Global Change Biology (2012), doi: 10.1111/j.1365-2486.2012.02756.x

Ocean acidification weakens the structural integrity of coralline algae FEDERICA RAGAZZOLA*†, LAURA C. FOSTER*†, ARMIN FORM†, PHILIP S.L. A N D E R S O N * , T H O R H . H A N S T E E N † and J A N F I E T Z K E † *Department of Earth Sciences, University of Bristol, Wills Memorial Building, Queen’s Road, BS8 1RJ, Bristol, UK, †GEOMAR, Helmholtz Centre for Ocean Research, Wischhofstraße 1-3, 24148, Kiel, Germany

Abstract The uptake of anthropogenic emission of carbon dioxide is resulting in a lowering of the carbonate saturation state and a drop in ocean pH. Understanding how marine calcifying organisms such as coralline algae may acclimatize to ocean acidification is important to understand their survival over the coming century. We present the first long-term perturbation experiment on the cold-water coralline algae, which are important marine calcifiers in the benthic ecosystems particularly at the higher latitudes. Lithothamnion glaciale, after three months incubation, continued to calcify even in undersaturated conditions with a significant trend towards lower growth rates with increasing pCO2. However, the major changes in the ultra-structure occur by 589 latm (i.e. in saturated waters). Finite element models of the algae grown at these heightened levels show an increase in the total strain energy of nearly an order of magnitude and an uneven distribution of the stress inside the skeleton when subjected to similar loads as algae grown at ambient levels. This weakening of the structure is likely to reduce the ability of the alga to resist boring by predators and wave energy with severe consequences to the benthic community structure in the immediate future (50 years). Keywords: climate change, coralline algae, long-term experiments, Ocean acidification, structural changes Received 25 January 2012; revised version received 2 May 2012 and accepted 13 May 2012

Introduction Since the industrial revolution, the carbon dioxide in the atmosphere has risen by over 100 ppm due to anthropogenic input (Solomon et al., 2007). The ocean has absorbed about one third of the anthropogenically derived CO2, which has resulted in a lowering of the carbonate saturation and a reduction of the average global surface ocean pH by 0.1 pH unit (Caldeira & Wickett, 2003), a process termed ‘ocean acidification’. Multi-model projections based on IPCC emission scenarios suggest an additional future reduction of 0.14 –0.35 pH units towards the end of the 21st Century (Solomon et al., 2007). These chemical changes are suggested to have direct implications for physiological processes such as photosynthesis, calcification, growth rates or internal pH regulation in a wide range of organisms (Orr et al., 2005; Turley et al., 2010; Lombardi et al., 2011). Benthic shelf habitats are ecological hotspots and among the most reproductive and diverse in the world. Their benthic communities support rich commercial fisheries and provide important ecosystem goods and service (Turley et al., 2010). Coralline algae represent a Correspondence: Federica Ragazzola, tel. + 44 0 117 954 5235, fax + 44 0 117 925 338, e-mail: [email protected]

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major component of most benthic ecosystems from the poles to the tropics, comprising up to half of the carbonate-secreting organisms in some cold water habitats (Freiwald & Henrich, 1994). They are particularly dominant in shallow water ecosystems and are important habitat builders as they create and stabilize reefs and greatly increase benthic diversity by providing hard substrate for other organisms to settle on (Foster, 2001; Steller et al., 2003). In addition, they are important contributors to the global inorganic carbon budget in shallow water ecosystems (Mackenzie et al., 2004). The susceptibility of calcifying organisms such as coralline algae to ocean acidification depends on the polymorph of CaCO3 utilized. Pure calcite is less soluble then aragonite, while the solubility of calcite increases with increasing mol% MgCO3 (Andersson et al., 2008). Coralline algae build their skeleton from high-Mg calcite, the most soluble form of carbonate, being 50% more soluble than calcite and 20% more than aragonite (Busenberg & Plummer, 1989; Feely et al., 2004). This higher solubility of their carbonate thallus may explain why they are absent in naturally acidified waters, such as volcanic vent systems, where other marine calcifiers are able to live and calcify (Hall-Spencer et al., 2008; Couto et al., 2010). In addition, at high latitudes, the saturation state is naturally lower, due to the increased uptake of CO2 1

