 2012). This likely resulted from biological draw-down of TA via calcification, despite the frequent bi-weekly water changes intended to counteract such processes. While this did reduce Ωar, positive rates of calcification were documented for all specimens across all tanks. Moreover, we found that calcification rates within our tanks were similar to values obtained from other mesocosm studies working with a variety of species (Sinutok et al. 2011; Comeau et al. 2013; Campbell et al. 2014; Hofmann et al. 2015). We further compared calcification rates from this study with in situ estimates from natural field populations in the Florida Keys (Vroom et al. 2003), and generally found good agreement. Vroom et al. (2003) documented H. tuna calcification rates of 4.4 mg CaCO3 thallus−1 d−1, while rates within our control tanks averaged 2.6 and 21.3 mg CaCO3 thallus−1 d−1 for H. simulans and H. opuntia, respectively.
Table 1

Calculated carbonate chemistry parameters from the measured parameters of pH, total alkalinity (TA), temperature, and salinity (n = 11 for each tank)



Temp (°C)


TA (µmol kg−1)

pCO2 (µatm)

CO2 (µmol kg−1)

HCO3 (µmol kg−1)



High pH, low temp

36.8 ± 0.1

28.39 ± 0.04

8.01 ± 0.01

1755.5 ± 62.1

490.5 ± 22.2

12.6 ± 0.6

1410.1 ± 52.0

3.15 ± 0.14

2.10 ± 0.09

High pH, high temp

37.5 ± 0.1

30.78 ± 0.07

7.99 ± 0.01

1696.6 ± 67.0

501.6 ± 27.0

12.2 ± 0.7

1346.5 ± 55.9

3.15 ± 0.16

2.12 ± 0.11

Low pH, low temp

36.8 ± 0.1

28.26 ± 0.04

7.63 ± 0.01

1827.2 ± 61.2

1401.9 ± 72.7

36.3 ± 1.9

1660.9 ± 57.7

1.53 ± 0.06

1.02 ± 0.04

Low pH, high temp

37.3 ± 0.1

30.92 ± 0.08

7.67 ± 0.02

1835.7 ± 62.8

1307.2 ± 86.1

31.7 ± 2.1

1631.5 ± 60.7

1.88 ± 0.14

1.27 ± 0.09

Treatment responses (photosynthesis, respiration, calcification, CaCO3 content)

After 4 weeks of exposure, additional incubations and buoyant weight measurements were conducted to examine shifts in NP, R, and long-term calcification (n = 3 for all response variables). Three chambers were submerged in each of the 12 tanks, filled with treatment seawater, and sealed containing several individuals of the same species. Thus, replicate specimens of each species within the same tank were pooled within the same chamber. Incubations were conducted for 60 min under experimental lighting conditions (200 µmol photons m−2 s−1) with adequate water motion provided by a magnetic stirrer. Water samples for oxygen determination were collected at the beginning and the end of all incubations. Overall, 72 separate incubations were conducted: 36 light incubations (3 species × 4 treatments × 3 replicates) and an additional 36 dark incubations. To examine calcification rates integrated over the entire exposure period, final buoyant weights of all specimens were recorded with an analytical scale, and long-term calcification (reported as mg CaCO3 g−1 d−1) was determined by comparing the initial and final buoyant weight measurements. Buoyant weight measurements from replicate specimens of the same species within a single tank were averaged to derive a tank mean, avoiding pseudoreplication (Hurlbert 1984; Cornwall and Hurd 2015). CaCO3 content (% dry wt) of all individuals was determined using a gravimetric acidification approach (van Tussenbroek and van Dijk 2007). After buoyant weight measurements, samples were dried to a constant weight for 72 h in a 60 °C convection oven. Segments from the intact aboveground thallus of each specimen were placed in separate pre-weighed glass vials, acidified with 10 % HCl for 1 h, rinsed with DI water to remove HCl traces, and then dried to a constant weight. CaCO3 content was determined from the dry weight difference between calcified and decalcified segments.

