Interactive comment on “ Calcification and inducible defence response of a calcifying organism could be maintained under hypoxia through phenotypic plasticity

Specific comments: Line 40: In general I agree that calcification costs energy, but in some organisms the energy-dependence has been postulated as low (e.g. in corals – see McCulloch et al. (2012)). So this may be true for gastropods, but not necessarily so for some other organisms. So this sentence needs to be balanced somewhat. Line 70: The hypotheses around phenotypic plasticity needs to be strengthened and clarified. What exactly is the phenotype that is plastic here? The capacity to form different types of mineral in the shell? Or simply that responses will differ between control and reduced O2 concentrations? Reading the discussion, I think the authors are misusing the term phenotypic plasticity. Demonstrating variability in responses of individuals within the same population to a stressor is not demonstrating phenotypic plasticity, nor is demonstrating a different response under different treatments between different individuals. Line 82: How was pH measured, and on what scale, using what buffers? More information needed here. How was salinity and temperature measured? I see some of these details in table S1, but there are required in the methods section. Statistical analysis: why use a permanova? I would expect each parameter to be separated analysed using univariate analyses as a first step. A justification for using permanova over an anova or linear model needs to be justified here. Line 191-192: But this statement is at odds with the findings of the permanova and the figures, that calcification was impacted by hypoxia in this study. Also, the end of the sentence that this could be due to phenotypic plasticity needs to be explained, as this makes no sense to me.


