Incorporation of Mg and Sr and oxygen and carbon stable isotope fractionation in cultured Ammonia tepida

Marine Micropaleontology 92-93 (2012) 16–28

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Marine Micropaleontology journal homepage: www.elsevier.com/locate/marmicro

Incorporation of Mg and Sr and oxygen and carbon stable isotope fractionation in cultured Ammonia tepida Paula Diz a,⁎, Christine Barras a, Emmanuelle Geslin a, Gert-Jan Reichart b, c, Edouard Metzger a, Frans Jorissen a, Jelle Bijma c a b c

Laboratoire des Bio-Indicateurs Actuels et Fossiles (BIAF), UPRES EA 2644, Université d'Angers, 2, Boulevard Lavoisier, 49045 Angers and (LEBIM), Ker Chalon, 85350 Ile D'Yeu, France Faculty of Geosciences, Organic Geochemistry, Utrecht University, Budapestlaan 4, Utrecht, 3584 CD, Netherlands Alfred Wegener Institute for Marine and Polar Research, Bremerhaven, Germany

a r t i c l e

i n f o

Article history: Received 15 April 2011 Received in revised form 22 March 2012 Accepted 29 April 2012 Available online 7 May 2012 Keywords: benthic foraminifera culturing oxygen and carbon stable isotopes Mg/Ca Sr/Ca size

a b s t r a c t The shallow water benthic foraminiferal species Ammonia tepida was cultured in controlled temperature conditions (20 °C) at three different salinities (29.8, 32.2, and 35.5). The calcite shells of single individuals of different sizes that calcified entirely in controlled experimental conditions were analysed for their magnesium and strontium concentrations (LA-ICP-MS) as well as for their oxygen and carbon isotopic composition (pooled individuals). The approach used in this study allows us to investigate the effect of seawater salinity, inter-test variability and test size on the studied parameters. Inter-test Mg/Ca variability is large (24–28%) in each salinity experiment. The size of the individuals seems to have an effect on Mg/Ca only in the lowest salinity experiment (29.8 salinity units). In contrast, Sr/Ca ratios show a strong size dependency in all salinity experiments, with Sr/Ca ratios decreasing with the size of the individuals. There is no response of Mg/Ca and Sr/Ca ratios to a 5 unit salinity increase. The carbon isotopic composition of cultured A. tepida shows an important size dependency with the smallest individuals (~200 μm) being around 1‰ 13C-depleted compared to the largest individuals (~600 μm). Interestingly, the oxygen isotopic composition of A. tepida, which does not show an ontogenetic effect, is ~0.30–0.45‰ enriched relative to the corresponding δ18Oeq. values. We hypothesise that heavier than predicted δ18O values may be the result of depleted carbonate ion concentrations in the microenvironment surrounding the foraminiferal shell during calcification (i.e., food cyst). © 2012 Published by Elsevier B.V.

1. Introduction The stable oxygen isotopic composition of foraminiferal test calcite has for several decades been the standard tool for the reconstruction of past seawater temperatures and ice volume (e.g., Shackleton, 1974; Bemis et al., 1998; Lea, 2003). The carbon isotopes analysed simultaneously are generally used to reconstruct the dynamics of deeper water masses and to gain insight into oceanic carbon cycling (e.g., Rohling and Cooke, 1999). Over the past years, Mg/Ca in foraminiferal calcium carbonate has emerged as a valuable independent proxy for seawater temperature (Nürnberg et al., 1996; Rosenthal et al., 1997; Lear et al., 2002; Martin et al., 2002; Rathmann et al., 2004; Rosenthal et al., 2006; Marchitto et al., 2007; Bryan and Marchitto, 2008). When combined with shell δ 18O this independent temperature proxy potentially allows the reconstruction of past seawater δ 18O (e.g., Pena et al., 2008). On the other hand, ⁎ Corresponding author at: Departamento de Xeociencias Mariñas e Ordenación do Territorio, Facultade de Ciencias do Mar, Universidade de Vigo, Campus LagoasMarcosende, 36310, Vigo, Spain. Tel.: +34 986811941; fax: +34 986812556. E-mail address: [email protected] (P. Diz). 0377-8398/$ – see front matter © 2012 Published by Elsevier B.V. doi:10.1016/j.marmicro.2012.04.006

foraminiferal Sr/Ca ratios are generally considered a potential tool to reconstruct past variations in seawater Sr/Ca (e.g., Martin et al., 1999; Elderfield et al., 2000; Shen et al., 2001; Raitzsch et al., 2010), which reflect changes in fluxes of Sr and/or Ca into or out of the ocean related to river input and aragonite precipitation (Lear et al., 2003). However, several secondary factors, such as salinity, carbonate ion concentrations and/or biological processes may also impact stable isotope fractionation and trace metal uptake in foraminiferal test carbonate, thereby complicating palaeoenvironmental reconstructions. Recent field and controlled growth studies with planktonic foraminifera (Ferguson et al., 2008; Kisakürek et al., 2008; DueñasBohórquez et al., 2009) indicated that Mg and Sr incorporation increases with increasing salinity. However, laboratory experiments with shallow water benthic foraminifera show contradictory results. Whereas Toyofuku et al. (2000, 2011) found that salinity does not exert and impact on the Mg/Ca content of hyaline calcareous foraminifera Planoglabratella opercularis (30–38 salinity range) and Ammonia “beccarii” (18.4–33.6 salinity range) or porcelaneous calcareous Quinqueloculina yabei, Dissard et al. (2010a) indicated a 3–5% increase in Mg/Ca ratios per salinity unit (20–40 salinity range) in Ammonia tepida (hyaline calcareous test).

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The seawater carbonate ion concentration is known to affect the stable isotopic composition of foraminiferal calcite. Culturing experiments with planktonic foraminifera showed that shell δ18O and δ 13C decreases with increasing [CO32 −] (Spero et al., 1997). It is likely that such a carbonate ion effect also influences benthic foraminiferal stable isotopes, but the response may not be linear over a wide pH range (Rollion-Bard et al., 2008). Field studies and laboratory experiments also indicate that low carbonate ion saturations result in decreased Mg and Sr incorporation in planktonic foraminifera (Kisakürek et al., 2008; Dueñas-Bohórquez et al., 2009). The effect of carbonate ion concentrations on benthic foraminifera remains inconclusive with field studies considering deep water species showing a negative effect on Mg/Ca ratios (Elderfield et al., 2006; Rosenthal et al., 2006; Yu and Elderfield, 2008) but culturing experiments with shallow water benthic foraminifera indicate the absence of a clear influence of carbonate ion concentrations or pH (Allison et al., 2010; Dissard et al., 2010b). Biological control on chamber formation and CaCO3 precipitation is an important factor impacting foraminiferal Mg/Ca, Sr/Ca, δ18O and δ13C signatures (e.g., Nürnberg et al., 1996; Lea et al., 1999; Bentov and Erez, 2006). The physiological control of foraminifera on Mg incorporation is apparent from the strong fractionation against seawater Mg (e.g., Rosenthal et al., 1997). For a given temperature, the Mg/Ca ratios of different benthic foraminiferal species may diverge substantially, being high in some species (e.g., Oridorsalis umbonatus, Rathmann and Kuhnert, 2008; Hyalinea balthica, Rosenthal et al., 2011) and much lower in others (e.g., Uvigerina spp., Bryan and Marchitto, 2008). Ontogenetic effects (Shen et al., 2001; Elderfield et al., 2002; Hintz et al., 2006a; Dueñas-Bohórquez et al., 2011), natural variability between shells of the same species deposited under similar environmental conditions (Nürnberg et al., 1996; Elderfield et al., 2000; Reichart et al., 2003; Rathmann et al., 2004; Anand and Elderfield, 2005; Hintz et al., 2006b; Sadekov et al., 2008), intra-shell and intrachamber variability (Allison and Austin, 2003; Eggins et al., 2004; Toyofuku and Kitazato, 2005; Kunioka et al., 2006; Allison and Austin, 2008; Sadekov et al., 2008; Allison et al., 2010) are other indications of the strong biological control on trace element incorporation into foraminiferal calcium carbonate. Also indirect biological factors, not directly related to biomineralisation, potentially influence stable isotopic fractionation in foraminifera. For instance, ontogenetic effects (e.g., Wefer and Berger, 1991; Schmiedl et al., 2004), respiration (Grossman, 1987; McConnaughey, 1989) and symbiont photosynthesis (e.g., Erez, 1978) could all result in an apparent disequilibrium with seawater δ18O during calcification, and/or a significant offset between foraminifera and the δ13C of the dissolved inorganic carbon (DIC). Also individuals grown under identical controlled conditions may show high variability of δ18O (Bemis et al., 1998; Wilson-Finelli et al., 1998; McCorkle et al., 2008; Barras et al., 2010; Filipsson et al., 2010). Culturing foraminifera under controlled laboratory conditions is the most promising approach to study species-specific biological effects and to quantify the influence of single environmental parameters on foraminiferal trace element ratios and stable isotopic compositions. Through carefully controlling other environmental factors, those can be excluded when reviewing the trace metal and isotopic data. Here, we cultured the shallow-water benthic foraminiferal species A. tepida at a single temperature (20 °C), at three different salinities (29.8, 32.2, and 35.5). Temperature, salinity, total alkalinity and δ18Osw were closely monitored and remained stable throughout the duration of the experiments. Mg/Ca and Sr/Ca compositions were measured by LA-ICP-MS on single chambers of individuals entirely calcified under controlled conditions. Since individual foraminiferal test were light in weight, the oxygen and carbon stable isotopic composition of A. tepida grown in the three different salinity experiments were measured by pooling a few specimens (2–12 individuals) according to their size. This experimental setup allows us to evaluate simultaneously the effect of salinity and shell size on foraminiferal Mg/Ca, Sr/Ca, δ18O and δ13C, and inter-test variability.

