Contrasting trends in element incorporation in hyaline and miliolid foraminifera 1

8 We analyzed trends in element incorporation between hyaline (perforate) and miliolid (imperforate) 9 foraminifera in order to investigate processes involved in calcification affecting element incorporation 10 into foraminiferal carbonate. For both groups, we observed similar trends in element incorporation with 11 pCO2, suggesting there some mechanisms to transports ions to the site of calcification are similar for 12 both calcification pathways, although the impact might be different across species. A previously 13 published trans-membrane transport model assumes foraminifera utilize Ca channels to transport 14 calcium to the site of calcification. These channels are somewhat a-specific, leading to (accidental) 15 transport of other free ions. By modelling the activity of free ions as a function of pCO2, we observed 16 that speciation of some elements (like Zn and Ba) are heavily influenced by the formation of carbonate 17 complexes. This leads to an increase in availability of free Zn and Ba with increasing pCO2, which leads 18 to more transport to the site of calcification and subsequently incorporation in the foraminiferal shell. 19 We further observed that incorporation of the trace elements studied here is positively correlated 20 between the hyaline test building species. This could be due to dissimilar activity and/or selectivity of 21 calcium channels between species, perhaps due to differences in size. For miliolid calcification, part of 22 the calcium is obtained not only through channels but by also included seawater vesicles, which leads 23 to similar element to calcium ratios between species and element partitioning which is more in line with 24 inorganic carbonates. 25 Biogeosciences Discuss., doi:10.5194/bg-2016-402, 2016 Manuscript under review for journal Biogeosciences Published: 27 September 2016 c © Author(s) 2016. CC-BY 3.0 License.


Introduction
On the broadest taxonomic scale, calcareous foraminifera, cosmopolitan unicellular protists, produce tests using either one of two fundamentally different mechanisms.These calcification strategies reflect the evolutionary separation of foraminiferal groups dating back to the Cambrian diversification, from where the imperforate miliolids and perforate hyaline foraminifera, developed independently (Pawlowski et al., 2003).The calcification process of the latter group has been studied more extensively than that of the miliolids (De Nooijer et al., 2014).Although many aspects of perforate calcification remain unsolved, there is consensus that chamber formation takes place extracellularly, but within a (semi-) enclosed space, generally termed the site of calcification (SOC).The first layers of calcite precipitate on an organic matrix (the POS or primary organic sheet) that serves as a template for the calcite layer that forms the chamber wall (Hemleben et al., 1977;Erez, 2003).To promote calcification, foraminifera furthermore need to remove Mg ions and/or protons (Zeebe and Sanyal, 2002) from the seawater entering the SOC.Many larger benthic foraminifera are hyaline species although the amount of Mg in their shells is often more than 10 times higher than that of planktonic and small benthic hyaline species, hence covering a large range in Mg/Ca values.
The calcification strategy of porcelaneous foraminifera is less well studied, which may be partly explained by their limited application in paleoceanography.Porcelaneous foraminifera use a different mode of calcification (Berthold, 1976;Hemleben et al., 1986;Debenay et al., 1998;De Nooijer et al., 2009) and produce shells without pores (hence, the term imperforate) consisting of tablets or needles (Debenay et al., 1998;Erez, 2003;Bentov and Erez, 2006).These calcitic needles (2-3µm) are precipitated intracellularly (Berthold, 1976), after which they are transported out of the foraminifer to form a new chamber (Angell, 1980).At the outer and inner layers of these chambers, the needles are arranged along the same orientation so that they form an optically homogenous surface, giving it a shiny (hence the term 'porcelaneous') appearance.In general the Mg/Ca values of the shells of porcelaneous foraminifera are high.
Remarkably, despite this large biological control, incorporation of minor and trace elements still reflects environmental conditions, in both hyaline and porcelaneous foraminiferal shells.For instance, the Mg/Ca of foraminiferal shells is primarily determined by seawater temperature (Allen and Sanders, 1994;Nürnberg et al., 1996) and seawater Mg/Ca (Chapter 3; Segev and Erez, 2006;Evans et al., 2015;Wit et al., in review).After correcting for the effect of the latter (if necessary) the use of foraminiferal Mg/Ca has been validated by its wide application as paleothermometer (Elderfield and Ganssen, 2000;Lear et al., 2000).Insight in vital effects (Erez, 2003) and inter-specific differences in trace element incorporation (Bentov and Erez, 2006;Toyofuku et al., 2011;Wit et al., 2012) is needed for making the Mg/Ca thermometer more robust.Systematic offsets between different species, interdependence of trace elements incorporated (Langer et al., in press.)and the different response of element incorporation on Biogeosciences Discuss., doi:10.5194/bg-2016-402, 2016 Manuscript under review for journal Biogeosciences Published: September 2016 c Author(s) 2016.CC-BY 3.0 License.element speciation (Chapter 6; Keul et al., 2013;Wit et al., 2013), potentially provides useful clues for determining which processes play an important role in the biomineralization pathways.
Here we present the results from a controlled growth experiment for which we used several (intermediate-and high-Mg) hyaline and miliolid species and an inter-species comparison of trace elements.We assessed the impact of bio-calcification on element incorporation as a function of pCO2 in order to contrast the impact of different calcification strategies.During foraminiferal calcification, incorporation of certain elements or fractionation of certain isotopes is shown to depend on the carbonate system, e.g.U/CaCALCITE (Russell et al., 2004;Keul et al., 2013) et al., 2000;Chapter 6) and δ 11 B to pH (Sanyal et al., 1996).Species-specific differences in partitioning and fractionation most likely primarily reflect differences in calcification strategy.Offsets are largest between hyaline and miliolid species, due to their fundamentally different calcification strategies (see for a summary, Toyofuku et al., 2011).Differences in chemical composition and their dependency on environmental variables can hence be used to identify key processes in miliolid and hyaline calcification.
We cultured eight benthic foraminiferal species (4 hyaline and 4 porcelaneous) under four different pCO2 conditions, analyzing incorporation of Mg, Sr, Na, Zn and Ba.Results are combined and compared with literature data, to identify processes involved in calcification.