2 F . R A G A Z Z O L A et al. into cold waters. Thus these waters will be the first habitat that will be exposed to undersaturated conditions with respect to aragonite and calcite driven by anthropogenic CO2 input (Orr et al., 2005). As a consequence, coralline algae living at high latitudes are likely to be particularly vulnerable to anthropogenic ocean acidification (Andersson et al., 2008). Here we present a long-term study to examine the impact of ocean acidification on Lithothamnion glaciale, a high latitude non geniculated cold water species with a high- Mg calcite skeleton (~12 mol% Mg). Laboratory experiments permit all the parameters to be strictly controlled and therefore allow the isolation of the effect of one parameter, in this case increased pCO2 in the water, to be studied. However, in its natural environment the alga is subjected to many physical challenges such as predator attacks and wave energy therefore we have conducted analyses of the skeleton stability and resistance to make estimations at the ecosystem level.

Materials and methods Specimens of L. glaciale Kjellman were collected in Kattegat (57° 0.84′ N, 11° 35.10′ E and 57° 0.38′ N, 11° 34.88′ E) at 20 m depth in June 2010 on board RC Littorina. The algae were sampled with a mini-dredge and for the experiments only healthy specimens were selected i.e. those with no sign of damage or bleaching. Thalli were placed in aerated 15-litre tanks with a solution of 0.5 g L1 Alizarin Red S staining (Fluka, Sigma-Aldrich, Steinheim, Germany) for 24 h at 8°C in 12:12 hours light-dark cycle (Blake & Maggs, 2003). After this period the algae were rinsed to remove the extra staining and placed in the aquaria.

Experimental set up The selected specimens were randomly assigned in 16, 5 L glass aquaria filled up with natural seawater (salinity 32) and bubbled with 4 different CO2 concentrations (422, 589, 755 and 1018 latm) for 3 months using a CO2 mixing-facility (KICO2 Kiel CO2 manipulation experimental facility, Linde Gas & HTK Hamburg, Germany). KICO2 determines the CO2 content of inflowing ambient air and automatically adds pure CO2 to produce the three different CO2-air mixtures used during this experiment. Ambient air was used as a pCO2 control. At the beginning, all aquaria with the algae were supplied with ambient air (with a pCO2 level of ~422 latm) for 1 month. After taking initial water samples for Total Alkalinity (At), and nutrients, the pCO2 concentration were slowly increased over 1 month, apart from the control, until the desired concentrations were reached. The water was exchanged approximately fortnightly (as required) so that the nutrient levels and alkalinity were kept constant. All aquaria belonging to the same treatment had the water changed at the same time from a water reservoir, which was adjusted to the

treatment pCO2 concentration by bubbling in CO2 over 24 h. The experimental condition were set at 7 ± 0.5 °C with 20 lmol photons m2 sec1 in 12 h light/ dark cycle. To keep the temperature constant, the aquaria were located inside water baths all connected to the same cooling unit (Aqua Medic Titan 4000, Bissendorf, Germany). Water motion and filtration in the aquaria was ensured by a submersible pump (crystal mini 20-50; Hydor, Bassano del Grappa, Italy). The irradiance was adjusted by dimming the light source with semi-transparent foil to in situ level found at 20 m at Kattegat during the summer season (20 lmol photons m2 sec1). The light source consisted of fluorescent tubes (39 W, Marine blue; Arcadia Products, Redhill, UK) above each water bath; the surface irradiance was measured once a month to monitor the decreasing in light intensity due to the aging of the light bulb.

Dissolution experiment Dead specimens were assigned in 5 L glass aquaria filled up with natural seawater (salinity 32) and bubbled with 422 and 1018 latm for 1 month using the same CO2 mixing-facility of the long-term experiment. After 1 month the specimens were collected and prepared for scanning electron microscopy (SEM).