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2. Material and methods 2.1. Laboratory experiments 2.1.1. Experimental protocol Sediment containing live A. tepida was collected on the 12th of March 2008 in the intertidal area of the Bay of Aiguillon (west coast of France) at low tide. Upon arrival in the laboratory on the same day, sediment samples were sieved with natural seawater (salinity of ~ 35.5) over 150 μm and 63 μm meshes. Sieving had two main purposes: 1) to obtain living individuals from the 63–150 μm for culturing experiments and 2) to remove macrofauna, in particular gastropods that were abundant in the samples. Prior to the start of the culturing experiments, benthic foraminifera from the 63–150 μm fraction were labelled with the fluorescent marker calcein (e.g., Hintz et al., 2004; 2006a; McCorkle et al., 2008; Barras et al., 2009; Dissard et al., 2009), to discriminate at the end of experiments between the introduced parental individuals and the individuals that calcified their entire test under experimental conditions. Calcein binds with calcium and incorporates into the mineral structure, so that chambers formed during calcein incubation fluoresce green under an epifluorescence microscope (Bernhard et al., 2004). A. tepida specimens along with sediment containing some other organisms (small nematodes, gastropods and diatoms) were maintained at room temperature (20–23 °C) in a solution of calcein and seawater (10 mg of calcein/L seawater) for 7 days. After one week, we carefully examined the fluorescence pattern of the incubated individuals under a stereomicroscope equipped with epifluorescence optics (OLYMPUS U-RFL-T, excitation at 470 nm, emission at 500 nm). We used cytoplasmic colour as an indicator of viability (e.g., Goldstein and Corliss, 1994; Le Cadre et al., 2003; Bernhard et al., 2004; Barras et al., 2009). Individuals with yellowishbrownish cytoplasm were considered alive, whereas white and/or transparent individuals were considered dead. Only live individuals with more than 2 calcein-labelled chambers, showing that they were active and calcifying during the 1 week pre-incubation period, were selected for our experiments. Well-labelled and living A. tepida were carefully picked with a brush and rinsed several times in microfiltred natural seawater to ensure no sediment particles attached to their tests. The natural seawater used in the experiments was collected two weeks before the beginning of the experiments (06/03/08) at 250 m water depth in the Bay of Biscay (43°40′N, 1°37′W). This water was filtered through a 0.45 μm cellulose filter and stored in an autoclaved tank. The salinity of this water, measured with a salinometer (Guildline Portasal), was 35.48. This constitutes the first of our three experimental waters with different salinities. By diluting this water with ultrapure deionised water (Milli-Q water, MILLIPORE Integral 3), the other two culture waters were set at salinities of 32.2 and 29.8. Previous laboratory experiments with A. tepida indicated that no sediment is needed to obtain growth and reproduction (Bradshaw, 1957, 1961; Stouff et al., 1999). Experiments were therefore performed without sediment, which has the advantage of minimising geochemical gradients or exchange of trace elements with the sediment. The experimental protocol used in this study is adapted from the “Petri Dish system” designed by Barras et al. (2010) to culture deep-sea benthic foraminifera under controlled stable geochemical conditions. The 120 mL polypropylene lid-equipped Petri dishes of 7.5 cm diameter and 3 cm were filled with 100 mL of the culture water. Thirty labelled foraminifera were added and the dishes were closed with an additional piece of PARAFILM “M” to reduce the risk of evaporation. Culture water was renewed every three to four days to minimise changes in seawater chemistry. Over the course of the experiments foraminifera were fed with living diatoms, Phaeodactylum tricornutum, cultured as explained by Barras et al. (2009). Experiments were run at 20 °C (±0.5 °C) with a 12 hour light–dark cycle.

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Experiments started on the 20th of March 2008. Eight days later (March 28th), we observed reproduction in all Petri dishes. All calcein-labelled (parental generation) foraminifera were removed from the experiments on the 2nd of April. Culturing of the new generation continued until a sufficient number of living individuals attained a size of >500 μm, comparable to the size reached by adult specimens in the field, and the experiment was ended on the 31st of May. Only specimens that calcified entirely under experimental conditions (the new generation) were used for trace element and stable isotopic measurements.

2.1.2. Monitoring of culture conditions During the experiments salinity and total alkalinity were measured bi-weekly, when renewing culture water. Water was removed using sterile syringes and all relevant parameters were measured within 10 min after collection. Salinity was determined by conductivity (Conductimeter WTW 330i), which was calibrated against the salinity of the three reservoirs at the beginning of the experiments. At the end of the experiments, precise salinity measurements (Guildline Portasal Salinometer) of the reservoir waters were performed. The total alkalinity of the culture waters was measured with a Ω Metrohm 785 DMP Titrino equipped with a pH glass electrode and an Ag/AgCl reference electrode. Precision of the analyses, based on duplicate analyses was, on average, better than 10 μmol kg − 1. Dissolved inorganic carbon (DIC) content and oxygen isotopic composition of the water were measured monthly, on March 28th, April 29th and May 31st coinciding with the time of parental reproduction, middle and end of the experimental period, respectively. Water samples used for DIC measurements were filtered over a 0.45 μm cellulose filter, poisoned with 100 μL of a saturated solution of HgCl2 and stored in brown gas-tight bottles at 4 °C until further analysis. Water samples for δ 18Ow measurements were stored in a similar way, albeit without poison. DIC was measured using a total organic carbon analyzer (Shimadzu, Model TOC-5050A). Average precision of the analyses was 10 μmol kg − 1. From total alkalinity and DIC, we calculated the pH, the degree of calcite saturation (Ω factor) and the carbonate ion concentration, using the programme CO2SYS (Lewis and Wallace, 1998) with the CO2 constants of Mehrbach et al. (1973) refitted by Dickson and Millero (1987) and the KSO4 constant of Dickson (1990). The oxygen isotope composition of the culture water was measured using a Finnigan GasBenchII following the protocol described by Nelson (2000). Each seawater sample δ 18O was measured twice and the mean and standard deviation were used to characterise δ18Ow for each salinity experiment. Data are expressed in ‰ relative to VSMOW. Precision of the measurements was 0.05‰ determined by repeated analysis of the laboratory standard RMW.

2.3. Minor element analyses of cultured Ammonia tepida Element/Ca ratios (i.e., Mg/Ca and Sr/Ca) of cultured benthic foraminifera were measured with laser ablation inductively coupled plasma-mass spectrometry (LA-ICP-MS). Measurements were carried out on the antepenultimate chamber of each individual. The reason for this is that the antepenultimate chamber is somewhat heavily calcified than later chambers, allowing longer laser ablation ICP-MS acquisition times and thus more robust analyses. A GeoLas 200 Q Excimer 193 nm deep ultra violet laser (Lambda Physik) was used to ablate craters of 60–80 μm in diameter depending on the size of the individuals. Energy density at the sample surface was kept at 4 mJ/cm 2 and the shot repetition rate at 6 Hz. The ablated material was transported on a continuous He flow and mixed with Ar before being injected into the ICP-MS plasma (Micromass Platform, Utrecht University). Element acquisition time during foraminiferal ablation was generally between 40 and 70 s and the wall of calcite shells of most of the chambers was ablated over the full shell thickness (Fig. 1). Elemental concentrations were calculated through calibration against NIST SRM 610 glass (Pearce et al., 2007). To check for matrix dependent fractionation a matrix matched inhouse Iceland spar calcite standard was regularly measured (GJR, Wit et al., 2010). 44Ca was used for quantification and 42Ca and 43Ca for internal monitoring. Calcium carbonate is well suited for LA-ICP‐MS analyses because Ca can be used as an internal standard at 40% weight percentage (Reichart et al., 2003). Concentrations of Mg and Sr were calculated using 24Mg and 88Sr. Concentrations and confidence intervals for each individual measurement was calculated using the Glitter computer programme (Glitter, Maquarie Research Limited, 1999–2000). The matrix matched in-house standard GJR showed relative standard deviations of 8.3% for Mg/Ca and 7.6% for Sr/Ca. Foraminiferal trace element data reduction involved initial screening for outliers. Elemental ratios of single measurements are indicated together with their associated analytical standard error. The average and standard deviation (S.D.) of Mg/Ca, Sr/Ca ratios of benthic foraminifera

a

Inside Standard deviation test of individual point

1.2

Average test concentration

1.0

}

0.8

Standard deviation of test average

0.6 0.4 0.2

background

test carbonate

background

0

2.2. Foraminiferal sample preparation for trace elements and stable isotope measurements

Time (arbitrary units)

b Sr/Ca (mmol/mol)

Trace element and stable isotope measurements were carried out on those individuals born and grown in experimental conditions. The largest and the smallest diameters of each test were determined using a micro-scale (resolution 10 μm) inserted in the ocular of a stereo microscope. Foraminifera were subsequently grouped according to their largest diameter in size classes of 50 μm: ≤150 μm, 150–200 μm, 200–250 μm up to the maximum size class of 700–750 μm. Before trace elemental and stable isotope analyses, foraminiferal tests were cleaned using a protocol adapted from Havach et al. (2001). Two successive rinses with Milli-Q water were used to remove salt. To remove organic matter (cyst, cytoplasm and organic linings) individual shells were placed in an ultrapure sodium hypochlorite (2.5–3% by weight) solution and left to react for about 1 h, followed by five successive rinses with Milli-Q water.

Outside test

1.4

Mg/Ca (mmol/mol)

18

Standard deviation of individual point

2.0 Average test 1.8 concentration 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 background 0

} Standard deviation of test average

test carbonate

background

Time (arbitrary units) Fig. 1. Laser ablation profiles of Ammonia tepida calcite, Mg/Ca (a) and Sr/Ca (b).

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falling in the same size range are also reported. In total, 103 measurements were obtained for the low salinity experiment (29.8), 57 for the intermediate salinity (32.2), and 105 for the high salinity (35.5) cultures.

