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PlantaDOI 10.1007/s00425-013-1982-1

OrIgInal artIcle

Nutrient availability affects the response of the calcifying chlorophyte Halimeda opuntia (L.) J.V. Lamouroux to low pH

Laurie C. Hofmann · Jasmin Heiden · Kai Bischof · Mirta Teichberg

received: 20 august 2013 / accepted: 13 October 2013 © Springer-Verlag Berlin Heidelberg 2013

nutrient conditions and lowest when phosphate was limit-ing. In general, we found that enzymatic responses were strongly influenced by nutrient availability, indicating its important role in dictating the local responses of the calci-fying primary producer H. opuntia to ocean acidification.

Keywords nitrate reductase · carbonic anhydrase · calcification · Ocean acidification · eutrophication · Photosynthesis

Introduction

Since the industrial revolution, the sea surface waters have dropped 0.1 pH units and are expected to drop a further 0.3–0.5 units by 2100 (caldeira and Wickett 2003; Feely et al. 2004; Orr et al. 2005). adding dissolved cO2 to sea-water increases the concentrations of bicarbonate (HcO3

2−) and hydrogen ions (H+), which leads to a decrease in pH, carbonate ions (cO3

2−), and subsequently aragonite satura-tion state (Fabry et al. 2008). By the end of this century, the atmospheric cO2 partial pressure could reach up to 1,000 ppm (Intergovernmental Panel on climate change, IPcc). Such atmospheric changes are expected to lead to a decline in cO3

2− ions of about 60 % in surface ocean waters (Brewer 1997; Feely et al. 2004). these changes in ocean water chemistry could have significant consequences for calcifying organisms, which use cO3

2− ions to build their calcified shells and skeletons.

Many calcifiers have been shown to be sensitive to decreases in the carbonate saturation state, as this impairs the formation of biogenic calcium carbonate (Orr et al. 2005). For coral reefs, an ecosystem dominated by cal-cifying organisms, the effect of ocean acidification is anticipated to be negative (langdon et al. 2000, 2003;

Abstract atmospheric carbon dioxide emissions cause a decrease in the pH and aragonite saturation state of surface ocean water. as a result, calcifying organisms are expected to suffer under future ocean conditions, but their physiolog-ical responses may depend on their nutrient status. Because many coral reefs experience high inorganic nutrient loads or seasonal changes in nutrient availability, reef organ-isms in localized areas will have to cope with elevated car-bon dioxide and changes in inorganic nutrients. Halimeda opuntia is a dominant calcifying primary producer on coral reefs that contributes to coral reef accretion. therefore, we investigated the carbon and nutrient balance of H. opuntia exposed to elevated carbon dioxide and inorganic nutrients. We measured tissue nitrogen, phosphorus and carbon con-tent as well as the activity of enzymes involved in inorganic carbon uptake and nitrogen assimilation (external carbonic anhydrase and nitrate reductase, respectively). Inorganic carbon content was lower in algae exposed to high cO2, but calcification rates were not significantly affected by cO2 or inorganic nutrients. Organic carbon was positively cor-related to external carbonic anhydrase activity, while inor-ganic carbon showed the opposite correlation. carbon diox-ide had a significant effect on tissue nitrogen and organic carbon content, while inorganic nutrients affected tissue phosphorus and n:P ratios. nitrate reductase activity was highest in algae grown under elevated cO2 and inorganic

l. c. Hofmann (*) · K. Bischof Marine Botany Department, Bremen Marine ecology center for research and education, University of Bremen, leobener Straße nW2, 28359 Bremen, germanye-mail: l.hofmann@uni-bremen.de

J. Heiden · M. teichberg leibniz center for tropical Marine ecology, Farenheitstraße 6, 28359 Bremen, germany

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leclercq et al. 2000; guinotte et al. 2003; albright et al. 2008, 2010; Jokiel et al. 2008; andersson et al. 2009). However, many tropical calcifying species have shown mixed responses to ocean acidification scenarios (anders-son et al. 2009; ries et al. 2009; Fabricius et al. 2011; Mcculloch et al. 2012). this wide variety of responses of calcifying organisms is likely due to differences in experi-mental design, temperature, light intensities, seawater chemistry, and nutrient regimes and history or food avail-ability (Kleypas et al. 2006; Hurd et al. 2009), as well as potential differences in population responses. It is there-fore important to conduct experiments that combine ocean acidification scenarios with other factors such as tem-perature, light, and nutrient or food availability, because changes in these parameters are occurring simultaneously with changes in seawater carbonate chemistry. Multiple stressors likely have cumulative or interactive impacts that cause more complex but meaningful responses at the physiological and ecological level than any single stressor (guinotte and Fabry 2008).