Monitoring of the carbonate system and nutrients Salinity, temperature and pH were measured once a week using a glass combination electrode (SenTix80; WTW, Weilheim, Germany) with an accuracy of ±0.005. Water samples for the Total Alkalinity (At) were collected every 2 weeks. Salinity was measured using a WTW conductometer (LF340) with a TetraCon 325 measuring cell (WTW). The value of At was measured in duplicate using a potentiometric, open-cell titration procedure according to Dickson et al. (2003). GF/F filtered seawater samples of 10–15 g were accurately weighed (1416B MP8-1; Sartorius AG, Go¨ttingen, Germany) and for the titration, an automatic titrator (Titrando 808; Deutsche Metrohm GmbH & Co. KG, Filderstadt, Germany) was used. The titrant was hydrochloric acid (HCl) with a concentration of 0.005 N. The average precision between duplicate measurements was  4 lmol kg1. The At measurements were corrected against Certified Reference Material provided by Andrew Dickson (Scripps Institution of Oceanography). The carbonate system (Table 1) was calculated from pH and At measurements using the thermodynamic constants of Mehrbach et al. (1973) as refitted by Dickson & Millero (1987) on the free scale and the carbonate chemistry calculation programme CO2SYS (Lewis & Wallace,1998). Water samples for nutrient measurements were taken every month: Nitrate, nitrite and phosphate were measured photometrically (U-2000; Hitachi, Tokyo, Japan) according to Hansen & Koroleff (1999) with precision levels of ±0.5 lmol kg1, ±0.02 lmol l1, and ±0.05 lmol l1 respectively. Ammonium was measured fluorometrically (SFM 25; Kontron Instruments, Neufahrn, Germany) according to Holmes et al. (1999) with a precision of ±0.08 lmol l1.

© 2012 Blackwell Publishing Ltd, Global Change Biology, doi: 10.1111/j.1365-2486.2012.02756.x

Carbonate system parameters during the 3 months incubation of Lithothamnion glaciale. All numbers are mean values (n = 4) ±standard deviation. The pH, salinity, temperature and total alkalinity (TAlk) were measured while other parameters were calculated.

38.9 29.6 118.10 174.8 ± ± ± ± 422 589 755 1018 1.77 1.31 1.12 0.98 2.8 2.08 1.78 1.55 76.6 106.4 77.8 83.9 ± ± ± ± 2076.7 2181.8 2226.5 2403.8 86.6 89.2 0.1 155.9 ± ± ± ± 2159.45 2216.7 2496.1 2285.37 101.2 122.7 96.0 97.5 ± ± ± ± 2311.5 2315.0 2355.1 2537.7 0.2 0.1 0.3 0.2 ± ± ± ± 7.7 7.6 7.7 7.7 32.2 31.3 31.5 31.6 0.05 0.03 0.07 0.07 ± ± ± ± 8.03 7.90 7.81 7.72 1 (410 latm) 2 (560 latm) 4 (840 latm) 3 (1120 latm)

pH (free scale) Treatment

Table 1 Carbonate system


± ± ± ±

1.2 0.1 0.7 0.7

T (°C)

TAlk (lmol kg1)

DIC (lmol kg1)

HCO3 (lmol kg1)


± ± ± ±

0.33 0.3 0.34 0.15


± ± ± ±

0.2 0.2 0.2 0.09

pCO2 (latm)


Growth rate Alizarin Red-S was used as a growth marker for the experiments. Longitudinal thallus sections were prepared and viewed under a dissecting microscope (Fig. 1). Four samples for each treatment were photographed (microscope: Zeiss Stemi 2000 C, digital camera: CANON Powershot A80 Krefeld, Germany) and prepared for SEM. Growth from the tip to the stained section was measured using ImageJ image analysis software (Rasband, 1997-2011).