Biogeosciences
We used an adapted version of the culture set-up descripted in Chapter 7. In short, four barrels each containing 100 L of seawater (5µm filtered), were connected to a Li-Cor CO2/H2O analyzer (LI-7000), to regulate the CO2 level in the barrels' head space.The set levels were maintained by addition of CO2 and/ or CO2-scrubbed air according to the monitored pCO2.The set-points for pCO2 were 350 (A), 450 (B), 760 (C) and 1400 (D) resulting in four batches of seawater differing only in their inorganic carbon chemistry.Salinity (34.0±0.2) was monitored with a salinometer (VWR CO310).The fluorescent compound calcein (Bis[N,N-bis(carboxymethyl)aminomethyl]-fluorescein) was added to the culture media (5 mg/L seawater) to enable determination of newly formed chambers during the culture experiment (Bernhard et al., 2004).Short-term exposure (<three weeks) to calcein has no detectable impact on the physiology of benthic foraminifera (Kurtarkar et al., 2015), and the presence of calcein has no effect on the incorporation of Mg and Sr in foraminiferal calcite (Dissard et al., 2009).Culture media was stored air-free in portions of 250 ml in Nalgene bottles with teflon lined caps at 4°C until further use.
Foraminifera were divided over the different treatments in duplicate and placed in 70 ml Falcon ® tissue bottles with gas-tight caps in a thermostat set at 25°C (Fig. 1).The thermostat was monitored by a temperature logger (Traceable Logger Trac, Maxi Thermal), monitoring the temperature every minute.
To create uniform light conditions, the thermostat was equipped with two LED shelfs, which resulted in high light conditions 12 hr/12hr.Culture media was replaced every four days, to avoid build-up of organic waste and to obtain stable seawater element concentrations and carbon chemistry.Foraminifera were fed after every water change with 0.5 ml of concentrated freeze-dried Dunaliella salina cells, prediluted with the corresponding treatment seawater.After 21 days, the experiment was terminated.
Foraminifera were rinsed three times with de-ionized water, dried at 40°C and stored in micropaleontology slides until further analysis at the Royal Netherlands Institute for Sea Research (NIOZ).

Seawater carbon parameters
At the start and termination of the experiment, 125 ml samples of the seawater at each of the different experimental conditions were collected to analyze dissolved inorganic carbon (DIC) and total alkalinity (TA) on a Versatile INstrument for the Determination of Titration Alkalinity (VINDTA) at the CNSI.
Using the measured DIC and TA values and the software CO2SYS v2.1, adapted to Excel by Pierrot et al. (2006) the other carbon parameters (including [CO3 2-] and Ωcalcite) were calculated.For this we used the equilibrium constants for K1 and K2 of Mehrbach et al. (1973), refitted by Dickson and Millero (1987) (Table 1).

