A reappraisal of the vital e ff ect in benthic foraminifera on Mg / Ca ratios : species specific uncertainty relationships

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Introduction
The ratio of magnesium to calcium in the calcite of benthic foraminifera (i.e.test Mg/Ca) is an important tool to reconstruct past bottom water temperatures.From basic thermodynamic principles it follows that the rate of substitution of Mg-ions for Ca in the of foraminiferal Mg/Ca.However, most foraminiferal species produce calcite with a Mg content that is approximately an order of magnitude lower than those from inorganic precipitation experiments (Bentov and Erez, 2006;Morse et al., 2007).This implies that there is a strong biological control on Mg incorporation.This difference in element (and isotope) composition between biologically and inorganically precipitated calcium carbonate is known as the vital effect.The vital effect varies between species, which is indicated by the large differences in Mg incorporation in foraminiferal test carbonate of different species at the same temperature.The variability in Mg/Ca between individual tests of the same species suggests that the biologically-induced offset might not be constant within one species.A number of field and culture studies have, furthermore, shown that the relation between temperature and test carbonate Mg/Ca differs between species and are therefore best described by species-specific calibrations (Lear et al., 2002;Anand et al., 2003;Rathmann et al., 2004;Elderfield et al., 2006;Rosenthal et al., 2011;Toyofuku et al., 2011;Wit et al., 2012).Most calibrations found so far are described by an exponential function linking Mg/Ca and temperature (Eq. 1) where T is the temperature in degrees Celsius and a and b are empirically derived species-specific constants.The pre-exponential constant a, equals the (hypothetical) Mg/Ca at 0 • C and the exponential constant b describes the steepness of the slope with increasing temperature and is often referred to as the sensitivity of the temperature relation.
Here a new Mg/Ca-temperature calibration based on cultured specimens of the benthic foraminifer Bulimina marginata is presented.This species lives in shallow to deep infaunal habitats and is capable of living under sub-oxic conditions.Therefore B. marginata is often found in large abundances at shelf sediments with high organic contents at intermediate water depths (e.g., Jorissen, 1987;Murray, 2006).A Mg/Catemperature calibration for B. marginata would allow the reconstruction of temperatures under sub-oxic conditions.This calibration could, therefore, enable reconstructions Introduction

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Full helping to unravel the paleo-environment during events with limiting oxygen availability.This requires, however, also insight into the factors potentially offsetting such a calibration (e.g. a vital effect) and the effect on the accuracy of this paleo-thermometer.

Methods
Living specimens of the benthic, symbiont-barren foraminifer Bulimina marginata were collected from two stations in the Bay of Biscay (450 m and 600 m deep).Isolated specimens were placed in culture set-ups between 4 and 14 • C at Utrecht University and the University of Angers.Growth was monitored through incorporation of the fluorescent marker Calcein.This compound is a suitable marker for recognizing newly formed calcite (Bernhard et al., 2004), and does not affect the incorporation of Mg and Sr in foraminiferal calcite (Dissard et al., 2009).Two different culture setups were used for this Mg/Ca-T calibration: ( 1) an open system using 250 ml water, of which the seawater was replaced weekly to bi-weekly and (2) a closed system with seawater circulation from a large reservoir (25 l) towards the different experiments (Barras et al., 2010).Seawater from both setups was sampled weekly to monitor temperature, salinity, alkalinity and pH/DIC of the media.Alkalinity, DIC/pH were used to calculate [CO 2− 3 ], using the CO2SYS software (Lewis and Wallace, 1998) (Table 1).
Experiments ran for 2-3 months to maximize the chance of sufficient calcite addition.Specimens were harvested by sieving over a 63 µm mesh with de-ionized water.
After terminating each experiment, specimens were cleaned for 20 min in 5 % NaClO to dissolve organic matter attached to the surfaces of the foraminiferal shells.Afterwards individual foraminifera were rinsed 3-6 times with MilliQ and 2 times with methanol (Utrecht samples) to prepare the samples for trace metal analysis (Barker et al., 2003;Wit et al., 2010;Rosenthal et al., 2011).
Elements were measured, on newly calcified chambers of adult foraminifera, as a ratio to calcium with laser ablation inductively coupled mass spectrometry (LA-ICP-MS), Introduction

