domain: sdu chim sdv

text:
M Mojtahid
email: meryem.mojtahid@univ-angers.fr

P Depuydt

A Mouret

S Le Houedec

S Fiorini

S Chollet

F Massol

F Dohou

H L Filipsson

W Boer

G-J Reichart

C Barras

Assessing the impact of different carbonate system parameters on benthic foraminifera from controlled growth experiments

Keywords: Carbonate system, Culture experiment, Sr/Ca, Benthic foraminifera, Ocean acidification, Biomineralisation Highlights

Insights into past marine carbon cycling and water mass properties can be obtained by means of geochemical proxies calibrated through controlled laboratory experiments with accurate seawater carbonate system (C-system) manipulations. Here, we explored the use of strontium/calcium ratio (Sr/Ca) of the calcite shells of benthic foraminifera as a potential seawater C-system proxy through a controlled growth experiment with two deep-sea species (Bulimina marginata and Cassidulina laevigata) and one intertidal species (Ammonia T6). To this aim, we used two experimental set-ups to decouple as much as possible the individual components of the carbonate system, i.e., changing pH at constant dissolved inorganic carbon (DIC) and changing DIC at constant pH. Four climatic chambers were used with different controlled concentrations of atmospheric pCO2 (180 ppm, 410 ppm, 1000 ppm, 1500 ppm). Our results demonstrated that pH did not influence the survival and growth of the three species.

However, low DIC conditions (879 µmol kg -1 ) negatively affected B. marginata and C. laevigata through reduced growth, whereas no effect was observed for Ammonia T6. Our results also showed that Sr/Ca was positively correlated with total Alkalinity (TA), DIC and bicarbonate ion concentration ([HCO3 -]) for Ammonia T6 and B. marginata; i.e., DIC and/or [HCO3 -] were the main controlling factors. For these two species, the regression models were coherent with published data (existing so far only for Ammonia T6) and showed overall similar slopes but different intercepts, implying species-specific effects. Furthermore, the Sr/Ca -Csystem relationship was not impacted by ontogenetic trends between chamber stages, which is a considerable advantage for paleo-applications. This applied particularly to Ammonia T6 that calcified many chambers compared to the two other species. However, no correlation with any of the C-system parameters was observed for Sr/Ca in C. laevigata. This might imply either a strong species-specific effect and/or a low tolerance to laboratory conditions leading to a physiological stress, thereby impacting the Sr incorporation into the calcite lattice of C. laevigata.

Introduction

In Earth's geological history, carbon cycle perturbations have had severe consequences (i.e., warming, acidification, and deoxygenation) and their interactive effects were often coupled with extinctions of major species' groups (e.g., [START_REF] Bijma | Climate change and the oceans -What does the future hold? Mar. Pollut. Bull., The Global State of the Ocean; Interactions Between Stresses, Impacts and Some Potential Solutions[END_REF]. Moreover, because the ocean is one of the largest carbon reservoirs, the strength of the production of deep and intermediate waters and their consequent carbon content, is a significant control on atmospheric pCO2 (e.g., [START_REF] Stocker | Influence of CO 2 emission rates on the stability of the thermohaline circulation[END_REF][START_REF] Ganopolski | Rapid changes of glacial climate simulated in a coupled climate model[END_REF][START_REF] Qin | Sustained deep Pacific carbon storage after the Mid-Pleistocene Transition linked to enhanced Southern Ocean stratification[END_REF].

Therefore, accurate reconstructions of past seawater carbonate chemistry are fundamental for i) understanding carbon cycle-climate interactions and feedbacks, either induced naturally or by anthropogenic carbon emissions (e.g., [START_REF] Hönisch | The Geological Record of Ocean Acidification[END_REF][START_REF] Bijma | Climate change and the oceans -What does the future hold? Mar. Pollut. Bull., The Global State of the Ocean; Interactions Between Stresses, Impacts and Some Potential Solutions[END_REF][START_REF] Martínez-Botí | Plio-Pleistocene climate sensitivity evaluated using highresolution CO 2 records[END_REF], and ii) elucidating critical processes and mechanisms in past ocean thermohaline circulation, which have global climate implications (e.g., [START_REF] Yu | Seawater carbonate ion-δ 13 C systematics and application to glacial-interglacial North Atlantic ocean circulation[END_REF]Yu et al., , 2014)).

Insights into past marine carbon cycling crucially depend on robust proxies for the seawater carbonate system (C-system), and well-designed experiments permit to deconvolve the covarying parameters (Zeebe and Wolf-Gladrow, 2001). In the ocean, calcifying microorganisms, such as foraminifera, incorporate various chemical elements from the seawater during calcification. Elements (El/Ca and isotopic ratios) that have been shown to be linked with the C-system in foraminifera are B/Ca and δ 11 B (e.g., [START_REF] Hönisch | Atmospheric Carbon Dioxide Concentration Across the Mid-Pleistocene Transition[END_REF][START_REF] Yu | B/Ca in planktonic foraminifera as a proxy for surface seawater pH[END_REF][START_REF] Yu | An evaluation of benthic foraminiferal B/Ca and δ 11 B for deep ocean carbonate ion and pH reconstructions[END_REF][START_REF] Howes | Decoupled carbonate chemistry controls on the incorporation of boron into Orbulina universa[END_REF][START_REF] Levi | Intrashell Variability of Trace Elements in Benthic Foraminifera Grown Under High CO 2 Levels[END_REF], Li/Ca and δ 7 Li (e.g., [START_REF] Vigier | Lithium isotopes in foraminifera shells as a novel proxy for the ocean dissolved inorganic carbon (DIC)[END_REF][START_REF] Roberts | Lithium isotopic composition of benthic foraminifera: A new proxy for paleo-pH reconstruction[END_REF], U/Ca (e.g., [START_REF] Raitzsch | U/Ca in benthic foraminifers: A proxy for the deep-sea carbonate saturation[END_REF][START_REF] Keul | Effect of ocean acidification on the benthic foraminifera Ammonia sp. is caused by a decrease in carbonate ion concentration[END_REF], Zn/Ca (e.g., [START_REF] Marchitto | Deep Pacific CaCO 3 compensation and glacial-interglacial atmospheric CO 2[END_REF]van Dijk et al., 2017b) and Sr/Ca (e.g., Dissard et al., 2010a;Dueñas-Bohórquez et al., 2011b;Diz et al., 2012;Yu et al., 2014;Keul et al., 2017). Still, many of these elemental ratios suffer either from analytical constraints (e.g., limited amount of B and Li in small-sized foraminifera usually used in paleoceanography) and/or a limited understanding of the fundamental mechanisms controlling element incorporation in carbonates (e.g., [START_REF] Kaczmarek | Boron incorporation in the foraminifer Amphistegina lessonii under a decoupled carbonate chemistry[END_REF].

When studying the impact of the C-system on trace and minor element incorporation, strontium has the advantage to be one of the major elements incorporated into foraminiferal calcite, making it analytically very robust with high accuracy and precision (e.g., Dueñas-Bohórquez et al., 2011a;de Nooijer et al., 2014). Foraminiferal Sr/Ca was first proposed as a temperature proxy, mostly from core-top calibration studies (e.g., [START_REF] Rathburn | Magnesium and strontium compositions of Recent benthic foraminifera from the Coral Sea, Australia and Prydz Bay, Antarctica[END_REF]Reichart et al., 2003;[START_REF] Mortyn | An evaluation of controls on planktonic foraminiferal Sr/Ca: Comparison of water column and core-top data from a North Atlantic transect[END_REF][START_REF] Rosenthal | Temperature and carbonate ion effects on Mg/Ca and Sr/Ca ratios in benthic foraminifera: Aragonitic species Hoeglundina elegans[END_REF]. Most of these studies, however, emphasized the possibility that Sr/Ca might reflect other parameters co-varying with temperature such as water depth, pressure, dissolution, and/or carbonate ion concentration [CO3 2-]. More recent controlled growth experiments showed a link with seawater Sr/Ca ratio (Langer et al., 2016) and a strong control of the C-system on foraminiferal Sr/Ca (e.g., Dissard et al., 2010a;Dueñas-Bohórquez et al., 2011b;Yu et al., 2014;Raitzsch et al., 2010;[START_REF] Allen | Trace element proxies for surface ocean conditions: A synthesis of culture calibrations with planktic foraminifera[END_REF]Keul et al., 2017;van Dijk et al., 2017b;[START_REF] Levi | Intrashell Variability of Trace Elements in Benthic Foraminifera Grown Under High CO 2 Levels[END_REF]. However, the conclusions of these studies are sometimes contradictory regarding the major C-system parameter controlling Sr incorporation. For instance, while the study of Dissard et al. (2010a) showed that the incorporation of Sr increases with increasing [CO3 2-] (increasing pH), Keul et al. (2017) suggested that DIC or [HCO3 -] are the main controlling factors. Inconsistencies in interpretations are mainly due to incomparable experimental designs. Indeed, the complexity of the C-system necessitates to experimentally deconvolve the impact of the co-varying C-system parameters (pCO2, bicarbonate ion concentration-[HCO3 -], carbonate ion concentration-[CO3 2-], pH, total alkalinity-TA, dissolved inorganic carbon-DIC, and calcium carbonate saturation state of seawater-Ωcc). Such designs are very complex to set-up, and therefore, except for Keul et al. (2017) and van Dijk et al. (2017b) (mostly focusing on Zn/Ca), to our knowledge, experimental studies investigating the link between foraminiferal Sr/Ca and the C-system employed classical C-system manipulation, i.e., either DIC (injection of CO2 enriched air or water or NaHCO3/Na2CO3 additions) or TA (changing pCO2 with HCl/NaOH additions) manipulation. In both cases, pH and [CO3 2-] co-vary and cannot be decoupled. Moreover, these studies were conducted on either planktonic, intertidal benthic and/or large tropical foraminifera, which could be impacted differently than deep-sea benthic foraminifera.

In this study, we used a decoupled C-system experimental design (where pH and [CO3 2-] are decoupled), similar to that of Keul et al. (2017) and van Dijk et al. (2017b) to investigate the relationship between Sr/Ca and the C-system in two species Bulimina marginata and Cassidulina laevigata (also referred to as Cassidulina carinata; [START_REF] Murray | Ecology And Applications of Benthic Foraminifera[END_REF]. For potential comparison with previous studies and to test reproducibility of the results, we added Ammonia molecular type T6 (Ammonia T6), recently named Ammonia confertitesta [START_REF] Hayward | Molecular and morphological taxonomy of living Ammonia and related taxa (Foraminifera) and their biogeography[END_REF], the same species used by Keul et al. (2017) and van Dijk et al. (2017b). To improve the stability and the monitoring of the set-up, our experiment was performed for the first time with a new generation of environmental ecological experiment simulators (Ecolab system; CEREEP -ECOTRON Île de France) using four climatic chambers with different concentrations of atmospheric pCO2 (180 ppm, 410 ppm, 1000 ppm, 1500 ppm). In addition to the Sr/Ca-Csystem relationship, we explored the survival and growth of foraminifera under different pH and DIC conditions and tested the effect of ontogeny between chamber stages on the incorporation of Sr into foraminiferal calcite.