Introduction
Calcification is a biomineralization process where many marine organisms, such as corals, molluscs, polychaetes and echinoderms, deposit carbonate minerals and form their calcareous shells.This process is highly 25 associated with the fitness and survival of calcifying organisms because shell growth not only allows continuous somatic growth, but also strengthens protection against physical and chemical damages.The protective role of shells is particularly important under life-threatening conditions (e.g.following non-lethal shell damage), where many calcifying organisms are able to produce stronger shells at a higher rate to augment physical protection (Cheung et al., 2004;Brookes and Rochette, 2007;Hirsch et al., 2014).Indeed, such inducible defence response via enhanced 30 calcification plays an important role in the survival of calcifying organisms (Harvell, 1990).
In view of the accelerated anthropogenic emission of carbon dioxide, however, calcification and hence defence response of calcifying organisms may be dampened by climate change stressors, such as ocean acidification and hypoxia (Bijma et al., 2013).While ocean acidification was expected to retard calcification (Orr et al., 2005), it is now realized that calcification is not primarily driven by the pH and carbonate saturation state of seawater (Roleda et 35 al., 2012), meaning that the impact of ocean acidification on calcifying organisms through the changes in seawater carbonate chemistry is less deleterious than previously thought (e.g.Garilli et al., 2015;Ramajo et al., 2016;Leung et al., 2017).Indeed, calcification is an energy-dependent physiological process actively regulated by calcifying organisms (Roleda et al., 2012).As such, hypoxia (i.e.dissolved oxygen concentration in seawater ≤ 2.8 mg O2 L -1 or ≤ 63 µmol L -1 ) can probably compromise calcification through its direct, adverse effect on aerobic metabolism and 40 hence production of metabolic energy (Wu, 2002;Leung et al., 2013a).Since calcification is an energy-demanding process (Palmer, 1992), the impaired aerobic metabolism under hypoxia could be the underlying mechanism causing the reduced calcification as previously observed (e.g.Cheung et al., 2008;Findlay et al., 2009;Wijgerde et al., 2014).
As the occurrence of hypoxia is predicted to become more prevalent in future marine ecosystems owing to ocean warming and human-induced eutrophication (Diaz and Rosenberg, 2008;Keeling et al., 2010;Bijma et al., 2013), the 45 impact of hypoxia on calcifying organisms would be continuously escalated.
However, few previous studies showed that some calcifying organisms are able to maintain calcification under hypoxia (Mukherjee et al., 2013;Frieder et al., 2014;Keppel et al., 2016), and even anoxia (Nardelli et al., 2014).These unexpected results suggest potential mechanisms which can compensate for the reduced metabolic energy under hypoxia in order to sustain calcification.It could be mediated by phenotypic plasticity, which involves 50 trade-offs between phenotypic traits in response to altered conditions (Malausa et al., 2005).For example, shell growth may be maintained under hypoxia at the expense of shell quality or other physiological processes via energy tradeoffs (Nisbet et al., 2012;Sokolova et al., 2012).Alternatively, the mineralogical properties of shells (e.g.carbonate polymorphs and organic matter content) can be modified by calcifying organisms, which possibly reduces the energy demand for calcification and thus favours shell growth when metabolic energy is reduced (Ramajo et al., 2015;Leung 55 et al., 2017).Whether calcifying organisms can exhibit such phenotypic plasticity to alleviate the impact of hypoxiainduced metabolic depression on calcification and defence response remains largely unknown and deserves a comprehensive investigation.
In this study, we examined how hypoxia affects calcification and defence response of a common calcifying polychaete (Hydroides diramphus), which is tolerant to hypoxia (Vaquer-Sunyer and Duarte, 2008;Leung et al., 60 2013b).Calcification was indicated by shell growth, while defence response by both shell growth and fracture toughness.We analysed the mineralogical properties of shells (organic matter content, calcite to aragonite ratio, magnesium to calcium ratio in calcite and relative amorphous calcium carbonate content) to indicate the possible changes in calcifying mechanism in response to hypoxia.Respiration rate and feeding rate were measured to reflect aerobic metabolism and energy gain, respectively.Given the possible impact of hypoxia on aerobic metabolism, we 65 hypothesized that (1) the mineralogical properties of newly-produced shells would be modified to reduce the energy demand for calcification so that shell growth can be sustained; (2) defence response would be undermined as the Biogeosciences Discuss., https://doi.org/10.5194/bg-2017-378Manuscript under review for journal Biogeosciences Discussion started: 4 October 2017 c Author(s) 2017.CC BY 4.0 License.reduced metabolic energy is possibly insufficient to enhance both shell growth and fracture toughness.If phenotypic plasticity can help alleviate the impact of hypoxia on calcification and even defence response without causing significant adverse effects by trade-offs, this suggests that calcifying organisms would be more robust to metabolic 70 stress conditions than previously thought.
Two dissolved oxygen levels of seawater were chosen to represent normoxia (~6.0 mg O2 L -1 ) and hypoxia (~2.0 mg O2 L -1 ).Normoxia (i.e.control) and hypoxia were achieved by aerating seawater with air and a mixture of 85 nitrogen and air, respectively (Leung et al., 2013b).Digital flow meters (Vögtlin Instruments, Switzerland) were used to adjust the flow rate of each gas (i.e.nitrogen and air) so that the desired dissolved oxygen concentration for hypoxia was maintained.To simulate the summer seawater temperature at the collection site, the containers in the following experiments were put into a water bath with water temperature maintained at 28°C using a heating bath circulator.
There were two contexts in this study to examine the defence response of H. diramphus: life-threatening and 90 unthreatened (i.e.control) conditions.To create the life-threatening conditions, non-lethal shell damage was made by carefully trimming the calcareous tube until the radioles were exposed, while the body was still fully covered.The polychaetes with "intact" (tube length: ~40 mm; body length: ~20 mm) and "damaged" (tube length: ~20 mm; body length: ~20 mm) tubes were then allowed to acclimate under either normoxia or hypoxia for another week before experimentation.Thus, there were four treatments based on the crossed combinations of dissolved oxygen levels 95 (Normoxia vs. Hypoxia) and contexts (Intact vs. Damaged).

Shell growth and shell properties
Shell growth was indicated by the change in tube length over time, where the newly-produced shells can be easily identified by the difference in colour from the original shells (Fig. A1).The rearing method for the polychaetes was previously described (Leung and Cheung, 2017).Briefly, polychaetes with their tube length measured were  A1.After measuring respiration rate and feeding rate (see the section below), the newly-produced shells were carefully removed for the analyses of mechanical and geochemical properties.Fracture toughness was measured using a micro-hardness tester (Fischerscope HM2000, Fischer, Germany) to indicate mechanical strength.A shell fragment was mounted firmly onto a metal disc using cyanoacrylate adhesives (n = 5 fragments from 5 individuals per treatment).