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Full   Reichart et al., 2003;and Wit et al., 2010 for a more detailed description).Element/Ca ratios were calibrated against the NIST 610 and an in-house calcite standard, verifying that differences in ablation energy do not affect measured elemental concentrations (Hathorne et al., 2008;Wit et al., 2010).Of all measured data, about 22 % was discarded because ablation profiles were too short (less than 20-30 pulses at 6 Hz), contamination values for Al were to high (> 20 ppm) or the standard deviation of the measurement was too high (> 70 ppm for Mg).High standard deviations in individual measurements are indicative for a heterogeneous distribution of Mg in the foraminiferal test.Elemental ratios with respect to Ca were based on the average of each ablation profile (Fig. 1).Individual foraminiferal Mg/Ca values were based on the average of 1-4 measured test-chambers.Samples were measured for size, in order to assess any ontogenetic effect within the Mg/Ca-temperature calibration, using an ocular with a build in scale bar, which was scaled on a 1 mm slide.Size was determined by measuring the height of each individual B. marginata.

Results
All culture experiments were monitored for stability of temperature, salinity, alkalinity and pH/DIC (Table 1).Individuals of Bulimina marginata calcified 1-4 new chambers in all experiments (Barras et al., 2010).The Mg/Ca of the newly formed calcite was measured by laser ablation ICP-MS (Fig. 1).Values for Mg/Ca ranged from 0.75-2.91mmol mol −1 for individual foraminifera (Table 2).Inter-individual variability is calculated as a standard deviation expressed as a percentage of the average and varied between 5 and 25 %. Figure 2 shows the averaged Mg/Ca values per experiment versus temperature (Table 2).Values for Mg/Ca do not increase with test size (R 2 = 0.03, p > 0.10, Fig. 3).and similar to values for other calcitic hyaline foraminifera (Lear et al., 2002;Anand et al., 2003;Rathmann et al., 2004;Elderfield et al., 2006;Rosenthal et al., 2011).
Analyses by Filipsson et al. (2010) suggested higher Mg/Ca values for B. marginata.Their results, however, were based on laser ablation rastering of the test surface.Since the outermost layer of calcite is commonly enriched in Mg (Fig. 1 and Hathorne et al., 2009), their results may not be representative for the average chamber wall Mg/Ca.Despite the different analytical procedures and much higher absolute Mg/Ca values, the obtained Mg/Ca-temperature calibration of Filipsson et al. (2010) does have a similar sensitivity as the one presented here.

Ontogeny
Since the response of Mg/Ca to T is relatively low, additional impacts (e.g.size effects), may have a relatively large impact on the Mg/Ca-temperature calibration presented here.A positive and significant correlation between oxygen isotope values and size for B. marginata, possibly related to changes in growth rates, has been reported (Barras et al., 2010;Filipsson et al., 2010).An effect of ontogeny on planktonic foraminiferal Introduction

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Full Mg/Ca has also been stipulated (N ürnberg et al., 1996;Wit et al., 2010;Due ñas-Boh órquez et al., 2011a).Ontogenetic effects for benthic foraminiferal Mg/Ca values are generally less well known, although Hintz et al. (2006) reported elevated Mg/Ca values for the mid-life stage of Bulimina aculeata by measuring Mg/Ca on both the whole foraminifer and on micro-dissected chambers.However, the Mg/Ca values reported by Hintz et al. (2006) were exceptionally high (up to 84 mmol mol −1 ), suggesting that a phase with elevated Mg concentrations biased their results.An ontogenetic trend observed in the results of the micro-dissection method should be directly comparable to laser ablation Mg/Ca measurements of individual chambers.The mid-life stage from Hintz et al. (2006) corresponds to the F-2 chambers from this study.Intra-test variability for the experiment at 14 • C was, therefore, tested by using an analysis of variance (ANOVA), because the experiment contained enough data for this analysis.The ANOVA was designed to test whether Mg/Ca values for F-2 were significantly elevated compared to the F, F-1 and F-3 chamber.
In our dataset, Mg/Ca for F-2 is not significantly different (F (3, 13) = 0.071, p > 0.10), nor is there a systematic difference in Mg/Ca with size (200-580 µm, R 2 = 0.03, p > 0.10), indicating that there is no significant size-related impact on the Mg/Ca of B. marginata (Fig. 3).The absence of an ontogenetic effect in Mg/Ca, while a significant effect on oxygen isotopes is recognized (Barras et al., 2010) fits the hypothesis that divalent cations (Ca and Mg) are transported to the site of calcification by a different mechanism as the DIC (Erez, 2003;De Nooijer et al., 2009b;Due ñas-Boh órquez et al., 2011b).variability in Mg/Ca is caused by a combination of (1) variability in culture conditions (e.g.temperature, salinity, seawater Mg/Ca and carbonate ion concentration) and (2) inherent biological effects (i.e. the vital effect).