2.4. Stable isotope measurements in cultured Ammonia tepida After measuring trace metal concentrations by LA-ICP-MS, stable oxygen and carbon isotope ratios of the same individuals were measured using a Thermo Finnigan MAT-253 mass spectrometer coupled online to an automated carbonate preparation device (Kiel III). In order to obtain reliable results for the smaller size fractions, several individuals of the same size had to be pooled. For larger size fractions, measurements were carried out on 1 or 2 individuals. Analyses were calibrated against NBS 19, and isotope values are reported in ‰ relative to the VPDB (Vienna PDB) scale. The external reproducibility of the standards is generally better than ±0.04‰ for δ 13C and ±0.07‰ for δ18O (samples > 17 μg). A somewhat lower reproducibility (±0.05‰ for δ 13C and ±0.2‰ for δ 18O) was achieved for samples weighing less than 10 μg, which was the case for three of the analyses.

3. Results 3.1. Salinity and oxygen isotopic composition of the experimental water High precision salinity measurements of the waters from the three reservoirs at the end of the experiments yielded values of 35.6, 32.3 and 29.9 indicating that the salinity difference between start and end of the experiments was less than 0.1 salinity unit. Experimental stability was confirmed by the more regular conductivity-converted-to-salinity measurements (Fig. 2a, left axis). The oxygen isotopic composition of the experimental seawater shows little variations over the course of the experiments (Fig. 2a, right axis). The mean and standard deviation of oxygen isotopic measurements in the experimental vessels were −0.53± 0.03‰ VSMOW (N= 6), −0.07 ± 0.02‰ VSMOW (N= 6) and 0.6 ± 0.03‰ VSMOW (N= 6) for the 29.8, 32.2 and 35.5 salinity experiments, respectively. Cultured water δ18O thus increased linearly with salinity, as expected from the fact that the different salinities were reached by admixing deionised water from the same source. The resulting relationship between culture water δ18O and salinity was δ18Osw (‰ VSMOW)= −6.54 (±0.13) + 0.2014 (±0.004) * salinity (R2 = 0.996, N = 18, p b 0.0001).

3.2. Carbonate system parameters Total alkalinity of the reservoirs and experimental water was stable over the course of the experiments (Fig. 2b, left axis). The experimental water of 35.5 salinity showed the highest total alkalinity, both in the seawater reservoir (2403 ± 9 μmol kg− 1; N = 43) and in the cultures themselves (2406 ± 33 μmol kg− 1; N = 39). Values are comparable to those of eastern North Atlantic surface water (Lee et al., 2006). For salinity 32.2, the reservoir was 2189 ± 15 μmol kg − 1 (N= 45) and the cultures were 2183± 36 μmol kg− 1 (N= 40). In the experiment with salinity 29.8, reservoir TAlk was 2060 ± 18 μmol kg− 1 (N= 38) and in cultures were 2050 ± 38 μmol kg− 1 (N= 45). The dissolved inorganic carbon (DIC) content of the culture water was measured three times and measurements indicated minor variability (Fig. 2b right axis). The calculated pH (total scale, Fig. 2c, left axis) of the reservoirs and culture water showed little variation over the course of the experiments. The average degree of calcite saturation (Ω) was 4.4, 5.2 and 5.2 for the experiments with salinities 29.8, 32.2 and 35.5 respectively ([CO32 −] 180, 212 and 220 μmol kg− 1), indicating that the culture water was oversaturated with respect to calcite for all three salinities (Fig. 2c, right axis).

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3.3. Minor element concentration in foraminiferal test carbonate Single test Mg/Ca and Sr/Ca ratios of A. tepida, sorted according to their size classes are plotted in Fig. 3. The inter-shell Mg/Ca variability is high in each salinity experiment (28%, 28%, and 24%, in experiments at salinities 29.8, 32.2 and 35.5). In order to evaluate the possible effect of size of the individuals on measured Mg/Ca ratios we performed least square linear regressions between test size and Mg/Ca ratios. Results indicate statistically significant relationships between size and Mg/Ca only for individuals growing in the lowest salinity experiment (see caption of Fig. 3). Aiming to determine whether the mean of Mg/Ca was statistically different between salinity experiments, one-way ANOVA and Tukey's pairwise comparison were performed. Results demonstrated that the average Mg/Ca is significantly higher (pb 0.005) in the lower salinity experiment (1.46 ± 0.41 mmol/mol, N = 103) than in the other two experiments (0.92 ± 0.26 mmol/mol, salinity 32.2, N = 57 and 1.08 ± 0.26 mmol/mol, salinity 35.5, N = 105). The inter-shell variability in Sr/Ca is generally lower (15%, 9% and 10% for 29.8, 32.2 and 35.5 salinity experiments respectively) than that of Mg/Ca. Incorporation of Sr into foraminiferal calcite is generally higher in small individuals than in larger individuals (Fig. 3d–f). The decrease of Sr/Ca ratios with test size increase seems more pronounced between 150–200 and 400–450 μm than for individuals of >500 μm. In order to test the possibility of Sr/Ca exponentially or linearly decreasing with test size we fitted linear and polynomial regressions through the data. Linear and polynomial regressions between test size and Sr/Ca ratios are statistically significant. For the experiments with salinities 32.2 and 35.5, only a minor improvement of r-squared and p values was obtained when using a polynomial regression fit (Fig. 3d–f, see figure caption), indicating that both regressions may be used to describe the relationship between size and Sr/Ca ratios in A. tepida. 3.4. Stable isotopes in cultured Ammonia tepida Carbon and oxygen stable isotope ratios of the cultured A. tepida are listed in Table 1. Four stable oxygen isotopic values obtained from small individuals (150–300 μm) of the lower salinity experiment were unexpectedly heavy (data marked with an asterisk in Table 1 and with crosses in Fig. 4a, d). These aberrant δ 18O (and the corresponding δ 13C) values are not considered reliable, possibly due to the low amount of carbonate available for analyses, and omitted from further consideration. The lack of δ 13CDIC measurements in the culturing water precludes the estimation of disequilibrium offsets between foraminiferal δ13C and dissolved inorganic carbon (DIC) δ13C. However, it is clear that A. tepida δ13C values show a strong size dependency, which is consistent in all three culture experiments (Fig. 4d–f, Table 1) with the smallest measured individuals (~200 μm) being depleted in the heavy isotope by ~1‰ compared to the largest individuals (~600 μm). In contrast, foraminiferal δ 18O values do not show any relation with test size (Fig. 4a–c, see also figure caption and Table 1). The variability in δ18O values in each salinity experiment was considerably higher (Table 1, Fig. 4a–c) than the external reproducibility (b0.07‰). The average δ18O values increase linearly with salinity increase by about 0.20‰ per salinity unit, which is in line with the relationship between culture water δ 18O and salinity (Fig. 5). The theoretical oxygen isotopic composition of carbonate precipitating in equilibrium with seawater (δ 18Oeq) can be calculated from the experimental δ18Ow and temperature (20 °C), using published palaeotemperature equations such as the Shackleton (1974) equation: T = 16.9–4.38 (δ18Oeq − δ18Ow) + 0.1(δ 18Oeq − δ 18Ow) 2 or the recently published (Toyofuku et al., 2011) temperature equation for the shallow water species A. “beccarii”: T = −4.49 (δ18Oforam − δ18Owater) obtained from culturing experiments. In our opinion, the specimens analysed by Toyofuku et al. (2011, see their Fig. 2) should be regarded as A. tepida

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20/03 24/03 28/03 02/04 07/04 11/04 15/04 18/04 22/04 25/04 29/04 02/05 06/05 09/05 13/05 18/05 21/05 25/05 28/05 31/05

δ18Osw (‰VSMOW)

a

Date of water renewal

b

20/03 24/03 28/03 02/04 07/04 11/04 15/04 18/04 22/04 25/04 29/04 02/05 06/05 09/05 13/05 18/05 21/05 25/05 28/05 31/05

c

Date of water renewal Fig. 2. Measured and calculated physico-chemical parameters during culturing experiments. Plot a) represents salinity (lines, left axis) and oxygen stable isotopic composition of the experimental water (right axis). In plot b) total alkalinity (lines, left axis) and dissolved inorganic carbon (DIC, symbols, right axis) of the experimental water are represented. Plot c) indicates the calculated pH of the experimental water in the total scale (grey symbols, left axis) and the degree of calcite saturation (black symbols, right axis) calculated from total alkalinity and DIC using the CO2SYS programme (see text for details). In plots a) and b) crosses and black lines refer to the reservoirs (i.e., solutions used to renew the water of the petri dishes) and grey lines represent the experimental water (petri dishes). In plots a)–c) triangles, circles and squares represent measurements in experiments of salinities 35.5, 32.2 and 29.8 respectively. The arrow at the bottom of plot c) indicates the time when reproduction was observed in the Petri dishes.

(Cushman) (see for example Jorissen, 1988, Plates 7 and 10). The standard mean ocean water δ18Ow (VSMOW) measurements were converted to VPDB units δ18Ow using the conversion factor (−0.27) of Hut (1987). The resulting δ18Oeq values are −0.35‰, −1.03‰ and −1.50‰ (Shackleton equation) and −0.29‰, −0.97‰, −1.42‰ (Toyofuku et al., 2011) for the cultured specimens at salinities 35.5; 32.2 and 29.8 respectively. These values would be lighter using the equation of Kim and O'Neil (1997) (−0.47‰, −1.14‰ and −1.60‰ respectively). Comparison between these theoretical values and the mean δ18O values (−0.01‰, −0.67‰, and −1.15‰, see Table 1) measured in our culture experiments reveals that A. tepida calcifies with a positive offset ranging from ~0.27 to 0.45‰ (Fig. 4a–c).