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Then, the fragment was indented by a Vickers 4-sided diamond pyramid indenter for 10 s in the loading phase (Peak load: 300 mN; Creep: 2 s).In the unloading phase, the load decreased at the same rate as the loading phase until the loading force became zero.At least five random locations on each fragment were indented.Vickers hardness (H) and elastic modulus (E) were calculated based on the load-displacement curve using software WIN-HCU (Fischer, Germany).Vickers hardness to elastic modulus ratio (H/E) was calculated to indicate the fracture toughness of shells 120 (Marshall et al., 1982).
Organic matter content was determined by mass loss upon ignition at 550°C in a muffle furnace for six hours (n = 5 replicates from 5 individuals per treatment).Given the limited amount of newly-produced shells, composite shell powder samples were made from 3 to 5 individuals from the same treatment for the analyses of the following geochemical properties.Carbonate polymorphs were analysed using an X-ray diffractometer (D4 ENDEAVOR, 125 Bruker, Germany).A small quantity of shell powder was transferred onto a tailor-made sample holder and then scanned by Co Kα radiation (35 kV and 30 mA) from 20° to 70° 2θ with step size of 0.018° and step time of 1 s (n = 3 replicates per treatment).Carbonate polymorphs were identified based on the X-ray diffraction spectrum using the EVA XRD analysis software (Bruker, Germany).Calcite to aragonite ratio was calculated using the following equation (Kontoyannis and Vagenas, 2000) coated by carbon (n = 3 replicates per treatment; 3 trials per replicate).The shell powder was irradiated by an electron beam with an accelerating voltage of 12 kV to obtain the energy spectrum with background correction.Elements were identified and magnesium to calcium ratio was calculated using software Genesis Spectrum SEM Quant ZAF (EDAX, USA).To determine relative amorphous calcium carbonate (ACC) content, 1 mg shell powder was mixed with 10 mg potassium bromide, followed by compressing the mixture into a disc using a manual hydraulic press (n = 3 replicates 140 per treatment).An infrared absorption spectrum ranging from 400 cm -1 to 4000 cm -1 with background calibration for the baseline was obtained using a Fourier transform infrared spectrometer (Avatar 370 DTGS, Nicolet, USA).The relative ACC content was estimated as the intensity ratio of the peak at 856 cm -1 to that at 713 cm -1 (Beniash et al., 1997).

Physiological performance 145
Following the 3-week exposure period, the respiration rate and feeding rate of polychaetes were measured Respiration rate was expressed as oxygen consumed per individual per hour.

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To measure feeding rate, five individuals which had been starved for one day to standardize their hunger level were put into a glass bottle containing 80 mL FSW with an initial concentration of ~1 × 10 6 cell mL -1 D.
tertiolecta (n = 5 replicate bottles per treatment).After feeding for one hour, 1 mL seawater was taken from the bottle and the microalgae were enumerated using a haemocytometer (n = 6 trials per bottle).Prior to counting, 1% Lugol's solution was used to fix the microalgae.Clearance rate was calculated using the following formula to represent feeding 160 rate (Coughlan, 1969): where CR is the clearance rate (mL ind -1 hr -1 ); V is the volume of seawater; n is the number of individuals; t is the feeding time; Co and Ct are the initial and final concentrations of microalgae, respectively.

Statistical analysis 165
Two-way permutational analysis of variance (PERMANOVA) was used to test the effects of hypoxia and non-lethal shell damage on the aforementioned parameters using software PRIMER 6 with PERMANOVA+ add-on.
after non-lethal shell damage (Fig. 1, Table A2).Hypoxia slightly hindered shell growth in both contexts.The fracture toughness of newly-produced shells was enhanced after non-lethal shell damage (c.f.control), while hypoxia had negligible effect (Fig. 2, Table A2).As for the geochemical properties of newly-produced shells, organic matter content was elevated after non-lethal shell damage, whereas the effect of hypoxia was indiscernible (Fig. 3a, Table A2).Calcite was the dominant carbonate polymorph and its proportion increased under hypoxia (Fig. 3b, Table A2).H. diramphus 175 produced high-Mg calcite (i.e.Mg/Ca > 0.04) and the magnesium content in calcite increased under hypoxia (Fig. 3c, Table A2).The relative ACC content was slightly elevated under hypoxia, meaning that less crystalline shells were produced (Fig. 3d, Table A2).Calcite/Aragonite, Mg/Ca in calcite and relative ACC content were not significantly affected by non-lethal shell damage.Respiration rate was reduced by hypoxia, but only slightly by non-lethal shell damage (Fig. 4a, Table A2).Clearance rate decreased not only under hypoxia, but also after non-lethal shell damage 180 under normoxia (Fig. 4b, Table A2).The survival rate of H. diramphus ranged from 93% to 100% across treatments after the 3-week exposure period (Fig. A2).

