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Ca values of coccoliths from cultured specimens of the species Emiliania huxleyi and Gephyrocapsa oceanica
Marine Micropaleontology 77 (2010) 119–124
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Marine Micropaleontology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a r m i c r o
Research paper
Mg isotopes and Mg/Ca values of coccoliths from cultured specimens of the species Emiliania huxleyi and Gephyrocapsa oceanica Kongtae Ra a,c,⁎, Hiroyuki Kitagawa a, Yoshihiro Shiraiwa b a b c
Graduate School of Environmental Studies, Nagoya University, Nagoya 464-8601, Japan Graduate School of Life and Environmental Sciences, Tsukuba University, Tsukuba 305-8572, Japan Marine Environment Pollution Prevention Research Department, Korea Ocean Research & Development Institute (KORDI), Ansan 426-744, Republic of Korea
a r t i c l e
i n f o
Article history: Received 22 January 2009 Received in revised form 7 August 2010 Accepted 9 August 2010 Keywords: Mg isotopes Mg/Ca Coccoliths Coccolithophores Temperature proxy
a b s t r a c t Coccoliths from cultured specimens of two species of coccolithophores (Emiliania huxleyi and Gephyrocapsa oceanica) were sampled during two growth phases (late exponential and stationary), and their Mg isotope values (δ26Mg) as well as Mg/Ca values were measured in order to investigate whether δ26Mg can be used as a temperature proxy. Mg/Ca values were positively related with temperature (~ 0.002 mmol/mol/°C), without statistically significant differences between the two growth phases and the two species. Both species were depleted in heavier Mg isotopes relative to the culture medium, and δ26Mg values were temperature dependent in both growth phases of E. huxleyi, although the δ26Mg values differed in the two growth phases. In G. oceanica, a weak correlation between δ26Mg values and temperature was seen in the late exponential growth phase only, and the δ26Mg values differed between growth phases. The large differences between δ26Mg values as measured in calcite formed during different growth phases indicate that Mg isotopes of coccoliths cannot be simply used as a temperature proxy. Our conclusions are preliminary and more data must be collected in order to fully evaluate the use of Mg isotopes of coccoliths as a temperature proxy. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
1. Introduction Coccolithophores (haptophyte algae) are the most important pelagic calcifying organism in the present oceans, and they produce minute calcite shields known as coccoliths (Westbroek et al., 1993). Coccolithophores carry out photosynthesis as well calcification, thus forming both organic matter and calcite. They are among the most important primary producers in open ocean, and constitute a significant components of the Earth's biogeochemical cycles (especially, the carbon and Ca cycles), owing to their great abundance, and fast turnover rates (Winter and Siesser, 1994; Bown, 1998). Coccolithophores first occurred in the Late Triassic (~ 220 Ma), and in the present ocean occur even in regions where planktic foraminifera are very rare or absent, including the subpolar regions (Bown, 1998, 2005; Thierstein and Young, 2004; Edvardsen and Medlin, 2007). Coccoliths calcite is more resistant to dissolution than foraminiferal calcite (Frenz et al., 2005; Chiu and Broecker, 2008). The geochemical records preserved in marine biogenic carbonate sediments provide valuable tools for investigating paleoclimates, including several proxies for temperature. Mg/Ca thermometry in foraminifera has been applied routinely to reconstruct changes in temperature over time, because Mg/Ca values of biogenic carbonate
⁎ Corresponding author. Tel.: + 82 31 400 6166; fax: + 82 31 406 4250. E-mail address: [email protected] (K. Ra).
are correlated with the temperature of the surrounding water (e.g., Nurnberg et al., 1996; Toyofuku et al., 2000; Barker et al., 2005). It is possible to use Mg/Ca thermometry over Plio-Pleistocene glacial/ interglacial timescales, because Mg/Ca of seawater is constant on such time scales due to the long residence times for Ca and Mg (106 and 107 years, respectively). This approach has also been applied to other carbonate materials such as ostracods, mollusks, corals, and coccolithophores (Wansard, 1996; Stoll et al., 2001; Watanabe et al., 2001; Anadon et al., 2002). In general, however, elemental ratios and isotopes proxies measured in coccoliths have not been used as mush those measured in foraminifera, coccoliths are much smaller (1–10 μm across) than planktic foraminifera, and have a much lower magnesium content. An exception is the study using coccoliths Mg/Ca as a temperature proxy (Stoll et al., 2001). In addition, other compounds have been used as temperature proxies, such as stable isotope ratios (Elderfield and Ganssen, 2000; Rosenthal et al., 2000) and alkenone biomarkers (UK37; Volkman et al., 1980, 1995; Brassell et al., 1986). The oxygen isotope values of coccoliths also record temperature changes, but oxygen isotope data are also influenced by the isotopic composition of seawater (thus global ice volume), and a wide range of vital effects has been described, ranging up to nearly 5‰ among different species under similar physical conditions (Dudley and Goodney, 1979; Ziveri et al., 2003a). The dissolved Mg isotopic composition in seawater appeared to be constant over a variety of locations and depths in the ocean (Galy
0377-8398/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.marmicro.2010.08.003
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et al., 2001; Chang et al., 2003, 2004; Young and Galy, 2004; de Villiers et al., 2005; Ra and Kitagawa, 2007) due to the long residence time of Mg (~ 13 Ma, Broecker and Peng, 1982). Attempts to reconstruct seawater temperature in the geological past based on Mg isotope analysis have lately attracted considerable attention. Mg isotope values have been reported for foraminiferal calcite (Chang et al., 2003, 2004) and aragonite deposited by sponges and corals (Wombacher et al., 2005), showing limited isotope variability, but the system is still not understood. The Mg isotope values of coccoliths may have potential as proxies for paleoceanography and paleoclimatology, but using coccoliths from bulk sediment (i.e., an uncontrolled mixture of general and species) as a proxy is problematic, due to the documented vital effects in other proxies measured in coccoliths. It is difficult to physically separate species of coccoliths from bulk sediments because of their small size (Stoll et al., 2007a). Thus, culture experiments of coccolithophores are very important for the testing of proxies and evaluation of differences between species. We present preliminary data on the Mg isotope values of coccoliths in two species (Emiliania huxleyi and Gephyrocapsa oceanica). We contribute information on species effects on the temperature dependence of Mg/Ca values and Mg isotopes measured on specimens cultured at different temperatures and sampled during two different growth phases. Our preliminary study shows that Mg isotopes do not show a simple relation with temperature in both species, and more research might document whether Mg-isotope records in coccoliths could be used as a temperature proxy. 2. Materials and methods 2.1. Culture experiments