4. Discussion 4.1. Mg and Sr incorporation in cultured Ammonia tepida 4.1.1. Comparison with other foraminiferal species Compared to most other foraminiferal species Mg/Ca ratios measured in A. tepida are low (i.e., 1.19± 0.39 mmol/mol, N = 265), especially for the relatively high experimental temperature (20 °C), which is in line with previous culturing studies on the same species (Dissard et al., 2010a; Toyofuku et al., 2011). A. tepida belongs to a group of benthic foraminifera with low-Mg calcite, which Cibicidoides pachyderma (Marchitto et al., 2007) and Uvigerina spp. (Bryan and Marchitto,

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a

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f

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Fig. 3. Mg/Ca and Sr/Ca ratios in Ammonia tepida. Trace elements expressed in (mmol/mol) were measured in the antepenultimate chamber of single individuals of cultured A. tepida and they are arranged by size classes. Grey circles and bars indicate single-shell (single-chamber) measurements and their analytical error (1-sigma). The average and standard deviation of Mg/Ca (a–c) and Sr/Ca (d–f) values measured in individuals within the same size class are indicated by black-filled circles and black bars. Dashes lines illustrate the linear (red) or polynomial (grey) fit of Mg/Ca and Sr/Ca values to foraminiferal size (middle point of the size range in μm). Equations of the statistically significant linear or polynomial relationships between Mg/Ca and Sr/Ca ratios and foraminiferal size together with r-squared correlation coefficients and p (significance of the relationship between parameters) are indicated: a) Mg/Ca= 1.8 (±0.1)− 0.0010 (±0.0003) × (size), R2 = 0.08, p= 0.003; d) Sr/Ca= 1.61 (±0.06) − 0.0006 (±0.0001) × (size), R2 = 0.12, p = 0.003 (linear fit); e) Sr/Ca = 1.51 (±0.05) − 0.0004 (±0.0001) × (size), R2 = 0.20, p = 0.0001 (linear fit), Sr/Ca: 1.86− 0.002 × (size)+ 0.000015 (size)2, R2 = 0.26, p b 0.0001 (polynomial fit); f) Sr/Ca = 1.75 (±0.04) − 0.0008 (±0.0001) × (size), R2 = 0.28, p b 0.0001 (linear fit); Sr/Ca = 2.07− 0.0028× (Size)+ 0.000029 (size)2; R2 = 0.32, p b 0.0001 (polynomial fit). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2008) also belong to. On the other hand, species such as Hyalinea balthica present higher Mg/Ca ratios and a strong positive response of Mg content to temperature increase (Rosenthal et al., 2011). Large differences in Mg incorporation between foraminiferal species reinforce

the nowadays generally accepted idea that calcification and Mg incorporation into foraminiferal calcite are under strong biological control (Angell, 1979; Elderfield et al., 1996; Bentov and Erez, 2006; Toyofuku et al., 2008; Bentov et al., 2009; de Nooijer et al., 2009).

22

Table 1 Oxygen and carbon stable isotopic composition of cultured A. tepida in the three experimental salinities arranged by size fractions. The weight and number of specimens (#) used in each measurement are also indicated. δ18O Experiment Size interval δ13C (‰ PDB) (‰ PDB) salinity 29.8 (μm) − 3.260*

0.200*

5.0

n.d

− 3.080*

0.900*

8.0

9

150–200 150–200 200–250 200–250 250–300 250–300 250–300 300–350

− 3.570* − 3.065*

− 0.172* 25.4 0.027* 35.8

17 9

− 3.203

− 1.074

48.6

11

350–400 350–400 350–400 400–450 400–450 400–450 400–450 450–500 450–500 450–500 450–500 500–550 500–550 550–600 550–600

− 2.618 − 2.663 − 2.747 − 2.559 − 2.262 − 2.553 − 2.604 − 2.677 − 2.332 − 2.406 − 2.486 − 2.564 − 2.184 − 2.144 − 1.943

− 1.236 − 1.009 − 1.005 − 0.770 − 1.121 − 1.267 − 0.987 − 1.104 − 1.235 − 1.160 − 1.259 − 1.312 − 1.490 − 1.116 − 1.313

28.6 45.8 22.9 26.2 20.9 35.7 24.2 32.9 30.7 39.4 31.9 38.2 21.4 41.9 38.2

4 5 3 3 2 3 2 3 2 2 2 2 1 2 1

−2.50 0.29

− 1.15 0.17

− 1.94 − 3.20 16

−0.77 − 1.49 16

Size interval δ13C δ18O Weight # individuals Experiment (μm) (‰ PDB) (‰ PDB) (μg) salinity 35.5

Size interval δ13C δ18O Weight # individuals (μm) (‰ PDB) (‰ PDB) (μg) 150–200 150–200 200–250 200–250 250–300 250–300 250–300 300–350 300–350 300–350 300–350 350–400 350–400 350–400 400–450 400–450 400–450

− 2.785 − 2.751 − 2.529 − 2.323 − 2.168 − 2.224 − 2.519 − 2.163 − 2.357 − 2.151 − 2.114 − 2.013 − 1.792 − 1.711 − 1.857 − 1.625 − 1.991

0.369 0.130 − 0.194 −0.119 − 0.004 − 0.079 − 0.231 0.086 0.012 0.205 0.069 − 0.001 0.163 0.168 0.093 0.065 − 0.252

17.0 18.0 18.0 19.0 17.0 18.0 29.0 21.0 20.0 22.0 26.0 23.0 24.0 22.0 37.0 32.0 31.0

12 8 6 7 5 5 7 4 4 3 4 2 2 2 3 2 2

450–500 450–500 450–500 450–500

− 1.609 − 1.650 − 1.711 − 1.734

0.183 0.547 − 0.163 − 0.157

32.0 17.0 21.0 21.0

2 1 1 1

550–600 550–600

− 1.335 − 1.453

− 0.374 − 0.274

31.0 28.0

1 1

250–300 250–300 250–300 300–350 300–350

− 2.660

−0.700

7.0

3

− 2.195 −2.241

− 0.621 − 0.541

35.0 17.0

4 3

350–400 350–400

−2.157 − 1.913

− 0.847 − 0.720

28.0 22.0

3 2

400–450 400–450 400–450

−1.965 − 1.944 − 1.466

− 0.555 − 0.381 − 0.850

23.0 25.0 25.0

2 2 2

450–500 450–500 450–500

− 2.105 − 1.491 − 2.366

− 0.872 − 0.399 − 0.542

31.0 36.0 30.0

2 2 2

500–550 500–550 550–600 550–600 600–650 600–650 650–700 700–750

− 2.051 − 1.484 −1.668 − 1.905 − 1.719 − 1.547 − 1.494 − 1.087

− 0.706 − 0.742 − 0.664 −0.906 − 0.944 − 0.649 − 0.708 − 0.374

34.0 30.0 21.0 25.0 26.0 27.0 29.0 45.0

2 1 1 1 1 1 1 1

Mean Standard deviation

− 1.87 0.39

− 0.67 0.17

Mean Standard deviation

− 2.02 0.40

0.01 0.22

Maximum Minimum N

− 1.09 − 2.66 19

− 0.37 − 0.94 19

Maximum Minimum N

− 1.34 − 2.78 23

0.55 − 0.37 23

n.d. indicates no data available and * indicates data not taken into consideration in the discussion or average calculations.

P. Diz et al. / Marine Micropaleontology 92-93 (2012) 16–28

Mean Standard deviation Maximum Minimum N

Weight # individuals Experiment (μg) salinity 32.2

P. Diz et al. / Marine Micropaleontology 92-93 (2012) 16–28

a

d

b

e

c

f

23

Fig. 4. Stable oxygen and carbon isotopic measurements of cultured A. tepida.Oxygen and carbon stable isotopic measurements in individuals or groups of individuals of A. tepida (see also Table 1) arranged by size classes are represented in plots a, d (salinity 29.8); b, e (salinity 32.2) and c, f (salinity 35.5), respectively. Measurements carried out in > 3, 2–3, and 1 individuals are indicated by black circles, grey filled squares and empty grey squares, respectively.Horizontal dashed lines in a–c indicate the oxygen isotopic composition of a carbonate precipitating in thermodynamic equilibrium with the experimental seawater. Lower and upper lines reflect values calculated with Kim and O'Neil (1997) and Shackleton (1974) equations respectively.Black lines illustrate the linear fit of δ13C values to foraminiferal size (middle point of the size range in μm). Foraminiferal δ18O values are independent of the size of the individuals (no statistically significant correlation coefficients, a–c): δ18O (‰) = − 0.002 (± 0.005) − 0.1 (± 0.2) × size (μm) for salinity 29.8 (N = 16, R2 = 0.23, p = 0.046).Benthic foraminiferal carbon isotopes show a substantial size effect (d–f) with the δ13C of A. tepida increasing with size increase. Regression lines of each salinity experiment are as follows: d) δ13C (‰) = − 4.0 (± 0.2) + 0.0032 (± 0.0006) × size (μm) for salinity 29.8 (N = 16, R2 = 0.673, p = 0.0001); e) δ13C (‰) = − 3.0 (± 0.2) + 0.0023 (± 0.0005) × size (μm) for salinity 32.3 (N = 19, R2 = 0.557, p = 0.0002); f) δ13C (‰) = − 3.22 (± 0.09) + 0.0032 (± 0.0002) × size (μm) for salinity 35.5 (N = 23, R2 = 0.89, p b 0.0001).Crosses in plots a) and d) contain isotopic values considered aberrant and excluded from the discussion and average calculations.

Another interesting feature of the Mg/Ca ratios is the large intershell variability observed separately in each of the experiments, which is highest in the lower salinity experiment (Fig. 3a–c). The

large variability in Mg/Ca content of foraminiferal shells seem to be a common feature of Ammonia species as suggested by the substantial intra-shell variability of Mg/Ca in Ammonia batavus (Allison and

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P. Diz et al. / Marine Micropaleontology 92-93 (2012) 16–28

Fig. 5. Oxygen isotopic composition of Ammonia tepida at the three different salinities. Grey line indicates the linear fit of the data: δ18O (‰) = −7.29 (± 0.35) + 0.206 (± 0.011) × salinity (N = 58, R2 = 0.868, p b 0.00001).