the effects of the combination of elevated tempera-ture and cO2 on calcifying organisms have already been widely investigated, particularly in coral reef research (reynaud et al. 2003; Hoegh-guldberg et al. 2007; Man-zello 2010; Sinutok et al. 2011; Mcculloch et al. 2012), but the combination of nutrient availability and cO2 is not as strongly studied (renegar and riegl 2005; Kley-pas et al. 2006; nelson 2009; russell et al. 2009; chauvin et al. 2011). nevertheless, this combination of factors is important to investigate, since the responses of calcifiers to elevated cO2 can be strongly influenced by nutrient or food availability (russell et al. 2009; Holcomb et al. 2010; chauvin et al. 2011; Findlay et al. 2011; Matthies-sen et al. 2012) and nutrient history (Fong et al. 2003), which can change seasonally (lapointe 1987). therefore, the response of calcifiers to elevated cO2 will likely differ regionally based on local and seasonal nutrient regimes, which has implications for management decisions (rus-sell et al. 2009).

the aragonite-depositing members of the green algal genus Halimeda (Caulerpales, Halimedaceae) have been shown to be susceptible to ocean acidification (robbins et al. 2009; Price et al. 2011; Sinutok et al. 2011) and can be overgrown by fleshy macroalgae under eutrophic conditions (Delgado and lapointe 1994; Hofmann et al. 2013a). However, Halimeda spp. are also stimulated by nutrient addition (Delgado and lapointe 1994; teich-berg et al. 2013), but to date, no studies have looked at the combined effects of elevated cO2 and inorganic nutrients on these algae. accumulation of dead Halimeda spp. skel-etons produce bank-like mounds (bioherms) containing high amounts of carbonate sediment (littler et al. 1988; rees et al. 2007). Halimeda is, therefore, an important

contributor to carbonate sediments in tropical environments (Hillis-colinvaux 1980; Drew 1983; Marshall and Davies 1988; Drew and abel 1988; Diaz-Pulido et al. 2007; rees et al. 2007). estimates suggest modern Halimeda bioherms accumulate globally 0.15–0.4 gt cacO3* year−1, which is a major part of the annual coral reef carbonate production (Milliman 1993; Hillis 1997).

Halimeda opuntia can be found widely distributed in coral reefs and lagoons throughout the tropics and sub-tropics, and is most abundant at shallow depth (van den Hoek et al. 1972, 1975; Kuenen and Debrot 1995). an increase in nutrients in tropical coastal waters, mainly due to terrestrial runoff, sewage and groundwater discharge, can greatly influence the coral reef ecosystem (Mccook 1999; Fabricius 2005 and references therein). Because carbon and nitrogen uptake and assimilation compete for energy reserves in algae (turpin et al. 1988; turpin 1991; Huppe and turpin 1994; andria et al. 1999), the aim of this study was to determine if the physiological response of H. opuntia to elevated cO2 is affected by nutrient availability. In addition to carbon, nitrogen and phosphorus content, the response of photosynthesis, calcification, and the activ-ity of two enzymes that regulate inorganic carbon uptake and nitrate assimilation (external carbonic anhydrase and nitrate reductase) were investigated.