Statistical procedures

Data were tested for normality and equal variances prior to statistical analysis. Data which did not conform to these assumptions were either log transformed or ranked prior to analysis. Species-specific distinctions in baseline biological parameters assessed during the pre-exposure incubations were tested with a one-way analysis of variance (ANOVA), in which species served as a fixed factor and G, NP, GP, and R served as dependent variables. Ranked data were analyzed with a Kruskal–Wallis test. Relationships between NP and G derived from pre-treatment incubations were tested with linear regression. Following the treatment incubations, a three-way ANOVA was used to test for differences in NP, R, calcification (Δ buoyant weights), and CaCO3 content across species and treatments. For all response variables, overall tank means were calculated and used as the independent unit of replication. Species, temperature, and pH level were designated as fixed factors within the analysis. Post hoc comparisons were conducted with a Holm–Sidak test (overall α adjusted to 0.05 to account for multiple comparisons).


Initial incubations (photosynthesis, respiration, instantaneous calcification)

Pre-treatment incubations demonstrated interspecific variation in baseline measurements of net photosynthesis, gross photosynthesis, respiration, and calcification (Fig. 1). Net photosynthesis (normalized to dry mass, mean ± SE) was highest in H. incrassata (20.9 ± 1.5 µmol O2 g−1 h−1), and comparatively lower in H. opuntia (11.7 ± 0.9 µmol O2 g−1 h−1) and H. simulans (14.6 ± 0.8 µmol O2 g−1 h−1) (ANOVA, F2,15 = 16.6, p < 0.001). Similar trends were documented for gross photosynthesis, with values of 25.0 ± 2.3, 14.2 ± 1.8, and 17.4 ± 0.5 µmol O2 g−1 h−1 for H. incrassata, H. opuntia, and H. simulans, respectively (ANOVA, F2,15 = 9.2, p = 0.011). Respiration was lower in H. opuntia (−2.1 ± 0.3 µmol O2 g−1 h−1), and higher in H. incrassata (−4.1 ± 0.3 µmol O2 g−1 h−1) and H. simulans (−3.8 ± 0.2 µmol O2 g−1 h−1) (ANOVA, F2,9 = 16.2, p = 0.002). Calcification (normalized to dry mass, mean ± SE) was higher in H. incrassata (40.1 ± 5.4 mmol CaCO3 g−1 h−1) and comparable between H. opuntia and H. simulans (21.7 ± 1.8 and 22.3 ± 5.3 mmol CaCO3 g−1 h−1, respectively) (Kruskal–Wallis, H = 7.4, p = 0.024). When all species were pooled, NP and G were linearly correlated (linear regression, F1,15 = 10.2, r2 = 0.42, p = 0.007; Fig. 2). Within species, H. opuntia displayed the strongest correlation between NP and G (r2 = 0.91, p = 0.012), while weaker correlations were displayed by H. incrassata (r2 = 0.2, p = 0.333) and H. simulans (r2 = 0.3, p = 0.31).
Fig. 1

Initial measurements of net photosynthesis, gross photosynthesis, respiration, and calcification in Halimeda opuntia (n = 5), Halimeda simulans (n = 5), and Halimeda incrassata (n = 6) prior to treatment exposure. Letters indicate significant differences among species (Holm–Sidak post hoc comparisons, α ≤ 0.05)

Fig. 2

Correlations between net photosynthesis and calcification in Halimeda opuntia (open circles), Halimeda simulans (gray circles) and Halimeda incrassata (black circles) prior to treatment exposure. Solid line represents linear fit to the dataset (all species pooled). Dashed lines represent 95 % confidence intervals

Treatment responses (photosynthesis, respiration, calcification, CaCO3 content)