curves of the cultured coccolithophores for both species at 20 °C. At each temperature, E. huxleyi and G. oceanica were sampled in the late exponential and stationary phases, before the culture medium was significantly altered with respect to such parameters as alkalinity and pH. Coccolithophore samples were collected in a pre-acid cleaned centrifuge tube by centrifuging. All culture experiments were done at Tsukuba University in Japan. 2.2. Mg purification in coccoliths Mg present outside the calcite is mainly present in phytopigments, and was extracted through solvent extraction, in order to determine Mg isotope values of chlorophyll (Ra, 2007). The residual fraction after extraction with 90% acetone was washed several times with quartzdistilled Milli-Q water. The potential Mg residue in the organic matter was oxidized and removed by boiling in alkaline peroxide solution (0.15% H2O2 in 0.1N NaOH). These procedures removed all non-calcite Mg from the calcite fraction. The purified coccoliths samples were dissolved in 3M HCl and loaded on the cation-exchange column in order to remove the matrix effects on the Mg isotope measurements (Galy et al., 2002; Chang et al., 2003). The purification of Mg in coccoliths was performed by a two-step purification method (AG50W-X12 resin, BioRad, USA). Mg was eluted together with Na and isolated from the K and Ca fractions in the first column (220 mm in length × 3 mm in diameter) by passing 3M HCl (Fig. 2a). The Mg fraction including Na was evaporated and redissolved in 0.4M HCl. In the second column (35 mm in length × 3 mm in diameter), Na was successfully removed with 20 ml of 0.4M HCl and then Mg was finally eluted with 2 ml of 6M HCl (Fig. 2b). The yield of Mg through the two columns was more than 99.9% (Ra and Kitagawa, 2007). The δ26Mg value of the WAKO mixed solution
The two coccolithophore species E. huxleyi and G. oceanica were selected because they are the predominant bloom-forming coccolithophre species in the oceans, thus affecting the global environment. Batch cultures of the two species of coccolithophores were carried out at three different temperatures (15, 20 and 25 °C) for E. huxleyi and at two different temperatures (20 and 25 °C) for G. oceanica, respectively. Artificial seawater medium was used as the culture medium, Marine Art SF (Senju Pharmaceutical Co., Japan) with an ESM medium in which soil extract was replaced with selenite (Danbara and Shiraiwa, 1999). Cultures were grown under a regime of 8 h darkness, 16 h light at a light intensity of 100 μmoles/m2/s. Sterilized air was introduced from the bottom of the vessel, in which the contents were gently mixed (aeration rate = 200 ml/min). The pH of the culture medium was adjusted to 8.2. The growth rates and cell number of coccolithophores were checked every 24 h. Fig. 1 shows growth
Fig. 1. Plots of coccoliths cell density vs. cultured time (h) on E. huxleyi (circle symbol) and G. oceanica (triangle symbol) culture experiment at 20 °C. The growth curve was obtained from regression fitting to polynomial model equations. The symbols show sampling at the late exponential (open) and stationary (filled grey) phases.
Fig. 2. Elution of Mg, Na, K and Ca in the first (a) and second (b) column chemistry using AG50W-X12 (IAPSO seawater; Na-107 μg, Mg-12.7 μg, K-4 μg, Ca-4 μg). Loading 0.3 ml of 3M HCl (a) and 0.2 ml of 0.4M HCl (b) samples.
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showed that no mass fractionation occurred during column separation. The purified Mg in coccoliths was dried in an evaporation chamber under an IR lamp and finally re-dissolved in 2% HNO3 for Mg isotope analyses with a multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS). All chemical experiments were done in a Class 100 clean room at Nagoya University in Japan. Mg and Ca concentrations were measured by ion chromatography (Dionex DX-100, USA). Mg isotope values were also measured in the culture medium and the original source of Mg (MgCl2∙H2O). 2.3. Mg isotope measurements The Mg isotope ratios in coccoliths were determined using a standard-sample bracketing technique with a MC-ICP-MS (IsoProbe, GV instruments, UK). Mg concentration was matched to less than ±5% at 0.5 ppm. Sample and standard solutions were injected into the ICP torch using a PFA micro-concentric nebulizer (Elemental Scientific Inc., USA). The Mg isotope ratios are expressed in d notation, thus as a part per thousand deviation from the isotope reference standard, NIST SRM 980, according to the following equation: x
δ Mg =
x
24 Mg= Mg
sample
#
x
24 Mg= Mg
SRM980
−1 × 1000
where x is 25 or 26. Typical internal precisions for δ25Mg and δ26Mg measurements were ±0.04‰ and ±0.08‰ (2σ), respectively. The long-term repeatability of δ26Mg was 0.06‰ (2σ; n = 88). Our standard was renormalized into the DSM3 scale (Ra and Kitagawa, 2007) due to the heterogeneity of SRM 980 (Galy et al., 2003). 3. Results and discussion 3.1. Mg/Ca values Mg/Ca values of coccoliths ranged from 0.029 to 0.051 mmol/mol for E. huxleyi and from 0.011 to 0.025 mmol/mol for G. oceanica (Table 1 and Fig. 3a), similar to values reported in the literature for low Mg coccoliths calcite of E. huxleyi from sediments traps from Bermuda, where values between 0.02 and 0.04 mmol/mol were measured (Stoll et al., 2007b). Our highest value (0.051 mmol/mol) for E. huxleyi is less than the lowest published value (about 0.11 mmol/mol) of Mg/Ca in cultured E. huxleyi (Stoll et al., 2001). The molar Mg/Ca value of the culture medium was 5.18, similar to the present ocean value of about 5 (Coggon et al., 2010), indicating that the Mg/Ca values of the culture medium did not cause the lower observed Mg/Ca value. It is somewhat difficult to directly compare our Mg/Ca values with those in other studies because the culture conditions were quite different. The lower Mg/Ca values of coccoliths measured in this study may be affected by the different culture experiments conditions such as light intensity, temperature, cultivation time, etc. Mg/Ca values of G. oceanica in both growth phases were significantly lower than those of E. huxleyi, suggesting the importance of vital effects in different species grown under similar culture
Fig. 3. Mg/Ca and Mg isotope values of E. huxleyi (circle symbol) and G. oceanica (triangle symbol) in coccoliths for coccolithophores. Samples were obtained in the late exponential phase (open) and stationary (solid) phases.
conditions. The changes in the relative carbonate condition of different species to bulk carbonate may cause significant changes in the Mg/Ca values, partially masking signal of temperature changes, as also suggested for the vital effects on the oxygen isotope composition of coccoliths (Dudley and Goodney, 1979; Dudley et al., 1980; Ziveri et al., 2003a). Our results showed that there is a linear increase of Mg/Ca in coccoliths over the temperature range of 10 °C for E. huxleyi and 5 °C for G. oceanica, so that the temperature dependence can be calculated as follows, E: huxleyi; ½Mg = Ca = 0:002 × TðCÞ−0:002
G: oceanica; ½Mg = Ca = 0:002 × Tð CÞ−0:024
Table 1 Mg isotopes and Mg/Ca values of coccoliths from cultured coccolithophore species (E. huxleyi and G. oceanica). Species
E. huxleyi
G. oceanica a
Temp. (°C)
Late exponential phase δ26Mg
2 S.D.
εMga
Mg/Ca
δ26Mg
Stationary phase 2S.D.