Austin, 2003, ion microprobe analyses) and the inter-sample variability observed in the Mg/Ca ratios of A. tepida (Toyofuku et al., 2011, atomic absorption spectrometry). The causes for inter-shell variability in Mg incorporation are not well known. For symbiont-bearing foraminifera, Mg/Ca variability has been explained by the presence of calcite layers of high (primary calcite) and low Mg (secondary calcite) (Bentov and Erez, 2006). In planktonic foraminifera intra-shell differences in Mg/Ca and Sr/Ca ratios were related to changes in the microenvironment surrounding the shells (Eggins et al., 2004) and to the distribution of the organic matrix within the calcareous test walls, with high Mg/Ca being associated with layers of organic components (Kunioka et al., 2006). The examination of laser ablation profiles through the test wall show no evidence of distinct elemental layering related to e.g., potential organic layers or bands (see example in Fig. 1). Therefore, inter-shell variability in our study is not explained by inhomogeneities between specimens or difference in integration. Furthermore, differences in the crystal structure of calcite could potentially also affect Mg/Ca variability (Hathorne et al., 2009). In comparison with Mg/Ca, the Sr/Ca ratios measured in the A. tepida shells, between 1.00 and 2.1 mmol/mol, are in the same range as found for planktonic foraminifera (e.g. 0.7–2.36 mmol/mol in Globigerina bulloides and Globorotalia truncatulinoides, Anand and Elderfield, 2005), small benthic foraminifera (e.g., 1.12–1.43 in Cibicidoides wuellerstorfi, McCorkle et al., 1995; Shen et al., 2001; 1.8–3.2 mmol/mol in Bulimina aculeata and Rosalina vilardeboana, Hintz et al., 2006a) and larger symbiont-bearing benthic foraminifera (e.g., 0.6–2.16 mmol/mol in Marginopora vertebralis, Amphisorus hemprichii, Neocalcarina calcar, Amphistegina lessoni, Raja et al., 2005). Comparable Sr/Ca values between different genera of foraminifera suggest that Sr2 + (opposed to Mg2 +), is not directly involved in the biomineralisation process in benthic foraminifera. 4.1.2. Influence of specimens size Test size has a clear effect on Sr/Ca ratios, with Sr incorporation in foraminiferal calcite decreasing with increasing test size (Fig. 3d–f). Comparable trends have been reported earlier by Elderfield et al. (2002) for some species of planktonic foraminifera. The latter authors explain that such a pattern is consistent with the idea of a growth rate control on Sr/Ca. At high carbonate precipitation rates (such as those occurring in small individuals) foraminifera are less capable of discriminating against Sr 2 + and Sr incorporation into foraminiferal calcite occurs under kinetic control, resulting in high Sr/Ca ratios. Increased Sr 2 + incorporation into calcite with increasing crystal growth rates has also been demonstrated in inorganic precipitation

experiments (Nehrke et al., 2007). As the test grows larger, calcification rates will slow down, and the kinetic control on Sr 2 + incorporation will diminish, leading to lower Sr/Ca ratios in the benthic foraminiferal tests. Kinetics may also explain the statistically significant effect of the size of the individuals on Mg/Ca ratios in A. tepida, observed only in the lower salinity experiment (Fig. 3a–c). Similarly, the larger intersample variability of Mg/Ca at low salinities may result from kinetics. A. tepida may not be able to fully control Mg 2 + intake at low salinities which results in larger inter-test variability compared to higher salinities. This lack of effectiveness to discriminate against Mg 2 + intake may have a larger effect on individuals with increased calcification rates, resulting in higher Mg/Ca values in the smallest foraminifera. There are only few studies studying Mg incorporation as a function of foraminiferal size and there is no general agreement about the influence of this factor on Mg/Ca ratios. Elderfield et al. (2002) showed a slight increase in Mg/Ca with specimen size in some species of planktonic foraminifera and Dueñas-Bohórquez et al. (2011) indicated a decrease in Mg/Ca ratios per ontogenetic stage in the last four chambers of Globigerinoides sacculifer. On the contrary, Hintz et al. (2006a) indicated enrichment in Mg/Ca at the middevelopmental stage (150–225 μm) in the cultured benthic foraminifera Bulimina aculeata. 4.1.3. Influence of salinity Several recent field studies (Ferguson et al., 2008) and laboratory experiments (Kisakürek et al., 2008; Dueñas-Bohórquez et al., 2009; Dissard et al., 2010a) showed that salinity, in addition to temperature, also influences Mg incorporation in foraminiferal test carbonate of both planktonic and shallow water benthic foraminifera, such that Mg/Ca-ratios increase with increasing salinity. In particular, the data presented by Dissard et al. (2010a) in the same species used here, A. tepida, indicate that Mg/Ca ratios increase linearly by 3.2–3.6% per salinity unit, albeit over a larger salinity gradient (salinity range of 20–40 at 10 and 15 °C). In contrast to these studies, Toyofuku et al. (2000, 2011) found no impact of salinity on Mg incorporation in cultured shallow water benthic foraminifera (salinities between 30 and 38 and between 18 and 34, respectively). Our results corroborate the conclusions of Toyofuku et al. (2000, 2011) in the sense that no systematic increase in Mg/Ca ratios of A. tepida is observed in response to a 5 unit salinity increase. Moreover, in the 29.8 salinity experiment the average Mg/Ca ratios are ~0.4–0.5 mmol/mol higher than at salinities 32.2 and 35.5 (Fig. 6a). As suggested by Toyofuku et al. (2011), the lack of a positive response of Mg/Ca of A. tepida to salinity and the low Mg content of their shells may be the result of the adaptation of this species to highly variable coastal environments. The different response observed by Dissard et al. (2010a) furthermore suggests that this adaptation may vary between locations. The differences in the average Sr/Ca ratios between the three salinity experiments, although statistically significant, are rather small (around 0.1 mmol/mol, Fig. 6b). This result contrasts with the linear increase of 0.8–1.3% per salinity unit indicated by Dissard et al. (2010a) for the same species (20–40 salinity units, 10 and 15 °C). Although the salinity range used by Dissard et al. (2010a) was larger, differential responses of the same species might suggest different adaptation of A. tepida strains harvested from different locations. 4.2. Oxygen and carbon isotopes in cultured Ammonia tepida The stable isotopic composition of cultured A. tepida reveals two main features. First, shell size exerts a strong control on benthic foraminiferal δ 13C but, unexpectedly, ontogeny does not appear to influence δ 18O (Fig. 4). And second, the δ 18O of A. tepida is on average 0.3–0.45‰ enriched relative to the oxygen isotopic composition of carbonate precipitating in equilibrium with the seawater (δ 18Oeq).

P. Diz et al. / Marine Micropaleontology 92-93 (2012) 16–28

a

b

Fig. 6. Mean and standard deviation of Mg/Ca (a), Sr/Ca (b) versus salinity in A. tepida.Grey circles represent single individual measurements and black squares and vertical bars the mean and standard deviation for each salinity experiment. Differences in the mean Mg/Ca and Sr/Ca values have been statistically tested using a One-way ANOVA and Tukey's pairwise comparison. Results indicate statistical different mean values for Mg/Ca and Sr/Ca between each salinity experiment (pb 0.005).

25

fractionation generally results in discrimination against the heavier isotope, i.e. 18O. When δ18O enrichment is observed in field studies, it is generally explained as the result of inaccurate temperature and/or salinity measurements, preferential growth of foraminifera in a particular season or the choice of the palaeotemperature equation used to calculate δ18Oeq values (Grossman, 1987). These mechanisms can, however, not explain the offset observed in the A. tepida cultured under stable temperature and known δ18Osw conditions. Moreover, the positive offsets are clear for the three different palaeotemperature equations used. We suggest that heavier than equilibrium δ18O values could be caused by an isotopic response in the microenvironment surrounding the foraminiferal shell during calcification. We observed that in our cultures A. tepida systematically surrounds its test with an organic cover (cyst) of brownish colour (see for example the cyst around cultured A. tepida in Dupuy et al., 2010, Fig. 3), which is likely formed by organic material secreted by the foraminifera itself (e.g., Langer and Gehring, 1993). The chemical conditions (carbonate ion concentration, calcite saturation state, pH and DIC) in the cyst microenvironments can be expected to differ significantly from the culture water (Fig. 2b–c). For example, organic matter degradation within the cyst may decrease pH (and carbonate ion concentrations) in the microenvironment between the foraminiferal shells and the cyst. This putative decrease in the carbonate ion concentration could enrich the calcite in the 18O as indicated in culturing experiments with planktonic foraminifera (Spero et al., 1997; Bijma et al., 1999; Lea et al., 1999; Zeebe et al., 1999). If we take the slope of the carbonate ion effect for the planktonic foraminifer G. bulloides (−0.004‰/μmol CO32 −; Spero et al., 1997) we can estimate the CO32 − gradient (ΔCO32 −) between the cyst-shell microenvironment and the overlying water. Thus, a positive disequilibrium of 0.30–0.45‰ in A. tepida would correspond to a gradient in the carbonate ion concentration between the cyst and the environmental seawater outside the cyst (ΔCO32 −) of ~ −100 μmol CO32 −. The effect of such cysts and their potential role in explaining heavier than predicted δ18O values in cultured A. tepida has to be tested in future laboratory experiments using i.e., micro-electrodes to determine chemical conditions inside the cyst. 4.3. Palaeoceanographic implications

