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Alkenones in Gephyrocapsa oceanica: Implications for studies of paleoclimate
Geochimicaet CosmochimicaActs,Vol. 59, No. 3, pp. 513-520, 1995 Copyright© 1995ElsevierScienceLtd Printed in the USA.All rightsreserved 0016-7037/95 $9.50 + .00
Pergamon
0016-7037(95) 00325-4
AIkenones in Gephyrocapsa oceanica: Implications for studies of paleoclimate JOHN K. VOLKMAN,1,3 STEPHANIEM. BARRETT,1 SUSANI. BLACKBURN,2 and ELISABETHL. SIKES3'4 tCSIRO, Division of Oceanography, GPO Box 1538, Hobart, Tasmania 7001, Australia 2CSIRO, Division of Fisheries, GPO Box 1538, Hobart, Tasmania 7001, Australia 3Antarctic CRC, G.P.O. Box 252C, Hobart, Tasmania 7001, Australia 'Australian Geological Survey Organisation, GPO Box 378, Canberra, ACT 2601, Australia
(Received April 11, 1994; accepted in revisedform August 25, 1994) A b s t r a c t - - Emiliania huxleyi is widely regarded as the most likely source of C37-C39 alkenones in presentday seawater and Recent sediments, but other sources are required to account for the presence of alkenones in sediments that predate the first occurrence of E. huxleyi about 265,000 years ago. Analysis of the lipids of a laboratory culture of the closely related marine coccolithophorid Gephyrocapsa oceanica (strain JB02) isolated from a massive bloom in Jervis Bay, eastern Australia showed that this species also synthesizes C37-C39 alkenones and esters of di- and tri-unsaturated C36 fatty acids. This confirms earlier predictions based on the fossil record that species of Gephyrocapsa should contain these distinctive biomarkers. In this strain of G. oceanica the sum of the C3s ethyl ketone concentrations is similar to, or greater than, that of the C37 methyl ketones over the temperature range 11-29°(2, whereas the reverse is true for Emiliania huxleyi. It should thus be possible to determine whether there is a contribution from Gephyrocapsa to the alkenones in seawater and sediments. The concentrations per cell of the major alkenones showed different responses to increasing growth temperature. The 37:3 and 38:3 methyl alkenones and 38:3 ethyl alkenone showed an approximately linear decrease in cellular concentration over the entire temperature range, whereas the 37:2 and 38:2 methyl alkenones and 38:2 ethyl alkenone concentrations showed almost no change from 11 to 20°C followed by a dramatic increase above 20°C. As a result, the ratio of di- to triunsaturated methyl alkenones as measured by U~'7changes greatly with growth temperature, but the relationship is different from that found for E. huxleyi in culture. The temperature response can be approximated by the linear relationship Uk'7 = 0.049T - 0.520 (r 2 = 0.89), although a better fit can be obtained using polynomial expressions. These data might account for some of the apparent anomalies in predictions of sea surface temperature (SST) derived from Emiliania-based alkenone-SST calibrations in those sediments which contain contributions from Gephyrocapsa.
INTRODUCTION
The cosmopolitan alga E. huxleyi is the dominant coccolithophorid in many of the world's oceans (Berge, 1962; Okada and Honjo, 1973, 1975; Okada and Mclntyre, 1977, 1979), and it is believed to be the main producer of alkenones and alkenoates in the open ocean (Volkman et al., 1980b; Marlowe et al., 1984a,b, 1990). This species exists as genetically different strains, and it is now apparent that these can have different lipid compositions which respond differently to temperature change (e.g., Sikes and Volkman, 1993; Conte and Eglinton, 1993; Conte et al., 1994). E. huxleyi first appeared in the fossil record in the late Quaternary about 265,000 years ago in oxygen isotope stage 8 (Thierstein et al., 1977) and became the dominant coccolithophorid about 85,000-73,000 years ago during oxygen isotope stages 5a at low latitudes and 4 at high latitudes (Hay, 1977; Thierstein et al., 1977). Mclntyre (1970) suggested that E. huxleyi evolved from the Gephyrocapsa group perhaps from G. ericsonii via G. protohuxleyi which have very similar coccolith structures. The close relationship between the two species is shown by the presence in some natural phytoplankton samples of dimorphic strains of E. huxleyi which bear plaeoliths of both E. huxleyi and G. oceanica (e.g., Winter et al., 1979). Marlowe et al. (1990) compared microfossil distributions with alkenone abundance in a large number of sediments obtained from DSDP cores. They showed that all of the Cenozoic (i.e., middle Eocene to Recent) sediments which conmined species from the Gephyrocapsaceae also contained al-
Some species of microalgae from the class Prymnesiophyceae, such as Emiliania huxleyi Hay et Mohler, contain an unusual group of very long chain C37-C39unsaturated methyl and ethyl ketones (alkenones), as well as C31, C33, C37, and C38 alkenes and esters of C36 fatty acids (Volkman et al., 1980a, b; Marlowe et al., 1984a,b, 1990). Brassell et al. (1986a,b) showed that alkenone abundance ratios in sediments changed in a systematic way with inferred sea surface temperature (SST). They proposed a new function termed uk7 defined as: uk7 = ([37:2] -- [37:4])/([37:2] + [37:3] + [37:4]), where the concentration of the C37 alkenone with " x " double bonds is denoted by [37:x]. The relationship between alkenone abundances and temperature was quantified by Prahl and Wakeham (1987) using data from a clonal culture of E. huxleyi (clone 55a, NEPCC) grown at different temperatures. Prahl et al. (1988) proposed that a simpler term designated U~'7which does not include the concentration of the tetra-unsaturated alkenone should be used since the Ca7:4 alkenone was not abundant in many sediment or seawater samples. That is, U~'7 = [37:2]/([37:2] + [37:3]). U'37 has proven to be a useful indicator of past sea surface temperature (SST) in low to mid latitudes. It has been applied to a wide variety of paleoceanographic topics from glacial scale SST changes to estimates of upwelling intensity and frequency of El Niflo events (see Brassell, 1993, for a review; Sikes and Keigwin, 1994). 513
514
J.K. Volkman ei al.