εMga
Mg/Ca
15 20 25 20 25
−1.88 −1.61 −1.24 −1.39 −1.11
0.13 0.08 0.16 0.14 0.13
−1.00 −0.73 −0.36 −0.77 −0.31
0.029 0.042 0.050 0.011 0.025
−2.61 −2.19 −1.67 −2.23 −2.19
0.12 0.13 0.12 0.12 0.11
−1.72 −1.30 −0.79 −1.35 −1.31
0.030 0.038 0.051 0.012 0.021
The Mg isotopic fractionation (εMg) between coccoliths and cultured medium is reported as: εMg = [(δ26Mgcoccoliths + 1000)/(δ26Mgmedium + 1000) − 1] × 1000.
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where T is culture temperature. The Mg/Ca values of both coccolithophore species show a general increase with increasing temperature (0.002 mmol/mol/°C). The temperature dependence of the Mg/Ca values was nearly identical for both species. 3.2. δ26Mg values of coccoliths Mg isotopes in coccoliths are isotopically lighter than in the culture medium (δ26Mg = −0.88‰), and all data (δ25Mg and δ26Mg) fall on a line with a slope of ~ 0.5, as predicted for mass-dependent fractionation. The Mg isotopic composition of the culture medium was measured before and after the culture experiments. No difference in δ26Mg values larger than the analytical error was observed. δ26Mg values of coccoliths for E. huxleyi ranged from −2.61‰ to −1.24‰, and have a positive relationship with temperature (Fig. 3b). A best fit line through the δ26Mg values suggests that the correlation with temperature is about 0.09‰/°C. The δ26 Mg measured in the exponential growth phase are consistently higher than those measured during the stationary growth phase. For the late exponential phase, δ26Mg values of coccoliths for G. oceanica were −1.39 ± 0.14‰ at 20 °C and −1.11 ± 0.13‰ at 25 °C, respectively, suggesting an apparent temperature relation of about 0.06‰/°C. In contrast, the δ26Mg values of coccoliths of G. oceanica show no significant temperature of coccoliths formed during the stationary phase. The δ26Mg values measured in coccoliths formed during exponential and stationary growth phase were different, with value of the exponential phase higher than those of the stationary phase. Culture studies indicate different non-equilibrium effects in the oxygen isotope fractionations in different species of coccolithophores (Ziveri et al., 2003b). Ca has an essential role during calcification. Mg is chemically similar to Ca hence it can substitute during calcification, but has no specific biological function. Our data show that the coccoliths were depleted in heavier Mg isotopes in the product phase, because the lighter isotopes always tend to diffuse faster than heavier ones. Continuous production of coccoliths might thus result in depletion of the heavier isotope inside the coccolithophore cell, causing the δ26Mg values of coccoliths formed during the stationary phase to be isotopically lighter than those formed during the late exponential phase. δ26Mg values thus might be affected by kinetic isotope fractionation. 3.3. Assessment the reliability of Mg isotopes of coccoliths as a temperature proxy The temperature dependence of Mg/Ca values during the late exponential and stationary growth phases for both coccolithophore species (E. huxleyi and G. oceanica) is the same (i.e., within the analytical error), and the slope of Mg/Ca with temperature is 0.002 mmol/mol/°C, for both species and both sampling times. Stoll et al. (2001) reported slopes of ~ 6% increase in Mg/Ca per degree Celsius temperature increase, as compared to out slope of Mg/Ca with temperature, i.e. ~ 7% increase per °C at both phases. Toyofuku et al. (2000) reported that Mg/Ca in two neritic high-Mg benthic foraminifera species increased by between 1.8 and 2.6% per °C, closer to the ~ 3% per °C observed for inorganic calcite (Oomori et al., 1987). In this study, however, the slope of Mg/Ca with temperature is 0.002 mmol/mol per °C, there times lower than the results in Stoll et al. (2001). They reported Mg/Ca values in coccoliths of E. huxleyi which were grown in culture at two different temperatures (17 and 27 °C), and did not describe the detailed explanation of culture conditions. It is difficult to directly compare our Mg/Ca values with those of Stoll et al. (2001), because the culture temperature is different and other condition may be different, which might have affected Mg/Ca. We conclude that Mg/Ca values of coccoliths respond more sensitively to temperature than those of foraminifera.
For E. huxleyi, the temperature relation was about 0.06‰/°C in the late exponential phase, 0.09‰/°C in the stationary phase, close to the value of 0.06‰/°C found for the exponential phase of G. oceanica, but this species shows no relation between temperature and δ26Mg in coccoliths samples during the stationary phase. Several authors have reported Ca isotopes values of coccoliths (Zhu and Macdougall, 1998; De La Rocha and DePaolo, 2000; DePaolo, 2004; Fantle and DePaolo, 2005; Gussone et al., 2006, 2007). The Ca isotopic composition, however, shows significant species-specific differences in coccolithophores, with a small but similar temperature relation found for E. huxleyi (0.027 ± 0.006‰/°C; Gussone et al., 2006) and Syracosphaera pulchra (0.029 ± 0.013‰/°C; Gussone et al., 2007). We found that δ26Mg values between two growth phases were quite different, by about 0.6‰ for E. huxleyi and by about 0.8‰ for G. oceanica at 20 °C. There is a correlation between δ26Mg and temperature for the two growth stages of coccoliths as temperature proxy thus might be limited due to the big differences in δ26Mg values by growth status, and the Mg isotopes of coccoliths may represent a complex relation between growth phase (i.e., rate of calcification), species, and temperature. The coccoliths production may be regulated by carbonate chemistry, and this factor may influence the Mg isotope values. Dissolved inorganic carbon (DIC) and total alkalinity (TA) are the most relevant parameters in biological processes. The initial DIC in the culture medium is 2.13 mM NaHCO3. Coccolithophores utilize DIC for photosynthesis and for calcification. Through the production of calcite coccoliths, DIC and TA are reduced, shifting the carbonate system towards lower partial pressure of carbon dioxide (CO2), higher pH and therefore lower carbonate ion concentration and calcite saturation state. As the coccoliths production in the two studied coccolithophore species has been shown to be sensitive to such change in carbonate chemistry. The total amount of DIC would start to decrease and finally calcification would stop at a low DIC concentration, without re-supply of DIC. In this study, the aeration makes it possible that carbonate chemistry keeps up with the biological activities, and here was no change in carbonate chemistry. Additionally, the ESM culture medium used in this study contains 8.2 mM of Tris as ingredient. Theoretically, the cultures grow under stable carbonate chemistry conditions, because of the aeration and the presence of the buffer. Nimer et al. (1995) formulated a model of inorganic carbon use, in which calcification and photosynthesis are linked to maintain intracellular pH. Hence, the difference in δ26Mg values collected during two different growth phases probably was not influenced by changes of carbonate system. Our results show a positive relationship between the Mg isotopic fractionation and growth rate. The Mg isotopic fractionation between coccoliths and the culture medium tends to become smaller as the growth rate increases, similar