Seawater element concentrations
At the start and end of the experiment and during replacement of the culture media, subsamples were collected in duplo using 50 ml LDPE Nalgene bottles and immediately frozen at -80⁰C.After transportation to the NIOZ, melted samples were acidified with 3 times Quartz distilled HCl to pH ~1.8 and the seawater composition of the samples was analyzed on an Element 2 sector field double focusing mass spectrometer (SF-ICP-MS) run in medium resolution mode.IAPSO Standard Seawater was used as a drift monitor.Analytical precision (relative standard deviation) was 3% for Ca, 4% for Mg, 1% Na, 1% for Sr and 5% Ba.We obtained average values of 5.25±0.06mol/mol for Mg/Ca, 44.6±0.6 mol/mol Na/Ca, 8.63±0.05mol/mol for Sr/Ca, and 9.04±0.47for µmol/mol Ba/Ca.
A subsample was analyzed using a commercially available pre-concentration system, SeaFAST S2.
With the SeaFAST system elements with low concentrations can be pre-concentrated to values above detection limit of the SF-ICP-MS.Accordingly, we measured Cd, Pb U, B, Ti, Mn, Fe, Co, Ni, Cu, and Zn.In short, 10ml of sample was mixed with an ammonium acetate buffer to pH 6.2 and loaded on a column containing NOBIAS chelating agent.After rinsing the column with a diluted ammonium acetate buffer the metals were eluted in 750 µl of quartz distilled 1.5 M HNO3 before being quantified on the SF-ICP-MS.Here we use the Zn data only, as this was analyzed in the foraminifera well.Analytical precision (relative standard deviation) was 5% for Zn.We obtained average values 15.3±0.5 µmol/mol for Zn/Ca for all treatments.Although these values are clearly above natural open ocean values, the concentrations are very uniform between treatments and when comparing start and end of the experiments.The contamination with Zn might hence have occurred already when filling the culture setup with the waters from the bay adjacent to the culture facility.In any concentrations are well below values considered toxic (Nardelli et al., 2016).

Cleaning methods
After termination of the experiment, foraminiferal shells were cleaned following an adapted version of Barker et al. (2003).Per treatment duplicate, all foraminifera were transferred to 10 ml PE vials.To each vial, 10 mL 1% H2O2 solution (buffered with 0.5M NH4OH) was added to remove organic matter.The vials were heated for 10 minutes in a water bath at 95 °C, and placed in an ultrasonic bath for 30 seconds (degas mode, 80kHz, 50% power), after which the oxidizing reagent was removed.These steps (organic removal procedure) were repeated five times.Foraminiferal samples were rinsed five times with ultrapure water, after which the vials were stored overnight in a laminar flow cabinet at room temperature to dry.Dried foraminifera were placed on double sided tape on LA-ICP-MS stubs.Pictures

