Organic Geochemistry 31 (2000) 799±811 www.elsevier.nl/locate/orggeochem Physiological responses of lipids in Emiliania huxleyi and Gephyrocapsa oceanica (Haptophyceae) to growth status and their implications for alkenone paleothermometry Masanobu Yamamoto a,*, Yoshihiro Shiraiwa b, Isao Inouye b aDepartment of Mineral and Fuel Resources, Geological Survey of Japan, 1-1-3 Higashi, Tsukuba, Ibaraki 305-8567, Japan bInstitute of Biological Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan Received 4 January 2000; accepted 7 June 2000 (returned to author for revision 11 April 2000) Abstract The physiological responses of alkenone unsaturation indices to changes in growth status of E. huxleyi and G. oceanica strains isolated from a water sample of the NW Paci®c were examined using an isothermal batch culture K0 system. In both E. huxleyi and G. oceanica the unsaturation index U37 changed during the growth period, but the eects of this change were dierent. This suggests that genotypic variation rather than the adaptation of the strains to the geographical environment of the sampling location is a major factor in determining the physiological responses to K0 K0 U37. Changes of U37 were associated with those of the unsaturation indices of C38 and C39 alkenones, the abundance ratios of lower to higher homologues of alkenones, the abundance ratios of saturated to polyunsaturated n-fatty acids, the abundance ratio of ethyl alkenoate to alkenones, and sterol contents. These associations might be attributable to the physiological response of lipids for maintaining their ¯uidity. The degree of unsaturation both in alkenones and n- fatty acids increased at day 8, possibly due to nutrient depletion. The ethyl alkenoate/total alkenone and ethyl alkenoate/C37 alkenone ratios increased abruptly at day 8 in both strains. These ratios should be useful in clarifying the relationship between the marine environment and its corresponding growth phase of batch culture. E. huxleyi and G. K0 K oceanica can be eectively distinguished using the U37-U38Et diagram. # 2000 Elsevier Science Ltd. All rights reserved. K0 Keywords: Alkenones; U37; Paleotemperature; n-Fatty acids; Long-chain alkenes; Sterols; Batch culture; Emiliania huxleyi; Gephyr- ocapsa oceanica; Coccolithophorids 1. Introduction Gephyrocapsa (Family Gephyrocapsae) and Chrysotila and Isochrysis (Family Isochrysidaceae) (Marlowe et al., Alkenone paleothermometry was proposed in the 1984; Volkman et al., 1995). In recent classi®cation sys- mid-1980s (Brassell et al., 1986; Prahl and Wakeham, tems, the former two genera are often classi®ed into the 1987), and has been widely applied to the assessment of Family Noelaerhabdaceae (e.g. Jordan and Kleijne, late Quaternary changes in sea surface temperature 1994). Although the phylogenetic relationship between (reviewed by Brassell, 1993; MuÈ ller et al., 1998). Long the Isochrysidaceae and Noelaerhabdaceae was uncer- chain alkenones are biolipids in a speci®c group of tain, the monophyly of Emiliania, Gephyrocapsa and Iso- haptophyte algae (Volkman et al., 1980), and until now chrysis was recently con®rmed using 18SrDNA sequence they were reported exclusively from Emiliania and analysis (Edvandersen et al., 2000). In open marine environments, alkenones are thought to be produced by Emiliania and Gephyrocapsa exclusively (Marlowe et al., * Corresponding author. Fax:+81-298-61-3666. 1984, 1990). The function and biosynthetic pathways of E-mail address: [email protected] (M. Yamamoto). these compounds, however, remain unknown. 0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0146-6380(00)00080-2 800 M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811 Alkenone paleothermometry uses the physiological dependent on growth status and show intraspeci®c response of the unsaturation degree of C37 alkenones to variability. K K0 growth temperature. The unsaturation degree is expres- There are large variations of U37 and U37-temperature K K0 sed as the unsaturation indices U37 and U37 (Brassell et relationships among both cultured strains and ®eld al., 1986; Prahl and Wakeham, 1987), which are de®ned samples, along with biases due to alkenone production K as U37=([C37:2Me][C37:4Me])/([C37:2Me]+[C37:3Me]+ depth, seasonal temperature change, water column K0 [C37:4Me]) and U37=[C37:2Me]/([C37:2Me]+[C37:3Me]), degradation and sedimentary alteration. For this rea- K K0 where [C37:2Me], [C37:3Me] and [C37:4Me] are the con- son, the correlation between the U37 or U37 from core centrations of di-, tri- and tetra-unsaturated C37 alke- top sediments and the measured temperature of the nones, respectively. Early studies demonstrated a linear overlying surface water (core-top calibration) has been relationship between alkenone unsaturation indices and assessed for each region (e.g. Sikes et al., 1991; Rosell- growth temperature in a batch culture experiment with Mele et al., 1995; Pelejero and Grimalt, 1997; Sonzogni E. huxleyi (strain 55a) from the NE Paci®c (Prahl and et al., 1997; MuÈ ller et al., 1998; Herbert et al., 1998; Wakeham, 