to what is observed in the nitrogen isotope values in marine diatoms (Wada and Hattori, 1978; Montoya and McCarthy, 1995). The difference in δ26Mg values in different growth phases thus may be a function of phytoplankton growth rates. It may cause less isotopic discrimination against the heavier isotopes, presuming kinetic isotope effects during active transport and uptake across cell membrane. Coccolithophores have relatively short reproduction times, hence coccolithophores are sensitive indicators of environmental parameters such as temperature, salinity, nutrient conditions as well as growth rates, various factors besides temperature must be considered, and species-specific variability can influence the Mg isotopic fractionation. A positive linear correlation to salinity and growth rate was also observed in the hydrogen isotopic composition of the coccolithophores E. huxleyi and G. oceanica, but there was no correlation to temperature (Schouten et al., 2006). Our results indicate that Mg isotope values in coccoliths cannot be used simply as a temperature proxy, and the ecological influence on the isotope signal may be assessed from the by comparing Mg/Ca and Mg isotope data. We do not yet know whether our experimental culture
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data can be applied to natural system. Stoll et al. (2007b) reported that coccoliths E. huxleyi collected in sediment traps are characteristic for low-Mg calcite, so that the higher value obtained in some cultured coccoliths are not representative of typical open ocean coccoliths. We need more data including data for other species of coccolithophores in order to ascertain whether or not Mg isotope values of coccoliths are useful as a temperature proxy, and questions about the importance of such factors as ecological bias, species offsets and environmental influences other than temperature should be addressed. 4. Conclusion We present Mg/Ca and Mg isotope values of coccoliths of two coccolithophore species (E. huxleyi and G. oceanica), cultured at different temperature. Species-specific temperature dependency on the Mg/Ca values in coccoliths are dependent upon temperature, and the Mg isotope values are correlated to temperature in some species in some growth phases. Mg/Ca values of coccoliths thus may be used as a proxy of temperature, because the two studied species show the same temperature sensitivity. The difference in absolute Mg/Ca values of the two species, however, complicated the use of Mg/Ca values as a paleothermometer. The differences in δ26Mg values between coccoliths samples during the late exponential and stationary phases for both species suggest that the Mg isotopes of coccoliths cannot simply be used as temperature proxy, and temperature relation differs for different species. Recently, new techniques have made it possible to separate monospecific coccoliths assemblages. Improving such techniques to separate of coccoliths belonging to different species from bulk sediments can make it possible to perform species-specific analysis. Further research, especially of the differences between species, is needed before Mg/Ca and Mg isotope values of coccoliths as studied in culture experiments can be used in paleoclimate studies. Acknowledgements The author dedicates this manuscript to the late Dr. Toshiyuki Masuzawa. This research has been partly supported by a scholarship received from MEXT (Japanese Government). We are grateful to the editor and the anonymous reviewer for their detailed comments on earlier versions of our manuscript. References Anadon, P., Ghetti, P., Gliozzi, E., 2002. Sr/Ca, Mg/Ca ratios and Sr and stable isotopes of biogenic carbonate from the Late Miocene Velona Basin (Central Apennines, Italy) provide evidence of unusual non-marine messinian conditions. Chemical Geology 187, 213–230. Barker, S., Cacho, I., Benway, H., Tachikawa, K., 2005. Planktonic foraminiferal Mg/Ca as a proxy for past oceanic temperature: a methodological overview and data compilation for the Last Glacial Maximum. Quaternary Science Reviews 24, 821–834. Bown, P.R., 1998. Calcareous nannofossil biostratigraphy. Chapman & Hall, London, p. 314. Bown, P.R., 2005. Calcareous nannoplankton evolution: a tale of two oceans. Micropaleontology 51, 299–308. Brassell, S.C., Eglinton, G., Marlowe, I.T., Pflaumann, U., Sarnthein, M., 1986. Molecular stratigraphy: a new tool for climatic assessment. Nature 320, 129–133. Broecker, W.S., Peng, T.H., 1982. Tracers in the Sea. Eldigo Press, New York. Chang, V.T.-C., Makishima, A., Belshaw, N.S., O'Nions, R.K., 2003. Purification of Mg from low-Mg biogenic carbonate for isotope ratio determination using multiple collector ICP-MS. Journal of Analytical Atomic Spectrometry 18, 296–301. Chang, V.T.-C., Williams, R.J.P., Makishima, A., Belshaw, N.S., O'Nions, R.K., 2004. Mg and Ca isotope fractionation during CaCO3 biomineralisation. Biochemical and Biophysical Research Communications 323, 79–85. Chiu, T.-C., Broecker, W.S., 2008. Toward better paleocarbonate ion reconstructions: new insights regarding the CaCO3 size index. Paleoceanography 23, 1–7. Coggon, R.M., Teagle, D.A.H., Smith-Duque, C.E., Alt, J.C., Cooper, M.J., 2010. Reconstructing past seawater Mg/Ca and Sr/Ca from Mid-Ocean Ridge Flank calcium carbonate veins. Science 327, 1114–1117.
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Magnesium isotope in chlorophyll and calcite of marine phytoplankton. Graduate School of Environmental Studies, Nagoya University. Ra, K., Kitagawa, H., 2007. Magnesium isotope analysis of different chlorophyll forms in marine phytoplankton using multi-collector ICP-MS. Journal of Analytical Atomic Spectrometry 22, 817–821. Rosenthal, Y., Lohmann, G.P., Lohmann, K.C., Sherrell, R.M., 2000. Incorporation and preservation of Mg in G. sacculifer: implication for reconstructing the temperature and 18O/16O of seawater. Paleoceanography 15, 135–145. Schouten, S., Ossebaar, J., Schreiber, K., Kienhuis, M.V.M., Langer, G., Benthien, A., Bijma, J., 2006. The effect of temperature, salinity and growth rate on the stable hydrogen isotopic composition of long chain alkenones produced by Emiliania huxleyi and Gephyrocapsa oceanica. Biogeoscience 3, 113–119. Stoll, H.M., Encinar, J.R., Alonso, J.I.G., Rosenthal, Y., Probert, I., Klaas, C., 2001. A first look at paleotemperature prospects from Mg in coccoliths carbonate: cleaning technique and culture measurements. Geochemistry Geophysics Geosystems 2, 1047. doi:10.1029/2000GC000144. Stoll, H.M., Shimizu, N., Arevalos, A., Martell, N., Banasiak, A., Zeren, S., 2007a. Insight on coccoliths chemistry from a new ion probe method for analysis of individually picked coccoliths. Geochemistry Geophysics Geosystems 8, Q06020. doi:10.1029/ 2006GC001546. Stoll, H.M., Ziveri, P., Shimizu, N., Conte, M., Theroux, S., 2007b. Relationship between coccoliths Sr/Ca ratios and coccolithophore production and export in the Arabian Sea and Sargasso Sea. Deep Sea Research Part II 54, 581–600. Thierstein, H.R., Young, J.R., 2004. Coccolithophores from molecular processes to global impact. Springer Verlag, p. 565. 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. Paleoceanograpy 15, 456–464. Volkman, J.K., Eglinton, G., Corner, E.D.S., Forsberg, T.E.V., 1980. Long-chain alkenes and alkenones in the marine coccolithophorid Emiliania huxleyi. Phytochemistry 19, 2619–2622.