Materials and methods

culture conditions

H. opuntia fragments were collected from a caribbean reef off the coast of curaçao at 15 m in March 2011. the algae were maintained in a large mesocosm tank for 2 months in the Marine experimental ecology Facility at the leibniz center for tropical Marine ecology in Bremen, germany. ambient abiotic conditions consisted of 27 °c, a salinity of 33 psu, 8.00 pH units, and 55 μmol photons m−2 s−1 of photosynthetically active radiation (Par) on a 12:12 h light/dark cycle. Irradiance was generated from two Marine white FMW39tS and one Marine blue FMB5at5 light bulbs (arcadia, redhill, UK). Salinity and pH were meas-ured using a tetracon 325 conductivity probe and a Sen-tix 81 pH electrode connected to a Multiline P4 multi-measuring device (WtW, Weilheim, germany). the pH meter was calibrated weekly using pH buffer solutions in ampules (SI analytics gmbH, Mainz, germany) and any offset of the electrode was corrected based on a tris buffer. light intensity was measured using an underwater lI-1400 lI-cOr sensor connected to a data logger (lI-cOr Bio-sciences, lincoln, USa). H. opuntia thalli were divided into four pieces at least 1 week prior to the beginning of the experiment.

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experimental design

the experiment was conducted using a randomized 2 × 2 factorial design to test the effects of nutrient (nitrate and phosphate) and cO2 addition on H. opuntia physiology. 16 aquaria (20 l) were randomly assigned 1 of 4 treat-ments: ambient cO2 and no nutrient addition (−cO2−nP), cO2 addition only (+cO2−nP), nutrient addition only (−cO2+nP), and cO2 and nutrient addition (+cO2+nP). One algal clump (5–10 g FW) was placed in each aquarium. nutrients were added at each weekly water change to start-ing concentrations of 100 μM KnO3 and 10 μM KH2PO4 to create nitrogen-limited conditions, as normal conditions were phosphorus limited. We chose nitrate concentrations double that of a eutrophied harbour in curaçao (Jan gast 1998) and maintained a 10:1 ratio of nitrate to phosphate. ambient inorganic nitrogen and phosphate concentrations at 15 m in curaçao where H. opuntia can be found grow-ing are around 0.26 and 0.14 μM, respectively (teichberg et al. 2013). carbon dioxide addition was controlled using the IKS aquastar system (Iks computer Systeme gmbH, Karlsbad, germany) which monitored cO2 bubbling via solenoid valves that were programed to open and close when the pH deviated from pH 7.75 (±0.05), a pH level corresponding to around 1,500 μatm cO2. all aquaria were constantly aerated with ambient air. artificial seawater was prepared with reverse osmosis water and red Sea reef Salt to a salinity of 33 psu. light conditions during the experi-ment were similar to culture conditions (50 μmol photons m−2 s−1). although light levels can reach 500 μmol pho-tons m−2 s−1 at 15 m depth in curaçao (Fricke et al. 2011), we cultured the H. opuntia under low light conditions, and their Ik values indicated that they had acclimated to the low light conditions (see results). the carbonate system of the seawater was determined by using pH and total alkalin-ity as two of the three carbonate system parameters. total alkalinity was determined by the gran titration method using a titroline alpha 05 plus titrator with an automated sample changer and Ioline Il-Micro pH electrode (SI ana-lytics). the remaining parameters (pcO2, HcO3

−, cO32−)

were calculated using cO2calc (robbins et al. 2010), using the dissociation constants for cO2 and KHSO4 from Mehr-bach et al. (1973) refit by Dickson and Millero (1987) and Dickson (1990), respectively.

growth and photochemistry

the fresh weight of each algal clump was measured weekly by blotting the algae dry with paper towels until no change in weight was detectable. relative growth rates (rgr) were calculated as RGR = (ln (FWt − FW0))/(t × 100) where FWt and FW0 are the fresh weight at time t (days) and time 0, respectively. Photosynthetic parameters, including

maximum quantum yield (Fv/Fm), relative electron trans-port rate (retr) and nonphotochemical quenching (nPQ) were measured weekly via pulse amplitude modulated (PaM) fluorometry with a DIVIng-PaM (Heinz Walz gmbH, effeltrich, germany). Segments of all algae were dark-adapted using the magnet sample holder accessory for the diving PaM. Photosynthesis-irradiance curves were obtained using dark-adapted algae (10 min dark adaptation) exposed to saturation pulses separated by 1 min intervals. Previous tests showed that the ground fluorescence recov-ered after 1 min. the retr was calculated according to the equation:

where Y is the photochemical yield, 0.5 is the factor accounting for the two quanta that must be absorbed for every electron transported due to the presence of two pho-tosystems, and I is the irradiance in μmol photons m−2 s−1. the photochemical parameters Ik, retrmax, and α (light saturation point, maximum relative electron transport rate, and photochemical efficiency, respectively) were calculated from a nonlinear regression analysis using the model from eilers and Peeters (1988).