Discussion
Hypoxia is expected to diminish the fitness and survival of marine organisms, probably leading to serious 185 ramifications on marine ecosystems (Wu, 2002;Diaz and Rosenberg, 2008).Nevertheless, many less mobile marine organisms (e.g.molluscs, polychaetes and echinoderms) are generally tolerant to hypoxia (Vaquer-Sunyer and Duarte, 2008), suggesting their potential capacity to accommodate its impacts.Despite the substantial reduction in respiration rate and feeding rate under hypoxia, we found that calcification and defence response of a calcifying polychaete were generally maintained, which could be associated with phenotypic plasticity.

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Since energy demand for calcification is enormous (Palmer, 1992), the reduction in energy gain by feeding and energy production by aerobic respiration under hypoxia would undermine both quality and quantity of shells produced by calcifying organisms (Cheung et al., 2008;Wijgerde et al., 2014).Under unthreatened conditions (i.e. without shell damage), we found that hypoxia slightly hinders the shell growth of H. diramphus, but does not affect the fracture toughness (i.e.mechanical strength) of newly-produced shells.The retarded shell growth under hypoxia 195 could be pertinent to the reduced feeding rate, and hence energy reserves for calcification.While energy gain by feeding is suggested to be fundamental for shell growth (Melzner et al., 2011;Thomsen et al., 2013;Leung et al., 2017), aerobic respiration is necessary to efficiently convent energy reserves into metabolic energy for various biological processes, including calcification.As such, the retarded shell growth is more likely ascribed to the hypoxiainduced metabolic depression, which reduces the amount of metabolic energy allocated to calcification.Unexpectedly,

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hypoxia did not weaken the mechanical strength of newly-produced shells.The quantity of organic matter (e.g.matrix proteins) occluded in the shell is the key factor affecting mechanical strength (Weiner and Addadi, 1997;Addadi et al., 2006;Marin et al., 2008).Since the organic matter content was not affected by hypoxia, mechanical strength can Biogeosciences Discuss., https://doi.org/10.5194/bg-2017-378Manuscript under review for journal Biogeosciences Discussion started: 4 October 2017 c Author(s) 2017.CC BY 4.0 License.
be maintained.Our results imply that similar amount of metabolic energy is allocated to the production of organic matter for the shell, while less to shell growth under hypoxia.This strategy (i.e.shell quality over shell quantity) is

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favourable under energy-limiting conditions because there is no exigency to expedite shell growth when risk is not imminent and the shell can already offer sufficient protection.
Under life-threatening conditions (i.e.following non-lethal shell damage), H. diramphus exhibited defence response, indicated by the production of tougher shells at a higher rate.As H. diramphus is sessile, enhancing the protective function of shells is probably the most effective defence response.Therefore, more organic matter was 210 produced and occluded in the newly-produced shell to augment mechanical strength.Additionally, the carbonate crystals in the shell appeared to be more compacted (Fig. 5), which could also strengthen the shell.Such inducible defence response is commonly exhibited by calcifying organisms because shell repair should be prioritized to restore and enhance protection (Cheung et al., 2004;Hirsch et al., 2013;Brom et al., 2015).However, trade-offs are involved to activate defence response, such as reduction in the less essential biological processes or activities (Rundle and 215 Brönmark, 2001;Trussell and Nicklin, 2002;Hoverman and Relyea, 2009;Babarro et al., 2016).For example, Brookes and Rochette (2007) showed that the calcification rate of a grazing gastropod is promoted under predation risk at the expense of grazing activity and somatic growth.Similar trade-offs were observed in H. diramphus (i.e.enhanced shell growth against reduced feeding rate).Nevertheless, defence response should still be prioritized to maximize the chance of survival, when survival is not guaranteed under life-threatening conditions (Bourdeau, 2009).