Variability, low sensitivity and temperature reconstructions
For our results, the first cause of variability in foraminiferal Mg/Ca can be quantified using the measured variability in seawater temperature, carbonate ion concentration, Mg/Ca and salinity (Table 1).For instance, the temperature for the experiment at 6 • C varied with a standard deviation of 0.5 Full It follows that at a low sensitivity this uncertainty in Mg/Ca translates in to a larger temperature uncertainty, while at higher sensitivities the uncertainty is much smaller (Fig. 4).
The second source for the large inter-individual variability is the vital effect, caused by variability in the efficiency and rate of various cell-physiological processes that constitute the calcification pathway (Erez, 2003;Bentov and Erez, 2006;De Nooijer et al., 2009b).The impact of these processes can be estimated by correcting the observed Mg/Ca values for the maximum analytical error and the environmentally-induced offsets calculated above.If the vital effect would be zero, every measured foraminiferal Mg/Ca value would fit the calibrated regression line (Fig. 5).Although impact of the vital effect can not be determined directly, we can estimate its magnitude.The two examples in Fig. 5 show how the three types of variability in foraminiferal test Mg/Ca are related to the measured Mg/Ca values.The total range in test carbonate Mg/Ca caused by uncertainties in the four culture parameters over this experiment explains part of the observed inter-individual variability (Fig. 2, Table 4).The remaining component, expressed as the distance of the corrected Mg/Ca to the calibration curve, reflects the offset caused by the vital effect (Fig. 5, Table 4).The estimated vital effect may be larger than plotted, since we assume that analytical and environment-induced offsets are all lower than the measured variability in foraminiferal Mg/Ca: i.e. they all work in the same "direction" (Fig. 5).
The impact of inter-individual variability on the accuracy of paleo-temperature reconstructions can be calculated by assuming the standard deviation in the Mg/Ca values (measure of variability) from this culture study to be applicable to other foraminiferal species as well.The average variability (standard deviation) in the temperature calibration for B. marginata is 16.3 % (Table 2).This percentage can be used to calculate a standard error (σ/ √ n) of the average Mg/Ca value at any given temperature for a number of foraminiferal Mg/Ca-temperature calibrations.This results in a temperature uncertainty (range of (Mg/Ca , which is independent of the absolute temperature, but is depending on the number of foraminiferal specimens

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Full With this equation, the number of specimens that need to be measured for a certain temperature uncertainty as a function of the sensitivity of the used Mg/Ca-temperature calibration (Fig. 6) can be determined.For species with a relatively low temperature sensitivity, relatively many specimens need to be analyzed for the same temperature uncertainty (Table 5).For example, 94 individuals of B. marginata need to be analyzed to obtain an uncertainty in temperature of 1 • C.

Controls on sensitivity
The sensitivity of a calibrated Mg/Ca-temperature relationship is crucial for the accuracy of reconstructed paleo-temperatures.Using a species with a low sensitivity can be compensated by increasing the number of specimens (Eq.5, Fig. 6).The accuracy will ultimately depend on the combination of analytical errors, the vital effect and absolute calcitic Mg concentration, as an offset in Mg/Ca will have a relatively large impact on