calcification and skeletal structure

calcification rates were calculated using the alkalinity anomaly technique, which is based on the assumption that every mole of precipitated cacO3 reduces the total alka-linity by two equivalents. H. opuntia fragments were incu-bated in 100 ml glass Schott bottles and the change in alka-linity from time 0 to 4 h after the incubation was used to estimate calcification based on the equation:

where G is the calcification rate in mmol cacO3 g FW−1 h−1, p is the density of seawater (1.025 kg l−1), ta0 is the initial total alkalinity of the seawater (μM), tan is the total alkalinity of the seawater after n hours of incu-bation (in our case 4 h), V is the volume of the incubation chamber (l), FW is the fresh weight of the algal clump (g), and t is the period of incubation (h). When G > 0, calcifi-cation occurs, and when G < 0, dissolution occurs. Due to the lack of stirring during our incubations, our calculations of calcification rate are likely underestimated, but we were interested in comparing rates between treatments.

tissue carbon, nitrogen and phosphorus content

the total carbon and nitrogen content in H. opuntia thalli were measured at the beginning and end of the experiment using a euroea 3000 elemental analyzer (eurovector, Milan, Italy). Halimeda tissue was dried at 60 °c for 48 h

rETR = Y × 0.5 × I

G = 0.5p × (TA0 − TAn) × V/(FW × t)

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and ground to a powder with a mortar and pestle. Separate tissue samples from the same individuals were also ana-lyzed for organic carbon by acidification of the inorganic fraction with 1 n Hcl (100 μl per 3 mg tissue).

total phosphorus was measured in the same ground tissue as above using the colorimetric molybdenum blue method (Koroleff 1983). approximately 20 mg of dried algal tissue was combusted in a muffle furnace at 500 °c for 5 h and dissolved in 5 ml of 0.2 n Hcl. Following a 30 min heating period in a drying oven at 80 °c, 10 ml of distilled water was added to each sample. the samples were shaken and allowed to settle overnight. the follow-ing day, 2 ml of supernatant was added to clean test tubes, followed by 8 ml of distilled water and 1 ml of reagent solution. after a 30 min reaction period, determination of phosphomolybdenum blue was determined colorimetrically on a UV-vis spectrophotometer (UV-2401 Pc, Shimadzu, Kyoto, Japan) at 885 nm.

enzyme activity (external carbonic anhydrase and in situ nitrate reductase)

external carbonic anhydrase activity (ecaa) was meas-ured according to Haglund et al. (1992). Fragments of H. opuntia thalli (50–200 mg dry weight) were placed in a glass test tube, to which 3 ml of assay buffer (50 mM tris, pH 8.5, 25 mM dithiothreitol, 25 mM isoascorbic acid, 5 mM eDta) and 2 ml of ice-cold cO2-saturated water were added to start the reaction. the time it took for the pH to drop 0.4 units (within the pH range of 8.1–7.1) was recorded. ecaa was calculated as (tb/ts − 1)/DW, where tb and ts are the time in seconds it took for the pH to drop 0.4 units in the blank (no algae) and the sample (with algae), respectively, and DW is dry weight of the algal sam-ple after drying at 60 °c for 48 h.

nitrate reductase activity (nra) was measured in situ according to the method by corzo and niell (1991). the assay buffer (0.1 M phosphate buffer containing 0.5 mM eDta, 0.1 % eDta, 30 mM KnO3, 10 μM glucose) was bubbled with n2 gas for 5–10 min prior to the assay. H. opuntia fragments were added to 5 ml amber vials con-taining 3 ml of assay buffer, and each assay was bub-bled again with n2 gas for 1 min. the algae were incu-bated in the dark at 30 °c for 30 min. the reaction was stopped by removing 1 ml of the assay volume and adding 200 μl of 4 % sulfanilimide in 3 n Hcl, and 300 μl of 0.1 % n-(1-napthyl) ethylenediamine dihydrochloride. the absorbance of each sample was measured at 540 nm after a 15-minute period to allow for complete color change. nra was normalized to chlorophyll a content in the incu-bated tissue. Following the enzyme assay, the algae frag-ments were incubated in 4 ml of 100 % dimethylfluoride at 4 °c in the dark. the extracted chlorophyll concentration

was measured spectrophotometrically according to Porra et al. (1989).