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Volkman, J.K., Barrerr, S.M., Blackburn, S.I., Sikes, E.L., 1995. Alkenones in Gephyrocapsa oceanica: implications for studies of paleoclimate. Geochmica et Coschimica Acta 59, 513–520. Wada, E., Hattori, A., 1978. Nitrogen isotope effects in the assimilation of inorganic nitrogenous compounds by marine diatoms. Geomicrobiology Journal 1, 85–101. Wansard, G., 1996. Quantification of paleotemperature changes during isotopic stage 2 in the La Draga continental sequence (NE Spain) based on the Mg/Ca ratio of freshwater ostracods. Quaternary Science Reviews 15, 237–245. Watanabe, T., Winter, A., Oba, T., 2001. Seasonal changes in sea surface temperature and salinity during the Little Ice Age in the Caribbean Sea deduced from Mg/Ca and 18O/ 16 O ratios in corals. Marine Geology 173, 21–35. Westbroek, P., Brown, C.W., van Dleijswijk, J., Brownlee, C., Brummer, G.J., Conte, M., Egge, J., Fernandex, E., Jordan, R., Knappertsbusch, M., Stefels, J., Veldhuis, M., van der Wal, P., Young, J., 1993. A model system approach to biological climate forcing. The example of Emiliania huxleyi. Global and Planetary Change 8, 27–46.
Winter, A., Siesser, W.G. (Eds.), 1994. Coccolithophore. Cambridge University Press, New York, p. 242. Wombacher, F., Eisenhauer, A., Böhm, F., 2005. Magnesium isotope compositions of modern sponges and corals. Geophysical Research Abstract 7, 06868. Young, E.D., Galy, A., 2004. The isotope geochemistry and cosmogeochemistry of magnesium. Reviews in Mineralogy and Geochemistry 55, 197–230. Zhu, P., Macdougall, J.D., 1998. Calcium isotopes in the marine environment and the oceanic calcium cycle. Geochimica et Cosmochimica Acta 62, 1691–1698. Ziveri, P., Kroon, D., Stoll, H., Ganssen, G., Brummer, G.-J., 2003b. Sepcies-specific coccoliths calcite chemistry response to monsoonal upwelling (northwestern Indian Ocean). Geophysical Research Abstract 5, 05205. Ziveri, P., Stoll, H., Probert, I., Klaas, C., Geisen, M., Ganssen, G., Young, J., 2003a. Stable isotope “vital effects” in coccoliths calcite. Earth and Planetary Science Letters 210, 137–149.
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Marine Micropaleontology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a r m i c r o
Research paper
Mg isotopes and Mg/Ca values of coccoliths from cultured specimens of the species Emiliania huxleyi and Gephyrocapsa oceanica Kongtae Ra a,c,⁎, Hiroyuki Kitagawa a, Yoshihiro Shiraiwa b a b c
Graduate School of Environmental Studies, Nagoya University, Nagoya 464-8601, Japan Graduate School of Life and Environmental Sciences, Tsukuba University, Tsukuba 305-8572, Japan Marine Environment Pollution Prevention Research Department, Korea Ocean Research & Development Institute (KORDI), Ansan 426-744, Republic of Korea
a r t i c l e
i n f o
Article history: Received 22 January 2009 Received in revised form 7 August 2010 Accepted 9 August 2010 Keywords: Mg isotopes Mg/Ca Coccoliths Coccolithophores Temperature proxy
a b s t r a c t Coccoliths from cultured specimens of two species of coccolithophores (Emiliania huxleyi and Gephyrocapsa oceanica) were sampled during two growth phases (late exponential and stationary), and their Mg isotope values (δ26Mg) as well as Mg/Ca values were measured in order to investigate whether δ26Mg can be used as a temperature proxy. Mg/Ca values were positively related with temperature (~ 0.002 mmol/mol/°C), without statistically significant differences between the two growth phases and the two species. Both species were depleted in heavier Mg isotopes relative to the culture medium, and δ26Mg values were temperature dependent in both growth phases of E. huxleyi, although the δ26Mg values differed in the two growth phases. In G. oceanica, a weak correlation between δ26Mg values and temperature was seen in the late exponential growth phase only, and the δ26Mg values differed between growth phases. The large differences between δ26Mg values as measured in calcite formed during different growth phases indicate that Mg isotopes of coccoliths cannot be simply used as a temperature proxy. Our conclusions are preliminary and more data must be collected in order to fully evaluate the use of Mg isotopes of coccoliths as a temperature proxy. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
1. Introduction Coccolithophores (haptophyte algae) are the most important pelagic calcifying organism in the present oceans, and they produce minute calcite shields known as coccoliths (Westbroek et al., 1993). Coccolithophores carry out photosynthesis as well calcification, thus forming both organic matter and calcite. They are among the most important primary producers in open ocean, and constitute a significant components of the Earth's biogeochemical cycles (especially, the carbon and Ca cycles), owing to their great abundance, and fast turnover rates (Winter and Siesser, 1994; Bown, 1998). Coccolithophores first occurred in the Late Triassic (~ 220 Ma), and in the present ocean occur even in regions where planktic foraminifera are very rare or absent, including the subpolar regions (Bown, 1998, 2005; Thierstein and Young, 2004; Edvardsen and Medlin, 2007). Coccoliths calcite is more resistant to dissolution than foraminiferal calcite (Frenz et al., 2005; Chiu and Broecker, 2008). The geochemical records preserved in marine biogenic carbonate sediments provide valuable tools for investigating paleoclimates, including several proxies for temperature. Mg/Ca thermometry in foraminifera has been applied routinely to reconstruct changes in temperature over time, because Mg/Ca values of biogenic carbonate
⁎ Corresponding author. Tel.: + 82 31 400 6166; fax: + 82 31 406 4250. E-mail address: [email protected] (K. Ra).