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Full foraminifera with low Mg concentrations.Species that have a similar sensitivity, have calibration curves with a comparable steepness (Fig. 7), but not necessarily the same y-axis intercept.There appear to be three distinct sensitivities in the Mg/Ca response to temperature (Fig. 7).The first group includes only the calibration for the miliolid benthic foraminifer Pyrgo spp. with a sensitivity of 0.16.The sensitivity of the second group varies between 0.09-0.13and contains the foraminifera Oridorsalis umbonatus, Cibicidoides spp., Globigerinoides ruber (and most other planktonic species) and Hyalinea balthica.The third group entails B. marginata, Uvigerina spp.and Ammonia beccarii, which all have Mg/Ca-T sensitivities ranging from 0.04-0.06.The miliolid benthic foraminifer Pyrgo spp. is the only species studied so far that belongs to the group with high sensitivity in Mg incorporation with respect to increasing temperature.Miliolid foraminifera are calcifying following a different calcification mechanism than hyaline species (low and intermediate group) (Erez, 2003;de Nooijer et al., 2009a).Although the exact influence of this difference in calcification mechanisms on the sensitivity is unknown, it is likely that the difference in sensitivity between hyaline and porcelaneous foraminifera is caused by their different calcification pathways.The mechanisms responsible for the distinction between low and intermediate groups are unknown, but may be related to their evolutionary history or ecological strategies.Discrimination against Mg during calcification seems taxonomically related at least at a high level, since all known miliolid species produce calcite with high Mg/Ca (> 50 mmol mol −1 ), while only some hyaline species are producing high Mg calcite (Toyofuku et al., 2000).For hyaline species, three different clades were recognized based on 26 SSU sequence analyses (Schweizer et al., 2008).Foraminifers with the lowest temperature sensitivity have representatives from all three clades, indicating that the Mg/Ca-T sensitivity may not be (fully) related to taxonomic relationships within foraminifera.
Alternatively, differences in ecological strategies might provide an explanation on the species specific Mg sensitivity to temperature.Foraminifera inhabit environments with contrasting food regimes.Both the total amount of organic matter as well as the timing Introduction

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Full of its availability in benthic habitats is known to determine foraminiferal species composition (e.g., Murray, 2006).The organic load, in turn, affects the chemical conditions of the pore water in which foraminifera calcify.In eutrophic habitats, the enhanced organic matter availability causes high total faunal respiration rates and organic matter oxidation, thereby elevating CO 2 levels and lowering the pH and CO 2− 3 concentration and thus the saturation state of the pore water with respect to calcium carbonate (Ω cc ).
In eutrophic microenvironments with lower Ω cc , calcification is thought to be impaired by the decreased availability of CO 2− 3 (Erez, 2003;Bentov and Erez, 2006;Dissard et al., 2010b).Since a higher amount of Mg in foraminiferal test carbonate increases its dissolution potential (Brown and Elderfield, 1996), it may be that active discrimination against Mg during calcification is proportional to the ambient average saturation state of the pore water with respect to calcite and hence be affected by the organic load of the sediment or the related ecological strategy as a response to the availability of food.
The shallow to deep infaunal species B. marginata is mainly found at sub-oxic conditions at locations relatively rich in organic matter (Jorissen, 1988, Jorissen et al., 1992).
The group of species with intermediate sensitivity of Mg incorporation to temperature change contains the benthic species Cibicidoides spp., O. umbonatus and H. balthica and the planktonic species G. ruber.Most species within the genus Cibicidoides are epifaunal to shallow infaunal living close to or at the sediment-water interface, commonly in mesotrophic to oligotrophic, well oxygenated environments with relatively stable physio-chemical parameters (Jorissen et al., 1998;Gooday et al., 2003).The benthic foraminifer O. umbonatus has an epifaunal to shallow infaunal depth habitat and is mainly found in oligotrophic deep-sea environments (Jorissen et al., 1998;Rathmann Introduction

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Full  , 2004).Hyalinea balthica is a shallow infaunal living benthic foraminifer, typical for upper bathyal environments with mesotrophic conditions, although in some studies an opportunistic behavior has been described (Hess and Jorissen, 2009;Rosenthal et al., 2011).The planktonic foraminifer G. ruber is a shallow dwelling (living in the upper 50 m of the water column), symbiont-bearing species, living preferentially in oligotrophic surface waters (Hemleben et al., 1989).Summarizing, all species of this group are typical for oligotrophic to mesotrophic environments.All species of the low sensitivity group show some characteristics typical of an opportunistic lifestyle.B. marginata may reach very high densities in eutrophic settings (e.g., Jorissen, 1987;Jorissen et al., 1992), and is one of the few deep-sea species which reproduce in laboratory conditions (Barras et al., 2010).Ammonia spp.contains dominant taxa in a wide range of coastal ecosystems, where they tolerate large salinity and temperature variations.U. peregrina and U. mediterranea show a reproductive and growth response to phytoplankton bloom events in the Bay of Biscay (Fontanier et al., 2003, 2006).In the literature, all these taxa are generally considered as opportunists.
It could be expected that species from eutrophic settings have a more opportunistic life strategy than species from mesotrophic or oligotrophic settings.Such an opportunistic life strategy would imply foraminifera to be able to react quickly to a number of environmental settings, of which some are hostile, ensuring a low Mg concentration (less soluble and more robust test) to be beneficial to this strategy.The most efficient way to guarantee a calcite test low in Mg is to lower the sensitivity to temperature, since temperature is the main environmental parameter influencing foraminiferal Mg incorporation.Mg/Ca values for the above used species should thus correlate well with the sensitivity of the species specific Mg/Ca-temperature calibration.Foraminiferal test Mg/Ca at a given temperature correlates significantly with the sensitivity of the calibration (Fig. 8), indeed indicating that a low sensitivity to temperature is a good strategy to keep Mg concentrations low, although the mechanisms behind this process still stays unknown.