The fact that ontogeny influences δ 13C but does not appear to influence δ 18O cannot be explained by field and modelling studies dealing with isotopic fractionation in benthic foraminifera. The relation between size and carbon and oxygen isotopes varies between species of benthic foraminifera. Isotopic values in some species of deep sea benthic foraminifera are apparently size independent (e.g., Hoeglundina elegans; Corliss et al., 2002) others present a strong ontogenetic enrichment in both δ 18O and δ 13C, with size of the individuals (e.g., Uvigerina mediterranea, Uvigerina peregrina, Bulimina aculeata; Dunbar and Wefer, 1984; Schmiedl et al., 2004; Fontanier et al., 2006; McCorkle et al., 2008; Schumacher et al., 2010). Recent culturing experiments have also shown size related effects on the δ 18O of B. aculeata/marginata (Barras et al., 2010; Filipsson et al., 2010). If higher metabolic rates and larger incorporation of isotopically light metabolic CO2 in smaller individuals (e.g., Grossman, 1984, 1987; McConnaughey, 1989) explain the strong ontogenetic effect in our A. tepida δ13C, the absence of this effect on δ18O is indeed surprising (Fig. 4a–c). We do not have a straightforward explanation for this. Higher than predicted δ 18O (i.e., heavier than equilibrium values) values in A. tepida are difficult to explain. Recently another culturing experiment also showed higher than predicted δ 18O values for B. aculeata/marginata at 14 °C (Filipsson et al., 2010). No suggestions were made to explain these surprising isotopic values. Positive δ 18O offsets (with respect to equilibrium values) in calcite of benthic foraminifera have never been attributed to vital effects in the literature (see review in Rohling and Cooke, 1999). Biologically controlled

The outcome of this research has direct implications on the use of stable isotopes in palaeoceanographic interpretations. Salinity does not seem to have an effect on the Mg/Ca of A. tepida over the range studied. This result supports previous ideas that the Mg/Ca in Ammonia species can be used to reconstruct temperature in shallow water marine environments (Toyofuku et al., 2011). Because inter-shell Mg/Ca variability is high, a relatively large number of measurements and/or specimens is necessary to obtain reliable Mg/Ca based seawater temperature estimates with microanalytical techniques (i.e., LA-ICP), as recently pointed out by Allison et al. (2010). Test size has an effect on Sr/Ca ratios and on the carbon isotopic signature of A. tepida, independent of seawater salinity. At low salinities, this size effect could also be important in Mg/Ca. As a result, Sr/Ca measurements and δ13C measurements in benthic foraminifera should be carried out on welldefined test size classes. 5. Conclusions We cultured the benthic foraminifera A. tepida at a single temperature (20 °C) and three different salinities (29.8, 32.2 and 35.5). The calcite of the individuals born and grown in controlled experimental conditions was investigated for their Mg/Ca, Sr/Ca ratios and stable isotopic composition for different ontogenetic stages. Sr/Ca ratios of A. tepida show a size dependency in the three salinity experiments, with Sr/Ca decreasing with increasing size of the specimens. The size effect on Mg/Ca is only evident in the low salinity

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P. Diz et al. / Marine Micropaleontology 92-93 (2012) 16–28

experiment and shows the same trend as Sr/Ca. The effect of size may be related to a kinetic control on Sr 2 + and Mg2 + incorporation into the calcite lattice with increased ion incorporation at increasing calcite growth rates. Mg/Ca ratios in A. tepida are generally low (for the high experimental temperature) and show relatively high inter-test variability (24–28%) supporting previous suggestions of the strong biological control on Mg 2 + incorporation into foraminiferal calcite. In contrast, Sr 2 + shows much lower inter specimen variability, and Sr/Ca values are comparable to other species of benthic foraminifera which suggests that Sr 2 + is not directly involved in the biological pumps functioning during the biomineralisation process. For the studied 5 unit salinity range (29.8–35.5), we do not get a clear relationship between Mg/Ca (or Sr/Ca) and salinity in A. tepida. These results confirm previous studies showing the lack of a salinity effect on Mg/Ca on this species over a large range of salinities (18.4–33.6, Toyofuku et al., 2011) and conflict with others showing a positive response of both Sr/Ca and Mg/Ca to salinity increase (Dissard et al., 2010a). Carbon isotopes of cultured A. tepida show a strong ontogenetic effect with on average a 1‰ δ 13C increase between small (200 μm) and large specimens (600 μm). We do not observe such an ontogenetic effect for oxygen isotopes. The oxygen isotopic composition of cultured A. tepida shows ~0.3– 0.45‰ enrichment compared to predicted equilibrium values. Enrichment in the heavy isotope may be explained by the effect of decreased carbonate ion concentration occurring in the microenvironment surrounding the cyst formed by A. tepida during the culturing experience. Confirming the existence of such a “cyst effect” on oxygen stable isotopes or other parameters (minor elements, carbon isotopes, etc.) may be important for the further development of culture-based proxy calibrations. Acknowledgements This study is a contribution to the PaleoSalt project funded by the European Science Foundation (ESF) under the EUROCORES Programme EuroCLIMATE through contract number ERAS-CT-2003980409 of the European Commission, DG Research, FP 02. The authors are grateful to Arnold van Dijk for running the isotope analysis. The help and advice of Gijs Nobbe are strongly acknowledged. Eric Mace performed high precision salinity measurements in INSU, Roscoff (France). P.D. was funded by a postdoctoral grant from the Conseil Général of the Vendée (France). Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.marmicro.2012.04.006. References Allison, N., Austin, W.E.N., 2003. The potential of ion microprobe analysis in detecting geochemical variations across individual foraminifera tests. Geochemistry, Geophysics, Geosystems 4, 8403 doi: 8410.1029/2002GC000430. Allison, N., Austin, W.E.N., 2008. Serial Mg/Ca and Sr/Ca chronologies across single benthic foraminifera tests. Chemical Geology 253, 83–88. Allison, N., Austin, W., Paterson, D., Austin, H., 2010. Culture studies of the benthic foraminifera Elphidium williamsoni: evaluating pH, Δ[CO32 −] and inter-individual effects on test Mg/Ca. Chemical Geology 274, 87–93. Anand, P., Elderfield, H., 2005. Variability of Mg/Ca and Sr/Ca between and within the planktonic foraminifers Globigerina bulloides and Globorotalia truncatulinoides. Geochemistry, Geophysics, Geosystems 6, Q11D15. http://dx.doi.org/10.1029/2004GC000811. Angell, R.W., 1979. Calcification during chamber development in Rosalina floridana. Journal of Foraminiferal Research 9, 341–353. Barras, C., Geslin, E., Duplessy, J.-C., Jorissen, F., 2009. Reproduction and growth of the deep-sea benthic foraminifer Bulimina marginata under different laboratory conditions. Journal of Foraminiferal Research 39, 155–165.