1987; Prahl et al., 1988), and this calibration TEMPUS Project Members, 1998). This is the typical has been used for assessing paleo-sea surface temperature. way of assessing paleoceanographic proxies. However, it K It remains to be resolved why, or by what mechanism, does not clarify what cause the variations of U37-and K0 alkenone unsaturation indices and growth temperature U37-temperature relationships in cultured strains and are correlated. In general, membrane lipids change their ®eld samples, or why these relationships show regional degree of unsaturation in response to varying growth variation. Answers to these questions would improve temperatures in order to maintain ¯uidity and rigidity of the simply empirical core-top calibration, and could the membrane. It is speculated that alkenones have the minimize the errors in the application of alkenone same function (Brassell et al., 1986). paleothermometry. To augment the future application After the initial calibration by Prahl and coworkers, of alkenone thermometry, there is thus a need for fur- K Volkman et al. (1995) found that the U37-temperature ther investigations of processes ranging from alkenone relationship in JBO2, a G. oceanica strain from the SW production to alkenone burial. Paci®c, diered from that suggested by Prahl's calibra- In this study we examined the physiological responses tion, especially in the range of temperatures lower than of alkenone unsaturation indices to changes in growth 20C. Sawada et al. (1996) reported that EH2, an E. status of E. huxleyi and G. oceanica isolated from a K huxleyi strain from the SW Paci®c, exhibits a U37-tem- water sample of the NW Paci®c using an isothermal perature relationship similar to that of strain JB02 (G. batch culture system. Our comparison of the con- oceanica), whereas GO1, a G. oceanica strain from the centration and compositional changes of alkenones and Mutsu Bay is similar to strain 55a (E. huxleyi). Conte et other lipids over the growth periods of these strains K al. (1998) demonstrated large variations in U37-tem- should help to clarify the physiological factors control- perature relationships among E. huxleyi and G. oceanica ling alkenone unsaturation indices. strains from various locations. These variations in cul- tured strains account for the range of variation of the K0 U37 in the particulate organic matter in water-column 2. Experiments samples from numerous locations (e.g. Conte et al., 1992; Conte and Eglinton, 1993; Sikes and Volkman, 2.1. Samples and culture experiments 1993; Ternois et al., 1997; Sawada et al., 1998). Conte et al. (1995) found that replicate isothermal Both E. huxleyi (E1A) and G. oceanica (G1A) strains cultures of the same strain showed signi®cant variability were collected o Ishigaki Island in the NW Paci®c in their biomarker pro®les, indicating that their synth- (24220N, 124200E) during March 1998 in conjunction esis ratios are in¯uenced by environmental and/or phy- with the CREST2 program. The measured temperature siological variables in addition to temperature. and salinity of the surface water at the sampling loca- Recently, Epstein et al. (1998) and Conte et al. (1998) tion were 23.12C and 34.75 psu, respectively, at the K0 demonstrated the changes of U37 with varying growth time of the sampling. A unialgal culture of E. huxleyi phase in batch culture experiments on strains of E. (E1A) was established by dilution of the seawater sam- huxleyi. They considered that nitrate de®ciency aects ple. G. oceanica appeared mixed with E. huxleyi in a K0 U37. Popp et al. (1998) used a continuous culture system crude culture. A single cell of G. oceanica was isolated K0 (chemostat culture) and found that the U37 values were using a micropipette, and was used to establish a uni- signi®cantly lower than those in batch culture systems, algal culture (G1A). For both species, taxonomic iden- K0 and that the U37 of the non-calcifying strain decreased ti®cation was con®rmed by scanning electron slightly with increasing growth rate, while the calcifying microscopy. strain showed no systematic change. These results sug- For stock cultures, both species were grown in a 100- K K0 gest that the U37- and U37-temperature relationships are ml Erlenmeyer ¯ask containing 50 ml of the ESM-nat- M. Yamamoto et al. / Organic Geochemistry 31 (2000) 799±811 801 ural seawater medium (Okaichi et al., 1982) under a 16- and passed through the detector twice at a scan speed of h light/8-h dark regime. Cultures were gently shaken by 0.17 cm/s before use. Approximately 0.3 mg of sample hand once a day to avoid settling at the bottom of ¯ask. was dissolved in 50±100 ml of dichloromethane, and a 4± For experimental cultures, a small portion of the algal 10 ml aliquot was applied using a 5-ml microsyringe. culture, usually at the late logarithmic phase, was After spotting, the rods were conditioned for 10 min at a transferred to a 500-ml Sakaguchi ¯ask containing 300 constant humidity of 65%, and subsequently suspended ml of the arti®cial seawater, Marine Art SF (Senju for 10 min in a developing tank.