TOMCAT (TOmographic Microscopy and Coherent rAdiology experimenTs) Synchrotron-based X-ray Tomographic Microscopy was use for a non destructive, high resolution quantitative volumetric investigation of L. glaciale. These measurements were performed at the TOMCAT beamline at the Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland (Stampanoni et al., 2006). For each tomographic scan, 1501 projections over 180 degrees were acquired at energy of 15 keV with UPLAPO 10 9 objective (field view of 1.5 9 1.5 mm2; pixel size 0.74 9 0.74 mm2). The exposure time ranged between 300 and 400 ms. Projections were rearranged into corrected sinograms, and reconstructed using optimized FFT transformations and gridding procedures (Marone et al., 2010). The final data were exported as TIFF images (8 bit 2 9 2 binned). Additional processing to produce 3D isosurface and sample thickness was done using Avizo (Mercury Computer Systems Ltd., Chelmsford, MA, USA). The cell density was measured using the image analysis and processing software ImageJ. (Rasband, 1997-2011)

Data analysis A linear regression analysis was performed between the growth rates and the different CO2 concentrations with R2 = 0.6485 and P = 0.0066, giving a significant linear decrease in growth rates towards higher CO2 concentrations. For the cell density the cell wall thickness (intra filament) and growth rate data One-Way Analysis of Variance (ANOVA) was performed. Post-Hoc Testing (Holm–Sidak) was used to identify which treatments were significantly different from each other. For the analysis of the cell wall thickness (inter filament) Kruskal–Wallis One-Way ANOVA on Ranks was performed. Post-Hoc testing (Mann and Whitney U) was used to identify which treatments were significantly different from each other. The P value was then adjusted using the Bonferroni method.

Finite element modelling To better understand the effect of the ultra structural changes on the structure robustness we applied a finite element analysis (FEA). This is a technique that reconstructs stress, strain and deformation in model structures (Zienkiewicz et al., 2005). Digital models either created from scanned data or built from scratch are assigned user-defined material properties

© 2012 Blackwell Publishing Ltd, Global Change Biology, doi: 10.1111/j.1365-2486.2012.02756.x

4 F . R A G A Z Z O L A et al.

Fig. 1 Growth and banding in Lithothamnion glaciale maerl (a) Maerl collected at Kattegat. (scale = 1 cm) (b) Example of thallus stained with Alizarin Red and grown, sectioned longitudinally to show naturally pigmented growth bands and position of Alizarin Red mark (arrow), which indicates apex of thallus at the time of staining. (scale bar = 1 mm).

and loading conditions that ideally reproduce biological loads. FEA software reduces the complex structures modelled into a series of discrete problems (the finite elements) that can be solved mathematically. The majority of biological FE work has utilized scanned data to create complex biological models (Rayfield, 2007). However, when engineers use FEA to test for mechanical performance; they make the simplest model necessary to answer the question at hand. In this view, making an overly complex biological model can obfuscate the variables of interest, and make it hard to ascertain what effects they have on the structure. Several recent biological FE studies have explored the utility of simple models built to address shifts in mechanical performance due to specific morphological features (Qasim et al., 2007; Anderson et al., 2011). For model simplicity and due to the lack of reference data, we

assumed that the algae skeleton is homogeneous which is the same procedure used for other heterogeneous material such bones (Dumont 2005; Rayfield, 2007). The aim of the FE model in this study is a comparative analysis focusing on shape, so simplifying the materials is a valid procedure. Since we assume that algae skeleton is homogeneous and the material properties are the same for the specimens in the different treatments, the stress patterns and the total strain energy values must be interpreted cautiously. However, FEA can provide a way to compare the relative robustness of different structures. To determine the impact of differences in the robustness of the material to strain, a ~ 80 lm by 80 lm of the coralline algae ultra-structure was constructed in Finite Element-software package Abaqus/CAE v.6.8-2 (Simula, USA, Dassault Syste´mes, //Simulia, Providence, RI, USA). The model was

Fig. 2 Scale model of Lithothamnion glaciale. Scale model of the ultra-structure of L. glaciale at 422 and 589 latm showing the filaments and growth direction. The individual cells are separated by the intra (red line) and inter (yellow line) and the cell wall (high-Mg calcite, in grey). The measurements were taken within the region of the newly calcified cells, approximately after three cells from the tip to minimize any impacts of the Alizarin stain. The SE image shows differences in the amount of carbonate deposition within the cell wall of the two treatments. The model and SE image highlight the differences in growth of the cells between the two treatments, with a significant thinning of the intra- and inter-wall thickness as well as increased cell size at 589 latm. © 2012 Blackwell Publishing Ltd, Global Change Biology, doi: 10.1111/j.1365-2486.2012.02756.x