After 4 weeks of exposure, net photosynthesis across all species and treatments was comparable to previously reported rates in the literature for this group (Jensen et al. 1985; Lapointe et al. 1987; Littler et al. 1988). In response to treatment conditions, NP increased with elevated temperature across all species (ANOVA, F1,33 = 9.46, p = 0.006; Fig. 3) and was unaltered by reduced pH (ANOVA, F1,33 = 0.01, p = 0.954). Net photosynthesis increased by 58, 38, and 37 % under elevated temperature for H. incrassata, H. simulans, and H. opuntia, respectively. Net photosynthesis (mean ± SE across treatments) was highest in H. incrassata (4.5 ± 0.3 µmol O2 g−1 h−1), and lowest in H. simulans (2.9 ± 0.3 µmol O2 g−1 h−1) and H. opuntia (1.5 ± 0.3 µmol O2 g−1 h−1) (ANOVA, F2,33 = 25.1, p < 0.001). Interactions between treatment factors (species, pH, temperature) were not significant. Respiration, across all species, was unaltered by temperature (ANOVA, F1,34 = 2.22, p = 0.15; Fig. 4) or pH (ANOVA, F1,34 = 0.10, p = 0.75). Comparing species, respiration was higher in H. incrassata (−3.6 ± 0.3 µmol O2 g−1 h−1) and lower in H. simulans (−2.4 ± 0.3 µmol O2 g−1 h−1) and H. opuntia (−2.1 ± 0.3 µmol O2 g−1 h−1).
Fig. 3

Net photosynthesis (n = 3) in Halimeda opuntia, Halimeda simulans, Halimeda incrassata after 28 d of exposure to four treatments. The statistical table displays ANOVA results (fixed factors: species, temperature, pH). Letters indicate significant differences within species (Holm–Sidak post hoc comparisons, α ≤ 0.05)

Fig. 4

Respiration (n = 3) in Halimeda opuntia, Halimeda simulans, and Halimeda incrassata after 28 d of exposure to four treatments. The statistical table displays ANOVA results (fixed factors: species, temperature, pH). Post hoc comparisons within species were not conducted

Excessive segment-shedding by H. incrassata limited our ability to obtain accurate calcification measurements via the buoyant weight technique, thus long-term calcification data are restricted to H. opuntia and H. simulans. In both species, calcification increased under elevated temperature (ANOVA, F1,23 = 5.4, p = 0.033, Fig. 5) and declined under reduced pH (ANOVA, F1,23 = 12.1, p = 0.003). Under control pH, elevated temperature increased calcification by 14 and 32 % for H. opuntia and H. simulans, respectively. Under reduced pH, elevated temperature increased calcification by 60 and 88 % in H. opuntia and H. simulans, respectively. Across all temperatures, declines in pH reduced calcification by 32 and 49 % in H. opuntia and H. simulans, respectively. Treatment interactions were not significant. Moreover, pairwise comparisons revealed that calcification rates under control (28 °C, pH 8.0) and forecasted conditions (31 °C, pH 7.6) were not significantly distinct for either H. opuntia (t test, t = 0.77, p = 0.48) or H. simulans (t test, t = 0.59, p = 0.59) Across all treatments, calcification was higher in H. opuntia (9.9 ± 0.9 mg CaCO3 g−1 d−1) than H. simulans (3.7 ± 0.7 mg CaCO3 g−1 d−1) (ANOVA, F1,23 = 50.9, p < 0.001).
Fig. 5

Calcification rates (measured via the buoyant weight technique, n = 3) of Halimeda opuntia and Halimeda simulans. Values from replicate specimens within a single tank were averaged to derive a tank mean. Excessive segment-shedding by Halimeda incrassata prevented accurate buoyant weight measurements. The statistical table displays ANOVA results (fixed factors: species, temperature, pH). Letters indicate significant differences among treatments (Holm–Sidak post hoc comparisons, α ≤ 0.05)

CaCO3 content varied by species (ANOVA, F2,35 = 14.4, p < 0.001) but was unaltered by pH (p = 0.22) or temperature (p = 0.28). Across all treatments, CaCO3 content varied from a minimum of 82.0 ± 0.7 % in H. incrassata, to a maximum of 86.9 ± 0.4 % and 85.3 ± 0.7 % in H. simulans and H. opuntia, respectively. Post hoc analysis revealed that H. incrassata was distinct from H. simulans and H. opuntia (which were statistically similar).