Barras, C., Duplessy, J.-C., Geslin, E., Michel, E., Jorissen, F.J., 2010. Calibration of δ18O of cultured benthic foraminiferal calcite as a function of temperature. Biogeosciences 7, 1349–1356. Bemis, B.E., Spero, H.J., Bijma, J., 1998. Reevaluation of the oxygen isotopic composition of planktonic foraminifera: experimental results and revised paleotemperature equations. Paleoceanography 13, 150–160. Bentov, S., Erez, J., 2006. Impact of biomineralization processes on the Mg content of foraminiferal shells: a biological perspective. Geochemistry, Geophysics, Geosystems 7, Q01P08. http://dx.doi.org/10.1029/2005GC001015. Bentov, S., Brownlee, C., Erez, J., 2009. The role of seawater endocytosis in the biomineralization process in calcareous foraminifera. Proceedings of the Natural Academy of Science 106, 21500–21504. Bernhard, J.M., Blanks, J.K., Hintz, C.J., Chandler, G.T., 2004. Use of the fluorescent calcite marker calcein to label foraminiferal tests. Journal of Foraminiferal Research 34, 96–101. Bijma, J., Spero, H.J., Lea, D.W., 1999. Reassessing foraminiferal stable isotope geochemistry: impact of the oceanic carbonate system (experimental results). In: Fischer, G., Wefer, G. (Eds.), Use of Proxies in Paleoceanography: Examples from the South Atlantic. Springer-Verlag, Berlin Heindelber, pp. 489–512. Bradshaw, J.S., 1957. Laboratory studies on the rate of growth of the foraminifer, “Streblus beccarii (Linné) var. tepida (Cushman)”. Journal of Paleontology 31, 1138–1147. Bradshaw, J.S., 1961. Laboratory experiments on the ecology of foraminifera. Contributions from the Cushman Foundation for Foraminiferal Research XII, pp. 87–106. Bryan, S.P., Marchitto, T.M., 2008. Mg/Ca‐temperature proxy in benthic foraminifera: new calibrations from the Florida Straits and a hypothesis regarding Mg/Li. Paleoceanography 23. http://dx.doi.org/10.1029/2007PA001553. Corliss, B.H., McCorkle, D.C., Higdon, D.M., 2002. A time series study of the carbon isotopic composition of deep-sea benthic foraminifera. Paleoceanography 17. http:// dx.doi.org/10.1029/2001PA000664. de Nooijer, L.J., Langer, G., Nehrke, G., Bijma, J., 2009. Physiological controls on seawater uptake and calcification in the benthic foraminifer Ammonia tepida. Biogeosciences 6, 2669–2675. Dickson, A.G., 1990. Standard potential of the reaction: AgCl(s) + 1/2 H2(g) = Ag(s) + HCl(aq), and the standard acidity constant of the ion HSO4− in synthetic seawater from 273.15 to 318.15 K. The Journal of Chemical Thermodynamics 22, 113–127. Dickson, A.G., Millero, F.J., 1987. A comparison of the equilibrium constants for the dissociation of carbonic acid in seawater media. Corrigenda. Deep Sea Research 36, 983 Deep-Sea Research 34, 1733–1743. Dissard, D., Nehrke, G., Reichart, G.J., Nouet, J., Bijma, J., 2009. Effect of the fluorescent indicator calcein on Mg and Sr incorporation into foraminiferal calcite. Geochemistry, Geophysics, Geosystems 10. http://dx.doi.org/10.1029/2009GC002417. Dissard, D., Nehrke, G., Reichart, G.-J., Bijma, J., 2010a. The impact of salinity on the Mg/Ca and Sr/Ca ratio in the benthic foraminifera Ammonia tepida: results from culture experiments. Geochimica et Cosmochimica Acta 74, 928–940. Dissard, D., Nehrke, G., Reichart, G.-J., Bijma, J., 2010b. Impact of seawater pCO2 on calcification and Mg/Ca and Sr/Ca ratios in benthic foraminifera calcite: results from culturing experiments with Ammonia tepida. Biogeosciences 7, 81–93. Dueñas-Bohórquez, A., da Rocha, R.E., Kuroyanagi, A., Bijma, J., Reichart, G.-J., 2009. Effect of salinity and seawater calcite saturation state on Mg and Sr incorporation in cultured planktonic foraminifera. Marine Micropaleontology 73, 178–189. Dueñas-Bohórquez, A., da Rocha, R.E., Kuroyanagi, A., de Nooijer, L.J., Bijma, J., Reichart, G.-J., 2011. Interindividual variability and ontogenetic effects on Mg and Sr incorporation in the planktonic foraminifer Globigerinoides sacculifer. Geochimica et Cosmochimica Acta 75, 520–532. Dunbar, R.B., Wefer, G., 1984. Stable isotope fractionation in benthic foraminifera from the Peruvian continental margin. Marine Geology 59, 215–225. Dupuy, C., Rossignol, L., Geslin, E., Pascal, P.-Y., 2010. Predation of mudflat meiomacrofaunal metazoans by a calcareous foraminifer, Ammonia tepida (Cushman, 1926). Journal of Foraminiferal Research 40, 305–312. Eggins, S.M., Sadekov, A., De Deckker, P., 2004. Modulation and daily banding of Mg/ Ca in Orbulina universa tests by symbiont photosynthesis and respiration: a complication for seawater thermometry? Earth and Planetary Science Letters 225, 411–419. Elderfield, H., Bertram, C.J., Erez, J., 1996. A biomineralization model for the incorporation of trace elements into foraminiferal calcium carbonate. Earth and Planetary Science Letters 142, 409–423. Elderfield, H., Cooper, M., Ganssen, G., 2000. Sr/Ca in multiple species of planktonic foraminifera: implications for reconstructions of seawater Sr/Ca. Geochemistry, Geophysics, Geosystems 1 1999GC000031. Elderfield, H., Vautravers, M., Cooper, M., 2002. The relationship between shell size and Mg/Ca, Sr/Ca, δ18O, and δ13C of species of planktonic foraminifera. Geochemistry, Geophysics, Geosystems 3. http://dx.doi.org/10.1029/2001GC000194. Elderfield, H., Yu, J., Anand, P., Kiefer, T., Nyland, B., 2006. Calibrations for benthic foraminiferal Mg/Ca paleothermometry and the carbonate ion hypothesis. Earth and Planetary Science Letters 250, 633–649. Erez, J., 1978. Vital effect on stable-isotope composition seen in foraminifera and coral skeletons. Nature 273, 199–202. Ferguson, J.E., Henderson, G.M., Kucera, M., Rickaby, R.E.M., 2008. Systematic change of foraminiferal Mg/Ca ratios across a strong salinity gradient. Earth and Planetary Science Letters 265, 153–166. Filipsson, H.L., Bernhard, J.M., Lincoln, S.A., McCorkle, D.C., 2010. A culture-based calibration of benthic foraminiferal paleotemperature proxies: δ18O and Mg/Ca results. Biogeosciences 7, 1335–1347.

P. Diz et al. / Marine Micropaleontology 92-93 (2012) 16–28 Fontanier, C., Mackensen, A., Jorissen, F.J., Anschutz, P., Licari, L., Griveaud, C., 2006. Stable oxygen and carbon isotopes of live benthic foraminifera from the Bay of Biscay: microhabitat impact and seasonal variability. Marine Micropaleontology 58, 159–183. Goldstein, S.L., Corliss, B.H., 1994. Deposit feeding in selected deep-sea and shallowwater benthic foraminifera. Deep Sea Research I 41, 229–241. Grossman, E.L., 1984. Carbon isotopic fractionation in live benthic foraminifera comparison with inorganic precipitate studies. Geochimica et Cosmochimica Acta 48, 1505–1512. Grossman, E.L., 1987. Stable isotopes in modern benthic foraminifera: a study of vital effect. Journal of Foraminiferal Research 17, 48–61. Hathorne, E.C., James, R.H., Lampitt, R.S., 2009. Environmental versus biomineralization controls on the intratest variation in the trace element composition of the planktonic foraminifera G. inflata and G. scitula. Paleoceanography 24. http:// dx.doi.org/10.1029/2009PA001742. Havach, S.M., Chandler, G.T., Willson-Finelli, A., Shaw, T.J., 2001. Experimental determination of trace element partition coefficients in cultured benthic foraminifera. Geochimica et Cosmochimica Acta 65, 1277–1283. Hintz, C.J., Chandler, G.T., Bernhard, J.M., McCorkle, D.C., Havach, S.M., Blanks, J.K., Shaw, T.J., 2004. A physicochemically constrained seawater culturing system for production of benthic foraminifera. Limnology and Oceanography: Methods 2, 160–170. Hintz, C.J., Shaw, T.J., Bernhard, J.M., Chandler, G.T., McCorkle, D.C., Blanks, J., 2006a. Trace/minor element:calcium ratios in cultured benthic foraminifera. Part II: ontogenetic variation. Geochimica et Cosmochimica Acta 70, 1964–1976. Hintz, C.J., Shaw, T.J., Chandler, G.T., Bernhard, J.M., McCorkle, D.C., Blanks, J.K., 2006b. Trace/minor element:calcium ratios in cultured benthic foraminifera. Part I: inter-species and inter-individual variability. Geochimica et Cosmochimica Acta 70, 1952–1963. Hut, G., 1987. Stable isotope reference samples for geochemical and hydrological investigations. In: Hut, G. (Ed.), Report on Consultants' Meeting, Vienna. IAEA, Vienna. 16–18 September 1985. Jorissen, F.J., 1988. Benthic foraminifera from the Adriatic Sea; principles of phenotypic variation. Utrecht Micropaleontology Bulletins 37, 1–174. Kim, S.-T., O'Neil, J.R., 1997. Equilibrium and nonequilibrium oxygen isotope effects in synthetic carbonates. Geochimica et Cosmochimica Acta 61, 3461–3475. Kisakürek, B., Eisenhauer, A., Böhm, F., Garbe-Schönberg, D., Erez, J., 2008. Controls on shell Mg/Ca and Sr/Ca in cultured planktonic foraminiferan, Globigerinoides ruber (white). Earth and Planetary Science Letters 273, 260–269. Kunioka, D., Shirai, K., Takahata, N., Sano, Y., Toyofuku, T., Ujiie, Y., 2006. Microdistribution of Mg/Ca, Sr/Ca, and Ba/Ca ratios in Pulleniatina obliquiloculata test by using a NanoSIMS: implication for the vital effect mechanism. Geochemistry, Geophysics, Geosystems 7, Q12P20. http://dx.doi.org/10.1029/2006GC001280. Langer, M.R., Gehring, C.A., 1993. Bacteria farming: a possible feeding strategy of some smaller, motile foraminifera. Journal of Foraminiferal Research 23, 40–46. Le Cadre, V., Debenay, J.-P., Lesourd, M., 2003. Low pH effects on Ammonia beccarii test deformation: implications for using test deformations as a pollution indicator. Journal of Foraminiferal Research 33, 1–9. Lea, D.W., 2003. Elemental and isotopic proxies of past ocean temperatures. In: Elderfield, H. (Ed.), Treatise on Geochemistry. Elsevier, pp. 365–390. Lea, D.W., Mashiotta, T.A., Spero, H.J., 1999. Controls on magnesium and strontium uptake in planktonic foraminifera determined by live culturing. Geochimica et Cosmochimica Acta 63, 2369–2379. Lear, C.H., Rosenthal, Y., Slowey, N., 2002. Benthic foraminiferal Mg/Capaleothermometry: a revised core-top calibration. Geochimica et Cosmochimica Acta 19, 3375–3387. Lear, C.H., Elderfield, H., Wilson, P.A., 2003. A Cenozoic seawater Sr/Ca record from benthic foraminiferal calcite and its application in determining global weathering fluxes. Earth and Planetary Science Letters 208, 69–84. Lee, K., Tong, L.T., Millero, F.J., Sabine, C.L., Dickson, A.G., Goyet, C., Park, G.-H., Wanninkhof, R., Feely, R.A., Key, R.M., 2006. Global relationships of total alkalinity with salinity and temperature in surface waters of the world's oceans. Geophysical Research Letters 33. http://dx.doi.org/10.1029/2006GL027207. Lewis, E., Wallace, D., 1998. Program Developed for CO2 System Calculations. In: Division, E.S. (Ed.), Publication No. 4735. Department of Applied Science Brookhaven National Laboratory Upton, New York, Oak Ridge, Tennesse. Marchitto, T.M., Bryan, S.P., Curry, W.B., McCorkle, D.C., 2007. Mg/Ca temperature calibration for the benthic foraminifer Cibicidoides pachyderma. Paleoceanography 22. http://dx.doi.org/10.1029/2006PA001287. Martin, P.A., Lea, D.W., Mashiotta, T.A., Papenfuss, T., Sarnthein, M., 1999. Variation of foraminiferal Sr/Ca over Quaternary glacial–interglacial cycles: evidence for changes in mean ocean Sr/Ca? Geochemistry, Geophysics, Geosystems 1. http://dx.doi.org/ 10.1029/1999GC000006. Martin, P.A., Lea, D.W., Rosenthal, Y., Shackleton, N.J., Sarnthein, M., Papenfuss, T., 2002. Quaternary deep sea temperature histories derived from benthic foraminiferal Mg/Ca. Earth and Planetary Science Letters 198, 193–209. McConnaughey, T.A., 1989. 13C and 18O isotopic disequilibrium in biological carbonates: I. Patterns. Geochimica et Cosmochimica Acta 53, 151–162. McCorkle, D.C., Martin, P.A., Lea, D.W., Klinkhammer, G.P., 1995. Evidence of a dissolution effect on benthic foraminiferal shell chemistry: δ13C, Cd/Ca, Ba/Ca, and Sr/Ca results from the Ontong Java Plateau. Paleoceanography 10, 699–714. McCorkle, D.C., Bernhard, J.M., Hintz, C.J., Blanks, J., Chandler, G.T., Shaw, T.J., 2008. The carbon and oxygen stable isotopic composition of cultured benthic foraminifera. Geological Society, London, Special Publications 303, 135–154.