Material and methods

2.1.Sampling of seawater and foraminifera for culturing experiments

Two hundred liters of natural seawater were sampled in open waters of the Bay of Biscay (45°18.86 N, 01°43.31 W; salinity = 34.7; DIC ~ 2200 µmol kg -1 ; TA ~ 2400 µmol kg -1 ; pH total scale = 7.95) the 29 th of November 2019 during the ORHAGO 19 cruise (IFREMER) onboard the R/V Côtes De La Manche. In the laboratory, seawater was filtered using 0.45 µm WHATMAN filters membranes and autoclaved (at 121 °C for 20 min) to limit bacterial proliferation. This process did not affect the seawater elemental chemistry since values found after the autoclave process (e.g., Srsw ~82 µmol kg -1 ) were very similar to values found for Atlantic waters (e.g., [START_REF] Wakaki | Precise and accurate analysis of deep and surface seawater Sr stable isotopic composition by double-spike thermal ionization mass spectrometry[END_REF] and no precipitation was observed. To prevent possible foraminiferal dissolution in the most extreme pCO2 condition (1500 ppm), we increased slightly the alkalinity and DIC of the seawater stock by adding Na2CO3 (200 μmol L - 1 ) to obtain DIC values of ~ 2350 µmol kg -1 , TA of ~ 2700 µmol kg -1 and pH total scale ~ 8.06.

Seawater was stored at 4°C until the experiments started.

Foraminiferal specimens were harvested alive from their natural environment. Bulimina marginata and Cassidulina laevigata were collected from the surface sediments of the Gullmar Fjord (58°16.86 N, 11°30.514 E; Sweden) at 30-50 m water depth, using the R/V Skagerak on the 14 th of January 2020. Although these species were harvested at shallow water depths, their depth range extends to outer continental shelf and slope environments in many areas (e.g., ~200-1500 m water depths; [START_REF] Mackensen | Cassidulina teretis Tappan and C. laevigata d'Orbigny; their modern and late Quaternary distribution in northern seas[END_REF][START_REF] Van Marle | Bathymetric distribution of benthic foraminifera on the Australian-Irian Jaya continental margin, eastern Indonesia[END_REF][START_REF] Murray | Ecology and palaeoecology of benthic foraminifera[END_REF][START_REF] Fontanier | Seasonal and interannual variability of benthic foraminiferal faunas at 550 m depth in the Bay of Biscay[END_REF], making them good candidates for paleoceanographic applications. In the following, we will refer to them as "deep-sea species". The shallow water species Ammonia T6 was harvested from the intertidal mudflat of Bourgneuf bay (France) on the 13 th of January 2020. Sediments were sieved in the field over 63 µm or 125 µm mesh sieves (using onsite seawater) to remove fine particles and 500 µm to remove macrofauna, all with the aim of concentrating foraminifera.

Once in the laboratory (LPG, Angers, France), the collected sediments were placed in 12°C incubators for 2-3 weeks in a solution containing calcein (10 mg L -1 of calcein stain in Instant Ocean artificial seawater-35 psu) with freeze-dried algae Chlorella. This way, specimens that calcified new chambers were labelled with calcein and appeared fluorescent under the epifluorescence microscope. This was used as a criterion to select and isolate only active specimens (i.e., specimens that grew new chambers) to be introduced later in the experiment.

Therefore, chambers that calcified afterwards in the controlled experiment would appear nonfluorescent (Supplementary material S1). A total of 1320 adult specimens of labelled Ammonia T6, 2176 adult specimens of labelled B. marginata and 1176 adult specimens of labelled C. laevigata were picked out from the 150-250 µm fraction of the sediment under epifluorescent stereomicroscope. Once harvested, the foraminifera were transported in Falcon vials to the Ecolab facility (CEREEP Ecotron, Ile de France), with calcein solution and a small quantity of in situ <38 μm sediment to reduce stress during transportation.

2.2.Experimental set-up and culturing in the Ecolab facility

The Ecolab is a modular structure that includes three gas-tight environmental chambers and one laboratory room (Supplementary material S2). Each environmental chamber (13 m 3 in volume) can be independently and precisely controlled for different conditions (e.g., air temperature, humidity, pCO2, lighting). For this study, we had access to two Ecolabs and used four climatecontrolled chambers with four concentrations of atmospheric pCO2 (180 ppm, 410 ppm, 1000 ppm, 1500 ppm). Temperature and humidity were set respectively at ~12 °C and ~90 % to limit evaporation. The experimental design is presented in Figure 1.

Besides precise monitoring and control of air temperature, humidity, pCO2, the large space provided permitted i) manipulation inside each chamber, ii) the use of a large quantity of seawater, iii) culturing a large number of foraminifera and iv) continuous monitoring of pH of the culture media. Indeed, in each climatic chamber, we placed two aquaria (A and B) filled with 12 L of seawater from our initial 200 L stock (no seawater was exchanged between the 12 L and 200 L tanks during the experiment). To ensure homogenization of seawater and equilibration with pCO2, two bubblers were placed in each aquarium. To further limit evaporation, the aquaria were covered by Plexiglas lids. Variability in pH was monitored at 5 min intervals using glass pH electrodes (Consort SP21) continuously immersed in each aquarium. Via an externally connected computer, this continuous measurement allowed early detection of any potential problem in the stability of the system. Absolute pH measurements in total scale were performed using a multimeter (cf. below).

Before adding the foraminiferal specimens, two weeks of pre-test phase were necessary to optimize the geochemistry and the stability of the C-system in the four climatic chambers.

During the pre-test phase, and after few days of equilibration with the ambient pCO2 conditions in each climatic chamber, the carbonate chemistry of seawater was modified chemically following the protocol of [START_REF] Keul | Effect of ocean acidification on the benthic foraminifera Ammonia sp. is caused by a decrease in carbonate ion concentration[END_REF]Keul et al. ( , 2017) ) to decouple as much as possible the covarying parameters of the C-system (Supplementary material S3; Table 1).

i)

Set-up A (DIC-stable manipulation): we added NaOH or HCl to obtain a range of pH and [CO3 2-] while maintaining constant DIC.

ii) Set-up B (pH-stable manipulation): we added NaHCO3 to modify DIC, TA and [CO3 2-], while maintaining a stable pH value.

In the case of the pCO2 condition at 180 ppm (set-up B), the targeted DIC value was below the DIC value of the seawater. Therefore, seawater had to be acidified first with HCl and bubbled with nitrogen to remove excess CO2 from the seawater. Then, the targeted pH and DIC were readjusted with adding NaHCO3. For all treatments, the added volumes of chemicals were estimated using Seacarb package in R software [START_REF] Gattuso | seacarb: seawater carbonate chemistry[END_REF].

After stabilization of seawater chemical parameters during the pre-test phase, foraminifera were put in small glass vials and introduced into the aquaria. Three glass vials (named Rnet, R1, R2)

containing each 55 and 49 specimens of Ammonia T6 and C. laevigata respectively, were put in each aquarium. For B. marginata, four glass vials (named Rnet, R1, R2, R3) containing 68 specimens each were put in each aquarium. We covered one of the glass vials (Rnet) with a 100 µm mesh net in case foraminifera were able to climb out of the glass vials. This was to ensure that in at least one of the glass vials, we could recover all specimens easily at the end of the experiment. All glass vials were not covered in case the net creates a microenvironment in which the seawater is not totally in equilibrium with the ambient water of the aquarium. Because Rnet, R1, R2, R3 are put together in the same aquarium, they are not true replicates and will be referred to hereafter as pseudo-replicates. The culturing experiment lasted ~45 days, during which the foraminifera were fed three times with 200 μL of frozen cells of the algae Phaeodactylum tricurnutum, diluted in seawater.

In the aquaria, pH, TA, DIC, salinity, and temperature of the seawater were measured daily or once every two days. For pH, salinity, and temperature, we used a multimeter WTW 3620 ID equipped with a Tetracon sensor for conductivity (and associated temperature) and a SENTIX sensor for pH (and associated temperature). Parallel measurements of seawater pH buffers (Tris purchased from the Certified Reference Materials Laboratory of SCRIPPS and AMP prepared according to the recipe of [START_REF] Dickson | Guide to Best Practices for Ocean CO 2 Measurements[END_REF]) allowed conversion of the measured potential difference values to pH values on the total scale. For DIC and TA measurements, we sampled ~45 mL of seawater in each aquarium using a 50 mL syringe. The sampled seawater was placed in borosilicate vials with PTFE/silicone septa, that we overfilled to avoid air bubbles, and closed inside the climatic chambers under the correspondent pCO2 conditions. Then, DIC was measured directly after sampling with a Shimadzu TOC-L analyzer. After DIC measurement, the seawater was analyzed for TA by automated titration with a Titrino 785 (Methrom) with certified 0.1 mol kg -1 HCl (and 0.6 mol kg -1 NaCl) as titrant (Dickson). The accuracy of DIC and TA analyses was verified with a reference seawater standard (Dickson batch 182). The precision of individual samples, reference seawater standard (Dickson batch 182) and in-house seawater standards assessed by three injections for DIC and by three replicate analyses per measurement for TA, was 0.6 % and 0.2 %, respectively. In our experiment, we used pH total scale, DIC, water temperature and salinity values to calculate C-system parameters using CO2 SYS program [START_REF] Lewis | Environmental System Science Data Infrastructure for a Virtual Ecosystem (ESS-DIVE)[END_REF] with equilibrium constants of [START_REF] Lueker | Ocean pCO2 calculated from dissolved inorganic carbon, alkalinity, and equations for K1 and K2: validation based on laboratory measurements of CO 2 in gas and seawater at equilibrium[END_REF].

Because only two C-system parameters are needed as inputs for the CO2 SYS program, TA measurements were only used as an additional control for the outputs of the CO2 SYS program.

For the elemental composition of seawater (Sr, Mg, Ca…), we sampled 5 mL of seawater using a syringe equipped with a cellulose acetate filter (0.2 μm). The filtered water samples were placed in plastic tubes, acidified with concentrated ultrapure nitric acid (HNO3), and stored at 4°C for later analyses.