k e n o n e s and alkenoates. From this, they c o n c l u d e d that species from the G e p h y r o c a p s a c e a e have produced alkenones for at least the last 45 million years. The family G e p h y r o c a p saceae extends back at least to the Cretaceous (Tappan, 1981)) and is characterized by a diversity o f species and subspecies many o f relatively short duration. The genus Gephyrocapsd is k n o w n from the Pliocene onwards, but relatively few species are extant. Species which are c o m m o n today include Gephyrocapsa oceanica, G. ericsonii. G. muelleri, and G. caribbeanica (e.g., Winter et al,, 1994; Okada and Mclnlyre, 1977). W e report here a study o f the lipids in the widely distributed haptophyte G. oceanica Kampter. This alga is c o m m o n in tropical to temperate waters, but it only rarely occurs in m o n o specific b l o o m s (Blackburn and Cresswell, 1993; W i n t e r et al., 1994). Its range is more restricted than E. huxleyi and it is usually found in warm marginal seas ( O k a d a and Honjo, 1975) and boundary regions in low latitudes ( B l a c k w e l d e r , 1984), although it is occasionally found in upwelling areas ( M i t c h e l l - I n n e s and Winter, 1987). In waters around Australia, it is the d o m i n a n t coccolithophorid in the Gulf o f Carpentaria, on the North W e s t S h e l f and in the Coral Sea (Hallegraeff, 1984); in s u m m e r higher concentrations occur at higher latitudes (Hallegraeff, 1984; Blackburn and Cresswell, 1993). Both E. huxleyi and G. oceanica o c c u p y similar biogeographic and ecological niches ( M c l n t y r e , 1967). Their coccolith structures are distinctive and so they are readily distinguished in seawater samples, but in s e d i m e n t s the effects o f dissolution can make coccolith identification quite difficult. The n e w data presented here show that G. oceanica contains alkenones and alkenoates and thus caution is n e e d e d w h e n alkenone distributions are used to infer absolute paleo-SST values.
METHODS Algal Samples Gephyrocapsa oceanica bloomed in Jervis Bay, New South Wales, eastern Australia (34°S) between December 16, 1992 and January 22, 1993. The bloom was clearly visible from aircraft and detectable in the visible band of the NOAA-11 satellite. Water temperatures were 18°C or higher and cell counts were up to 1.8 x 107 cells L The coccospheres were remarkably uniform in size with a diameter of 7-8/~m, and the coccoliths showed a characteristic bridge of two elements arching across a single pore. This size uniformity and oceanographic data suggest that the bloom was seeded from a small genetically similar stock derived from oceanic waters outside the bay. Full details of the bloom and description of the alga can be found in Blackburn and Cresswell (1993). Cultures were established from cells isolated from water samples collected on December 31, 1992. A clonal culture designated strain JB02 was lodged with the CSIRO Algal Culture Collection at the CSIRO Division of Fisheries in Hobart and used in these studies. Insufficient cells were available from the natural samples for detailed lipid analysis since these were only provided for species identification. Culture Conditions Cultures were grown at selected temperatures between I I and 29°C in 200 mL Erlenmeyer flasks containing 150 mL GPM medium ( Loeblich, 1975) with selenium added as H2SeO~ at I x 10 8 M. Initial inocula were temperature-adjusted log phase cultures. The cultures were maintained under cool-white fluorescent lighting at 80 #E m 2 s-~ under a 12:12 light:dark cycle; light levels were measured with a QSL-100 (Biospherical Instruments) light meter. Temperatures
were measured with a thermometer calibrated to _+0. I °C. Subsamples (2 mL) were fixed in Lugol's iodine solution and cell counts were measured with a Neubauer haemocytometer on a Zeiss Axioplan microscope (errors +_6%). The alga was cultured on three occasions. In a preliminary study, one culture was grown at 20°C and analysed for the presence of alkenones. When the results proved positive, two experiments were carried out to examine how the biochemical composition changed with growth temperature. In Experiment 1, algae from a single starter culture were grown at 11.1, 15.7, 20, 24, and 29°C, whereas in Experiment 2 cultures were grown at II .9, 15.9, 18.1, and 20°C.
Lipid Extraction and Fractionation Cultures were harvested at late logarithmic phase 9 - 1 2 days after inoculation by filtering through a 47 mm diameter glass fibre filter (Whatman GFF, nominal pore size 0.7 ffm) and extracted immediately with chloroform-methanol-water (1:2:0.8, 5 X 5 mL) using ultrasonication. Chloroform and purified water (Milli-Q ® system) were added to the combined extracts to give a final chloroform-methanol-water ratio of 1:1:0.9, to initiate phase separation. The upper aqueous phase containing salts and water-soluble material was discarded and the total solvent-extractable lipids (TSE) were recovered in the lower chloroform phase. The solvents were then removed under vacuum, and the lipids stored under nitrogen at -20°C. In Experiments I and 2 the TSE was transesterified directly with methanol-chloroform-HCl (10:1:1, v/v/v; 3 mL) at 80°C for 2 h. After addition of 1 mL of Milli-Q® water, the lipids were extracted with hexane-chloroform (4:1, v/v; 3 x 3 mL). The samples were treated with bis(trimethylsilyl)trifluoroacetamide (BSTFA) immediately before GC analysis to convert compounds containing free hydroxyl groups (e.g., sterols) to their trimethylsilyl-ether derivatives. This extract contained fatty acid methyl esters as well as the alkenones. Note that any ethyl esters of the 36:2 fatty acid are converted to the methyl ester by this procedure. Samples were stored under nitrogen at -20°(?.