Statistical analysis

Statistical analysis was conducted using SPSS Statistics Version 20.0. all response variables measured at the end of the experiment were analyzed using a multivariate analy-sis of variance (ManOVa) with cO2 and nutrients as the independent factors. anOVa assumptions were met by log transforming the data when they did not satisfy tests for normal distribution.

Results

Seawater chemistry

table 1 shows the mean seawater chemistry conditions for each treatment. the mean temperature and salinity were the same in all treatments, while nutrient concentration and carbon chemistry differed. all treatments had high alkalin-ity values due to the artificial sea salt used for preparing the seawater. treatments without cO2 enrichment had lower alkalinity values than those with cO2 enrichment due to the oversaturation of cacO3 in the artificial seawater that caused precipitation onto the aquaria walls. the addition of nitrate and phosphate to the seawater may have caused some of the observed differences in alkalinity between nutrient enriched and unenriched treatments with similar cO2 levels.

growth and photochemistry

relative growth rates (% day−1) were very low throughout the experiment (Fig. 1a). there was a clear effect of nutri-ents on growth rates (table 2), as the algae grown under low nutrients increased growth rates over time, whereas the nutrient enriched algae decreased growth rates over time, regardless of cO2 concentration. at the same time, high diatom growth occurred in all aquaria treated with high nutrients.

Maximum photochemical quantum yield (Fv/Fm) of H. opuntia ranged from 0.70 to 0.76, but was not significantly affected by inorganic nutrients or cO2 (table 2; Fig. 1b). light curves with H. opuntia retr as a function of light intensity are shown for each week at each cO2 and nutrient treatment in Fig. 1c–f. after 4 weeks, retrmax, alpha, and Ik were also not significantly affected by cO2 or nutrients, although after 20 days there was a general trend that H. opuntia grown under elevated cO2 had higher retrmax and Ik values regardless of nutrient concentration compared to ambient cO2-grown algae (Fig. 1g–h). nonphotochemical

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quenching was higher in algae grown under elevated cO2 than algae grown under normal cO2 conditions, and the same pattern was observed in the xanthophyll pool (Fig. 2).

calcification and tissue carbon, nitrogen, and phosphorus

calcification rates were not significantly affected by cO2 or inorganic nutrients (tables 2, 3). the relationship between calcification rates and retrmax was affected by the experi-mental treatments (Fig. 3), as algae grown under elevated cO2 conditions generally had higher retrmax values but lower calcification rates.

the percentage of algal tissue made up of cacO3, was significantly affected by cO2 but not nutrients (tables 2, 3) and this subsequently affected the total c:n and inorganic:organic carbon ratios. the percentage of tissue made of cacO3 was highest in algae grown under normal cO2 conditions and lowest in the tissue of algae grown under elevated cO2 conditions (table 3). Organic car-bon and nitrogen content were higher in the elevated cO2 treatments regardless of nutrient level, while tissue phos-phorus was significantly higher in algae grown under nutrient enrichment, resulting in n:P ratios <10 (table 3). corg:n and n:P molar ratios ranged from 30.8 ± 1.6 to 33.7 ± 0.69 and 9 ± 1 to 41 ± 4, respectively.

enzyme activity (external carbonic anhydrase and in situ nitrate reductase)

nitrate reductase activity was affected by a significant interaction between cO2 and inorganic nutrients (table 2). the effect of nutrients on ecaa was significant only under elevated cO2 conditions, as the ecaa of algae grown under elevated cO2 without nutrient enrichment was 192 % (±29) that of the ecaa in algae grown under control con-ditions (Fig. 4). On the other hand, in situ nra decreased under elevated cO2, and increased under elevated cO2 plus inorganic nutrients, in relation to the control treat-ment (−cO2−nP). We also found significant correlations between ecaa and tissue carbon (Fig. 5). Inorganic carbon and ecaa were negatively correlated, while organic car-bon was positively correlated to ecaa (Pearson’s correla-tion = −0.453, p = 0.045; Pearson’s correlation = 0.536, p = 0.020, respectively).