LA-ICP-MS
Element concentrations of individual fluorescent chambers were analyzed by Laser Ablation-ICP-MS (Reichart et al., 2003;Van Dijk et al., in review).To determine foraminiferal element concentrations, the laser system (NWR193UC, New Wave Research) at the Royal NIOZ was equipped with a 2-volume cell 2 (New Wave Research), characterized by a wash-out time of 1.8 seconds (1% level) and hence allowing detection of variability of obtained element to Ca ratios within chamber walls.Single chambers were ablated in a helium environment using a circular laser spot with a diameter of 80 μm (M.vertebralis) or 60 μm (other species).We ablated all calcein-stained chambers twice, except for the first 1-2 chambers that formed during the experiment to avoid contamination of calcite of chambers formed prior to the experiments that may be overlapped by the first labelled chambers (Fig. 2).
All foraminiferal samples were ablated with an energy density of 1±0.1 J/cm -2 and a repetition rate of 6 Hz.The resulting aerosol was transported on a helium flow through an in house build smoothing device, being mixed with a nitrogen flow (2 L/min), before entering the quadrupole ICP-MS (iCAP-Q, Thermo Scientific).Monitored masses included 7 Li, 11 B, 23 Na, 24 Mg, 25 Mg, 27 Al, 43 Ca, 44 Ca, 66 Zn, 88 Sr and 137 Ba.
Contrary to 67 Zn and 68 Zn, 66 Zn is free of interferences when measuring calcium carbonate and SRM NIST glass standards (Jochum et al., 2012).Potential contamination or diagenesis of the outer or inner layer of calcite was excluded by monitoring the Al signal.At the start of each series, we analyzed SRM NIST612 and NIST610 glass standard in triplicate (using an energy density of 5±0.1 J/cm-2), JCt-1 (coral carbonate) and two in-house standards, namely NFHS (NIOZ Foraminifera House Standard; Mezger et al., in review) and the Iceland spar NCHS (NIOZ Calcite House Standard).We further analyzed JCp-1 (Giant clam) and MACS-3 (Synthetic Calcium Carbonate) at the start of each series, and to monitor drift after every ten samples.All element to calcium ratios were calculated with an adapted version of the MATLAB based program SILLS (Guillong et al., 2008).SILLS was modified to evaluate LA-ICP-MS measurements on foraminifera, allowing import of Thermo Qtegra software sample list, laser data reduction and laser LOG files.Major adaptions include improved automated integration and evaluation of (calibration and monitor) standards, quality control report of the monitor standards and export in element to calcium ratios (mol/mol).Calibration was performed against the MACS-3 carbonate standard, with 43 Ca as an internal standard and we used the multiple measurements of MACS-3 for a linear drift correction.Relative analytical precision (relative standard deviation (RSD) of all MACS-3 analyses) is 3% for 23 Na, 3% for 24 Mg, 3% for 25 Mg, 4% for 66 Zn, 3% for 88 Sr and 3% for 137 Ba.In total, 961 analyses were performed on 251 specimens covering eight species cultured in four experimental conditions (see  ]) using a two-sided T-test with 95% confidence levels.This also allows for the calculation of 95% confidence intervals over the average per treatment.Pairwise comparisons were made for per E/Ca per species and culture conditions using ANOVA.Groups that showed significant difference were assigned different letters.When comparing partition coefficients to other studies, E/CaSW data was, in some studies, not measured.In these cases, we used average seawater E/CaSW to calculate DE (see also supplementary Table 1).

Inter-species differences in element incorporation
In Table 3 we present all the elemental data for the eight species investigated in this controlled pCO2 culture experiment.Mg/CaCALCITE of Mg in hyaline species varies between 25.9-141.3mmol/mol Mg/Ca.In contrast, Mg/CaCALCITE of miliolid species ranges from 121.3-149.3mmol/mol.This large spread in foraminifera E/Ca of hyaline species is also observed for Sr (1.7-3.1 mmol/mol), Na (3.4-19.5 mmol/mol), Zn (9.0-97.0µmol/mol) and Ba (2.7-20.1 µmol/mol), while miliolids only vary over a narrow range (Sr = 2.0-2.2mmol/mol; Na = 3.8-5.8mmol/mol; Zn = 53.0-140.8µmol/mol; Ba = 18.0-29.0µmol/mol).When comparing Mg incorporation to that of the other elements studied here (Ba, Zn, Sr and Na) between species (treatment B; Table 3), we observe a positive relation between DSr (p<0.0025),DNa (p<0.0005),DBa (p<0.05) and DZn (p<0.005) for hyaline species (Table 4).In general hyaline species are enriched similarly in all elements (Fig. 3).Compared to porcelaneous species, the hyaline shell building species which incorporate the most Mg (>100 mmol/mol Mg/Ca) incorporate more Na, and Sr, while incorporating less Zn and Ba.Element incorporation across miliolid species is less variable then observed for hyaline species and in general partition coefficients for these species seem closer to inorganic values (Fig. 3).Including data from literature (both culture and field calibrations; see supplementary Table S1), preferable in which both Mg/Ca and at least one other element (Na, Sr, Ba or Zn) is measured, shows that the relation based on the Caribbean species studied here is also more general applicable when including more species (DSr = p<0.005;DNa = p<0.0005);DBa = p<0.005;DZn = p<0.01),even though this compiled data (labeled 'All studies' in Table 4) covers a wide range in environmental and experiment conditions.