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We expected that defence response would deteriorate under hypoxia in view of the substantial energy demand for shell production.Contrary to this prediction, H. diramphus can still produce tougher shells at a higher rate, meaning that the effect of hypoxia on defence response is not discernible.This unexpected finding not only reveals the strong tolerance of H. diramphus to hypoxia, but also suggests potential mechanisms that enable efficient calcification under hypoxia despite the reduced metabolic energy.We propose that changing mineralogical properties could help 225 compensate for the reduced metabolic energy in order to sustain defence response.In fact, the mineralogical properties were altered consistently in response to hypoxia, irrespective of context.For instance, hypoxia resulted in a greater proportion of calcite in the shell.When metabolic energy is reduced, precipitation of calcite is favourable because it requires less metabolic energy and allows faster shell growth than that of aragonite (Weiner and Addadi, 1997;Hautman, 2006;Ries, 2011).In addition, we found that more magnesium ions were incorporated into the newly-

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produced shell under hypoxia.It is evident that the incorporation of magnesium ions into calcite is actively regulated through various biological mechanisms, such as active extrusion of excess magnesium ions at the calcification site (Bentov and Erez, 2006).The elevated Mg/Ca in calcite under hypoxia signifies that the energy-requiring regulation of magnesium ions was relaxed.Furthermore, crystallization of amorphous calcium carbonate was slightly reduced by hypoxia, indicated by the higher relative ACC content.Since crystallization requires metabolic energy for the 235 transport of carbonate ions (Addadi et al., 2006;Weiner and Addadi, 2011), our results suggest that metabolic energy allocated to crystallographic control also decreased.Given the aforementioned changes in mineralogical properties, the energy cost for sustaining shell growth could be lessened.Such plastic response, also shown in other calcifying Biogeosciences Discuss., https://doi.org/10.5194/bg-2017-378Manuscript under review for journal Biogeosciences Discussion started: 4 October 2017 c Author(s) 2017.CC BY 4.0 License.
organisms under metabolic stress conditions (Ramajo et al., 2015;Leung et al., 2017), may explain why the defence response of H. diramphus can generally be maintained under hypoxia.