At the end of the experiment, each glass vial containing foraminifera was filtered through a 38 μm sieve with natural seawater. Specimens from each vial were transferred to a falcon tube and fixed with ethanol (96 %). All vials and sieves were checked under stereomicroscope for the presence of attached specimens.

2.3.Foraminiferal and seawater elements/Ca analyses

In the laboratory, all specimens were checked under epifluorescent microscope to assess their survival (i.e., the percentage of specimens with a full yellow/orange cytoplasm) and growth (i.e., the percentage of specimens that calcified new chambers) at the end of the experiment.

Specimens that calcified one or more chambers during the experiments were cleaned with 5 % NaOCl (~1 h incubation) to remove organic material. Specimens were rinsed before and after with ultra-pure water. The newly formed chambers were analyzed for elemental concentrations using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS).

For Ammonia T6, measurements were performed at LPG (Nantes, France) using an Excimer laser Analyte.G2 (Photon Machine) at a wavelength of 193 nm, coupled with a quadrupole ICP-MS (Varian). The ablation was conducted in a helium atmosphere. Pulse repetition rate was set at 5 Hz, with a fluence of 0.91 J cm -2 (10 % of the laser energy output).

Depending on the size of the analyzed chamber, ablation spots were 50 or 65 µm in diameter.

43 Ca isotope was used as an internal standard, and the SRM NIST 610 glass standard (National Institute of Science and Technology, USA) [START_REF] Jochum | Determination of Reference Values for NIST SRM 610-617 Glasses Following ISO Guidelines[END_REF] was used for the calibration of minor and trace elements. The NIST standard was measured every 10-15 samples with a fluence of 0.91 J cm -2 , a repetition rate of 5 Hz and ablation spots of 85 µm (cf. supplementary material S4a for the total operating parameters). The abundance of the following isotopes was wavelength) coupled with a quadrupole ICP-MS (iCAP-Q, Thermo Fisher Scientific). Single chambers of B. marginata were ablated with a laser energy of 1 J cm -2 , a repetition rate of 6

Hz, and an ablation spot size of 40 µm in diameter, whereas single chambers of C. laevigata were ablated with a laser energy of 0.5 J cm -2 , a repetition rate of 4 Hz, and a spot size of 40 µm (cf. supplementary material S4a for the total operating parameters). At the start of each series, SRM NIST 610 calibration and quality control reference material carbonate standards were analyzed, including JCt-1 (giant clam), JCp-1 (coral, Porites sp.; [START_REF] Okai | Preparation of a New Geological Survey of Japan Geochemical Reference Material: Coral JCp-1[END_REF], MACS-3 (synthetic calcium carbonate) and a foraminifera standard NFHS-2-NP [START_REF] Boer | A New Calcium Carbonate Nano-Particulate Pressed Powder Pellet (NFHS-2-NP) for LA-ICP-OES, LA-(MC)-ICP-MS and µXRF[END_REF]. Carbonate standards were ablated with the same laser settings as used for the foraminifera. SRM NIST 610 was ablated using an energy of 4 J cm -2 . SRM NIST 610 was preferred over MACS-3 for calibration, because MACS-3 was found to be too inhomogeneous at a spot size of 40 µm. NFHS-2-NP standard was used to monitor drift after every fourth sample. NFHS-2-NP is the preferred homogeneous reference material for drift monitoring at NIOZ as it shows lower gas blanks for some elements than SRM NIST 610 (e.g., Na). The calibrated data of NFHS-2-NP using SRM NIST 610 without drift correction were used to calculate drift correction factors. The first four NFHS-2-NP standards directly measured after the calibration standards were assumed to undergo no drift. Although instrumental drift was small, drift corrections were made to improve the accuracy. The isotopes of 24 Mg, 25 Mg, 43 Ca, 66 Zn, and 88 Sr were measured for B. marginata and 25 Mg, 43 Ca, 66 Zn, and 88 Sr for C. laevigata using 43 Ca as an internal standard. Backgrounds were measured for 60 s and the number of shots was 120. Raw counts were converted to element concentrations using an adapted version of the MATLAB based program Signal Integration for Laboratory Laser Systems [START_REF] Guillong | SILLS: A MATLABbased program for the reduction of laser ablation ICP-MS data of homogeneous materials and inclusions[END_REF]. This modification allows to import Thermo Qtegra software sample list and ICP-MS data files, screen shots, and laser LOG files (as described in [START_REF] Mezger | Salinity controls on Na incorporation in Red Sea planktonic foraminifera[END_REF]van Dijk et al., 2017a). The region of interest of the signal is automatically set by the Matlab scripts and fine-tuned manually using the intensity signals and screen shots of the ablation foraminifera (made automatically every 1.5 s). Repeatability, expressed as the relative standard deviation (RSD), of all NFHS-2-NP analyses (average of the five sessions; 325 data points), was 2 % for 25 Mg/Ca, 11 % for Zn/Ca, and 2% for Sr/Ca, and accuracies were 94 %, 99 %, and 102 % respectively (Supplementary material S4b; c).

For the three species, integration profiles were manually selected, by discarding the initial part of the signals showing Al and/or Mg peaks, which can be used as a sign of contamination, and by monitoring the decrease in Ca counts. In total, 436 ablation measurements were performed on chambers of Ammonia T6 that calcified under experimental conditions, 285 on B. marginata and 288 on C. laevigata. In all, 13 %, 17 % and 46 % of these profiles, for Ammonia T6, B. marginata, and C. laevigata respectively, were not included in the final analyses (cf. raw data https://doi.org/10.17882/89623) because they were either too short or because there was doubt on the fluorescence of the newly formed chambers, especially for C. laevigata. As such, and for each condition, between 6 and 57 measurements were obtained for each species (Table 2). When several chambers were calcified under controlled conditions, it was possible to perform LA-ICP-MS measurements on successively added chambers, from chamber n (latest calcified chamber) to older ones (n-1, n-2…). This allowed evaluating a potential ontogenetic effect.

Measurements of the elemental composition of seawater (Sr, Mg, Ca…) were performed by an ICP-OES ICAP 6300 Thermo-Fischer in LPG (Nantes) (Table 1). Samples were diluted 1:50 with 1 % HNO3. Calibration was performed with seven multi-element standards, covering the concentration range of the samples, prepared by properly diluting single-element stock solutions (1000 μg g -1 ). All standards and the blank were measured at the beginning, midpoint, and end of the analytical run; three replicate readings were performed on standards, blank and samples.

The partitioning coefficients of strontium (DSr) and magnesium (DMg) were calculated following the equations: DEl = (El/Ca)calcite/(El/Ca)seawater (Table 1).

Scanning Electron Microscope (SEM) pictures (Supplementary material S5) were obtained at LPG (Angers, France) using a Tabletop Microscope Hitachi TM4000Plus.

Statistical analyses

To compare survival and growth and El/Ca ratios (individual chamber analyses) between the treatments and the pseudo-replicates (Figures 2; 5 and 6), we tested first the data for normality (Shapiro-Wilk's test). Because a normal distribution was not observed for all data sets, we performed pairwise comparison with Kruskal Wallis (i.e., a non-parametric test that does not assume a normal distribution of the underlying data). These tests were performed using Past software [START_REF] Hammer | PAST: Paleontological statistics software package for education and data analysis[END_REF]). Dixon's test [START_REF] Dixon | Analysis of Extreme Values[END_REF][START_REF] Dixon | Ratios Involving Extreme Values[END_REF] was performed to identify outliers in the growth percentages between experimental conditions presented in Figure 3.

Linear regression analyses between foraminiferal El/Ca (individual chamber analyses) and the C-system parameters were performed using the statistics environment R (R Development Core

Team, 2012; http://www.R-project.org) (Table 3). To test the differences between regression models, we performed a one-way ANCOVA using Past software [START_REF] Hammer | PAST: Paleontological statistics software package for education and data analysis[END_REF]) (Table 4). Spearman's (non-parametric) rank-order correlation coefficient [START_REF] Spearman | The Proof and Measurement of Association between Two Things[END_REF] was calculated using Past software [START_REF] Hammer | PAST: Paleontological statistics software package for education and data analysis[END_REF] to assess the presence/absence of ontogenetic trends in foraminiferal El/Ca (individual chamber analyses) according to the position of the newly formed chambers (Table 5). A p-value of 0.05 was considered as significant for all statistical tests.

Results

Stability of the experimental set-up

In each environmental chamber, the measured pCO2 was similar to the nominal pCO2 (<1.5% difference) and showed stable values over the experimental period with a relative standard deviation (RSD) of <5% (Table 1). Air temperature was stable in each environmental chamber with a RSD of ≤6% and ranged between chambers from an average of 11.7±0.7 to 12.2±0.12 °C (Table 1). In each aquarium, temperature and salinity values were stable with a RSD of <2.5% (Table 1). Between the eight aquaria, the culture media temperatures and salinities varied from an average of 11.3±0.2 to 11.8±0.1 °C and 34.9±0.1 to 35.5±0.1 respectively (Table 1).

The measured C-system parameters (DIC, TA and pH total scale) were stable in each of the eight conditions with a <5% RSD (Table 1; Supplementary material S3). For set-up A, DIC was stable at an average of 2387±48 µmol kg -1 , TA and the calculated [HCO3 -] slightly varied between 2539±28 and 2912±15 µmol kg -1 and between 2018±41 and 2306±25 µmol kg -1 , respectively and a wide range was obtained for pH total scale (7.56±0.02 -8.40±0.04), the calculated [CO3 2-] (55±3 -328±29 µmol kg -1 ) and the calculated Ωcc (1.3±0.1 -7.8±0.8). For set-up B, pH total scale was constant at an average of 8.02±0.04 while a wide range was obtained for DIC (879±22 -7131±71 µmol kg -1 ), TA (1029±13 -7812±15 µmol kg -1 ), [HCO3 - ] (819±30 -6615±68 µmol kg -1 ), [CO3 1).