Element/Ca as a function of ocean acidification
In both porcelaneous and hyaline species we find an increase of Zn/CaCALCITE and Ba/CaCALCITE with pCO2, while foraminiferal Sr/Ca, Mg/Ca and Na/Ca remain similar across the experimental conditions (Fig. 4 and Table 5).Sensitivity of both foraminiferal Zn/Ca and Ba/Ca to changes in seawater pCO2 differs between the studied porcelaneous and hyaline species.When pCO2 changes from 350 to 1200 ppm, Zn/Ca of hyaline foraminifera increase by a factor of 3.7 (A.carinata) or 4.5 (A.gibbosa) while miliolid foraminiferal Zn/Ca increases only by 1.3 (M.vertebralis), 1.8 (A.angulatus) and 2.1 (L.bradyi).Also sensitivity of foraminiferal Ba/Ca to the same change in pCO2 shows a similar pattern, with Ba/Ca of hyaline species increasing by a factor of 3.6 (A.carinata) or 3.7 (A.gibbosa), while miliolid species increase Ba/Ca only with a factor of 1.8 (M.vertebralis), 1.6 (A.angulatus) or 2.1 (L.bradyi).

Trends in element incorporation
Both miliolid and hyaline foraminifera promote calcification by increasing their internal pH (De Nooijer et al., 2009).Still, they might use different mechanisms to take up the ions (Ca 2+ and CO3 2-) necessary for chamber formation, which is reflected in the different trends observed here.Element incorporation in hyaline foraminifera is highly interdependent, i.e. species with increased Mg content also incorporate more Sr, Na, Ba and Zn (Fig. 3).This observation suggests that uptake of all these elements is controlled by the same process, which may be the transmembrane transport of calcium ions to the site of calcification.Such transport likely involves Ca 2+ channels (Nehrke et al., 2013), capable of transferring other ions, like e.g.Mg, Sr and Na (Hess and Tsien, 1984;Allen and Sanders, 1994;Sather, 2005).This may result in an interdependence between all these elements studied such as observed here for the hyaline species if the selectivity for Ca 2+ of these channels vary between species.In contrast, miliolid species, building porcelaneous shells show much less inter-species variation in element incorporation and ratios between incorporated elements is thus relatively similar between species (Fig. 3).This may be explained by calcification from an internal reservoir, such as intracellular vacuoles containing (modified) seawater (Hemleben et al., 1986;Erez, 2003).The fact that the Mg partitioning in this foraminiferal group is similar to the inorganic partition coefficient may indicate that the carbonate is directly precipitated from seawater, without major removal of Mg 2+ ions.The relative similarity in partition coefficients of other elements between miliolid species are generally in line with an inorganiclike calcite precipitation, with only minor alteration of the elemental composition of the calcifying fluid by ion channels.

Effect of ocean acidification on Element/Ca
For neither miliolid nor hyaline species, foraminiferal Mg/Ca, Na/Ca and Sr/Ca systematically change with pCO2.The impact of pH (and/or [CO3 2-]) on Mg/CaCALCITE and Sr/CaCALCITE in foraminifera has been the subject of discussion (e.g., Elderfield et al., 1996;Dissard et al., 2010).In low-Mg benthic species, both Mg/CaCALCITE and Sr/CaCALCITE do not seem to depend on inorganic carbon system parameters, e.g.pH or [CO3 2-] ( Allison et al., 2011;Dueñas-Bohórquez et al., 2011).However, for several planktonic species pH does influence Mg/CaCALCITE and Sr/CaCALCITE (Lea et al., 1999;Russell et al., 2004;Evans et al., 2016).The effect of pH on Sr/CaCALCITE might be explained via increased growth rates due to pH-associated changes in [CO3 2-] ( Dissard et al., 2010).However, due to the limited experimental set-up, we are not able to disentangle the effects of the different carbon parameters in this study.Still, here we show that incorporation of Mg, Sr and Na of the selected larger benthic hyaline and miliolid foraminifera are not significantly impacted when cultured over a range of pCO2 and thus [CO3 2- ] and pH values.Observed offsets in studies using acid titration (Lea et al., 1999;Russell et al., 2004;Dueñas-Bohórquez et al., 2011;Evans et al., 2016) to alter the carbonate system might be related to changes in alkalinity rather than pCO2 or DIC.In the experimental setup here alkalinity was kept constant between the different treatments, but pH, DIC and carbonate ion concentration varied as a function of pCO2.
In contrast, foraminiferal Zn/Ca and Ba/Ca are significantly impacted by pCO2 for all species studied here (Table 5; Fig. 4).Although Hönisch et al. (2011) suggested that the impact of carbonate chemistry on Ba incorporation is negligible, their data does suggest a trend over the same interval in pH as studied here.In hyaline foraminifera, Zn/Ca and Ba/Ca increases more as a function of pCO2 (factor of 3.7-4.5 and 3.6-3.7,respectively when pCO2 increases from 350 to 1200 ppm) compared to the miliolid species (1.3-2.1 and 1.6-2.1 times, respectively).In the culture set-up used, increasing pCO2 increases DIC, reduces pH and thereby decreases seawater [CO3 2-].Speciation of Zn, Ba and also other elements, like U (Keul et al., 2013), is primarily controlled by seawater [CO3 2-].Using the PHREEQC (Parkhurst and Appelo, 1999) and the standard llnl database, the speciation of all elements studied here (Mg, Na, Sr, Zn and Ba) for our different seawater treatments were modelled.We observed a decrease of free ions (Zn 2+ and Ba 2+ ) and an increase in Ba and Zn carbonate complexes (BaCO3 0 and ZnCO3 0 ), with increasing pCO2 (Fig. 5), while the activity of Mg 2+ , Na + and Sr 2+ remained unaffected.This suggests that element incorporation in foraminiferal calcite might be depending on the bioavailability of free ions, which in the case of Ba and Zn, changes with pCO2.Discuss., doi:10.5194/bg-2016Discuss., doi:10.5194/bg- -402, 2016 Manuscript under review for journal Biogeosciences Published: 27 September 2016 c Author(s) 2016.CC-BY 3.0 License.