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Despite the benefit of changing mineralogical properties as the plastic response, trade-offs against other phenotypic traits are inevitably incurred (Malausa et al., 2005;Leung et al., 2013b).For instance, shell solubility increases due to the higher relative ACC content and Mg/Ca in calcite (Fernandez-Diaz, 1996;Ries, 2011;Fitzer et al., 2014).In other words, while the changes in mineralogical properties may allow sustained shell growth and mechanical strength under hypoxia, the chemical stability of shells may be weakened.Nevertheless, the benefit of 245 defence response probably outweighs the cost of this trade-off under life-threatening conditions, and therefore H.
Based on the present findings, we support the paradigm that calcification is mainly driven by the physiology of calcifying organisms rather than the seawater carbonate chemistry (Pörtner, 2008;Roleda et al., 2012).For example, the shell growth of H. diramphus decreased when the carbonate saturation state slightly increased under hypoxia.This 250 is contradictory to the paradigm that calcification generally increases with carbonate saturation state, vice versa (Orr et al., 2005).Indeed, most calcifying organisms do not directly utilize carbonate ions, but bicarbonate ions, as the substrate for calcification, meaning that formation of calcareous shells is not a chemical reaction between calcium and carbonate ions (Pörtner, 2008;Roleda et al., 2012;Bach, 2015).This concept based on physiology explains why many calcifying organisms can maintain or even enhance calcification when carbonate saturation state is reduced (e.g.Ries
Hypoxia can last for a long period of time (e.g.month) as observed in many coastal and marine waters worldwide (Helly and Levin, 2004;Diaz and Rosenberg, 2008), and is predicted to be more prevalent in future due to ocean warming and human-induced eutrophication (Bijma et al., 2013).In order to maintain populations under 260 hypoxia, calcifying organisms have to counter its impact on calcification.Despite the impaired aerobic metabolism, this study revealed that hypoxia only mildly hampers the shell growth of a calcifying polychaete, whereas its defence response can be sustained (i.e.harder shells produced at a higher rate).This is likely mediated by phenotypic plasticity, such as modifying mineralogical properties of shells to reduce the energy demand for calcification.While some potential trade-offs are incurred, such plastic response could be the cornerstone of calcifying organisms to acclimate 265 to metabolic stress conditions, and hence sustain their populations and ecological functions in coastal and marine ecosystems.
acknowledge the staff in Adelaide Microscopy for their assistance.
100 randomly and individually transferred into a 2 mL labelled microcentrifuge tube with the radioles pointing upward (n Biogeosciences Discuss., https://doi.org/10.5194/bg-2017-378Manuscript under review for journal Biogeosciences Discussion started: 4 October 2017 c Author(s) 2017.CC BY 4.0 License.= 10 individuals per replicate).A small hole was drilled at the bottom of each microcentrifuge tube to allow water exchange.The microcentrifuge tubes were glued together to maintain an upright position and then put into a lidded glass bottle containing 180 mL filtered seawater (FSW) (pore size: 0.45 µm) with dissolved oxygen concentration manipulated (n = 3 replicate bottles per treatment).Two holes were drilled on the lid so that the FSW in the bottle was 105 continuously aerated with air (normoxia) or a mixture of nitrogen and air (hypoxia) to maintain the desired dissolved oxygen concentration over time.20 mL algal suspension containing I. galbana and D. tertiolecta (1:1, v/v) at ~1 × 10 6 cells mL -1 was daily provided as food.The microcentrifuge tubes were cleaned and seawater was renewed once every three days to prevent accumulation of metabolic waste.The tube length of each individual was measured under a microscope on Day 1, Day 11 and Day 21 to estimate shell growth.The survival rate of polychaetes was determined 110 after Day 21.The seawater parameters throughout the 3-week exposure period are shown in Table using the method described inLeung et al. (2013a) with minor modifications.Briefly, five random individuals were transferred into an airtight syringe containing ~35 mL FSW with dissolved oxygen concentration adjusted to the treatment level, and allowed to rest for 15 min (n = 5 replicate syringes per treatment).Then, the initial dissolved oxygen concentration of FSW was measured using an optical dissolved oxygen probe (SOO-100, TauTheta 150 Instruments, USA).The air inside the syringe was then fully expelled and the tip of the syringe was sealed by Blu Tack to ensure an airtight condition.After one hour, the final dissolved oxygen concentration of FSW was recorded when it becomes steady by gently stirring the FSW inside the syringe.Blank samples without individuals were prepared to correct the background change in dissolved oxygen concentration, which fluctuated less than 1%.

Figure 1
Figure 1 Cumulative change in the tube length of H. diramphus in different treatments across the 3-week

Figure 3
Figure 3 Geochemical properties of H. diramphus shells, including (a) organic matter content, (b) calcite to aragonite ratio, (c) magnesium to calcium ratio in calcite and (d) relative amorphous calcium carbonate content, in different treatments (mean + S.E.; n = 3, except n = 5 for organic matter content).

Figure 5
Figure 5 SEM images of the inner surface of H. diramphus shells produced in different treatments, indicating the shell integrity.Scale bar: 20 µm.

Figure A1 A
Figure A1 A micrograph showing the newly-produced shell and original shell of H. diramphus, where the former is easily distinguished from the latter by the white colour.

Figure
Figure A2 Survival rate of H. diramphus in different treatments after the 3-week exposure period (mean + S.E.; n = 3). : XC/XA is the calcite to aragonite ratio.Magnesium to calcium ratio was determined by energy dispersive X-ray spectroscopy under the Philips XL 30 field emission scanning electron microscope.A small quantity of shell powder was transferred onto a stub and 135 Biogeosciences Discuss., https://doi.org/10.5194/bg-2017-378Manuscript under review for journal Biogeosciences Discussion started: 4 October 2017 c Author(s) 2017.CC BY 4.0 License.

Table A2 PERMANOVA table showing the effects of dissolved oxygen (DO) and context on shell growth, fracture toughness, organic matter content, calcite/aragonite, Mg/Ca in calcite, relative ACC content, respiration rate and clearance rate.
Biogeosciences Discuss., https://doi.org/10.5194/bg-2017-378Manuscript under review for journal Biogeosciences Discussion started: 4 October 2017 c Author(s) 2017.CC BY 4.0 License.