Comparisons of the relative effects of OA and elevated temperature on the physiological performance of Halimeda spp. suggests that warmer thermal environments might serve to increase calcification rates, and thereby mitigate the negative effects of declines in Ωar. In contrast with prior work (Sinutok et al. 2011, 2012), elevated temperatures enhanced net photosynthesis across both pH treatments (Fig. 3; Table 2). Given the empirical positive correlation between oxygen production and calcification (Fig. 2), this work suggests that for some groups of marine calcifiers, certain aspects of climate change (rising temperatures) may increase photosynthesis, promote calcification, and offset future OA. These results (1) highlight the important role that other environmental parameters play in governing the effects of OA and (2) suggest that organismal thermal optima (as influenced by both species-specific tolerances and current environmental conditions) need to be considered within the framework of climate change biology.
Table 2

Results of three-way ANOVA on rates of photosynthesis, respiration, and calcification after 4 weeks of treatment exposure to the crossed factors of species (Halimeda incrassata, Halimeda simulans, Halimeda opuntia), temperature (28 vs 31 °C), and pH (7.6 vs 8.0)

Source of variation




Post hoc results

Net photosynthesis






Hi > Hs > Ho










31 > 28

Species × pH





Species × temperature





pH × temperature





Species × pH × temperature











Hi > Ho, Hs











Species × pH





Species × temperature





pH × temperature





Species × pH × temperature











Ho > Hs





8.0 > 7.6





31 > 28

Species × pH





Species × temperature





pH × temperature





Species × pH × temperature





Statistical comparisons for calcification based on buoyant weights are restricted to H. opuntia and H. simulans due to high segment shedding in H. incrassata. Significant results are highlighted in bold. Post hoc comparisons were conducted with a Holm–Sidak test (α = 0.05)

Baseline measurements of physiological performance (photosynthesis, respiration, and calcification) reveal a number of interspecific distinctions. As normalized to dry weight, H. incrassata displayed elevated rates of NP and instantaneous G compared to H. opuntia and H. simulans (Fig. 1). These findings characterize H. incrassata as a relatively rapid calcifier and identify this species as a productive contributor to CaCO3 stocks within coastal environments. The reason underlying these distinctions remains unresolved; however, we note that relative to other species, H. incrassata typically displays a growth pattern with high rates of both segment production and segment shedding (personal observation). The elevated photosynthesis and calcification displayed by H. incrassata may be required to support rapid rates of segment turnover. Furthermore, it was evident that while uninfluenced by pH or temperature, CaCO3 content was lowest in H. incrassata relative to the other two species, thus high rates of segment production/turnover may result in lower %CaCO3 of individual segments, despite comparatively high rates of calcification (Fig. 1). Similar interspecific distinctions have been previously documented, where species with high CaCO3 content displayed increased sensitivity to OA (Campbell et al. 2014). Similar conclusions have been reached in other comparative studies (Price et al. 2011), suggesting that the factors influencing CaCO3 content (e.g., segment ultrastructure and utricle organization) may influence OA sensitivity. In the current study, both H. simulans and H. opuntia displayed similar CaCO3 content and responded similarly to OA. Future work would benefit from additional comparative studies that include a wide range of species spanning varying degrees of CaCO3 content. These details highlight the need for work that concurrently quantifies OA-induced shifts in both CaCO3 content and calcification.

Net photosynthesis and instantaneous G were positively correlated (Fig. 2); however, the strength of this relationship varied by species. The strongest positive correlation was displayed by H. opuntia, followed by H. incrassata. Relationships between NP and G were weak for H. simulans. When pooled, the correlation between NP and G likely resulted from photosynthetic mediation of calcification rates, as similarly demonstrated for the deep-water species H discoidea, H. cryptica, H. copiosa, and H lacrimosa (Jensen et al. 1985). As calcification occurs within semi-enclosed inter-utricular spaces, photosynthesis serves to aid CaCO3 precipitation via CO2 removal, and pH/Ωar elevation. The reason for the lack of a strong positive correlation between NP and G for H. simulans remains unknown; however, recent research has suggested that calcification in Halimeda may not be entirely driven by abiotic CaCO3 precipitation, and may be partially controlled by biological processes. The presence of organic matrices, acidic polysaccharides, and carbonic anhydrase enzymes all serve to initiate and promote crystal growth adjacent to the utricle wall (Wizemann et al. 2014). Thus, we recognize the potential role that these factors may have played in our instantaneous measurements, suggesting that interspecific distinctions in the correlation between NP and G may be attributable to varying degrees of biological control of calcification.