Survival and growth

Many specimens of the three species survived and grew new chambers in all experimental conditions (Fig. 2a). Nevertheless, lower survival and growth percentages on average were found for B. marginata (55±10 % and 20±15 %, respectively) and C. laevigata (66±19 % and 16±15 %, respectively) compared to Ammonia T6 (87±16 % and 84±11 %, respectively) (Fig. 2a). When considering the eight experimental conditions together, there was no significant difference between the pseudo-replicates in terms of survival and growth, except for R1 where Ammonia T6 recorded the lowest values (Fig. 2b;c). Survival percentages were similar in the eight conditions for the three species. While the growth percentages of Ammonia T6 were similar in the different conditions (> 80%, except at condition A180 recording values of ~79 %), the lowest growth percentages were recorded in the low DIC condition B180, for both B. marginata and C. laevigata (Fig. 3). These observations were confirmed statistically by the Dixon's test identifying the A180 growth value of Ammonia T6 and the B180 growth values of the too deep-sea species as outliers. Although survival and growth rates of the three species in Rnet were not significantly different from the other pseudo-replicates when considering the eight conditions together (Fig. 2), we observed that no specimens of C. laevigata survived and calcified new chambers in the Rnet of the low DIC condition B180. In the same condition (B180)

and pseudo-replicate (Rnet), the survival rate of B. marginata was not significantly different from the other conditions and pseudo-replicates (69 %), but the lowest growth (1.6 %) was found compared to the other pseudo-replicates of B180 (~12 %). For Ammonia T6, the net had no effect on its survival and growth in the B180 condition. In terms of shell aspect however, the three species showed high degrees of dissolution in the B180-Rnet condition whereas no consistent dissolution patterns were found in the other replicates and conditions (cf. SEM images Supplementary material S5).

Amongst the total Ammonia T6 specimens cultured in our experiment, ~68% calcified between one and five chambers, and ~16% between six and eleven chambers (cf. details in Fig. 3a). For some specimens of C. laevigata and B. marginata that calcified only one chamber, the identification of the newly calcified chamber under epifluorescence microscope was problematic. This was because the fluorescence of the old calcein-labelled chambers (i.e., not formed during our experiment) reflected on the last one (i.e., formed during our experiment), which explains the presence of doubtful chambers (~2 % on average for B. marginata and ~10 % for C. laevigata; cf. "1?" in Figs. 3b;c). Amongst the total B. marginata specimens cultured in our experiment, ~18 % on average calcified one to two chambers and ~2 % three to five chambers. For C. laevigata, ~5 % calcified one chamber and only ~1 % calcified two chambers.

There is no clear pattern in terms of the number of calcified chambers between the eight experimental conditions for each species (Fig. 3).

Link between foraminiferal Sr/Ca and C-system

To analyze the correlation between foraminiferal Sr/Ca for each species and the individual Csystem parameters, linear regression analyses were first performed using data from both setups A and B. The results of the regression analyses (intercepts and slopes) and the statistical outputs (p, and R 2 ) are summarized in Table 3. For Ammonia T6 and B. marginata, all the regressions were strongly significant (p <0.05), except for Sr/CaB. marginata vs. pH and explained, depending on the C-system parameter, between 5% and 53% of the observed variability in Sr/Ca. For C. laevigata, although the regressions were significant (p <0.05) except for Sr/CaC. laevigata vs. pCO2 and pH, they only explained 6% of the observed variability in Sr/Ca.

To narrow down the main controlling parameters, Figure 4 presents linear regression models for each set-up and the results of the regression analyses (intercepts and slopes) and the statistical outputs (p, and R 2 ) are summarized in Table 3. The results of Ammonia T6 and B. marginata showed significant correlations (p <0.05) in set-up B between Sr/Ca and all the measured parameters. These correlations present a high R 2 (>0.60), except for pH. Significant correlations (p <0.05) were also found in set-up A between Sr/Ca and all the measured parameters for Ammonia T6, although with lower R 2 (<0.24). For B. marginata, no significant correlations (p <0.05) were found in set-up A with any of the C-system parameters except for the DIC but with a very weak R 2 (= 0.04). For TA, DIC and [HCO3 -], all data points from setup A fall on the regression lines of set-up B. For C. laevigata, no trends were recorded between Sr/Ca and individual parameters of the C-system.

The ANCOVA tests analyses showed no significant difference between intercepts of Ammonia T6 regression models (Sr/Ca vs [HCO3 -] and DIC) in our study and that of Keul et al. (2017) (F=2.1, p=0.15), but shows a significant difference in the slopes (F=40.4, p=3.8 10 -10 ) (Table 4). The ANCOVA tests analyses showed a significant difference between intercepts of Ammonia T6 and B. marginata (this study) regression models (F=200.7, p=8.7 10 -40 ). There was a statistically significant, but weak, difference between the slopes of the two species (F=5.4, p=0.02) (Table 4).

Variability in Sr/Ca and Mg/Ca ratios between chamber stages and replicates

Here we evaluate the ontogenetic effect based on chamber stages since we did not obtain reproduction. For Ammonia T6, we estimated that adding on average five to six chambers increased the diameter from ~150 to 250 µm. For B. marginata, adding on average two to three chambers increased the maximum length from ~150 to 300 µm. For C. laevigata, adding on average one chamber increased the diameter from ~150 to 200 µm (cf. supplementary material S1).

To evaluate a potential ontogenetic effect (chamber stages) on Sr and Mg incorporation, we present in Figure 5 the average of Sr/Ca and Mg/Ca ratios calculated for the different chambers of each species. In Supplementary material S6, we present the same results for Sr/Ca displayed for each condition. The Spearman's rank-order correlation coefficients calculated between these ratios and the position of the newly formed chambers, all treatments combined (Table 5),

showed no significant correlations for B. marginata and C. laevigata (p >0.05) and significant correlations for Ammonia T6 (p>0.05) but with very low coefficients (r = 0.18 and -0.13 for Sr/Ca and Mg/Ca respectively). This indicates the absence of ontogenetic trends in Sr/Ca and Mg/Ca ratios. Caution should be taken for B. marginata and C. laevigata that calcified fewer chambers compared to Ammonia T6.

In figures 6a and 6b, we plotted Sr/Ca ratios against Mg/Ca ratios by species and pseudoreplicate, using respectively all measurements, and average data from each experimental condition. The results demonstrated that Ammonia T6 had higher Sr/Ca ratios and lower Mg/Ca ratios than the two other species showing equivalent absolute El/Ca values. There was also no significant correlation between Sr/Ca and Mg/Ca ratios for each species.

Discussion

Survival and growth of benthic foraminifera

Many specimens of the three cultured benthic species survived and calcified new chambers in the eight controlled conditions, with however higher survival and growth percentages for Ammonia T6 compared to the two other species studied (Fig. 2). It is well known that shallowwater foraminifera adapt more easily (growth and reproduction) to laboratory cultures (e.g., Raitzsch et al., 2010;Dueñas-Bohórquez et al., 2011b;[START_REF] Van Dijk | Chemical Heterogeneity of Mg, Mn, Na, S, and Sr in Benthic Foraminiferal Calcite[END_REF][START_REF] Geerken | High precipitation rates characterize biomineralization in the benthic foraminifer Ammonia beccarii[END_REF] than most deep-sea species. This is likely because shallow-water taxa have a natural adaptability to stressful and variable conditions characterizing their natural environments (e.g., variable salinity and temperature) (e.g., [START_REF] Bradshaw | Laboratory experiments on the ecology of foraminifera[END_REF][START_REF] Murray | Ecology and palaeoecology of benthic foraminifera[END_REF][START_REF] Pascal | Bacterivory in the common foraminifer Ammonia tepida: Isotope tracer experiment and the controlling factors[END_REF][START_REF] Geslin | Survival, reproduction and calcification of three benthic foraminiferal species in response to experimentally induced hypoxia[END_REF] whereas deep-sea species live in more stable settings (e.g., high pressure, stable salinity, and temperature). Unlike many other deep-sea foraminiferal taxa, B. marginata usually shows a rather good adaptation to laboratory conditions (e.g., [START_REF] Barras | Reproduction and growth of the deep-sea benthic foraminifer Bulimina marginata under different laboratory conditions[END_REF][START_REF] Barras | Calibration of δ 18 O of laboratorycultured deep-sea benthic foraminiferal shells in function of temperature[END_REF][START_REF] Barras | Experimental calibration of manganese incorporation in foraminiferal calcite[END_REF][START_REF] Filipsson | A culture-based calibration of benthic foraminiferal paleotemperature proxies: δ 18 O and Mg/Ca results[END_REF][START_REF] Wit | Calibration, validation and application of foraminiferal carbonate based proxies. Reconstructing temperature, salinity and sea water Mg/Ca[END_REF][START_REF] Geslin | Survival, reproduction and calcification of three benthic foraminiferal species in response to experimentally induced hypoxia[END_REF][START_REF] Nardelli | Experimental evidence for foraminiferal calcification under anoxia[END_REF]. Cassidulina laevigata has only been used to a limited extent in culture studies and it has typically low tolerance to anoxic conditions (e.g., [START_REF] Nardelli | Experimental evidence for foraminiferal calcification under anoxia[END_REF].

Amongst the eight controlled conditions, we expected possible negative effects (i.e., decalcification, reduced growth) on the cultured species under low pH conditions A1000 (pH=7.73) and A1500 (pH=7.56). Instead, no difference (according to Dixon's test) was observed compared to most other conditions with "normal" or high pH values (Fig. 3).

Responses of calcifying benthic foraminifera to pH changes are not consistent and laboratory studies show that calcification/dissolution rates under varying pH concentrations differ between and within species. Several studies observe continuing growth under low pH conditions while reporting a reduction in shell weight, size or the number of newly added chambers in several species, including Ammonia group tepida (e.g., Dissard et al., 2010a;[START_REF] Knorr | Response of the miliolid Archaias angulatus to simulated ocean acidification[END_REF][START_REF] Prazeres | Ocean acidification induces biochemical and morphological changes in the calcification process of large benthic foraminifera[END_REF][START_REF] Guamán-Guevara | Impacts of ocean acidification on intertidal benthic foraminiferal growth and calcification[END_REF][START_REF] Oron | Differential Sensitivity of a Symbiont-Bearing Foraminifer to Seawater Carbonate Chemistry in a Decoupled DIC-pH Experiment[END_REF]. On the contrary, other studies do not report any low pH-related negative effects on several species of benthic foraminifera, including B.

marginata (e.g., [START_REF] Mcintyre-Wressnig | Non-lethal effects of ocean acidification on the symbiont-bearing benthic foraminifer Amphistegina gibbosa[END_REF], 2014;[START_REF] Haynert | Response of benthic foraminifera to ocean acidification in their natural sediment environment: a long-term culturing experiment[END_REF][START_REF] Wit | A short-term survival experiment assessing impacts of ocean acidification and hypoxia on the benthic foraminifer Globobulimina turgida[END_REF][START_REF] Weinmann | Community responses of intertidal foraminifera to pH variations: a culture experiment with propagules[END_REF]. While enhanced dissolution due to acidification is mainly an inorganic process, [START_REF] Toyofuku | Proton pumping accompanies calcification in foraminifera[END_REF] suggest that the calcification in some species might be largely unaffected by decreasing pH as they find, together with other studies (e.g., [START_REF] Glas | Calcification acidifies the microenvironment of a benthic foraminifer (Ammonia sp.)[END_REF], that Ammonia sp. reduces its surrounding pH during chamber formation until 6.9 and even 6.3.