Discussion

the results from our inorganic nutrient and cO2 enrich-ment experiment show that nutrient status influences the response of H. opuntia to elevated cO2. the algae without nutrient enrichment were phosphorus limited (n:P > 30) under high cO2, while the nutrient enriched algae were Ta

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Fig. 1 (a) Mean (±Se, n = 4) relative growth rate, (b) maximum quantum yield of photosystem II (Fv/Fm), retr versus irradiance after 1 (c), 2 (d), 3 (e), and 4 (f) weeks of exposure to the experimental treatments, (g) light saturation point (Ik) and (h) maximum relative electron transport rate (retrmax) of H. opuntia over time for each treatment

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nitrogen limited (n:P < 10). cO2 enrichment caused an increase in phosphorus limitation, an increase in external ca activity, and a decrease in nitrate reductase activity in the −nP treated algae. In contrast, cO2 enrichment did not affect the n:P ratios, but increased nr activity and tissue

nitrogen in +nP treated algae. the variable effect of nutri-ent status on the response of H. opuntia to elevated cO2 suggests seasonal changes and local nutrient status will play a role in the response of this alga to elevated seawater cO2.

Table 2 results from ManOVa tests, including F-ratios, degrees of freedom (in parentheses) and p values significant at the 95 % confidence level

response variable cO2 effect Inorganic nutrient effect Interactive effect (cO2 × nP)

ecaa – – –

nra – F (1, 11) = 8.8, p = 0.01 F (1, 11) = 9.2, p = 0.01

growth – F (1, 11) = 8.9, p = 0.01 –

Ik – – –

retrmax – – –

alpha – – –

nitrogen F (1, 11) = 7.0, p = 0.02 – –

Phosphorus – F (1, 11) = 24.5, p = 0.0004 –

Organic carbon F (1, 11) = 5.5, p = 0.04 – –

Inorganic carbon F (1, 11) = 6.1, p = 0.03 – –

net calcification – – –

Fig. 2 Mean (±Se, n = 4) nonphotochemical quenching of H. opuntia exposed to the four experimental treatments as a function of mean (±Se, n = 4) photochemical yield. Inlaid fig-ure is a boxplot of the median, minimum, maximum, and first and third quartiles of the xanthophyll pool of H. opuntia in each treatment

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Table 3 Mean (±Se, n = 4) calcification rates, calcium carbonate (cacO3), organic carbon (corg), nitrogen (n) and phosphorus tissue content and organic carbon to nitrogen (corg:n) and nitrogen to phos-

phorus (n:P) molar ratios of H. opuntia after 4 weeks of exposure to the experimental treatments

Unshared superscripts indicate significant differences (p < 0.05)

treatment calcification rate (μmol cacO3 g FW−1 h−1)

cacO3 (% DW) corg (% DW) n (% DW) P (% DW) corg:n n:P

−cO2−nP 0.32 ± 0.09 90.9 ± 0.8a 5.5 ± 0.5a 0.57 ± 0.08a 0.06 ± 0.01a 33 ± 3 20 ± 1

−cO2+nP 1.57 ± 0.26 90.1 ± 1.2a 5.7 ± 0.2a 0.57 ± 0.04a 0.15 ± 0.01b 34 ± 0.7 9 ± 1

+cO2−nP 0.87 ± 0.30 82.1 ± 1.2b 7.4 ± 0.8b 0.80 ± 0.09b 0.05 ± 0.01a 31 ± 2 41 ± 4

+cO2+nP 0.31 ± 0.27 84.2 ± 2.4b 6.1 ± 0.5b 0.67 ± 0.05b 0.14 ± 0.01b 32 ± 2 12 ± 1