Analysis by Capillary Gas Chromatography (GC) All samples were analysed with a Shimadzu 9A gas chromatograph equipped with a flame ionisation detector and cooled OCI-3 on-column injector (SGE, Australia). Samples were dissolved in chloroform to which a known amount of 23:0 fatty acid methyl ester was added as internal standard. The sample was injected at 50°C onto a nonpolar methyl silicone fused-silica capillary column (50 m × 0.32 mm i.d., Hewlett Packard). After one minute, the oven temperature was raised to 120°C at 30°C per min and then to 320°C at 3°C per rain. This final temperature was then maintained for 20 rain. Hydrogen was used as the carrier gas and the detector temperature was 330°C. These analysis conditions provided excellent separation of all of the very long-chain compounds present (Fig, 1 ). Peak areas were quantified with DAPA software (DAPA Scientific Pty. Ltd., Kalamunda, Western Australia) and converted to concentrations using appropriate response and recovery factors.
Analysis by Gas Chromatography-Mass Spectrometry (GCMS) GC-MS analyses were performed with an HP 5890 Series II GC and 5790 MSD fitted with a cooled on-column injector. The nonpolar column and chromatography conditions were the same as those described above except that the oven temperature was raised to 315°C and helium was used as the carrier gas, Electron impact mass spectra were acquired and processed with an HP 59970A Computer Workstation. Typical MSD operating conditions were: electron multiplier 2000 volts; transfer line 315°C; electron impact energy of 70 eV; 0.8 scans per second; mass range 40-600 Da. Compounds were identified by comparison with compounds previously isolated from E, hux-
leyi. RESULTS AND DISCUSSION The transesterified T S E fractions contained long-chain alkenones, alkenoates, and a simple distribution o f sterols con-
Use of alkenones in paleoclimate studies
38:2 • 38:3
515
The major sterol in each culture was 24-methylcholesta5,22E-dien-3/~-ol. This is the predominant sterol in many haptophytes, including closely related E. huxleyi (Volkman et al., 1980b), but it is also abundant in some other microalgae such as diatoms (Volkman, 1986). The concentration of this sterol per cell varied from 59 to 155 fg/cell (Table 1 ), with a trend towards highest cellular content at either extreme of the temperature range (Fig. 2a). Other unidentified sterols represented less than 2% of the total sterols at all temperatures.
sponding compounds isolated from E. huxleyi and they had identical mass spectra so we assume that the double bonds have the same positions and trans-geometry previously established for some of the alkenones by De Leeuw et al. (1980) and Reckha and Maxwell (1988). The concentrations of the major alkenones showed different responses to increasing growth temperature (Fig. 2). The concentration of the triunsaturated 37:3 methyl alkenone and 38:3 ethyl alkenone showed a quasi-linear decrease over the entire temperature range (Fig. 2c,e). In contrast, both the 37:2 methyl alkenone and 38:2 ethyl alkenone concentrations showed almost no change from 11 to 20°(2 followed by a dramatic increase above 20°C (Fig. 2d,f ). The abundances of the C38 methyl alkenones (Fig. 2g,h ) followed the same trends as the corresponding C37 alkenones. The mean ratio of triunsaturated C37 methyl alkenone to C38 ethyl alkenone was 1.08 and the range was only 0.90 to 1.34, apart from the culture at 24°C which had an inexplicably high ratio of 2.35 (Table 2). The ratio of diunsaturated C37 methyl alkenone to C38 ethyl alkenone was remarkably constant from 11 to 20°(2 (mean 0.41 ; range 0.35 to 0.48), but almost doubled at temperatures higher than this (Table 2). Prahl et al. (1988) observed that the ratio of total C37 methyl alkenones to C38 methyl plus ethyl alkenones in Emiliania changed little with growth temperature suggesting that this may be a characteristic feature of this alga. The ratio in strain JB-02 of Gephyrocapsa was also remarkably constant (mean: 0.70; range: 0.59-0.81; Table 2), but significantly different from that found by Prahl et al. (1988) for E. huxleyi ( mean: 1.46; range: 1.18-1.71; Table 2) over a similar range of temperatures. Data presented by Conte et al. (1994) for twelve strains of E. huxleyi grown at 15°(2 suggest that significant differences can also occur between strains of the same species (mean: 1.16; range 0.86-2.16), but these data still do not overlap with those for this strain of G. oceanica. In all strains of E. huxleyi analysed to date the C37 methyl alkenones are more abundant than the C3s ethyl alkenones whereas the reverse is true for the strain of G. oceanica analysed here (mean: 0.81; range 0.69-0.95; Table 2). However, the data presented by Conte et al. (1994) for twelve strains grown at 15°C shows that the ratio varies between strains (mean: 1.7; range 1.3-2.8; Table 2) and further research on Gephyrocapsa strains may reveal a similar degree of variability. If so, this could limit the use of apparent differences in these distributions between species to identify different sources of alkenones.