Our results are in line with these findings, suggesting that there is a strong organismal control over biomineralisation during foraminiferal calcification that possibly makes them more adaptive and robust in coping with acidification (e.g., [START_REF] Toyofuku | Proton pumping accompanies calcification in foraminifera[END_REF][START_REF] Kawahata | Perspective on the response of marine calcifiers to global warming and ocean acidification-Behavior of corals and foraminifera in a high CO 2 world "hot house[END_REF]. While this is certainly true for Ammonia T6 showing successful calcification in most conditions (> 80% of the cultured specimens calcified), caution should be taken for the two deep-sea species of which only ~20% of the specimens grew in culture. It is therefore possible that the minority that did calcify were also those able to withstand/resist the varying pH conditions. Still, the survival and growth percentages that we recorded for B. marginata for example are in line with other culture experiments (e.g., ~30-40 % and 15-30 %, respectively; [START_REF] Geslin | Survival, reproduction and calcification of three benthic foraminiferal species in response to experimentally induced hypoxia[END_REF], meaning that this is a common feature in culture regardless of the controlled parameters. For a broader perspective, the fact that the studied species calcified well under low pH conditions might be coherent with geological records showing the resistance of some species to high pCO2 time periods (e.g., [START_REF] Foster | Surviving rapid climate change in the deep sea during the Paleogene hyperthermals[END_REF], although these occurred over evolutionary timescales, and were associated with a covariance in other factors.

In the low DIC condition B180 (DIC=880 µmol kg -1 , TA=1029 µmol kg -1 ), characterized by a normal pH (7.99), B. marginata and C. laevigata calcified fewer chambers (Fig. 3b-c).

Ammonia T6, however, did not show any significant difference compared to most other "normal" or high DIC conditions (Fig. 3a). These results seem to indicate a higher vulnerability of the two deep-sea species to low DIC concentrations than the shallow water species Ammonia T6 (Fig. 3a). Yet, [START_REF] Keul | Effect of ocean acidification on the benthic foraminifera Ammonia sp. is caused by a decrease in carbonate ion concentration[END_REF] report the lowest growth rate and size-normalized weight of Ammonia T6 under low DIC conditions (246 µmol kg -1 ), although lower than in our case study. This might indicate a DIC threshold value below which Ammonia struggles to calcify.

Additionally, and only in the Rnet replicate of the B180 condition, the three species showed severe signs of dissolution (Supplementary material S5), especially for B. marginata and C. laevigata specimens that were very fragile and sometimes broke during manipulation. This indicates that the net aggravated the impact of low DIC seawater on benthic foraminifera, most likely by creating a more isolated micro-environment where the seawater DIC is depleted faster.

One could argue that we decreased/increased the seawater DIC in our experiment beyond typical modern values in the ocean (~1800 -2300 µmol kg -1 ; e.g., [START_REF] Wu | What drives the latitudinal gradient in open-ocean surface dissolved inorganic carbon concentration?[END_REF]. This is first justified by the fact that low/high DIC concentrations were probably encountered in the past in open marine waters during specific climatic periods (e.g., [START_REF] Vigier | Lithium isotopes in foraminifera shells as a novel proxy for the ocean dissolved inorganic carbon (DIC)[END_REF][START_REF] Zeebe | History of carbonate ion concentration over the last 100 million years II: Revised calculations and new data[END_REF], although the recent reconstructions of [START_REF] Zeebe | History of carbonate ion concentration over the last 100 million years II: Revised calculations and new data[END_REF] suggest that long-term DIC and TA were similar to modern values across the past 100 Myr regardless of the varying atmospheric pCO2. Furthermore, in coastal waters, the carbonate chemistry is substantially more dynamic than the open ocean due, amongst others, to seasonal fluctuations and freshwater discharges (e.g., [START_REF] Borges | Carbonate chemistry in the coastal zone responds more strongly to eutrophication than ocean acidification[END_REF][START_REF] Rheuban | Quantifying the Effects of Nutrient Enrichment and Freshwater Mixing on Coastal Ocean Acidification[END_REF]. The DIC and TA of nearriver waters are largely governed by the type of bedrock of the catchment area, with high values (>3000 µmol kg -1 ) associated with limestone or carbonate bedrock and low values (<1000 µmol kg -1 ) linked to sandstones and volcanic rocks (e.g., [START_REF] Mcgrath | The Inorganic Carbon Chemistry in Coastal and Shelf Waters Around Ireland[END_REF]. Additionally, there is a close connection to salinity, especially for waters with low DIC and TA concentrations that increase with increasing salinity in the outer estuary (e.g., [START_REF] Mcgrath | The Inorganic Carbon Chemistry in Coastal and Shelf Waters Around Ireland[END_REF]. As such, and unlike deep-sea species, the low vulnerability of the intertidal Ammonia T6 to low DIC concentrations in our experiment might be due to organismal adaptability in its natural environment with variable salinities and freshwater input.

Effect of the C-System on Sr incorporation

Our results showed that Sr/Ca in Ammonia T6 and B. marginata vary in a similar fashion (i.e., trends) with respect to the C-system parameters as the Sr/Ca in Ammonia T6 of Keul et al. (2017) grown in a comparable decoupled C-system experiment (Fig. 4). In the decoupled Csystem study of van Dijk et al. (2017b), Sr/Ca values of Ammonia T6 did not show any correlation with the C-system. However, when considering the partition coefficient (DSr), van Dijk et al. (2017b) found similar trends (Supplementary material S7). Because Sr/Ca in foraminifera correlates with Sr/Casw (e.g., Langer et al., 2016), the discrepancy in the trends between Sr/Ca and DSr in the study of van Dijk et al. (2017b) might be due to the unstable Sr/Casw values between the experimental conditions (4.4 -6.4 mmol mol -1 ) (cf. additional discussion in Supplementary material S8). The overall consistency between the results issued from comparable C-system experiments validates the positive correlation between foraminiferal Sr/Ca and the C-system, at least for Ammonia T6 and B. marginata. However, caution should be taken in paleo-applications with respect to spatio-temporal Sr/Casw variability (e.g., Lebrato et al., 2020), even on time-scales shorter than Sr residence time in seawater (~3 Myrs; [START_REF] Richter | Simple models for the geochemical response of the ocean to climatic and tectonic forcing[END_REF].

Sr/Ca ratio in C. laevigata did not record any C-system dependency (Fig. 4), even though we had a conservative approach by discarding the Sr/Ca values of the doubtful chambers from the calculation of regression models. We can hypothesize that this species poorly tolerated our laboratory conditions (e.g., lack of sediment, high range of pH and DIC, etc.). This might have resulted in a physiological stress that impacted biomineralisation pathways and incorporation of Sr into the calcite. We can also assume a strong species-specific effect, affecting not only the nature of the correlation with the forcing parameter (e.g., slope, intercept, linear, exponential, etc.), such as in the case of Mg/Ca vs temperature (e.g., [START_REF] Toyofuku | Evaluation of Mg/Ca thermometry in foraminifera: Comparison of experimental results and measurements in nature[END_REF][START_REF] Lowenstein | The Use of Mg/Ca as a Seawater Temperature Proxy[END_REF], but also the existence/absence of such relationship (i.e., the mechanism that drives the Sr/Ca-C-System relationship in the other species is not present in C. deviation and confirm whether it is a common pattern for C. laevigata once put in laboratory conditions, or whether this was specific to our experimental conditions.

DIC and/or [HCO 3 -] as the main controlling factors for Sr/Ca ratio

We can assume that if a certain C-system parameter is controlling incorporation of Sr into foraminiferal calcite, the correlation between Sr/Ca and this parameter should be similar in both set-ups A and B. As such, for Ammonia T6 and B. marginata, we can exclude pCO2, [CO3 2-] and Ωcc (Fig. 4). pH can also be excluded because, although a trend can be observed in treatment A, the R² value is very small (Fig. 7a; Table 3) indicating that there is no predictive power of pH on foraminiferal Sr/Ca. This leaves us with [HCO3 -], TA and DIC as potential controlling factors. In set-up A (i.e., stable DIC), all [HCO3 -] and TA values fall within a narrow range, which is not surprising since HCO3 -is the major constituent of both TA and DIC. Despite the narrow range of values, we can observe a decreasing trend of Sr/Ca with the slight increase in [HCO3 -] in set-up A for Ammonia T6, oppositely to set-up B (Fig. 4). However, because the R² value is very small, and because this trend is not existent for B. marginata (Table 3), we cannot exclude [HCO3 -]. Furthermore, since all [HCO3 -] and TA values from set-up A fall overall on the regression line of set-up B (Fig. 4), this means that [HCO3 -], TA and/or DIC might control Sr/Ca incorporation (high R 2 values; Fig. 7a). The data presented here cannot empirically disentangle the effects of these parameters. However, because TA has no ''physical reality" which could be used by an organism, we can narrow it down to DIC and/or [HCO3 -] as the main driving forces behind the Sr incorporation in the calcite of Ammonia T6 and B. marginata. This is in line with the conclusions of Keul et al. (2017) for Ammonia T6.

When plotting Sr/Ca values against DIC and [HCO3 -] for Ammonia T6 and B. marginata, the regression models were overall similar and consistent with that published by Keul et al. (2017) for Ammonia T6 (Fig. 7b). Furthermore, there was no significant difference between the intercepts of Ammonia T6 regression models from both studies (Table 4). Though, a significant difference in the slopes was found and might be explained either by i) slight instabilities in the chemical system of Keul et al. ( 2017) (e.g., differences between nominal and measured pCO2

and between set-ups A and B) that we were able to improve thanks to the Ecolab system, or ii) by the fact that the DIC ranges were slightly different, with Keul et al. (2017) reaching lower DIC values than in our experiment. The regression models of Ammonia T6 and B. marginata (this study) showed overall parallel trends but a significant offset between the main values of Sr/Ca (Fig. 7b). This is a common feature in most elemental incorporation in foraminiferal test during calcification, due to biological processes that are often species dependent (e.g., [START_REF] Toyofuku | Mg/Ca and δ 18 O in the brackish shallow-water benthic foraminifer Ammonia 'beccarii[END_REF]van Dijk et al., 2017a;[START_REF] Barras | Experimental calibration of manganese incorporation in foraminiferal calcite[END_REF]. This means that there is a species-specific effect that will affect absolute values estimations (i.e., different intercept) in paleo-reconstructions but not the relative DIC and/or [HCO3 -] variations (i.e., similar slope).