Biogeosciences
During inorganic precipitation, carbonate complexes (e.g.MgCO3 0 ) are easily incorporated into the calcite crystal lattice.However, foraminifera build their test from ions available at the site of calcification, which is well separated from the surrounding seawater (De Nooijer et al., 2009).During calcification, Ca 2+ is proposed to be transported from seawater to the SOC via ion channels (Nehrke et al., 2013).This so-called trans-membrane transport (TMT) through Ca 2+ channels has also been found for other marine organisms, including coccolithophores (Gussone et al., 2006).These Ca 2+ channels may not discriminate perfectly between Ca ions and elements like Sr and Ba (Allen and Sanders, 1994), causing accidental transport of these elements into the SOC.How much of a certain element will enter the SOC in this way, depends on 1) the selectiveness of the channels and the characteristics of the transported ions, 2) the element to calcium ratio in the foraminiferal microenvironment and 3) the concentration gradient between seawater and the SOC.For instance, ions such as Mg 2+ are heavily fractionated against during TMT, which is reflected by the low DMg found in most species.The large range in Mg/Ca values in hyaline species suggests that TMT plays an important, but also variable, role in calcification of these species.The availability of some free ions, like Ba and Zn, changes as a function of pCO2 due to the formation of carbonate complexes (Fig. 5).When Zn and Ba form stable complexes with carbonate ions they are no longer available for (sporadic) transport through the Ca 2+ channels, decreasing the availability at the site of calcification and subsequently, incorporation into the foraminiferal calcite (Fig. 6).
In summary, the amount of Zn and Ba available at the site of calcification is proportional to the concentration of the ratio between Ca 2+ and free Zn 2+ and Ba 2+ in the foraminiferal microenvironment.
In turn, the amount of free Zn and Ba ions in seawater is controlled by their respective concentration in seawater concentration, as well as [CO3 2-].Foraminiferal Mg/Ca, Na/Ca and Sr/Ca is not detectably affected by [CO3 2-], since these elements do not form carbonate complexes over the range of [CO3 2-] studied here.