After treatment exposure, net photosynthesis was unaltered by pH (Fig. 3), suggesting that NP is not limited by dissolved inorganic carbon in these species. CO2 supply from the external environment, calcification, and carbonic anhydrase enzymes are likely sufficient to meet photosynthetic demand for this group. Moreover, our findings contrast with prior work suggesting that low pH impairs photosynthesis. While this trend has been demonstrated in several studies (Price et al. 2011; Sinutok et al. 2011, 2012), the mechanisms underlying such responses remain elusive. Hofmann et al. (2014, 2015) and Meyer et al. (2015) found no trend of CO2-induced declines in photosynthesis for H. opuntia. Similarly, Vogel et al. (2015b) found few effects of acidification on net photosynthetic rates of H. opuntia or H. digitata near natural CO2 seeps in Papua New Guinea. The current study found no evidence of OA-induced photosynthetic impairment, nor any evidence that increased temperature (singularly or synergistically with OA), negatively affected oxygen production. While surprising, these contrasting conclusions may largely result from methodological distinctions, as prior studies examining the concurrent effects of temperature and OA either (1) worked with isolated segments (which may not accurately represent responses from the entire individual) or (2) drew conclusions from treatment levels beyond those tested in the current study (e.g., temperatures >31° and pH <7.6). Furthermore, most prior work involved the use of Pacific species, which may be acclimated to cooler waters relative to their Caribbean counterparts. Thus, conclusions in regard to the physiological effects of temperature and OA may strongly depend on organismal acclimation, and regional distinctions in thermal environments. We demonstrated that elevated temperature enhanced NP, suggesting that while extreme temperatures may reduce oxygen output (via photosystem damage), moderate increases within the organismal tolerances may prove beneficial. Temperature-driven increases in NP have been documented for some corals (e.g., Acropora intermedia); however, variable and opposing responses from other taxonomic groups exposed to similar experimental conditions have been reported (Anthony et al. 2008).

Calcification was adversely affected by declines in pH, similar to the results of prior work. Campbell et al. (2014) reported that calcification rates declined by 15 and 50 % for H. opuntia and H. simulans, respectively, over comparable shifts in carbonate chemistry. Similarly, Comeau et al. (2013) reported that calcification declined by 11 % in H. minima as pCO2 doubled over ambient conditions, highlighting that decreased carbonate ion availability could inhibit CaCO3 deposition. However, variable effects have been reported (Price et al. 2011), with some species displaying relatively minor responses to shifts in carbonate chemistry. Campbell et al. (2014) reported that H. incrassata was unaffected by increases in pCO2, similar to the results of Comeau et al. (2013) for H. macroloba. Ries et al. (2009) demonstrated that while net calcification declined in H. incrassata in response to Ωar reduction, these effects were only detected in the highest pCO2 treatment (pCO2 = 2593 ppm, Ωar = 0.9), with minor responses as Ωar declined from 3.1 to 1.8. In the current study, net calcification remained positive for both H. opuntia and H. simulans, despite relatively high CO2 environments. Thus, it appears that for Halimeda spp., while pH reduction can influence calcification, moderate declines tend to impose minimal effects. These conclusions are further supported by findings by Vogel et al. (2015b), who (1) documented the occurrence of multiple species of Halimeda near volcanic CO2 seeps, (2) demonstrated that net calcification was unaltered by elevated pCO2 in H. opuntia and H. digitata, and (3) showed that light calcification rates were even enhanced near seeps, despite a near 0.4 unit reduction in pH. Moreover, it is interesting to note that while tropical CO2 seeps harbor multiple species of Halimeda (Vogel et al. 2015b), temperate species are notably excluded from similar seeps within the colder Mediterranean (Hall-Spencer et al. 2008). While such distinctions are likely due to multiple factors, we suggest that they may be partially attributable to the broader role of oceanographic parameters such as temperature.