Alkenone Distributions
Alkenoates
Partial gas chromatograms showing the distributions of alkenones at the two extreme growth temperatures clearly show a trend from dominance of triunsaturated alkenones at temperatures around 11°C to an almost complete predominance of diunsaturated alkenones at 29°C (Fig. 1 ). The major methyl alkenones had 37 and 38 carbon atoms, whereas ethyl alkenones had 38 and 39 carbon atoms, with the former in much greater abundance in both cases (Table 1). Small amounts of C39 methyl alkenones were also detected by GC (Fig. 1) and GC-MS, but their abundance was too small to quantify accurately. The alkenones co-eluted with the corre-
G. oceanica also contained high concentrations of esters of a diunsaturated C36 fatty acid, particularly at the lower growth temperatures where the abundance of 36:2 methyl ester (after transesterification) was almost as high as the 2 major alkenones (Fig. 1 ). Small amounts of the 36:3 esters were also found (Table 1 ). Concentrations of the total 36:2 methyl plus ethyl esters ranged from 183 fg/cell at l l . I ° C to 85 fg/cell at 18.1°(2 (Table 1 ), and showed a slight decline with increasing growth temperature (Fig. 2b). Since samples were analysed after direct transmethylation, information on the possible occurrence of ethyl esters in these samples was not ob-
• 37:3
• 38:3 j
/e 37:: • 38:3\
•38:2 •39:3
\
• //•39:2 ~ , ~ . ~ • 39:2
• 37:2
;8:2
o38:3
• 38:2\ , / • 37:3~ ~J
•
38:2
/ .38:3~• 74
76 Time(mins)
/~,/•jj39:2• 39:2 78
FIG. I. Partial capillary gas chromatograms showing the distribution of long-chain lipids (alkenones and alkenoates) in G. oceanica grown at low (I 1.1"(2) and high (29°(2) temperatures. Concentration data are shown in Table !. • methyl alkenones; • ethyl alkenones; & methyl esters (but note that these samples were transesterifiedso that ethyl esters have been converted to methyl esters). The tri-unsaturated alkenones are abundant at the low temperature, but are almost undetectable at the high growth temperature. sisting almost entirely of a single sterol (Table 1 ). Long-chain alkenes of the type found in some strains of E. huxleyi (Volkman et al., 1980a) were not detected at any temperature. Details of each distribution are given below. Sterois
516
J. K. V o l k m a n et al.
Table 1. Cellular concentrations ofalkenones, esters and sterols in Gephyrocapsa oceanica (fg/cell)
Growth temperature (°C) Experiment number
11.1 1
11.9 2
15.7 1
15.9 2
18.1 2
20 1
20 2
24 1
29 1
131
109
111
73
59
79
71
113
1~;5
206 25 62 6 4 303
203 34 55 9 5 306
118 28 30 4 2 182
77 25 4 2 130
57 25 14 3 1 100
79 33 16 4 1 133
69 37 18 5 1 130
47 131 9 14 201
9 377 2 46 434
213 71 39 12 335
169 80 29 11 289
131 78 19 10 238
85 62 13 8 168
53 53 7 7 120
59 82 6 7 154
56 77 8 9 150
20 183 2 14 219
9 420 30 459
183 12 195
114 6 120
158 6 164
107 4 111
85 2 87
100 2 102
100 3 103
89 tr 89
103 103
833
715
584
409
307
389
383
509
996
Sterols: 24-mcthylcholesta-5,22E-dien-3~-ol
Methyl alkenones : 37:3 37:2 38:3 38:2 39:3 subtotal:
22
Ethyl alkenones: 38:3 38:2 39:3 39:2 subtotal
Fatty acid esters*: 36:2 36:3 subtotal: Total alkenones and alkenoates
*determined as the methyl ester after transesterification,
but likely to be mainly ethyl esters. Trace amounts of C39 methyl alkenones were
found in some cultures (Fig. 1)
tained. However, direct GC analysis of the total extract of the 20°C sample from the preliminary experiment showed that the fatty acid occurred almost entirely as the ethyl ester with less than 5% as the methyl ester. Thus, the data in Table 1 for the 36:2 "methyl ester" represents the sum of the methyl and ethyl esters, with the latter likely to be the major form. The ratio of tri- to di-unsaturated esters changes systematically with temperature (Table 1 ), but the 36:2 esters always greatly predominate over the 36:3 esters even at the lowest growth temperatures. The ratio of the abundances of these esters to the alkenones is considerably higher in Gephyrocapsa than in Emiliania at all growth temperatures (Prahl et al., 1988). Others have constructed temperature calibrations based on alkenoate and alkenone abundances in Emiliania ( e . g . , AA36, see Conte et al., 1992; Conte and Eglinton, 1993), but from our limited data for Gephyrocapsa it seems likely that different temperature relationships will be found between the two genera of algae.
Calibration of U~ against Culture Temperature In Fig. 3, we have plotted U~'7values against temperature for the G. oceanica culture data. The data can be fitted by a linear relationship of the form U~'7 = 0.049T - 0,520 (r: = 0.89). This relationship is statistically different at the 95% confidence interval by 2-way t test from that established for E. huxleyi clone 55a in culture where U~'7 = 0.033T + 0.043 (Prahl and Wakeham, 1987). In particular, U~'7values for any given temperature are higher for the E. huxleyi clone than for G. oceanica (by 0.1 at 28°C and 0.4 at 11°C). The slope of the line is significantly different and the error envelopes of the two relationships do not overlap. Note that these changes in U~'7 with temperature in G. oceanica are not due to a simple conversion of one alkenone to another, but reflect complicated changes in cellular abundance. From l 1 to 20°C, the change is mainly due to a decrease in the abundance of the 37:3 alkenone, but at higher
temperatures the change is mainly due to a dramatic increase in the cellular content of the 37:2 alkenone (Fig. 2). For this reason, it is perhaps not surprising that a residuals analysis indicates nonlinearity in the Gephyrocapsa dataset. This can be seen visually in Fig. 3 where all the cold and warm water data points fall above the linear relationship and all the points at intermediate temperatures fall below. A better fit to the data can be achieved by applying a polynomial such as a Richard's growth curve to the dataset. This function is mathematically bounded by 0 and l - - a s is U~'v--and reproduces observations from field data that the change in alkenone ratios is much less at extremes of the temperature range (Sikes and Volkman, 1993). The relationship U~'7 = [21 + e -3°3~r-2522)] 0o5 reduces the least mean squared residuals from 0.007 for the linear regression to 0.002. However, we stress that other polynomials may be more applicable and a choice between them must await better information about the biophysical function of alkenones in these algae. For practical purposes, the data are reasonably represented by a linear regression at temperatures above 1I°C and for paleoclimate studies a linear function is certainly more suitable given the uncertainties involved. M. Conte (pets. commun., 1994) also found that U~'7changes in an approximately linear fashion for a different strain of Gephyrocapsa isolated from the subtropical western Pacific although the intercept differs from that found here. In Fig. 4 we have plotted U~'7values for seawater samples published by Prahl and Wakeham (1987), Prahl et al. ( 1988 ), Conte and Eglinton (1993), and Sikes and Volkman (1993) against surface seawater mixed layer (SML) temperatures. The combined dataset represents approximately the entire range of SML temperatures in the open ocean and includes seawater samples from the eastern Pacific, Caribbean, North Atlantic, equatorial Pacific, and Southern oceans. It is clear from these data that U~'7changes systematically with temperature although there is variability where data from different geographical regions having similar SML temperatures are
Use of alkenones in paleoclimate studies
36:2 m d ~ d estmr |aftur
tnmmmWffoellonl
~
j
200 150
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15 25 T*mlNmnur* I°C)
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35
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5
Temperature
25 (°C)
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517
water or sediment samples (e.g., Conte and Eglinton, 1993; Rostek et al., 1993), and in such cases it might be possible to estimate the relative contributions of the two algae from linear addition of the different alkenone profiles making use of the fact that the C37:C38 alkenone ratios or alkenone:alkenoate ratios appear to be different in these species (Table 2). This requires that the whole distribution of C37C39 alkenones and alkenoates must be quantified, not just the C37 components as done in most studies at present.