are correlated with the temperature of the surrounding water (e.g., Nurnberg et al., 1996; Toyofuku et al., 2000; Barker et al., 2005). It is possible to use Mg/Ca thermometry over Plio-Pleistocene glacial/ interglacial timescales, because Mg/Ca of seawater is constant on such time scales due to the long residence times for Ca and Mg (106 and 107 years, respectively). This approach has also been applied to other carbonate materials such as ostracods, mollusks, corals, and coccolithophores (Wansard, 1996; Stoll et al., 2001; Watanabe et al., 2001; Anadon et al., 2002). In general, however, elemental ratios and isotopes proxies measured in coccoliths have not been used as mush those measured in foraminifera, coccoliths are much smaller (1–10 μm across) than planktic foraminifera, and have a much lower magnesium content. An exception is the study using coccoliths Mg/Ca as a temperature proxy (Stoll et al., 2001). In addition, other compounds have been used as temperature proxies, such as stable isotope ratios (Elderfield and Ganssen, 2000; Rosenthal et al., 2000) and alkenone biomarkers (UK37; Volkman et al., 1980, 1995; Brassell et al., 1986). The oxygen isotope values of coccoliths also record temperature changes, but oxygen isotope data are also influenced by the isotopic composition of seawater (thus global ice volume), and a wide range of vital effects has been described, ranging up to nearly 5‰ among different species under similar physical conditions (Dudley and Goodney, 1979; Ziveri et al., 2003a). The dissolved Mg isotopic composition in seawater appeared to be constant over a variety of locations and depths in the ocean (Galy
0377-8398/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.marmicro.2010.08.003
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et al., 2001; Chang et al., 2003, 2004; Young and Galy, 2004; de Villiers et al., 2005; Ra and Kitagawa, 2007) due to the long residence time of Mg (~ 13 Ma, Broecker and Peng, 1982). Attempts to reconstruct seawater temperature in the geological past based on Mg isotope analysis have lately attracted considerable attention. Mg isotope values have been reported for foraminiferal calcite (Chang et al., 2003, 2004) and aragonite deposited by sponges and corals (Wombacher et al., 2005), showing limited isotope variability, but the system is still not understood. The Mg isotope values of coccoliths may have potential as proxies for paleoceanography and paleoclimatology, but using coccoliths from bulk sediment (i.e., an uncontrolled mixture of general and species) as a proxy is problematic, due to the documented vital effects in other proxies measured in coccoliths. It is difficult to physically separate species of coccoliths from bulk sediments because of their small size (Stoll et al., 2007a). Thus, culture experiments of coccolithophores are very important for the testing of proxies and evaluation of differences between species. We present preliminary data on the Mg isotope values of coccoliths in two species (Emiliania huxleyi and Gephyrocapsa oceanica). We contribute information on species effects on the temperature dependence of Mg/Ca values and Mg isotopes measured on specimens cultured at different temperatures and sampled during two different growth phases. Our preliminary study shows that Mg isotopes do not show a simple relation with temperature in both species, and more research might document whether Mg-isotope records in coccoliths could be used as a temperature proxy. 2. Materials and methods 2.1. Culture experiments
curves of the cultured coccolithophores for both species at 20 °C. At each temperature, E. huxleyi and G. oceanica were sampled in the late exponential and stationary phases, before the culture medium was significantly altered with respect to such parameters as alkalinity and pH. Coccolithophore samples were collected in a pre-acid cleaned centrifuge tube by centrifuging. All culture experiments were done at Tsukuba University in Japan. 2.2. Mg purification in coccoliths Mg present outside the calcite is mainly present in phytopigments, and was extracted through solvent extraction, in order to determine Mg isotope values of chlorophyll (Ra, 2007). The residual fraction after extraction with 90% acetone was washed several times with quartzdistilled Milli-Q water. The potential Mg residue in the organic matter was oxidized and removed by boiling in alkaline peroxide solution (0.15% H2O2 in 0.1N NaOH). These procedures removed all non-calcite Mg from the calcite fraction. The purified coccoliths samples were dissolved in 3M HCl and loaded on the cation-exchange column in order to remove the matrix effects on the Mg isotope measurements (Galy et al., 2002; Chang et al., 2003). The purification of Mg in coccoliths was performed by a two-step purification method (AG50W-X12 resin, BioRad, USA). Mg was eluted together with Na and isolated from the K and Ca fractions in the first column (220 mm in length × 3 mm in diameter) by passing 3M HCl (Fig. 2a). The Mg fraction including Na was evaporated and redissolved in 0.4M HCl. In the second column (35 mm in length × 3 mm in diameter), Na was successfully removed with 20 ml of 0.4M HCl and then Mg was finally eluted with 2 ml of 6M HCl (Fig. 2b). The yield of Mg through the two columns was more than 99.9% (Ra and Kitagawa, 2007). The δ26Mg value of the WAKO mixed solution
The two coccolithophore species E. huxleyi and G. oceanica were selected because they are the predominant bloom-forming coccolithophre species in the oceans, thus affecting the global environment. Batch cultures of the two species of coccolithophores were carried out at three different temperatures (15, 20 and 25 °C) for E. huxleyi and at two different temperatures (20 and 25 °C) for G. oceanica, respectively. Artificial seawater medium was used as the culture medium, Marine Art SF (Senju Pharmaceutical Co., Japan) with an ESM medium in which soil extract was replaced with selenite (Danbara and Shiraiwa, 1999). Cultures were grown under a regime of 8 h darkness, 16 h light at a light intensity of 100 μmoles/m2/s. Sterilized air was introduced from the bottom of the vessel, in which the contents were gently mixed (aeration rate = 200 ml/min). The pH of the culture medium was adjusted to 8.2. The growth rates and cell number of coccolithophores were checked every 24 h. Fig. 1 shows growth
Fig. 1. Plots of coccoliths cell density vs. cultured time (h) on E. huxleyi (circle symbol) and G. oceanica (triangle symbol) culture experiment at 20 °C. The growth curve was obtained from regression fitting to polynomial model equations. The symbols show sampling at the late exponential (open) and stationary (filled grey) phases.
Fig. 2. Elution of Mg, Na, K and Ca in the first (a) and second (b) column chemistry using AG50W-X12 (IAPSO seawater; Na-107 μg, Mg-12.7 μg, K-4 μg, Ca-4 μg). Loading 0.3 ml of 3M HCl (a) and 0.2 ml of 0.4M HCl (b) samples.
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showed that no mass fractionation occurred during column separation. The purified Mg in coccoliths was dried in an evaporation chamber under an IR lamp and finally re-dissolved in 2% HNO3 for Mg isotope analyses with a multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS). All chemical experiments were done in a Class 100 clean room at Nagoya University in Japan. Mg and Ca concentrations were measured by ion chromatography (Dionex DX-100, USA). Mg isotope values were also measured in the culture medium and the original source of Mg (MgCl2∙H2O). 2.3. Mg isotope measurements The Mg isotope ratios in coccoliths were determined using a standard-sample bracketing technique with a MC-ICP-MS (IsoProbe, GV instruments, UK). Mg concentration was matched to less than ±5% at 0.5 ppm. Sample and standard solutions were injected into the ICP torch using a PFA micro-concentric nebulizer (Elemental Scientific Inc., USA). The Mg isotope ratios are expressed in d notation, thus as a part per thousand deviation from the isotope reference standard, NIST SRM 980, according to the following equation: x
δ Mg =
x
24 Mg= Mg
sample
#
x
24 Mg= Mg
SRM980
−1 × 1000
where x is 25 or 26. Typical internal precisions for δ25Mg and δ26Mg measurements were ±0.04‰ and ±0.08‰ (2σ), respectively. The long-term repeatability of δ26Mg was 0.06‰ (2σ; n = 88). Our standard was renormalized into the DSM3 scale (Ra and Kitagawa, 2007) due to the heterogeneity of SRM 980 (Galy et al., 2003). 3. Results and discussion 3.1. Mg/Ca values Mg/Ca values of coccoliths ranged from 0.029 to 0.051 mmol/mol for E. huxleyi and from 0.011 to 0.025 mmol/mol for G. oceanica (Table 1 and Fig. 3a), similar to values reported in the literature for low Mg coccoliths calcite of E. huxleyi from sediments traps from Bermuda, where values between 0.02 and 0.04 mmol/mol were measured (Stoll et al., 2007b). Our highest value (0.051 mmol/mol) for E. huxleyi is less than the lowest published value (about 0.11 mmol/mol) of Mg/Ca in cultured E. huxleyi (Stoll et al., 2001). The molar Mg/Ca value of the culture medium was 5.18, similar to the present ocean value of about 5 (Coggon et al., 2010), indicating that the Mg/Ca values of the culture medium did not cause the lower observed Mg/Ca value. It is somewhat difficult to directly compare our Mg/Ca values with those in other studies because the culture conditions were quite different. The lower Mg/Ca values of coccoliths measured in this study may be affected by the different culture experiments conditions such as light intensity, temperature, cultivation time, etc. Mg/Ca values of G. oceanica in both growth phases were significantly lower than those of E. huxleyi, suggesting the importance of vital effects in different species grown under similar culture
Fig. 3. Mg/Ca and Mg isotope values of E. huxleyi (circle symbol) and G. oceanica (triangle symbol) in coccoliths for coccolithophores. Samples were obtained in the late exponential phase (open) and stationary (solid) phases.