That said, and although the trends observed here are robust, the sensitivity of the Sr/Ca to DIC and/or [HCO3 -] seems to be low (~0.04 mmol mol -1 /500 µmol kg -1 DIC), and as such, it does not form a strong paleo-proxy, especially for small shifts in these parameters. This low sensitivity, however, can be partly counterbalanced by the fact that Sr is analytically robust (e.g., Dueñas-Bohórquez et al., 2011a;de Nooijer et al., 2014), and with very little inter-and intra-individual variability as discussed in section 4.4. Keul et al. (2017) explored the transmembrane transport model to explain the impact of DIC and/or [HCO3 -] on Sr/Ca in Ammonia T6. According to these authors, pCO2 and HCO3 -enter the biomineralisation space (i.e. site of calcification), created by the pseudopodial network (i.e., protective envelop), where they are converted to CO3 2-. Keul et al. (2017) further hypothesized that in order to keep Ω constant, foraminifera have to decrease Ca causing increasing Sr/Ca in the calcite (Keul et al., 2017). Alternatively, [START_REF] Toyofuku | Proton pumping accompanies calcification in foraminifera[END_REF] emphasized that HCO3 -is externally transformed into CO2 due to active proton pumping, meaning that biomineralisation does not rely on the availability of CO3 2-. Once inside the biomineralisation space, calcification is driven by rapid transformation of CO2 into CO3 2-due to the high pH sustaining CaCO3 precipitation by reacting with Ca 2+ [START_REF] Toyofuku | Proton pumping accompanies calcification in foraminifera[END_REF]. Therefore, we can suppose that the more DIC and/or HCO3 -is available, the more CO2 diffuses inside the biomineralisation space, which will increase precipitation rate of calcite. This has been shown for inorganic precipitation of calcite, where the rate increases with increasing DIC and with it Sr incorporation into calcite (e.g., Lorens, 1981;[START_REF] Alkhatib | Calcium and strontium isotope fractionation in aqueous solutions as a function of temperature and reaction rate; I. Calcite[END_REF]. We can suppose that this mechanism might be responsible for higher Sr/Ca ratios in foraminiferal calcite at higher DIC and/or HCO3 - concentrations. Alternatively, if we consider the seawater vacuolization model (i.e., closed pool of seawater) (e.g., [START_REF] Erez | The Source of Ions for Biomineralization in Foraminifera and Their Implications for Paleoceanographic Proxies[END_REF][START_REF] Evans | Assessing foraminifera biomineralisation models through trace element data of cultures under variable seawater chemistry[END_REF], when there is more DIC, potentially more calcite can be formed necessitating utilizing more of the Ca pool, resulting in higher Sr/Ca ratio in foraminiferal shells. Mean DSr values in the present study for the three species (~0.13-0.21 mmol mol -1 ) plot near the highest DSr values measured for inorganic precipitation calcite (e.g., 0.02-0.14; Lorens, 1981;[START_REF] Tesoriero | Solid solution partitioning of Sr 2+ , Ba 2+ , and Cd 2+ to calcite[END_REF][START_REF] Nehrke | Dependence of calcite growth rate and Sr partitioning on solution stoichiometry: Non-Kossel crystal growth[END_REF], meaning probably a non-negligible role of inorganic processes (i.e., a relatively weak biological control) as hypothesized by [START_REF] Evans | Assessing foraminifera biomineralisation models through trace element data of cultures under variable seawater chemistry[END_REF].

To test whether other divalent cations display the same relationship to DIC and/or [HCO3 -], we measured alongside with Sr/Ca in Ammonia T6, Ba/Ca, Zn/Ca and Mg/Ca and in B. marginata, Mg/Ca. None of these ratios showed a relationship with any of the C-system parameters (Supplementary material S9). This may imply other biomineralisation pathways for these specific elements. Although it was only possible to measure Mg/Casw (~5260 mmol mol -1 ) in our culture study, it is well known that, in contrast to DSr, perforate foraminiferal DZn and DMg are below inorganic calcite values (e.g., Rosenthal et al., 1997;van Dijk et al., 2017b) . Some studies reported a certain link between Mg/Ca and the C-system, but this link is not systematic, especially for the Ammonia group tepida, where several culture studies show no effect of the carbonate chemistry on Mg/Ca (e.g., Dissard et al., 2010;Dueñas-Bohórquez et al., 2011b;[START_REF] Allison | Effects of seawater pH and calcification rate on test Mg/Ca and Sr/Ca in cultured individuals of the benthic, calcitic foraminifera Elphidium williamsoni[END_REF]van Dijk et al., 2017b). It appears therefore that the mechanisms responsible for the partitioning against Mg and Zn have very different sensitivities to certain key parameters than the mechanisms responsible for Sr incorporation.

Foraminiferal DBa, on the other hand, lies on the high side of the range of inorganic precipitation [START_REF] Lea | Assessing the reliability of paleochemical tracers: Barium uptake in the shells of planktonic foraminifera[END_REF], similarly to Sr and in consistency with inorganic calcite precipitation according to [START_REF] Evans | Assessing foraminifera biomineralisation models through trace element data of cultures under variable seawater chemistry[END_REF]. Following the same logic, Sr and Ba should display similar relationships to the C-system. Yet, in symbiont-bearing high Mg content hyaline tropical foraminifera, van Dijk et al. (2017a) reported the same behavior of Zn and Ba with increasing pCO2, and concluded that their incorporation in foraminiferal calcite might be depending on the availability of free ions and the formation of carbonate complexes, unlike the unaffected Sr/Ca since the availability of Sr 2+ does not change with pCO2. This shows the high complexity and variety of possible biomineralisation pathways that seem to differ between species, and for each element. More decoupled C-system experimental studies with benthic foraminifera are necessary to test and refine the proposed biomineralisation models.

Sr/Ca and Mg/Ca ratios between and within specimens and species

Despite stable culturing conditions, the variability observed in Sr/Ca ratios between and within specimens (relative standard errors of 1.4, 1.3 and 2 % on average) was much smaller than the variability observed in Mg/Ca ratios (relative standard errors of 19.1, 11.3 and 12.4 % on average) for Ammonia T6, B. marginata and C. laevigata respectively (Fig. 6). This is in line with previous findings showing generally uniform Sr/Ca ratios within the carbonate tests of foraminifera compared to Mg/Ca and seems to be inherent to the process of bio-calcification itself in the incorporation of these two elements (e.g., [START_REF] Eggins | Mg/Ca variation in planktonic foraminifera tests: implications for reconstructing palaeo-seawater temperature and habitat migration[END_REF]Dueñas-Bohórquez et al., 2011a;de Nooijer et al., 2014) and the potential Mg-related diagenetic contamination even in the inner layers of the calcite for fossil specimens (e.g., [START_REF] Sexton | Microstructural and geochemical perspectives on planktic foraminiferal preservation: "Glassy" versus "Frosty[END_REF][START_REF] Schneider | Diagenetic alteration of benthic foraminifera from a methane seep site on Vestnesa Ridge (NW Svalbard)[END_REF]. This implies that Sr is analytically very robust, with high accuracy and precision (i.e., only few ablation spots are sufficient to estimate the average with low uncertainty), not easily contaminated, and more importantly with little variability in the shells.

To test for a potential ontogenetic effect on Sr and Mg incorporation into foraminiferal tests, and since we did not obtain reproduction allowing to investigate different size fractions, we analyzed the ratios in the successive chambers in single specimens. No detectable ontogenetic effect was observed in Sr/Ca and Mg/Ca ratios for the last chamber stages of the three studied species (Fig. 5; Table 4), suggesting that i) there was no significant chamber stage-related impact on Sr/Ca and Mg/Ca within the growth range obtained (~150-300 µm) and ii) precipitation rates were probably similar during production of the last chambers. This in line with [START_REF] Geerken | High precipitation rates characterize biomineralization in the benthic foraminifer Ammonia beccarii[END_REF] who showed, using Sr-labeling, that precipitation rate is uniform among specimens and within chamber walls of Ammonia beccarii. Our results strongly suggest that we can exclude possible effects of ontogeny, at least between different chamber stages, to explain the differential incorporation of Sr/Ca in our experimentation, which is crucial for paleo-reconstructions of the C-system. For Ammonia spp. and Bulimina spp., the lack of ontogenetic trends in foraminiferal Sr/Ca and Mg/Ca has been reported in several studies investigating this effect in successive chambers in single specimens (e.g., [START_REF] Wit | A reappraisal of the vital effect in cultured benthic foraminifer Bulimina marginata on Mg/Ca values: assessing temperature uncertainty relationships[END_REF]de Nooijer et al., 2014). Some studies also report a lack of ontogenetic trend in Sr/Ca and Mg/Ca of Bulimina spp. between individual specimens from different size fractions (e.g., ~100-500 µm, 300-600 µm; [START_REF] Hintz | Trace/minor element:calcium ratios in cultured benthic foraminifera. Part II: Ontogenetic variation[END_REF]Wit et al., 2012, respectively), whereas Diz et al. (2012) report a size-related trend Sr/Ca for Ammonia sp. between 150-600 µm in size. However, when considering only sizes between 150 and 250 µm in the study of Diz et al. (2012), equivalent to the approximate range of growth of Ammonia T6 in our study (adding on average 5 to 6 chambers), the trend is no longer clear. For application of Sr/Ca ratios in paleostudies, this may imply that measurements can be performed regardless of the chamber stage in single specimens, using laser ablation or even bulk analyses, provided the use of a restricted size fraction.