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the increase in retrmax under elevated cO2 alone that we observed at the end of our experiment contradicts the results of Price et al. (2011), who found that elevated cO2 (pH 7.7, pcO2 946 ppm) decreased retrmax. However, their experiment lasted only 2 weeks, while our significant cO2 effect was first visible after 2 weeks. this discrepancy provides an example of how time is an important factor to consider in ocean acidification experiments. Furthermore, their experiment was conducted under high natural light

intensities and a higher temperature (28 °c), which when combined with elevated cO2, may have had a photoinhibi-tory effect, as elevated cO2 has been shown to increase the susceptibility of some macroalgae to UV stress (gao and Zheng 2010; liu et al. 2012). Our data also support this hypothesis, as the algae exposed to elevated cO2 had the highest nonphotochemical quenching and xanthophyll pools. the elevated nonphotochemical quenching and xan-thophyll pools are an indication that H. opuntia attempted to dissipate excess light energy and reducing pressure in the electron transport chain. Such a response has also been observed in Emiliania huxleyi and natural phytoplankton assemblages (gao et al. 2012; rokitta et al. 2012), but under normal and high light conditions. Our results show that the same photochemical response was observed in H. opuntia even at very low light levels.

In our experiment, growth rates were very low most likely due to low light levels and high turnover of old calci-fied material that was lost from the algal thalli during the course of the experiment (pers. observation). nevertheless, we were able to observe new segments being produced throughout the experiment, indicating that the algae were growing despite losing old segments. Furthermore, the Ik values ranged between ca. 40 and 60 μmol photos m−2 s−1, showing that the algae had acclimated to the light condi-tions in culture. growth rates were negatively affected by inorganic nutrients, which may have been a result of high phosphate concentrations, as previous studies have shown that high phosphate levels can increase segment death and decrease growth and biomineralization in other calcify-ing green algae (Delgado and lapointe 1994; Demes et al.

Fig. 3 relationship between mean (±Se, n = 4) calcification rates and mean (±Se) retrmax values after 4 weeks of exposure for each treatment

Fig. 4 Mean (±Se, n = 4) external carbonic anhydrase activity (ecaa) and nitrate reductase activity (nra), standardized to grams of organic carbon (corg)

Fig. 5 correlations between organic (corg) and inorganic (cinorg) carbon and external carbonic anhydrase activity (Pearson’s correla-tion = 0.536, p = 0.020; Pearson’s correlation = −0.453, p = 0.045, respectively)

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2009). In our high cO2 treatment with elevated nitrate and phosphate, diatom growth was extremely high. Based on chlorophyll c2:b ratios (data not shown) and visible obser-vations, the growth of diatoms in the +cO2+nP treatment indicated that these organisms were able to exploit the excess inorganic carbon, nitrogen and phosphorous levels to increase their growth. an increase in diatom abundance, photosynthesis and growth under elevated cO2 has previ-ously been reported in the lab at 1,000 ppmv cO2 and in the field along a natural cO2 gradient (Johnson et al. 2011; Wu et al. 2010). Furthermore, the success of diatoms at the expense of H. opuntia under high phosphate conditions is consistent with a higher distribution and abundance of calcified rhizophytic algae reported in phosphate limited regions compared to phosphate rich environments with silicalastic sediments (Demes et al. 2010 and references therein). therefore, elevated inorganic nutrients, particu-larly phosphate, in combination with elevated cO2 could alter the competitive interactions between H. opuntia and its diatom epiphytes by giving the diatoms a competitive advantage.

the most striking effect of the combination of cO2 and inorganic nutrient enrichment on H. opuntia was observed in ecaa and nra. When the algae were exposed to only cO2 enrichment, retr, organic carbon, nitrogen and n:P ratios increased, while nra decreased. In this treatment, we also saw a simultaneous increase in ecaa. the effect of elevated cO2 on nitrate reductase activity among higher plants, seagrasses and algae is highly variable (Purvis et al. 1974; Hocking and Meyer 1991; geiger et al. 1999; gordillo et al. 2001, 2006; Xia and gao 2005; alexan-dre et al. 2012; Hofmann et al. 2012, 2013b). In the case where nra decreases, Stitt and Krapp (1999) suggest that elevated cO2 could increase the preference for ammo-nium over nitrate due to the decrease in photorespiration and associated recycling of ammonium that occurs under elevated cO2. this process results in the formation of glu-tamine and subsequent repression of nra. Such a response would save energy due to the lower energy requirements of ammonium assimilation compared to nitrate (losada and guerrero 1979; Syrett 1981) and would allow the alga to allocate more energy to producing carbonic anhydrase for regulating inorganic carbon uptake and assimilation. Indeed, we saw a positive correlation between ecaa and organic carbon, and observed an increase in ecaa, retr and organic carbon in the treatment where nra decreased. In our study, the algae grown under +cO2−nP conditions were phosphorus limited, as the mean n:P ratio was greater than 30:1, which is the threshold for phosphate limita-tion (ryther and Dunstan 1971; Smith 1984). as nra is heavily dependent on phosphate availability (nicholas and Scawin 1956), phosphorus limitation was most likely the driving factor of the decrease in nra under high cO2 in