conditions. The changes in the relative carbonate condition of different species to bulk carbonate may cause significant changes in the Mg/Ca values, partially masking signal of temperature changes, as also suggested for the vital effects on the oxygen isotope composition of coccoliths (Dudley and Goodney, 1979; Dudley et al., 1980; Ziveri et al., 2003a). Our results showed that there is a linear increase of Mg/Ca in coccoliths over the temperature range of 10 °C for E. huxleyi and 5 °C for G. oceanica, so that the temperature dependence can be calculated as follows, E: huxleyi; ½Mg = Ca = 0:002 × TðCÞ−0:002
G: oceanica; ½Mg = Ca = 0:002 × Tð CÞ−0:024
Table 1 Mg isotopes and Mg/Ca values of coccoliths from cultured coccolithophore species (E. huxleyi and G. oceanica). Species
E. huxleyi
G. oceanica a
Temp. (°C)
Late exponential phase δ26Mg
2 S.D.
εMga
Mg/Ca
δ26Mg
Stationary phase 2S.D.
εMga
Mg/Ca
15 20 25 20 25
−1.88 −1.61 −1.24 −1.39 −1.11
0.13 0.08 0.16 0.14 0.13
−1.00 −0.73 −0.36 −0.77 −0.31
0.029 0.042 0.050 0.011 0.025
−2.61 −2.19 −1.67 −2.23 −2.19
0.12 0.13 0.12 0.12 0.11
−1.72 −1.30 −0.79 −1.35 −1.31
0.030 0.038 0.051 0.012 0.021
The Mg isotopic fractionation (εMg) between coccoliths and cultured medium is reported as: εMg = [(δ26Mgcoccoliths + 1000)/(δ26Mgmedium + 1000) − 1] × 1000.
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where T is culture temperature. The Mg/Ca values of both coccolithophore species show a general increase with increasing temperature (0.002 mmol/mol/°C). The temperature dependence of the Mg/Ca values was nearly identical for both species. 3.2. δ26Mg values of coccoliths Mg isotopes in coccoliths are isotopically lighter than in the culture medium (δ26Mg = −0.88‰), and all data (δ25Mg and δ26Mg) fall on a line with a slope of ~ 0.5, as predicted for mass-dependent fractionation. The Mg isotopic composition of the culture medium was measured before and after the culture experiments. No difference in δ26Mg values larger than the analytical error was observed. δ26Mg values of coccoliths for E. huxleyi ranged from −2.61‰ to −1.24‰, and have a positive relationship with temperature (Fig. 3b). A best fit line through the δ26Mg values suggests that the correlation with temperature is about 0.09‰/°C. The δ26 Mg measured in the exponential growth phase are consistently higher than those measured during the stationary growth phase. For the late exponential phase, δ26Mg values of coccoliths for G. oceanica were −1.39 ± 0.14‰ at 20 °C and −1.11 ± 0.13‰ at 25 °C, respectively, suggesting an apparent temperature relation of about 0.06‰/°C. In contrast, the δ26Mg values of coccoliths of G. oceanica show no significant temperature of coccoliths formed during the stationary phase. The δ26Mg values measured in coccoliths formed during exponential and stationary growth phase were different, with value of the exponential phase higher than those of the stationary phase. Culture studies indicate different non-equilibrium effects in the oxygen isotope fractionations in different species of coccolithophores (Ziveri et al., 2003b). Ca has an essential role during calcification. Mg is chemically similar to Ca hence it can substitute during calcification, but has no specific biological function. Our data show that the coccoliths were depleted in heavier Mg isotopes in the product phase, because the lighter isotopes always tend to diffuse faster than heavier ones. Continuous production of coccoliths might thus result in depletion of the heavier isotope inside the coccolithophore cell, causing the δ26Mg values of coccoliths formed during the stationary phase to be isotopically lighter than those formed during the late exponential phase. δ26Mg values thus might be affected by kinetic isotope fractionation. 3.3. Assessment the reliability of Mg isotopes of coccoliths as a temperature proxy The temperature dependence of Mg/Ca values during the late exponential and stationary growth phases for both coccolithophore species (E. huxleyi and G. oceanica) is the same (i.e., within the analytical error), and the slope of Mg/Ca with temperature is 0.002 mmol/mol/°C, for both species and both sampling times. Stoll et al. (2001) reported slopes of ~ 6% increase in Mg/Ca per degree Celsius temperature increase, as compared to out slope of Mg/Ca with temperature, i.e. ~ 7% increase per °C at both phases. Toyofuku et al. (2000) reported that Mg/Ca in two neritic high-Mg benthic foraminifera species increased by between 1.8 and 2.6% per °C, closer to the ~ 3% per °C observed for inorganic calcite (Oomori et al., 1987). In this study, however, the slope of Mg/Ca with temperature is 0.002 mmol/mol per °C, there times lower than the results in Stoll et al. (2001). They reported Mg/Ca values in coccoliths of E. huxleyi which were grown in culture at two different temperatures (17 and 27 °C), and did not describe the detailed explanation of culture conditions. It is difficult to directly compare our Mg/Ca values with those of Stoll et al. (2001), because the culture temperature is different and other condition may be different, which might have affected Mg/Ca. We conclude that Mg/Ca values of coccoliths respond more sensitively to temperature than those of foraminifera.