STRUCTURAL WEAKENING OF CORALLINE ALGAE 5 constructed in 2D to mimic our model (Fig. 2). To derive a solution for the model, Abaqus automatically assigned a uniform thickness to the third dimension, which is defined by the user. In this case, the thickness is 1 lm. The models are differentiated based on measurements made from SE images of the cell size and wall thicknesses. The size was approximately 80 lm 9 80 lm, with 422 latm being 78.29 9 72.46 lm and 589 being 77.2 by 74.99 lm. This was done to prevent the last cell being incomplete which would affect the resulting strain in the model. To test whether or not this slight variation in size significantly impacted the results, the model was also run in which the size was standardized between models by having increased calcite wall thickness at the bottom and right of the model. The material was assumed to be linear elastic isotrophic homogeneous material with a Young’s module of 36 GPa and Poisson of 0.31 (calcite) (Tanur et al., 2010). A mesh model of the structure using 4-node bilinear, plane stress, quadrilateral elements was produced. A load pressure of 20 000 Pa along 40 lm of the external top and left hand side of the ultra-structure was applied (equating to approximately half height and length of the model). This is meant to mimic loads due to wave action or predatory animals applying crushing forces to a corner of the structure. The general stress patterns (von Mises) over the entire model and the total strain energy were calculated. The former gives a good indication of where failures may occur when critical stresses are reached and which regions of the structure are most susceptible to failure. Strain energy is a measure of the work expended in deforming the structure for a given load (Dumont et al., 2009). For mineralized structures where maintaining stiffness is important, strain energy has been suggested as a useful performance variable. For a given load per volume, the more strain energy in the system, the more deformation has occurred and the more potential energy there is to convert into free surface energy during fracture, which can lead to catastrophic failure (Gordon, 1978). However, both stress and strain energy are scale-dependent measures, related to the surface area and volume of the models respectively. Recent theoretical work on FEA has shown that to analyse differences in stress patterns between shape alone, it is necessary to scale the models to keep the ratio between surface area and force constant across all models. This can be done most simply by multiplying the load input into the second model by the ratio of the surface areas in the models (Zienkiewicz et al., 2005). Although this will eliminate affects on stress due to size differences, it is not clear if this is always desirable. In the case of the coralline sections, the reduction in calcite surface area between models is potentially important and should not be removed. To examine differences between these interpretations, the 589 latm model was analysed two ways, once using the same load as the 422 latm model (20 kPa) and once by scaling the input load by the surface area ratio (10.17 kPa) (Table 2). Total strain energy is dependent on the volume of the material being loaded. To take into account differences in calcite volume between models when comparing we applied a transformation published by Dumont et al. (2009). After strain energy was calculated, the values for the heightened CO2 models were scaled to the ratios of loads and volumes

Table 2 Total strain energy Model (latm)

Loads (kPa)

Total strain energy (pJ)

Increase in TSE (%)

422 589 589*

20 20 10.17

0.00163 0.0112 0.0101

– 687 619

*Total strain energy corrected for volume of calcite. between models using the following equation (as outlined by Dumont et al., 2009): U B0 ¼ ðVB =VA Þ1=3 ðFA =FB Þ2 UB where UB′ is the total strain energy of the new scaled model B′, VA and VB the volume of the two models (A and B), FA and FB the force value loaded in model A and B, and UB the total strain energy of model B. The volume of calcite in the model was 242.91 and 130.08 lm3 for 422 and 589 latm, respectively, calculated by the total volume minus the volume of the cells. The results of these for the total strain energy (corrected for volume) are shown in Table 2 along with the percentage increase in Total Strain Energy in the 589 latm models. In addition, we constructed two additional models: 589 latm models using the wall thickness of 422 latm but the same cell size found at 589 latm, and 589 latm models using the cell size of 422 latm but with the wall thickness found at 589 latm.