d 37:2methyldkenone
37:3methylalkenone
250 200 i ,(
Caveats on the use of U~'7 calibrations in different water masses and sediments o f varied geological ages
400. ~ 350 300 250 200~
150 100
°
!
:
50, 0 5
15
25 TemplxltUm IoC|
35
1 go 60. 0 5
..
..m 15
25
Temporeture
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PC)
f
38:2 ~
38:3 ethyl dkenone 250
dkenone
500
200 i <
150 100 .i
50.
~
0 5
15
25 Temperature (oC)
100. O~ 5
35
15
25
Temperature
35
(°C)
h
38:2 methyl dkenon4
38:3 memyl alkenone
-- 50]
70
40 O
10 ¸ 0 5
ill .
15
25 Tenq~rlturl (*C)
35
5
iI
15 Temperlaure
25 (°C)
35
FIG. 2. Cellular concentrations (femtogram per cell) of individual lipids plotted against culture temperature. (a) 24-methylcholesta5,22-dien-3~/-ol;(b) 36:2 fatty acid esters; (c) 37:3 methyl alkenone; (d) 37:2 methyl alkenone; (e) 38:3 ethyl alkenone; (f) 38:2 ethyl alkenone; (g) 38:3 methyl alkenone; and (h) 38:2 methyl alkenone.
combined. It is intriguing that the slope of 0.049 for the linear temperature relationship for G. oceanica is remarkably similar to the value of 0.041 found by Sikes and Volkman (1993) and 0.037 by Brassell (1993) for seawater samples containing mainly E. huxleyi, although it must be pointed out that the intercepts on the temperature axis are quite different. The temperature responses of G. oceanica and the E. huxleyi strain analysed by Prahl et al. (1988) in culture enclose most of the data derived from natural seawater samples. It seems reasonable that while some of the variation is due to differences between strains of Emiliania, some of the scatter must reflect contributions from more than one species. Some authors have noted the presence of Gephyrocapsa in their
Studies by Jasper and Gagosian (1989), McCaffrey et al. (1990), Conte et al. (1992), Freeman and Wakeham (1992), Sikes and Volkman (1993), and others indicate that a single calibration of U~ with SST is not appropriate for all geographic areas, presumably due to the presence of different strains having different biochemical compositions. Indeed, genotypic and phenotypic variations in E. huxleyi have been well documented (Brand, 1982; Van Bleijswijk et al., 1991 ), with at least four genetically distinct morphotypes of E. huxleyi being recognised (Young and Westbroeck, 1991 ), as have differences in lipid compositions between strains (Conte et al., 1994; J. K. Volkman et al., unpubl, data). Calibrations based on field data are the most appropriate for studies of ancient sediments. For sediments in which E. huxleyi is the dominant coccolithophorid, the choice of calibration will probably be determined by the likely SST temperature range present and type of oceanic regime. For example, calibrations derived from Southern Ocean data (Sikes and Volkman, 1993 ) are clearly quite inappropriate for the Black Sea (Freeman and Wakeham, 1992). For sediments where Gephyrocapsa is dominant the choice is not so clear, and the difficulty is compounded in sediments which predate the first appearance of E. huxleyi since calibrations for E. huxleyi are probably not appropriate. Sediments older than 85000 years can also be expected to yield less certain SST estimates since E. huxleyi may be a contributor but not be the dominant source of alkenones. If our data for G. oceanica are typical for the genus Gephyrocapsa then the calibration determined in our work could be used. However, Brand (1982) has documented genetic differences in G. oceanica, and recent studies of other strains of Gephyrocapsa indicate that a range of responses is likely as with E. huxleyi (M. Conte, pers. commun., 1994). Fortunately, the differences in inferred SST values above 25°C may not be very large irrespective of which calibration is used, but at temperatures from 10 to 25°C the difference may be 6°(2 or more (Fig. 4). G. oceanica is most common in waters of temperatures above 20°C whereas other species may predominate at lower temperatures. Clearly a degree of caution is needed, and field calibrations are essential, when using U~'7to infer absolute paleo-SST values. Fortunately the slopes of the various calibrations are not greatly different so that estimated changes in paleo-SST are similar despite large differences in inferred absolute values. One must also consider what temperature is actually being measured using the alkenone calibrations. It is obvious that the alkenone composition reflects the temperature at which
518
J . K . Volkman et al. Table 2. Ratios of the abundances of selected alkenones in Gephyrocapsa oceanica and EmUiania huxleyi
Grov~ Temp. *C
37:3 methyl 37:2methyl + 38:3 ethyl 4-38:2 ethyl
Z37 methyl + 238 ethyl
237 methyl + Y38 alkenones
k' U37
Ref.