For E. huxleyi, the temperature relation was about 0.06‰/°C in the late exponential phase, 0.09‰/°C in the stationary phase, close to the value of 0.06‰/°C found for the exponential phase of G. oceanica, but this species shows no relation between temperature and δ26Mg in coccoliths samples during the stationary phase. Several authors have reported Ca isotopes values of coccoliths (Zhu and Macdougall, 1998; De La Rocha and DePaolo, 2000; DePaolo, 2004; Fantle and DePaolo, 2005; Gussone et al., 2006, 2007). The Ca isotopic composition, however, shows significant species-specific differences in coccolithophores, with a small but similar temperature relation found for E. huxleyi (0.027 ± 0.006‰/°C; Gussone et al., 2006) and Syracosphaera pulchra (0.029 ± 0.013‰/°C; Gussone et al., 2007). We found that δ26Mg values between two growth phases were quite different, by about 0.6‰ for E. huxleyi and by about 0.8‰ for G. oceanica at 20 °C. There is a correlation between δ26Mg and temperature for the two growth stages of coccoliths as temperature proxy thus might be limited due to the big differences in δ26Mg values by growth status, and the Mg isotopes of coccoliths may represent a complex relation between growth phase (i.e., rate of calcification), species, and temperature. The coccoliths production may be regulated by carbonate chemistry, and this factor may influence the Mg isotope values. Dissolved inorganic carbon (DIC) and total alkalinity (TA) are the most relevant parameters in biological processes. The initial DIC in the culture medium is 2.13 mM NaHCO3. Coccolithophores utilize DIC for photosynthesis and for calcification. Through the production of calcite coccoliths, DIC and TA are reduced, shifting the carbonate system towards lower partial pressure of carbon dioxide (CO2), higher pH and therefore lower carbonate ion concentration and calcite saturation state. As the coccoliths production in the two studied coccolithophore species has been shown to be sensitive to such change in carbonate chemistry. The total amount of DIC would start to decrease and finally calcification would stop at a low DIC concentration, without re-supply of DIC. In this study, the aeration makes it possible that carbonate chemistry keeps up with the biological activities, and here was no change in carbonate chemistry. Additionally, the ESM culture medium used in this study contains 8.2 mM of Tris as ingredient. Theoretically, the cultures grow under stable carbonate chemistry conditions, because of the aeration and the presence of the buffer. Nimer et al. (1995) formulated a model of inorganic carbon use, in which calcification and photosynthesis are linked to maintain intracellular pH. Hence, the difference in δ26Mg values collected during two different growth phases probably was not influenced by changes of carbonate system. Our results show a positive relationship between the Mg isotopic fractionation and growth rate. The Mg isotopic fractionation between coccoliths and the culture medium tends to become smaller as the growth rate increases, similar to what is observed in the nitrogen isotope values in marine diatoms (Wada and Hattori, 1978; Montoya and McCarthy, 1995). The difference in δ26Mg values in different growth phases thus may be a function of phytoplankton growth rates. It may cause less isotopic discrimination against the heavier isotopes, presuming kinetic isotope effects during active transport and uptake across cell membrane. Coccolithophores have relatively short reproduction times, hence coccolithophores are sensitive indicators of environmental parameters such as temperature, salinity, nutrient conditions as well as growth rates, various factors besides temperature must be considered, and species-specific variability can influence the Mg isotopic fractionation. A positive linear correlation to salinity and growth rate was also observed in the hydrogen isotopic composition of the coccolithophores E. huxleyi and G. oceanica, but there was no correlation to temperature (Schouten et al., 2006). Our results indicate that Mg isotope values in coccoliths cannot be used simply as a temperature proxy, and the ecological influence on the isotope signal may be assessed from the by comparing Mg/Ca and Mg isotope data. We do not yet know whether our experimental culture
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data can be applied to natural system. Stoll et al. (2007b) reported that coccoliths E. huxleyi collected in sediment traps are characteristic for low-Mg calcite, so that the higher value obtained in some cultured coccoliths are not representative of typical open ocean coccoliths. We need more data including data for other species of coccolithophores in order to ascertain whether or not Mg isotope values of coccoliths are useful as a temperature proxy, and questions about the importance of such factors as ecological bias, species offsets and environmental influences other than temperature should be addressed. 4. Conclusion We present Mg/Ca and Mg isotope values of coccoliths of two coccolithophore species (E. huxleyi and G. oceanica), cultured at different temperature. Species-specific temperature dependency on the Mg/Ca values in coccoliths are dependent upon temperature, and the Mg isotope values are correlated to temperature in some species in some growth phases. Mg/Ca values of coccoliths thus may be used as a proxy of temperature, because the two studied species show the same temperature sensitivity. The difference in absolute Mg/Ca values of the two species, however, complicated the use of Mg/Ca values as a paleothermometer. The differences in δ26Mg values between coccoliths samples during the late exponential and stationary phases for both species suggest that the Mg isotopes of coccoliths cannot simply be used as temperature proxy, and temperature relation differs for different species. Recently, new techniques have made it possible to separate monospecific coccoliths assemblages. Improving such techniques to separate of coccoliths belonging to different species from bulk sediments can make it possible to perform species-specific analysis. Further research, especially of the differences between species, is needed before Mg/Ca and Mg isotope values of coccoliths as studied in culture experiments can be used in paleoclimate studies. Acknowledgements The author dedicates this manuscript to the late Dr. Toshiyuki Masuzawa. This research has been partly supported by a scholarship received from MEXT (Japanese Government). We are grateful to the editor and the anonymous reviewer for their detailed comments on earlier versions of our manuscript. References Anadon, P., Ghetti, P., Gliozzi, E., 2002. Sr/Ca, Mg/Ca ratios and Sr and stable isotopes of biogenic carbonate from the Late Miocene Velona Basin (Central Apennines, Italy) provide evidence of unusual non-marine messinian conditions. Chemical Geology 187, 213–230. Barker, S., Cacho, I., Benway, H., Tachikawa, K., 2005. Planktonic foraminiferal Mg/Ca as a proxy for past oceanic temperature: a methodological overview and data compilation for the Last Glacial Maximum. Quaternary Science Reviews 24, 821–834. Bown, P.R., 1998. Calcareous nannofossil biostratigraphy. Chapman & Hall, London, p. 314. Bown, P.R., 2005. Calcareous nannoplankton evolution: a tale of two oceans. Micropaleontology 51, 299–308. Brassell, S.C., Eglinton, G., Marlowe, I.T., Pflaumann, U., Sarnthein, M., 1986. Molecular stratigraphy: a new tool for climatic assessment. Nature 320, 129–133. Broecker, W.S., Peng, T.H., 1982. Tracers in the Sea. Eldigo Press, New York. Chang, V.T.-C., Makishima, A., Belshaw, N.S., O'Nions, R.K., 2003. Purification of Mg from low-Mg biogenic carbonate for isotope ratio determination using multiple collector ICP-MS. Journal of Analytical Atomic Spectrometry 18, 296–301. Chang, V.T.-C., Williams, R.J.P., Makishima, A., Belshaw, N.S., O'Nions, R.K., 2004. Mg and Ca isotope fractionation during CaCO3 biomineralisation. Biochemical and Biophysical Research Communications 323, 79–85. Chiu, T.-C., Broecker, W.S., 2008. Toward better paleocarbonate ion reconstructions: new insights regarding the CaCO3 size index. Paleoceanography 23, 1–7. Coggon, R.M., Teagle, D.A.H., Smith-Duque, C.E., Alt, J.C., Cooper, M.J., 2010. Reconstructing past seawater Mg/Ca and Sr/Ca from Mid-Ocean Ridge Flank calcium carbonate veins. Science 327, 1114–1117.
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