Amongst the three species, Ammonia T6 recorded the highest Sr/Ca ratios on average and the lowest Mg/Ca ratios (Fig. 6a; b). Lower incorporation of Mg by Ammonia spp. than Bulimina spp. is a common feature reported in several studies (e.g., Dissard et al., 2010b;[START_REF] Filipsson | A culture-based calibration of benthic foraminiferal paleotemperature proxies: δ 18 O and Mg/Ca results[END_REF][START_REF] Toyofuku | Mg/Ca and δ 18 O in the brackish shallow-water benthic foraminifer Ammonia 'beccarii[END_REF][START_REF] Wit | A reappraisal of the vital effect in cultured benthic foraminifer Bulimina marginata on Mg/Ca values: assessing temperature uncertainty relationships[END_REF][START_REF] Groeneveld | Assessing proxy signatures of temperature, salinity, and hypoxia in the Baltic Sea through foraminifera-based geochemistry and faunal assemblages[END_REF]. This seems also to be the case for other elements such as Mn [START_REF] Barras | Experimental calibration of manganese incorporation in foraminiferal calcite[END_REF]. In our case study, this feature seems to be inversed for Sr between Ammonia T6 and B. marginata. Several studies argue that Sr/Ca of foraminiferal calcite is associated with increased shell Mg content, distorting the calcite lattice and weakening the discrimination against Sr (e.g., Mewes et al., 2015;Langer et al., 2016;Yu et al., 2019). Yet, our results showed no significant correlation between Mg/Cacc and Sr/Cacc ratios for the three cultured species (Fig. 6a;b). It is possible that the close relationship between Sr incorporation and Mg content is only robust for high Mg content species (e.g., large tropical hyaline foraminifera) vs low Mg species (e.g., temperate small benthic foraminifera) (van Dijk et al., 2017a), and not systematic within the low Mg group. The absence of correlation between Sr/Ca and Mg/Ca values for our low Mg species seems to confirm that the mechanism responsible for the partitioning against Mg is sensitive to other factors than that for Sr incorporation. This has been also hypothesized for the planktonic foraminifer G. sacculifer (e.g., Dueñas-Bohórquez et al., 2011a).

Conclusions

-Growth and survival of Ammonia T6, B. marginata and C. laevigata were unaffected by a pH as low as 7.5 under open marine-like DIC and TA conditions. However, at a normal pH (~8.0), growth of B. marginata and C. laevigata was negatively impacted by low DIC conditions (879 µmol kg -1 ). The absence of such an impact on Ammonia T6 growth demonstrates the high tolerance of this shallow water species to extreme DIC conditions, probably as a result of its variable natural environment. The decoupling of DIC and pH impacts are in line with the transport of CO2 to the site of calcification as proposed in [START_REF] Toyofuku | Proton pumping accompanies calcification in foraminifera[END_REF].

-There was a clear positive correlation between foraminiferal Sr/Ca and the C-system for Ammonia T6 and B. marginata. Sr/Ca ratio in C. laevigata however did not record any C-system dependency. We hypothesized either i) a strong species-specific effect that should be considered in paleo-studies or ii) a poor tolerance to laboratory conditions which resulted in a physiological stress that impacted Sr incorporation.

-Our results confirmed that DIC and/or [HCO3 -] are the driving forces behind Sr incorporation in the calcite of Ammonia T6 and B. marginata. The regression models showed similar slopes, confirming the reproducibility of such complicated experimentations, but different intercepts, suggesting that there is a species-specific effect between these two species that needs to be considered in paleo-applications. These paleo-applications would be limited by the low sensitivity of the Sr/Ca ratio to DIC and/or [HCO3 -].

-The inter-chamber and inter-individual variability of Mg/Ca was larger than that of Sr/Ca for the three cultured species, which is in line with previous studies. No detectable ontogenetic effect was observed in Sr/Ca and Mg/Ca ratios for the last chamber stages of the three studied species. Hence, Sr/Ca is analytically robust, not easily contaminated, and measurements can be made regardless of the chamber stage (within a narrow size range), which can be advantageous for paleo-applications. "Initial" refers to the total performed measurements. "Final" refers to the measurements selected after discarding the profiles with very short counting time (chambers that broke rapidly), with high contamination, and doubtful chambers in terms of calcification (cf. 1n? in Fig. 3). "Not available" refers to the absence of the replicate, and "Not measured" refers to the fact that the measurements were not performed.

Table 3. Linear regression analyses between foraminiferal Sr/Ca and the respective C-system parameters. Listed are the intercepts with the y-axis and the slope together with the associated standard errors (SE) for these parameters, and the p-values, and R 2 of the regression analyses. comparing Ammonia T6 (this study) and Ammonia T6 (Keul et al., 2017) regression models and Ammonia T6 and B. marginata (this study) regression models. , average percentages of specimens with full cytoplasm) and growth (i.e., average percentages of specimens that calcified i new chambers) for each species, all replicates and conditions combined. (b; c) Average survival and growth for each species and each replicate, all conditions combined. Rnet, R1, R2 and R3 are the used pseudo-replicates (Fig. 1). Aq corresponds to specimens found at the end of the experiment outside of the glass vials in the aquarium seawater. The number of data points (i.e., specimens) is indicated on top of the histograms. The slight difference in the number of data points (indicated on top of the histograms) between survival and growth data is due to the loss of some specimens between the two manipulations. The absence of data is either due to the initial absence of the replicate or the non-processing of the data. The pairwise significant differences (p< 0.05) are indicated with a bracket and an asterisk (Kruskal Wallis test). ] and DIC for our study and the study of Keul et al. (2017). Linear regression parameters of the calibration equations from the study of Keul et al. (2017) were recomputed from raw data because of an error in the publication. The dotted lines represent 95% confidence level.

  [START_REF] Guamán-Guevara | Impacts of ocean acidification on intertidal benthic foraminiferal growth and calcification[END_REF] report increased mortality of Elphidium williamsoni under low pH conditions (7.3≤pH≤7.7), and[START_REF] Charrieau | Decalcification and survival of benthic foraminifera under the combined impacts of varying pH and salinity[END_REF] further document severe decalcification for Ammonia sp. and Elphidium crispum (7.25≤pH≤7.5).

  laevigata). Such a drastic species-specific effect would be an issue for paleo-reconstructions based on foraminiferal Sr/Ca since typically culture calibrations are based on very few species and the results are extrapolated to other species hypothesizing that, although the absolute values change, the relative response to the environmental parameter would be similar. Based on that, Mojtahid et al. (2017) hypothesized that the low Sr/Ca in C. laevigata recorded during the last Heinrich event (~16.5 cal ka BP) indicated a change in water mass carbonate chemistry in the northeast Atlantic. Based on our findings, this hypothesis might not be valid. Instead, the recorded anomaly in Sr/Ca in C. laevigata might indicate a small-scale change of Sr/Casw, deviating from seawater Sr conservative behavior (e.g., Lebrato et al., 2020). More experimental and core top studies involving C. laevigata should help elucidating the observed

  , meaning a high discrimination against Zn and Mg by exerting perhaps a strong biological control. Moreover, van Dijk et al. (2017b) showed that Zn/Ca in Ammonia T6 decreased with increasing [CO3 2-]. Because we used a similar decoupled experiment as van Dijk et al. (2017b), this discrepancy can partly be explained by the fact that Zn concentration of the culture media in the study of van Dijk et al. (2017b) was increased by ~15 times compared to open ocean seawater [Zn]

Figure 1 .

 1 Figure1. Schematic of the experimental design. Four climate-controlled chambers with four concentrations of atmospheric pCO2 (180 ppm, 410 ppm, 1000 ppm, 1500 ppm) were used. Temperature and humidity were set respectively at ~12 °C and ~90 %. In each chamber, two aquaria corresponding to set-up A (DIC-stable manipulation) and set-up B (pH-stable manipulation) were present. In each aquarium, we put several pseudo-replicates of specimens of the three cultured species. Cf. Material and methods for the details.

Figure 2 .

 2 Figure 2. Survival and growth. (a) Average survival (i.e., average percentages of specimens with full cytoplasm) and growth (i.e., average percentages of specimens that calcified i new chambers) for each species, all replicates and conditions combined. (b; c) Average survival and growth for each species and each replicate, all conditions combined. Rnet, R1, R2 and R3 are the used pseudo-replicates (Fig.1). Aq corresponds to specimens found at the end of the experiment outside of the glass vials in the aquarium seawater. The number of data points (i.e., specimens) is indicated on top of the histograms. The slight difference in the number of data points (indicated on top of the histograms) between survival and growth data is due to the loss of some specimens between the two manipulations. The absence of data is either due to the initial absence of the replicate or the non-processing of the data. The pairwise significant differences (p< 0.05) are indicated with a bracket and an asterisk (Kruskal Wallis test).

Figure 3 .

 3 Figure3. Total percentages of specimens that calcified 1 to 11 new chambers in each condition for Ammonia T6, B. marginata and C. laevigata respectively, all replicates combined. Growth refers to the percentages of specimens that calcified i new chambers. "1?" indicates a doubt in the determination of the newly calcified chamber. Be aware of the different scaling between the graphs. The number of data points is indicated on top of the histograms.

Figure 4 .

 4 Figure 4. Foraminiferal Sr/Ca (mmol mol -1 ) versus individual C-system parameters for Ammonia T6, B. marginata and C. laevigata. Squares represent the mean values. Lines represent linear regressions. Ammonia T6 data from the study ofKeul et al. (2017) are plotted with Ammonia T6 from this study. The error bars in the data ofKeul et al. (2017) represent the maximum dispersion of points around the means (triangles). Dashed lines represent linear regressions.

Figure 5 .

 5 Figure 5. Box plots showing Sr/Ca and Mg/Ca ratios (mmol mol -1) measured in foraminiferal calcite as a function of the position of the newly formed chambers; "n-6" being the oldest and "n" the last chamber formed. The number of data points is indicated on top of the histograms. The pairwise significant differences (p< 0.05) are indicated with a bracket and an asterisk (Kruskal Wallis test).

Figure 6 .

 6 Figure 6. Sr/Ca and Mg/Ca (mmol mol -1 ) measured in the calcite formed in the experiment of the three cultured benthic species. a) all data points from the eight treatments and different pseudo-replicates are plotted together, b) average values per treatment. Error bars correspond to standard errors. (c-e) Sr/Ca and Mg/Ca plotted for each replicate and each cultured species. The number of data points is indicated on top of the box plots. The pairwise significant differences (p< 0.05) are indicated with a bracket and an asterisk (Kruskal Wallis test).

Figure 7 .

 7 Figure 7. Linear regression analyses. a) radar chart representing the R 2 values of the regression analyses between foraminiferal Sr/Ca and respective C-system parameters. The R 2 values reported byKeul et al. (2017) are also plotted, b) Calibration equations of foraminiferal Sr/Ca vs [HCO3 -] and DIC for our study and the study ofKeul et al. (2017). Linear regression parameters of the calibration equations from the study ofKeul et al. (2017) were recomputed from raw data because of an error in the publication. The dotted lines represent 95% confidence level.

  measured:24 Mg,25 Mg,27 Al,43 Ca,44 Ca, 66 Zn,88 Sr, and138 Ba and used to calculate elemental concentrations using average cosmic isotope ratios. The quality of trace elemental analyses of the foraminiferal calcite was further checked using the matrix-matched calcite standardMACS- 3 (MicroAnalytical Carbonate Standard, United States Geological Survey, 2012), and one NIOZ foraminifera in-house standard, NFHS, measured every 10-15 samples. Standardization was performed by bracketing analyses of SRM NIST 610 associated with each block of 10-15 samples. Raw counts were converted to element concentrations using the Glitter data reduction software developed by the GEMOC ARC National Key Centre and CSIRO and assuming 56 wt% CaO in CaCO3. Repeatability, expressed as the relative standard deviation (RSD), of all MACS-3 analyses (average of the four sessions; 44 data points), was 3 % for 25 Mg/Ca, 6 % for Zn/Ca, 3 % for Sr/Ca, and 3 % Ba/Ca, and accuracies were 99 %, 128 %, 104 %, and 105 % respectively (Supplementary material S4b; c).