Results Alizarin Red S was used as a growth marker for the experiments. The staining resulted in a clearly identifiable band in only 30% of the tips examined whereas in the others the stain was not always distinguishable from the rest of the tissue. The low percentage of successfully stained thalli was previously recorded in Lithothamnion glaciale by Blake and Maggs (Blake & Maggs, 2003). During the experiment, coralline algae continued to grow under all exposed CO2 scenarios, even including undersaturated conditions. Comparison between treatments reveals that growth was significantly different (P755vs1018 < 0.05, df = 3) between the control (422 latm) and the highest pCO2 treatment (1018 latm) while differences between the intermediate treatments were not significant. Growth rates of L. glaciale exposed to 1018 latm were 32% lower than those grown under present day pCO2 condition (Fig. 3a). There is a significant change in the internal structure between the control and the high pCO2 treatments: the cells became larger due to a significant elongation coupled with an increase in width (Fig. 2), resulting in a decreased cell density (all pairwise P < 0.001, df = 3; P755vs1018 = NS,

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6 F . R A G A Z Z O L A et al. df = 3) (Fig. 3b), while both the intra- (all pairwise P < 0.05, df = 3; P755vs1018 = NS, df = 3) and interfilament (all pairwise P < 0.05, df = 3; P755vs1018 = NS, df = 3; P755vs1018 = NS, df = 3) cell walls became thinner (Fig. 3c and d). For the specimens cultured at nonmanipulated conditions (control, 422 latm) the cell density and growth rates were similar to those observed in the field with the typical ultrastructure of summer cells (Halfar et al., 2000; Foster, 2001; Blake & Maggs, 2003; Adey et al., 2005; Kamenos et al., 2008; Martin & Gattuso, 2009). The major ultra-structural changes start to occur in waters that are still saturated (422–589 latm: 38.2% decrease in inter filament cell wall thickness compared to 44.5% decrease between 422 and 1018 latm). The transition from slightly saturated (755 latm; ΩAr = 1.12) to undersaturated conditions (1018 latm; ΩAr = 0.98) seems to have no further effect on cell density and inter-filament wall thickness suggesting that a limit of its physiological adaptation capabilities has been reached. The importance of the cuticle and the epithallium was highlighted during the perturbation experiment, in which four dead specimens were exposed to 1018 latm for 1 month (Fig. 4). The surface layers of cells of this specimen were completely corroded within a month, and dissolution within the thallus undermined the structure. From the FEA (Fig. 5) applied to the scale model (Fig. 2), the resultant strain energy was an order of magnitude higher in the 589 latm experiment specimen, indicating that the algae stores more strain energy per volume and has more internal energy available to propagate cracks and cause catastrophic breakage when damaged (Gordon, 1978). Addition modelling showed that the thinning of the wall and the change in cell size, contributed to a change in the total strain of similar magnitude.

Discussion The long-term experiment, using summer conditions to provide optimum growth condition, indicates that there is a negative linear correlation between growth rates and decreasing saturation state. Unexpectedly, even after 3 months the samples exposed to undersaturated water continue to grow and calcify, albeit at lower rates. Elevated CO2 levels resulted in a statistically significant decrease in growth rates only at the highest pCO2 tested (1018 latm), with aragonite saturation state (ΩAr) = 0.98. No significant difference was found in the intermediate pCO2 tested probably due to the low number of replicates. This result is in disagreement with previous studies (Bu¨denbender et al., 2011) where they found that L. glaciale was able to maintain the net calcification rates, during summer conditions, even at a ΩAr

Fig. 3 Parameters of growth of Lithothamnion glaciale under the four different pCO2: (a) Linear growth rate (b) cell density (c) inter-filament (d) cell wall thickness. In the growth rates graph (a) the pink area indicates the range of growth rates previously published measured both in cultured (Blake & Maggs, 2003) and in natural environment (Freiwald & Henrich, 1994; Halfar et al., 2000) using different techniques. All parameters (a–d) show a significant decrease with increasing pCO2. The parameters were calculated using ImageJ analyses of SE images (a, c, d) and TOMCAT images (b). Number of replicates n = 4; horizontal error bars display the standard deviation of latm CO2 during the incubation period. Vertical error bars display the standard deviation of measurements within the same treatment. The dotted line indicates the linear trend.