our study. Finally, the increase in nra, retr, organic car-bon, and nitrogen caused by cO2 enrichment in the nitro-gen-limited algae could be explained by an increased effi-ciency of nitrogen uptake and assimilation in the presence of more inorganic carbon.

In contrast to previous work on Halimeda spp., which found that Halimeda decreased calcification rates and increased shedding rates, but did not show changes in skeletal cacO3 composition under elevated cO2 (Price et al. 2011; Sinutok et al. 2011), we did not observe lower calcification rates, but did see less inorganic carbon in H. opuntia exposed to elevated cO2 compared to algae grown under control conditions. the contrasting results are most likely due to differences in experimental light and seawater chemistry conditions, as we had low light and high alkalin-ity conditions in our experiment. However, it is also possi-ble that different populations of H. opuntia respond differ-ently to elevated cO2. It has already been established that different Halimeda spp. respond differently to increasing cO2 (Price et al. 2011; Sinutok et al. 2011), but future stud-ies should also investigate if there are differences in popu-lation responses.

the very high calcification rate observed in our −cO2+nP treatment was likely due to the extremely high aragonite saturation state of the seawater under these condi-tions. Borowitzka and larkum (1976) also found that calci-fication rates of H. tuna were constant between pH 7.0–8.5, and even observed an increase in calcification after short term exposure to pH 6.5 and attributed it to higher photo-synthetic rates. Despite similar calcification rates among the different cO2 treatments, the amount of cacO3 in the algal skeleton and the inorganic:organic carbon ratios were lower in the elevated cO2 treatments compared to ambient cO2 conditions, highlighting the importance of aragonite saturation state for skeletal mineralogy. Such a decrease of inorganic carbon in the skeleton could make the alga more palatable and an easier and more nutritional target for graz-ers. In addition to having potential effects on grazers, the changes in skeletal composition that were observed in this study and others may have important implications for coral reef accretion, as Halimeda spp. are important reef building organisms due to the contribution of their dead skeletons to coral sands (Drew 1983). If their skeletons have lower inorganic content under elevated cO2 conditions, the rela-tive contribution of Halimeda spp. skeletons to tropical car-bonate sands would decrease under predicted future ocean conditions.

carbon, nitrogen and phosphorus availability control important aspects of H. opuntia physiology, and cO2 ele-vation that is likely to occur in the future combined with localized nutrient inputs will have profound impacts on enzymatic activity and carbon and nitrogen assimilation in macroalgae. Under low light conditions, when nutrients

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status changes, H. opuntia is able to alter its enzymatic activity and subsequently carbon and nitrogen assimila-tion while maintaining healthy calcification rates under elevated cO2. However, when nutrients are saturating and light limiting, which occurs under eutrophic conditions, non-calcifying organisms may benefit at the expense of cal-cifiers. although we did not directly test this hypothesis in our study, the high diatom growth in our +cO2+nP treat-ment hinted to such an effect. Furthermore, this trend has been reported by russell et al. (2009), who reported that the combination of elevated cO2 and inorganic nutrients altered subtidal rocky habitats by decreasing the competi-tiveness of crustose coralline algae compared to algal turfs. It will be necessary to conduct further studies to determine how Halimeda spp. will compete with non-calcifiers under elevated cO2 and inorganic nutrients, and how the pres-ence/absence of grazers also affects their competition.

Acknowledgments the authors would like to thank christian Brandt and andrian Basilico for help with the experimental set-up, Dorothea Dasbach for tissue carbon and nitrogen analysis and Mat-thias Birkicht for dissolved inorganic nutrient analysis. Funding for this project was provided by the german Federal Ministry of educa-tion and research (BMBF) through the cooperative research project Biological Impacts of Ocean acidification (BIOacID).

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