27

Mehrbach, C., Culberson, C.H., Hawley, J.E., Pytkowicz, R.M., 1973. Measurement of the apparent dissociation constants of carbonic acid in seawater at atmospheric pressure. Limnology and Oceanography: Methods 18, 897–907. Nehrke, G., Reichart, G.J., Van Cappellen, P., Meile, C., Bijma, J., 2007. Dependence of calcite growth rate and Sr partitioning on solution stoichiometry: non-Kossel crystal growth. Geochimica et Cosmochimica Acta 71, 2240–2249. Nelson, S.T., 2000. A simple, practical methodology for routine VSMOW/SLAP normalization of water samples analyzed by continuous flow methods. Rapid Communications in Mass Spectrometry 14, 1044–1046. Nürnberg, D., Bijma, J., Hemleben, C., 1996. Assessing the reliability of magnesium in foraminiferal calcite as a proxy for water mass temperatures. Geochimica et Cosmochimica Acta 60, 803–814. Pearce, N.J.G., Perkins, W.T., Westgate, J.A., Gorton, M.P., Jackson, S.E., Neal, C.R., Chenery, S.P., 2007. A compilation of new and published major and trace element data for NIST SRM 610 and NIST SRM 612 glass reference materials. Geostandards Newsletter 21, 115–144. Pena, L.D., Cacho, I., Ferreti, P., Hall, M.A., 2008. El Niño–Southern Oscillation‐like variability during glacial terminations and interlatitudinal teleconnections. Paleoceanography 23. http://dx.doi.org/10.1029/2008PA001620. Raitzsch, M., Dueñas-Bohórquez, A., Reichart, G.-J., de Nooijer, L.J., Bickert, T., 2010. Incorporation of Mg and Sr in calcite of cultured benthic foraminifera: impact of calcium concentration and associated calcite saturation state. Biogeosciences 7, 869–881. Raja, R., Saraswati, P.K., Rogers, K., Iwao, K., 2005. Magnesium and strontium compositions of recent symbiont-bearing benthic foraminifera. Marine Micropaleontology 58, 31–44. Rathmann, S., Kuhnert, H., 2008. Carbonate ion effect on Mg/Ca, Sr/Ca and stable isotopes on the benthic foraminifera Oridorsalis umbonatus off Namibia. Marine Micropaleontology 66, 120–133. Rathmann, S., Hess, S., Kuhnert, H., Mulitza, S., 2004. Mg/Ca ratios of the benthic foraminifera Oridorsalis umbonatus obtained by laser ablation from core top sediments: relationship to bottom water temperature. Geochemistry, Geophysics, Geosystems 5, Q12013. http://dx.doi.org/10.1029/2004GC000808. Reichart, G.-J., Jorissen, F., Anschutz, P., Mason, P.R.D., 2003. Single foraminiferal test chemistry records the marine environment. Geology 31, 355–358. Rohling, E.J., Cooke, S., 1999. Stable oxygen and carbon isotopes in foraminiferal carbonate shells. In: Sen Gupta, B.K. (Ed.), Modern Foraminifera. Kluwer Academic Publishers, Cornwall, pp. 239–258. Rollion-Bard, C., Erez, J., Zilberman, T., 2008. Intra-shell oxygen isotope ratios in the benthic foraminifera genus Amphistegina and the influence of seawater carbonate chemistry and temperature on this ratio. Geochimica et Cosmochimica Acta 72, 6006–6014. Rosenthal, Y., Boyle, E., Slowey, N., 1997. Temperature control on the incorporation of magnesium, strontium, fluorine, and cadmium into benthic foraminiferal shells from Little Bahama Bank: prospects for thermocline paleoceanography. Geochimica et Cosmochimica Acta 61, 3633–3643. Rosenthal, Y., Lear, C.H., Oppo, D.W., Linsley, B.K., 2006. Temperature and carbonate ion effects on Mg/Ca and Sr/Ca ratios in benthic foraminifera: aragonitic species Hoeglundina elegans. Paleoceanography 21, PA1007 doi:1010.1029/2005PA001158. Rosenthal, Y., Morley, A., Barras, C., Katz, M.E., Jorissen, F., Reichart, G.-J., Oppo, D.W., Linsley, B.K., 2011. Temperature calibration of Mg/Ca ratios in the intermediate water benthic foraminifer Hyalinea balthica. Geochemistry, Geophysics, Geosystems 12. http://dx.doi.org/10.1029/2010GC003333. Sadekov, A., Eggins, S.M., De Deckker, P., Kroon, D., 2008. Uncertainties in seawater thermometry deriving from intratest and intertest Mg/Ca variability in Globigerinoides ruber. Paleoceanography 23. http://dx.doi.org/10.1029/2007PA001452. Schmiedl, G., Pfeilsticker, M., Hemleben, C., Mackensen, A., 2004. Environmental and biological effects on the stable isotope composition of recent deep-sea benthic foraminifera from the western Mediterranean Sea. Marine Micropaleontology 51, 129–152. Schumacher, S., Jorissen, F.J., Mackensen, A., Gooday, A.J., Pays, O., 2010. Ontogenetic effects on stable carbon and oxygen isotopes in tests of live (Rose Bengal stained) benthic foraminifera from the Pakistan continental margin. Marine Micropaleontology 76, 92–103. Shackleton, N.J., 1974. Attainment of isotopic equilibrium between ocean water and the benthonic foraminifera genus Uvigerina: isotopic changes in the ocean during the last glacial. Colloques Internationaux du C.N.R.S., 219, pp. 203–208. Shen, C.-C., Hastings, D.W., Lee, T., Chiu, C.-H., Lee, M.-Y., Wei, K.-Y., Lawrence, E.R., 2001. High precision glacial–interglacial benthic foraminiferal Sr/Ca records from the eastern equatorial Atlantic Ocean and Caribbean Sea. Earth and Planetary Science Letters 190, 197–209. Spero, H.J., Bijma, J., Lea, D.W., Bemis, B.E., 1997. Effect of seawater carbonate concentration on foraminiferal carbon and oxygen isotopes. Nature 390, 497–500. Stouff, V., Lesourd, M., Debenay, J.-P., 1999. Laboratory observations on asexual reproduction (Schizogony) and ontogeny of Ammonia tepida with comments on the life cycle. Journal of Foraminiferal Research 29, 75–84. Toyofuku, T., Kitazato, H., 2005. Micromapping of Mg/Ca values in cultured specimens of the high-magnesium benthic foraminifera. Geochemistry, Geophysics, Geosystems 6, Q11P05. http://dx.doi.org/10.1029/2005GC000961. Toyofuku, T., Kitazato, H., Kawahata, H., Tsuchiya, M., Nohara, M., 2000. Evaluation of Mg/Ca thermometry in foraminifera: comparison of experimental results and measurements in nature. Paleoceanography 15, 456–464. Toyofuku, T., de Nooijer, L.J., Yamamoto, H., Kitazato, H., 2008. Real-time visualization of calcium ion activity in shallow water benthic foraminiferal cells using the flourescent indicator Fluo-3 AM. Geochemistry, Geophysics, Geosystems 9, Q05005. http://dx.doi.org/10.1029/2007GC001772.

28

P. Diz et al. / Marine Micropaleontology 92-93 (2012) 16–28

Toyofuku, T., Suzuki, M., Suga, H., Sakai, S., Suzuki, A., Ishikawa, T., de Nooijer, L.J., Schiebel, R., Kawahata, H., Kitazato, H., 2011. Mg/Ca and δ18O in the brackish shallow-water benthic foraminifer Ammonia ‘beccarii’. Marine Micropaleontology 78, 113–120. Wefer, G., Berger, W.H., 1991. Isotope paleontology: growth and composition of extant calcareous species. Marine Geology 100, 207–248. Wilson-Finelli, A., Chandler, G.T., Spero, H.J., 1998. Stable isotope behavior in paleocenographically important benthic foraminifera: results from microcosm culture experiments. Journal of Foraminiferal Research 28, 312–320.

Wit, J.C., Reichart, G.-J., Jung, S.J.A., Kroon, D., 2010. Approaches to unravel seasonality in sea surface temperatures using paired single-specimen foraminiferal δ18O and Mg/Ca analyses. Paleoceanography 25. http://dx.doi.org/10.1029/2009PA001857. Yu, J., Elderfield, H., 2008. Mg/Ca in the benthic foraminifera Cibicidoides wuellerstorfi and Cibicidoides mundulus: temperature versus carbonate ion saturation. Earth and Planetary Science Letters 276, 129–139. Zeebe, R.E., Bijma, J., Wolf-Gladrow, D.A., 1999. A diffusion–reaction model of carbon isotope fractionation in foraminifera. Marine Chemistry 64, 199–227.