© 2012 Blackwell Publishing Ltd, Global Change Biology, doi: 10.1111/j.1365-2486.2012.02756.x




Fig. 4 SE image of the epithallium surface of Lithothamnion glaciale: (a) 422 latm showing healthy epithallium (b) 1018 latm showing the cuticle lifting of from the epithallium in some part of the surface (c) dead specimen exposed to 1018 latm for 1 month showing severe dissolution and complete disappearance of the epithallium.

of 0.96. One explanation for the different result is that L. glaciale was cultured in 24 h light, according to the arctic summer conditions. However, L. glaciale is mainly a subarctic species and thus rarely lives in regions where continuous daylight occurs. Most of the work previously done on coralline algae response to elevated CO2 have been conducted in short-term experiment but an organisms response to short-term elevated CO2 exposure can strongly differ from its response to longterm exposure as shown for the temperate coralline algae Lithothamnion cabiochae (Martin & Gattuso, 2009) and for the cold water coral Lophelia pertusa (Form & Riebesell, 2012). Lithothamnion glaciale shows a major change of the ultra-structure already at 589 latm with a combination of the cell wall-thinning and increased cell size. The cell-shape displays significant elongation at higher pCO2 (Fig. 2) caused by an increase in width, and resulting in decrease in cell density. The final morphology of an organism depends on a coordination of patterns of cell division, cell elongation and cell differentiation. The filament elongation of Lithothamnion species occurs in the meristem cells soon after division (Adey & McKibbin, 1970) with some additional elongation during the summer in the underlying perithallial cells. In our experiment the decrease in growth is related to an increase in cell size, in length but also in width. However, the increased cell size occurs at higher CO2 in which in there is a slower growth rate, thus implying a possible interference with the cell division rates and not only with the mechanisms of calcification. The opposite trend, with a thickening of the cell wall and increase in cell density, was found in the temperate articulated alga Corallina elongata within a higher temperature, lower pH hydrothermal setting (Couto et al., 2010). These contradictory findings may be explained be a product of the peculiar environment of hydro-

thermal vents. In particular where low pH occurs with increased temperature this may enhance calcification processes and growth rates if the algae are living at lower than optimum temperatures (Martin & Gattuso, 2009). For the two higher pCO2 levels (755 and 1018 latm) there is no further significant difference in thickness and cell density. The reduction of inter-wall thickness plateaus at 0.7 lm suggesting that to maintain cell wall functions a minimum thickness is required. One possible way for the coralline algae to acclimatize to the changed environmental conditions could be a plastic response of the carbonate skeleton by producing less soluble forms of CaCO3 with reduction of the mol% Mg (Ries, 2011). Coralline algae calcify inside the cell wall, therefore, like most calcifying species, the carbonate skeleton is isolated from the external seawater. In L. glaciale, the interface with the surrounding environment is the cuticle and the underlying epithallium. These structures have long been considered to have a protective function (Cabioch & Giraud 1986) and this is also shown in our long-term experiment where they prevent the skeleton from dissolving in under-saturated conditions. During the dissolution experiment, dead Lithothamnion glaciale was heavily corroded after a month with sign of dissolution across the entire structure, this strong corrosion in such a short period could be also due to the thallus morphology of the cultured species (small branches) since the level and the speed of corrosion of the skeleton is not only controlled by the carbonate mineralogy but it is also strongly influenced by the skeletal structure such as crystal size, organization of the skeletal framework and the permeability within the skeleton (Ru¨diger & Wefer, 1986). Previous studies have linked the decrease of abundance (recruits) of coralline algae and lower calcification/ growth with impact of the lower saturation state on calcification processes (Kuffner et al., 2008; Hall-Spencer

© 2012 Blackwell Publishing Ltd, Global Change Biology, doi: 10.1111/j.1365-2486.2012.02756.x