0.81 0.95 0.70 0.69 0.77 0.79 0.80 0.88 0.90
0.66 0.76 0.60 0.59 0.67 0.70 0.68 0.79 0.81
0.11 0.14 0.19 0.25 0.31 0.29 0.35 0.74 0.98
(1) (1) (1) (1) (1) (1) (1) (1) (1)
1.18 1.34 1.51 2.26 1.22 1.o3 0.91 1.71 1.58
0.29 0,38 0.56 0.25 0.34 0.28 0.35 0.73 0.86
(2) (2)
Gephyrocapsa oceanica 11.1 11.9 15.7 15.9 18.1 20 20 24 29
0.98 1.20 0.90 0.91 1.08 1.34 1.23 2.35 1.00
Emiliania huxleyi 8 10 15 15 15 15 15 20 25
0.35 0.43 0.36 0.40 0.47 0.40 0.48 0.72 0.90
3.76
1.50
2.72
1.61 1.21 1.26
1.86 1.13 0.94
1.69 1.19 1.14
(2)
(3) (4) (4) (4) (2) (2)
(1): This work;(2): PRAHLet al. (1988); (3): clone92d, MARLOWEet al. (1984b); (4): motile,sessileand coccolithformsof a singleclone,VOLKMANel al. (1980a).
the o r g a n i s m grows, w h i c h m a y not necessarily be w h e r e the algae ( o r their r e m a i n s ) are found. C o c c o l i t h o p h o r e s are typically m o s t abundant in well-illuminated surface m i x e d layers w h e r e they can flourish even with e x t r e m e l y low levels o f the m a j o r nutrients ( B r a n d et al., 1983; Balch et al., 1991 ), and thus the appropriate temperature is that o f the surface m i x e d layer ( S M L ) . This is usually e q u a t e d with the sea surface temperature ( S S T ) for practical purposes, although it m a y be inappropriate in those situations w h e r e a subsurface maxim u m in c o c c o l i t h o p h o r e a b u n d a n c e occurs (e.g., Prahl e t a ] . , 1993 ). Such areas are typically found in oligotrophic regions w h e r e c o c c o l i t h o p h o r e s can be important constituents o f the phytoplankton. Seasonal c h a n g e s in SST and their relation-
ship to algal production must also be c o n s i d e r e d (e.g., C o n t e and Eglinton, 1993). A possible c o m p l i c a t i o n arises in the case o f large " m i l k y w a t e r " b l o o m s w h e r e d e t a c h e d coccoliths substantially reflect i n c o m i n g light ( b y up to 2 5 % ) and thus appear as white or grey patches in A V H R R satellite i m a g e s o f the sea surface (e.g., A c k l e s o n et al., 1988; Balch et al., 1991). U n d e r these b l o o m conditions ( a m i s n o m e r since cell a b u n d a n c e s in the water are rarely very h i g h ) a w a r m i n g o f the sea surface temperature by as m u c h as 5°C c o m p a r e d with the surrounding seawater can occur ( A c k l e s o n et al., 1988). In this e x a m p l e , the SST increased at 0.32°C per day during the bloom. Surface w a r m i n g due to near-surface light scattering and absorption
o Gephyrocapsa O: 0,~
- - Richer(Is growth curve I - - iinear regression
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:
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Gephyrocapsa
~+
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+
0.4
+
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02
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,;
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~o
Temperature (°C) FIG. 3. Plot of U~'7 against temperature for G. oceanica cultures. Two lines of best fit are shown. The linear relationship has the equation U~7 = 0.049T - 0.520 (r2 = 0.89) and the nonlinear curve is described by the Richard's growth curve: U~7 = [21 .+_ e-3.O3(T-25.22)]
+
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0.05.
/
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i
10
15
20
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30
Temperature (°C) FIG. 4. Plot of U~'7against temperature for the G. oceanica cultures and surface seawater data compiled from the literature (see text). Also shown is the linear fit to the Gephyrocapsa data (U~'7 = 0.1M9T 0.520) and the linear relationship established by Prahl and Wakeham (1987) for clone 55a of E. huxleyi in culture.
Use of alkenones in paleoclimate studies was also observed in an extensive bloom of E. huxleyi which occurred in the northeastern Atlantic Ocean in June 1991 (Holligan et al., 1993). It is not yet clear if such events are important determinants of local SST. However, coccolithophore blooms have certainly been common in the geological past, and thus the temperature recorded by alkenones in sediments may differ from that of seawater outside of the area covered by the bloom. It may thus be useful to measure the total abundance of alkenones per unit mass of sediment as a proxy for algal biomass (e.g., Prahl et al., 1988) in order to identify whether bloom conditions might have prevailed when the sediments were deposited. The difficulty here is that the effects of degradation of alkenones, particularly in oxic surface sediments, must be assessed (Prahl et al., 1989). As with any technique, possible limitations in the alkenone calibrations must be recognized and taken into account when estimates of paieo-SST are made. The great advantage of this approach is that it provides complementary data to techniques based on oxygen isotopes and foraminiferal assemblages (both of which also have restrictions on their use). Further studies in which several of these techniques are used in combination (e.g., Jasper and Gagosian, 1989; Sikes and Keigwin, 1994; Rostek et al., 1993) will undoubtedly lead to more reliable reconstructions of past climate changes. CONCLUSIONS
C37-C39 alkenones and esters of 36:2 and 36:3 fatty acids have been identified in the lipids of a laboratory culture of the coccolithophorid Gephyrocapsa oceanica isolated from a bloom in Jervis Bay, eastern Australia. This confirms earlier predictions based on the fossil record that species of Gephyrocapsa should contain these distinctive biomarkers. The distribution of alkenones in G. oceanica is broadly similar to that in E. huxleyi, but differences in the ratio of C37:C38alkenones or alkenones: alkenoates might make it possible to distinguish between contributions from the two species in seawater and sediments. The ratio of di- to tri-unsaturated alkenones changes systematically with growth temperature, but the relationship is different from those found for strains of E. huxleyi. These data indicate that Emiliania-based alkenone-SST calibrations should be used with caution to infer paleo-SST values from sediments which predate the first appearance of E. huxleyi or in areas where Gephyrocapsa may be important, such as warm marginal seas and subtropical waters. Acknowledgments--We thank Andrew Revill for valuable comments on the manuscript and for his help in preparing the figures. John Wilkin provided very useful assistance with data fitting, Barbara Molfino provided information on the evolution of Emiliania and Tom Truli and Peter Sedwick provided helpful comments on the manuscript. We thank Jervis Bay Sea Sports for providing the G. oceanica bloom sample and Suzanne Norwood for expert assistance with algal culture. Fred Prahl, Manreen Conte, and an anonymous reviewer provided very helpful and detailed comments on an earlier version of the manuscript. M. Conte is also thanked for providing access to unpublished data. This work was supported by the Antarctic Scientific Advisory Committee and the Australian Research Council. Editorial handling: S. Wakeham REFERENCES
Ackleson S., Balch W. M., and Holligan P. M. (1988) White waters of the Gulf of Maine. Oceanography 1, 18-22.