Element incorporation in hyaline species
Between hyaline species, we observe simultaneous increases in all elements incorporated and this trend is confirmed when including published data for other species compiled from previous studies (Fig. 4 and supplementary Table S1).Interestingly, the two hyaline species that are most enriched in all elements studied (Mg, Na, Sr, Zn and Ba) are also the foraminiferal species with the largest average adult test size (H.antillarum and P. acervalis) for which data is available (this study).The other hyaline species, A. carinata and A. gibbosa, have considerably smaller maximum shell sizes and lower Mg/Ca, Sr/Ca, etc. values.Two processes involving these calcium channels could possibly explain the observed size trend in hyaline species.First, larger foraminifera have a smaller surface area to volume ratio and, therefore, proportionally less Ca 2+ channels, assuming the density of these channels per surface area remains similar.This would imply that fewer channels need to transport more ions for a given volume of CaCO3 precipitated, which may in turn, possibly reduce selectivity between Ca 2+ and other divalent cations.
Secondly, a larger foraminifer will need more overall Ca 2+ compared to smaller species for the production of a single new chamber, since the volume of the chamber walls increases with the size of the individual.This increased uptake of Ca 2+ from the microenvironment around the foraminifer, may cause a lower concentration of Ca 2+ in the direct surroundings of the foraminifer compared to the other ions, which may subsequently translate into an increased transport of ions other than Ca 2+ to the site of calcification.
A consequence of these hypotheses is that juvenile or smaller adults should have lower partition coefficients than fully grown adults.Although some studies have shown a size effect for several elements (e.g.Elderfield et al., 2002), other studies show no major effect of size on element partitioning (e.g.Friedrich et al., 2012;Evans and Müller, 2013).The moderate trend observed within species, in comparison to the large differences observed here between species, may indicate that species control channel density per surface area as a function of average shell size of the species.Alternatively, the maximum size of a species may be accompanied by a difference in their calcification mechanism (e.g. the relative contribution of TMT in element uptake) explaining inter-species differences in element partitioning.From an evolutionary point of view the latter explanation seems more likely.

Mechanisms for element uptake in miliolid foraminifera
In contrast to hyaline species, the miliolid species build porcelaneous shells that show much less interspecies variation in element composition (Fig. 3).While hyaline species calcify in a (semi-)enclosed space, miliolids precipitate their calcite intracellularly in vesicles in which they promote calcification by increasing pH (De Nooijer et al., 2009).This suggests that these species calcify directly from seawater (Ter Kuile and Erez, 1987).The fact that the Mg partitioning is close to the inorganic partition coefficient in this foraminiferal group (Fig. 3) reflects that the carbonate is directly precipitated from intracellular seawater, without major alteration of the original [Mg 2+ ].The relative similarity in partition coefficients between different porcelaneous shell building species is in line with primarily inorganic precipitation, with only minor alteration of the elemental composition of the calcifying fluid by ion channels.However, the observed correlation between pCO2 and Ba and Zn (Fig. 4) suggests that Ca channels still play a (modest) role in supplying Ca 2+ to the miliolid SOC.The contribution of Ca 2+ through TMT is likely smaller than in hyaline species, since they already obtain calcium by including seawater in their compared to perforate species explains the observed lower sensitivity of e.g.foraminiferal Zn/Ca and Ba/Ca to changes in seawater [CO3 2-] in miliolid species (Fig. 4).This approximately 2 times lower sensitivity of porcelaneous foraminifera compared to hyaline species suggests that miliolid foraminifera acquire half of the necessary Ca 2+ through Ca-channels, and the other half directly from vacuolized seawater.Element incorporation in miliolid foraminifera will therefore be mainly governed by their respective concentrations in seawater, and to a lesser extent by the selectivity for Ca 2+ / permeability for other ions during TMT.

Conclusions
Trends in element incorporation in larger benthic foraminifera can be explained by a combination of differences in calcification strategy and seawater chemistry.Carbonate ion concentration in seawater determines bioavailability of some ions (e.g.Zn 2+ and Ba 2+ ), which are transported through Ca-channels to the site of calcification.For hyaline foraminifera, we observed increased element incorporation for larger species compared to smaller species, which can be explained by more intense activity of these channels and the relative concentration in seawater during calcification.For miliolid foraminifera, only half of the needed Ca is acquired through these Ca 2+ channels, while the other half is obtained by including small vesicles of seawater, leading to element partitioning to be more in line with inorganic calcite.
were taken of individual foraminifera with a ZEISS Axioplan 2 fluoresence microscope equipped with Biogeosciences Discuss., doi:10.5194/bg-2016-402,2016 Manuscript under review for journal Biogeosciences Published: 27 September 2016 c Author(s) 2016.CC-BY 3.0 License.appropriate excitation and emission optics and a ZEISS Axiocam MRc 5 camera, to assess the number of chambers added during the experiment based on the incorporation of calcein.

548Figure 1 .
Figure 1.Photograph of the culture set-up.Treatment with corresponding set-points are A=350, 549

Figure 3 .
Figure 3. Partition coefficient of Na, Sr, Zn and Ba versus DMg of hyaline (red symbols) and 556