8 F . R A G A Z Z O L A et al.

Fig. 5 Mechanical effects on Lithothamnion glaciale structure. 2D finite element (FE) models based on experimental results of coralline growth. Both models are subjected to the same loads and boundary conditions mimicking biological loads (black arrows). The resultant von Mises stresses are scaled the same for both models for comparative purposes (warm colours indicate high stress and cool colours low stress, and grey indicating extreme values). The two models show a significant difference in stress patterns. (a) Model based on coralline grown under 422 latm CO2 concentration shows that the stress is dispersed throughout the structure, thus helping to prevent large scale failure if any of these points result in fractures. (b) Model based on coralline growth under 589 latm CO2 concentrations shows significantly higher von Mises stress throughout the structure. Unlike the 422 latm model, the stress is focused along a wide band of the structure, creating a potential zone of weakness which coupled with higher strain energy storage could lead to catastrophic structural failure if a critical stress is reached.

et al., 2008; Martin et al., 2008; Fabricius et al. 2011) and space competitions. However, since we have shown that L. glaciale continues to calcify in undersaturated

conditions, it may not only be the calcification processes per se but also the interaction of the calcification rate with the ability of the structure to withstand bioerosion and physical erosion i.e. the net production vs. net loss. Our data show that there is no significant change in the growth rate between current pCO2 and 589 latm, but there is a significant change in the ultrastructure. The FEA modelling of the changes in the ultrastructure shows that the stresses are an order of magnitude higher in the 589 latm model compared to 422 latm, and that the distribution of stress is drastically different. The stress in 422 latm is evenly spread throughout the model with relatively few ‘hotspots’ of high stress and as such, critical failure is likely to be minimal and isolated. In contrast the 589 latm model, the stresses are not evenly distributed and therefore a critical structural failure is more likely to occur along the centre of the model in potentially a single event. These results are further supported by the difference in strain energy between models. The 589 latm model shows higher levels of strain energy during loading, potentially indicating a less efficient structure (Dumont et al., 2009). This is the case regardless of whether the models are scaled for volume or not (Table 2). The combined slower growth rate and poor structural integrity could result in a net loss of material. This loss of structural integrity has important implications for the ecology of high latitude carbonate-dominated ecosystem because the overall structure is weakened and hence more vulnerable to wave action, bioerosion and grazing activity, i.e. boring by predators. This overall weakening and increased vulnerability has two main implications. The first is the increased likelihood of fracturing of the skeleton and damage to the epithallium, which at high pCO2 protects the organism from dissolution. Second, it could alter the dynamic balance between (bio) erosion and growth, which is strictly linked to the vitality of various calcifying species (Glynn, 1997). Since coralline algae provide the basis of many ecological communities and are vitally important in habitat formation, the loss of strength will have a negative consequence not just for the algae but also for the entire community. Benthic communities are important for regional fisheries, seabirds and marine mammals and the effects of ocean acidification will also have serious economical implications (Costanza et al., 1997). As CO2 concentration of 589 latm are predicted to be reached within the next 50 years, major changes to coralline algae are likely to impact their performance as ecosystem engineers and thus affect their ecosystem function and service, including benthic diversity, community structure and faunal richness, in the immediate future.

© 2012 Blackwell Publishing Ltd, Global Change Biology, doi: 10.1111/j.1365-2486.2012.02756.x


Foster MS (2001) Rhodoliths: between rocks and soft places. Journal of Phycology, 37, 659–667.

This study is a contribution to the BIOACID (sub project 3.2.4 “Impact of ocean acidification on coralline red algae”) joint project, funded by the German Ministry of Research and Technology. This research project has also been supported by the LASSO project. These tomographic analyses were performed on the TOMCAT beamline at the Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland. European Commission under the 7th Framework Programme: Research Infrastructures. SLS grant Agreement Number no. 20110822. We are grateful to Julie Fife at Swiss Light Source whose outstanding efforts have made these measurements possible. LCF would like to acknowledge support from the Holmes Fellowship and NERC grant NE/ F017383/1. We would like to thank Janina Bu¨scher for her assistance with the culturing and Ute Schuldt from the electron microscopy lab at Inst. of Geosciences/Kiel University.

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© 2012 Blackwell Publishing Ltd, Global Change Biology, doi: 10.1111/j.1365-2486.2012.02756.x

Ocean acidification weakens the structural integrity of coralline algae.

The uptake of anthropogenic emission of carbon dioxide is resulting in a lowering of the carbonate saturation state and a drop in ocean pH. Understand...
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