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McCaffrey M. A., Farrington J. W., and Repeta D. J. (1990) The organic geochemistry of Peru margin surface sediments--I. A comparison of the C~7 alkenone and historical El Nifio records. Geochim. Cosmochim. Acta 54, 1671 - 1682. Mclntyre A. (1967) Coccoliths as paleoclimatic indicators of Pleis tocene glaciation. Science 158, 1314-1317. Mclntyre A. (1970) Gephyrocapsa protohuxleyi sp. n. a possible phyletic link and index fossil for the Pleistocene. Deep-Sea Res. 17, 187-190. Mitchell-lnnes B. A. and Winter A, (1987) Coccolithophores: a major phytoplankton component in mature upwelled waters off the Cape Peninsula, South Africa in March, 1983. Mur. BioL 95, 2 5 30. Okada H. and Honjo S. ( 1973 ) The distribution of oceanic coccolithophorids in the Pacific. Deep Sea Res. 20, 355 374. Okada H. and Honjo S. (1975) Distribution of coccolithophores in marginal seas along the western Pacific Ocean and in the Red Sea. Mar. Biol. 31, 271-285. Okada H. and McIntyre A. (1977) Modern coccolithophores of the Pacific and North Atlantic Ocean. Micropaleontology 23, 1-55. Okada H. and Mclntyre A. (1979) Seasonal distribution of modem coecolithophores in the western North Atlantic Ocean. Mar. Biol. 54, 319-328. Prahl F. G. and Wakeham S. G. (1987) Calibration of unsaturation patterns in long-chain ketone composition for palaeotemperature assessment. Nature 33tl, 367-369. Prahl F. G., Muelhausen L. A., and Zahnle D. A. (1988) Further evaluation of long-chain alkenones as indicators of paleoceanographic conditions. Geochim. Cosmochim. Acta 52, 2303-2310. Prahl F. G., De Lange G. J., Lyle M., and Sparrow M. A. (1989) Post-depositional stability of long-chain alkenones under contrasting redox conditions. Nature 341, 434-437. Prahl F. G., Collier R. B., Dymond J., Lyle M., and Sparrow M. A. (1993) A biomarker perspective on prymnesiophyte productivity in the northeast Pacific Ocean. Deep-Sea Res. 40, 2061-2076. Rechka J. A. and Maxwell J. R. (1988) Charaeterisation of alkenone temperature indicators in sediments and organisms. In Advances in Organic Geochemistry 1987 (ed. L. Mattavelli and L. Novelli ), pp. 727-734. Pergamon Press.
Rostek F., Ruhland G., Basssinot F. C., Muller P. J., Labeyrie L. D., Lancelot Y., and Bard E. (1993) Reconstructing sea surface temperature and salinity using 6 ~sO and alkenone records. Nature 364, 319-321. Sikes E. L. and Keigwin L. D. (1994) Equatorial Atlantic sea surface temperature for the last 30 kyr: A comparison of U~'7, 3 ~sO and foraminiferal assemblage temperature estimates. Paleoceanogruphy 9, 31-45. Sikes E. L. and Volkman J. K. (1993) Calibration of alkenone unsaturation ratios (U~'7) for palaeotemperature estimation in cold polar waters. Geochim. Cosmochim. Acta 57, 1883-1889. Tappan H. (1980) The Paleobiology of Plant Protists. Freeman. Thierstein H. R., Geitsenauer K. R., and Molfino B. (1977) Global synchroneity of late Quaternary coccolith datum levels: validation by oxygen isotopes. Geology 5, 400-404. Van Bleijswijk J., et al. ( 1991 ) Distribution of two types of Emiliania huxlevi (Prymnesiophyceae) in the northeast Atlantic region as determined by immunofluorescence and coccolith morphology. Z Phycol. 27, 566-570. Volkman J. K. (1986) A review of sterol markers for marine and terrigenous organic matter. Org. Geochem. 9, 83-100. Volkman J. K., Eglinton G., Comer E. D. S., and Forsberg T. E. V. (1980a) Long chain alkenes and alkenones in the marine coccolithophorid Emiliania huxleyi. Phytochemistry 19, 2619-2622. Volkman J. K., Eglinton G,, Corner E. D. S., and Sargent J. R. (1980b) Novel unsaturated straight-chain C37-C3~ methyl and ethyl ketones in marine sediments and a coccolithophorid Emiliania huxleyi, In Advances in Organic Geochemistry 1979 (ed. A. G. Douglas and J. R. Maxwell ), pp. 219-227. Pergamon Press. Winter A., Reiss Z., and Luz B. (1979) Distribution of living coccolithophore assemblages in the Gulf of Elat ("Aqaba). Mar Micropaleont. 4, 197-223. Winter A., Jordan R. A., and Roth P. (1994) Biogeography of living coccolithophores in ocean waters. In Coccolithophores (ed. A. Winter and W. G. Sieser), pp. 161-177. Cambridge University Press. Young J. R. and Westbroek P. ( 1991 ) Genotypic variation in the coccolithophorid species Emiliania huxleyi. Mar. Micropaleontol. 18, 5-23.