Thermal conditions likely play a key role toward regulating OA responses for Halimeda. Surprisingly, we showed that increased temperature elevated calcification by a similar magnitude to pH-induced declines in calcification; hence, OA and moderate warming influenced calcification in opposing directions, and the effects of temperature offset low pH. Interaction terms were not significant, thus even though OA had similar effects in both the 28 and 31 °C treatments (Fig. 5), differences in calcification between control (pH 8.0, 28 °C) and forecast (pH 7.6, 31 °C) scenarios were not detected. These results contrast with reports that warming amplified OA effects in corals (Anthony et al. 2008; Dove et al. 2013) and calcified algae (Sinutok et al. 2011, 2012). Given the correlation between G and NP (Fig. 2), we propose that thermally induced increases in NP enhanced calcification and compensated for declines in Ωar. Such shifts in metabolic performance may be due to increases in the efficiency of internal and externally bound carbonic anhydrase enzymes that facilitate the conversion of HCO3 to CO2 to support photosynthetic carbon demand (De Beer and Larkum 2001). Additionally, note that temperature itself can increase seawater Ωar (Table 1), thus thermodynamic effects may also partially account for increased calcification. Overall, the current work provides a descriptive mechanism in which thermal increases in metabolic functioning, combined with thermodynamic effects, can mitigate future OA for some calcified groups.

Temperature-related increases in calcification have been reported for several corals, namely Siderastrea siderea (Castillo et al. 2014), Porites rus (Edmunds et al. 2012), Porites spp. (Lough and Barnes 2000), and Pocillopora meandrina (Muehllehner and Edmunds 2008). While the current study represents the first demonstration that elevated temperature increases calcification in a widely distributed group of green algae, we recognize that temperature effects can be complex, particularly when taxon-specific thermal response curves are considered. Work with other groups of calcified algae (crustose corallines) consists of mixed reports, with temperature either increasing (Johnson and Carpenter 2012) or decreasing (Martin and Gattuso 2009; Diaz-Pulido et al. 2012) net calcification under OA scenarios. As suggested by Edmunds et al. (2012), varying outcomes may strictly depend on the range (and magnitude) of temperature manipulation as it relates to performance curves; experiments conducted across the ascending portion of the temperature-calcification curve report OA effects that are different from experiments conducted across the descending portion. Clearly, threshold temperatures exist, whereby additional warming serves to disrupt metabolic processes, and establish parabolic relationships between temperature and calcification (Castillo et al. 2014). We suggest that future work may benefit by coupling manipulative OA and warming experiments to detailed investigations of temperature performance curves. Such actions may place detected responses within a physiological context (e.g., organismal tolerances), and resolve varying conclusions among discrete experiments. For instance, prior work with the reef-building coral Siderastrea siderea has demonstrated that while forereef colonies displayed declines in calcification with warming temperatures, backreef and nearshore colonies remained unaffected, attributable to organismal acclimation to environmental fluctuations (Castillo et al. 2012). While temperature has largely been viewed as an abiotic parameter that exacerbates the detrimental effects of OA, the current work contributes to an increasing number of reports suggesting that moderate increases in temperature may serve to improve metabolic performance and mitigate the detrimental effects of shifts in seawater chemistry.


We thank Lane Johnston for assistance in the laboratory. This work was made possible through support from the Smithsonian Hunterdon Oceanographic Endowment and the Competitive Grants Program for Science. This is contribution no. 1013 from the Smithsonian Marine Station at Fort Pierce, FL.

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Justin E. Campbell
    • 1
  • Jay Fisch
    • 2
  • Chris Langdon
    • 2
  • Valerie J. Paul
    • 1
  1. 1.Smithsonian Marine StationFort PierceUSA
  2. 2.Corals and Climate Change Laboratory, Rosenstiel School of Marine and Atmospheric ScienceUniversity of MiamiMiamiUSA