The newly formed chambers of B. marginata and C. laevigata were ablated and measured at NIOZ using LA-ICP-MS. The system used consists of a NWR193UC (Elemental Scientific Lasers) with an ArF Excimer laser (Coherent Excistar XS) with deep UV

(193 nm

  2-] (52±8 -457±18 µmol kg -1 ), and Ωcc (1.2 ±0.2 -10.9±0.7) (Table1). Srsw, Mgsw, Casw, Sr/Casw and Mg/Casw values were stable in each aquarium and similar between the eight aquaria (81.8±0.5 -83.1±0.6 µmol kg -1 , 51.8±0.3 -52.8±0.4 mmol kg -1 , 9.9±0.05 -10.1±0.05 mmol kg -1 , 8.23 -8.31 mmol mol -1 and 5199 -5346 mmol mol -1 , respectively) (Table

  Statistical analyses showed however a significant difference (p = 0.01) between Sr/Ca values in Rnet and R1 for Ammonia T6 but since there was no significant difference between Rnet and R2, we cannot conclude on an effect of the covering net. For C. laevigata, Sr/Ca values in Rnet were significantly different from R1 and R2 but with p values close to 0.05, meaning that the difference was very low. For Mg/Ca ratios in the same species, R2 was significantly different from Rnet and R1.

	The variability of
	Sr/Ca in Ammonia T6, B. marginata and C. laevigata (relative standard errors of 1.4, 1.3 and 2
	% on average, respectively) was much smaller than the variability observed in Mg/Ca (relative

standard errors of

19.1, 11.3 and 12.4 % on average, respectively)

.

Figures

6 (c-e

) presents box plots of Sr/Ca and Mg/Ca ratios per species and pseudo-replicate, considering the eight experimental conditions together. In general, the results displayed similar values between replicates for each species.

Table 1 .

 1 Average values of the C-system parameters, climatic chambers parameters, Sr, Ca and Mg concentrations in seawater, Sr/Ca, Mg/Ca in both seawater and foraminifera and DSr and DMg (calculated partition coefficients) for each experimental condition. SD: Standard deviation (1SD). SE: Standard error. SD values of the C-system and seawater parameters are based on the dispersion of datasets (i.e., measurements performed every 1-2 days) relative to the mean in each experimental condition. SE values for El/Ca concentrations in foraminifera represent standard errors of the mean of the dataset (i.e., all final laser ablation measurements; cf. Table2) in each experimental condition and for each species.

Table 2 .

 2 Number of LA-ICP-MS Sr/Ca measurements per species, condition and replicate.

Table 4 .

 4 Results of ANCOVA test analyses between foraminiferal Sr/Ca and [HCO3 -]

Table 5 .

 5 Spearman

		Sr/Ca Mg/Ca Sr/Ca Mg/Ca	Sr/Ca	Mg/Ca
	r	0.189 -0.136 0.026	-0.074	0.081	-0.119
	p	0.0002 0.009	0.664	0.216	0.353	0.173

's (non-parametric) rank-order correlation coefficients (r) and the p-values between foraminiferal El/Ca (individual chamber analyses) and the position of the newly formed chambers (from the oldest to the latest formed), all treatments combined.

Ammonia T6

(n-6 to n chamber)

B. marginata (n-3 to n chamber)

C. laevigata (n-2 to n chamber)

Acknowledgments

The study was funded by the CNRS-INSU-LEFE/IMAGO-CYBER program (STING project), the Region Pays de Loire programs (Rising Star project TANDEM) and the Ecotron Ile De France incentive research projects (ECOFOR project). Salary and research support for the PhD student (Second author) were provided by the French Ministry of Higher Education and Research. The authors warmly thank the Ecotron Ile De France team for their extraordinary commitment and for giving us access to laboratory facilities. We acknowledge the support for the Captain and the crew of R/V Skagerak and the staff at Kristineberg Marine Station. We thank A. Elofsson and K. Ljung for help with the fjord sampling. H.L. Filipsson acknowledges support from the Swedish Research Council VR (2017-04190). We thank F. Rihani and S. Quinchard (University of Angers) for their help with foraminifera picking and experimental work, C. La (University of Nantes) for her assistance with LA-ICPMS analyses, Y. Coupeau (IFREMER) for providing us with seawater from the Bay of Biscay, and Nina Keul for

providing us the raw data of the publication Keul et al. (2017). Raw data is available on SEANOE data repository (https://doi.org/10.17882/89623).

Supplementary material S5. SEM pictures of the three studied species in various pseudoreplicates, at the initial state (before experimentation), and at B180, A410 and A1500 conditions. All pictures represent living specimens that did not calcify new chambers (because most calcifying specimens were used for LA-ICPMS analyses) except for the Ammonia T6 specimens outlined in yellow. The dark spots on some specimens represent the food gathered around them. Supplementary material S6. Box plots showing Sr/Ca (mmol mol -1 ) measured in foraminiferal calcite as a function of the position of the newly formed chambers in each experimental condition; "n-6" being the oldest and "n" the last chamber formed. The number of data points is indicated on top of the histograms. Supplementary material S7. Plots of DSr values vs [HCO3 -] and Mg/Cacc comparing our data of Ammonia T6 with data reported in different culture studies using Ammonia spp. and some planktonic species.

Supplementary material S8. Additional discussion on Sr partition coefficient (DSr).

To consider the differences in Sr/Casw, we used DSr to compare Sr incorporation in our cultured foraminifera with the existing literature. For B. marginata and C. laevigata, no studies to our knowledge report DSr values. However, the mean DSr values for these two species in our experiment (0.14-0.18 and 0.13-0.15 respectively) were consistent with the values (0.10-0.20) reported for other deep-sea species (Uvigerina spp., Cibicidoides spp., Melonis barleeanum and Hoeglundina elegans) collected from core top studies [START_REF] Mccorkle | Evidence of a dissolution effect on benthic foraminiferal shell chemistry: δ 13 C, Cd/Ca, Ba/Ca, and Sr/Ca results from the Ontong Java Plateau[END_REF][START_REF] Elderfield | A biomineralization model for the incorporation of trace elements into foraminiferal calcium carbonate[END_REF]Rosenthal et al., 1997;Reichart et al., 2003;[START_REF] Lear | A Cenozoic seawater Sr/Ca record from benthic foraminiferal calcite and its application in determining global weathering fluxes[END_REF]Yu et al., 2014).

Mean DSr values of Ammonia T6 in the present study (~ 0.14-0.20) were overall in the range of several published values for the Ammonia group tepida (~ 0.15-0.20) (e.g., Dissard et al., 2010aDissard et al., , 2010b;;Raitzsch et al., 2010;Dueñas-Bohórquez et al., 2011;de Nooijer et al., 2014;Langer et al., 2016). These are however significantly lower that the published values of Keul et al. (2017) (~ 0.17-0.29 mmol mol -1 ) and van Dijk et al. (2017b) (~ 0.21-0.31 mmol mol -1 ) using the same experimental set-up, and the same molecular type of Ammonia (Supplementary material S7). Because we record similar Sr/Cacc values (Fig. 7b) than Keul et al. (2017), this difference is mathematically explained by the fact that their culture media had a lower Sr/Casw (Sr/Casw ~ 4.4 -6.9 mmol mol-1) compared to the present study and the studies of Dissard et al. (2010) (Lebrato et al., 2020). Experimental studies show that Sr/Ca in foraminifera linearly correlates with Sr/Casw (e.g., Raitzsch et al., 2010;Langer et al., 2016). From this linear relationship, we would expect lower Sr/Cacc values in the study Keul et al. (2017) compared to our study and the previous publications, and constant DSr values, whereas we obtain exactly the opposite (i.e., similar Sr/Cacc and different DSr values). Two potential factors could explain these differences:

i) The higher temperatures used in Keul et al. (2017) (26°C) and van Dijk et al. (2017b) (25°C) experiments, might have accelerated the incorporation of Sr into foraminiferal calcite leading to the observed higher DSr values. This would be in line with Lorens (1981) emphasizing that temperature increases calcite growth rate that allows Sr to incorporate more easily. Furthermore, our Ammonia T6 DSr values were coherent with several studies culturing Ammonia spp. between 10 and 18°C (Dissard et al., 2010a(Dissard et al., , 2010b;;Raitzsch et al., 2010;Dueñas-Bohórquez et al., 2011), a temperature range close to our setting (Supplementary material S7). However, other culture studies show a weak/undetectable link between Sr/Ca and temperature (e.g., [START_REF] Russell | Effects of seawater carbonate ion concentration and temperature on shell U, Mg, and Sr in cultured planktonic foraminifera[END_REF][START_REF] Kısakürek | Controls on shell Mg/Ca and Sr/Ca in cultured planktonic foraminiferan, Globigerinoides ruber (white)[END_REF]Diz et al., 2012;[START_REF] De Nooijer | Ba incorporation in benthic foraminifera[END_REF][START_REF] Geerken | Impact of salinity on element incorporation in two benthic foraminiferal species with contrasting magnesium contents[END_REF], and for Ammonia T6 specifically, the culture study of Yu et al. (2019) (referred to as A. aomoriensis clade T6) report that temperature has no significant effect on Sr/Ca, and salinity has only a weak effect (~1% per salinity unit).

iii) We could suspect a possible effect of the shell Mg content that distort the calcite lattice and weakened the discrimination against Sr (e.g., Mewes et al., 2015;Langer et al., 2016;Yu et al., 2019). Yet, our results show no significant correlation between Mg/Cacc and Sr/Cacc ratios for the three cultured species as discussed before (Fig. 6a; b), and no correlation seems to exist either for Ammonia spp. between DSr values and Mg/Cacc (Supplementary material S77). This needs to be further experimented in order to better emphasize biomineralization processes in foraminifera for Sr incorporation. Supplementary material S9. Foraminiferal Mg/Ca, Zn/Ca and Ba/Ca (mmol mol -1 ) versus individual C-system parameters for Ammonia T6 and B. marginata. Lines represent linear regressions.