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Full However, also for some species of the intermediate sensitivity group an opportunistic behavior is suspected.This is clearly the case for Hyalinea balthica, which has been described with very high densities in eutrophic submarine canyon environments (Hess and Jorissen, 2009).But also Cibicidoides species are sometimes occurring in high densities, and dominate the foraminiferal fauna (e.g., Koho et al., 2008).Conversely, both G. ruber and O. umbanatus are always considered as oligotrophic taxa, without any opportunistic tendency.Summarizing, there is indeed a tendency for the low sensitivity taxa to be more opportunistic than the intermediate sensitive taxa, but the separation between the two groups is not as clear as we would hope.A better knowledge about the ecological strategies of these species is necessary to confirm that the lower temperature sensitivity is indeed the result of a more opportunistic lifestyle.

Conclusions
LA-ICP-MS-measured Mg/Ca in cultured B. marginata correlates with temperature, although the sensitivity of Mg incorporation to temperature is low.The calibration is not hindered by any ontogenetic effects.The inter-individual variability within this calibration is too large to be caused by variations in culture parameters over the course of the experiment, but is tied to an intrinsic "vital effect" within the calcification process.
This inter-individual variability influences the practicality of the Mg/Ca thermometer, especially impacting calibrations with a low sensitivity.Foraminifera with this low sensitivity are, therefore, not ideal for reconstructing paleo-temperatures, due to associating large uncertainties or large sample sizes needed for an accurate reconstruction of temperature.
Although the biochemical mechanism responsible for the low sensitivity is yet unknown, it appears that foraminiferal species with this low sensitivity (B.marginata, A. beccarii and Uvigerina spp.) are living in more eutrophic environments.Foraminifera mainly living in oligotrophic to mesotrophic environments should, therefore, be used when reconstructing temperatures with the help of the Mg/Ca temperature proxy.

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Full CaCO 3 lattice increases with temperature.This explains the temperature dependency Figures Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Measured inter-individual variability in foraminiferal test carbonate Mg/Ca of cultured foraminifera is larger than for other elements (Dissard et al., 2010a; Due ñas-Boh órquez et al., 2011a, b).The standard deviation in Mg/Ca between individuals is an order of magnitude larger than what can be explained on the basis of the analytical uncertainty (on average 11 % of the mean, Fig. 1).Besides the relatively small analytical errors, Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |analyzed (N).Calculating the temperature uncertainty as a function of the number of specimens analyzed for a different species, the relation between temperature uncertainty and sensitivity can be expressed as: Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | et al.
Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Examples of such taxa are Cibicidoides spp., O. umbonatus, G. ruber but especially H. balthica and Pyrgo spp.

Fig. 1 .
Fig. 1.Laser ablation profiles for Mg/Ca measured on benthic foraminifer B. marginata (upper panel) and the inhouse GJR standard (lower panel).The absence of peak values for Mg/Ca in the GJR standard at the start and end of the ablation rule out any instrumental cause for these values.

Table 1 .
Average and standard deviation of the main seawater parameters for all temperature experiments.
Experiments contain samples from cultures at 1 Utrecht University, 2 University of Angers or 3 combined samples.

Table 3 .
Standard deviation (as percentages of the average) based on the measured standard deviation of each parameter during the experiments.Standard deviations from Table1are used and converted to Mg/Ca values using the same Mg/Ca-parameter relations