Pergamon
0016-7037(95) 00325-4
AIkenones in Gephyrocapsa oceanica: Implications for studies of paleoclimate JOHN K. VOLKMAN,1,3 STEPHANIEM. BARRETT,1 SUSANI. BLACKBURN,2 and ELISABETHL. SIKES3'4 tCSIRO, Division of Oceanography, GPO Box 1538, Hobart, Tasmania 7001, Australia 2CSIRO, Division of Fisheries, GPO Box 1538, Hobart, Tasmania 7001, Australia 3Antarctic CRC, G.P.O. Box 252C, Hobart, Tasmania 7001, Australia 'Australian Geological Survey Organisation, GPO Box 378, Canberra, ACT 2601, Australia
(Received April 11, 1994; accepted in revisedform August 25, 1994) A b s t r a c t - - Emiliania huxleyi is widely regarded as the most likely source of C37-C39 alkenones in presentday seawater and Recent sediments, but other sources are required to account for the presence of alkenones in sediments that predate the first occurrence of E. huxleyi about 265,000 years ago. Analysis of the lipids of a laboratory culture of the closely related marine coccolithophorid Gephyrocapsa oceanica (strain JB02) isolated from a massive bloom in Jervis Bay, eastern Australia showed that this species also synthesizes C37-C39 alkenones and esters of di- and tri-unsaturated C36 fatty acids. This confirms earlier predictions based on the fossil record that species of Gephyrocapsa should contain these distinctive biomarkers. In this strain of G. oceanica the sum of the C3s ethyl ketone concentrations is similar to, or greater than, that of the C37 methyl ketones over the temperature range 11-29°(2, whereas the reverse is true for Emiliania huxleyi. It should thus be possible to determine whether there is a contribution from Gephyrocapsa to the alkenones in seawater and sediments. The concentrations per cell of the major alkenones showed different responses to increasing growth temperature. The 37:3 and 38:3 methyl alkenones and 38:3 ethyl alkenone showed an approximately linear decrease in cellular concentration over the entire temperature range, whereas the 37:2 and 38:2 methyl alkenones and 38:2 ethyl alkenone concentrations showed almost no change from 11 to 20°C followed by a dramatic increase above 20°C. As a result, the ratio of di- to triunsaturated methyl alkenones as measured by U~'7changes greatly with growth temperature, but the relationship is different from that found for E. huxleyi in culture. The temperature response can be approximated by the linear relationship Uk'7 = 0.049T - 0.520 (r 2 = 0.89), although a better fit can be obtained using polynomial expressions. These data might account for some of the apparent anomalies in predictions of sea surface temperature (SST) derived from Emiliania-based alkenone-SST calibrations in those sediments which contain contributions from Gephyrocapsa.
INTRODUCTION
The cosmopolitan alga E. huxleyi is the dominant coccolithophorid in many of the world's oceans (Berge, 1962; Okada and Honjo, 1973, 1975; Okada and Mclntyre, 1977, 1979), and it is believed to be the main producer of alkenones and alkenoates in the open ocean (Volkman et al., 1980b; Marlowe et al., 1984a,b, 1990). This species exists as genetically different strains, and it is now apparent that these can have different lipid compositions which respond differently to temperature change (e.g., Sikes and Volkman, 1993; Conte and Eglinton, 1993; Conte et al., 1994). E. huxleyi first appeared in the fossil record in the late Quaternary about 265,000 years ago in oxygen isotope stage 8 (Thierstein et al., 1977) and became the dominant coccolithophorid about 85,000-73,000 years ago during oxygen isotope stages 5a at low latitudes and 4 at high latitudes (Hay, 1977; Thierstein et al., 1977). Mclntyre (1970) suggested that E. huxleyi evolved from the Gephyrocapsa group perhaps from G. ericsonii via G. protohuxleyi which have very similar coccolith structures. The close relationship between the two species is shown by the presence in some natural phytoplankton samples of dimorphic strains of E. huxleyi which bear plaeoliths of both E. huxleyi and G. oceanica (e.g., Winter et al., 1979). Marlowe et al. (1990) compared microfossil distributions with alkenone abundance in a large number of sediments obtained from DSDP cores. They showed that all of the Cenozoic (i.e., middle Eocene to Recent) sediments which conmined species from the Gephyrocapsaceae also contained al-
Some species of microalgae from the class Prymnesiophyceae, such as Emiliania huxleyi Hay et Mohler, contain an unusual group of very long chain C37-C39unsaturated methyl and ethyl ketones (alkenones), as well as C31, C33, C37, and C38 alkenes and esters of C36 fatty acids (Volkman et al., 1980a, b; Marlowe et al., 1984a,b, 1990). Brassell et al. (1986a,b) showed that alkenone abundance ratios in sediments changed in a systematic way with inferred sea surface temperature (SST). They proposed a new function termed uk7 defined as: uk7 = ([37:2] -- [37:4])/([37:2] + [37:3] + [37:4]), where the concentration of the C37 alkenone with " x " double bonds is denoted by [37:x]. The relationship between alkenone abundances and temperature was quantified by Prahl and Wakeham (1987) using data from a clonal culture of E. huxleyi (clone 55a, NEPCC) grown at different temperatures. Prahl et al. (1988) proposed that a simpler term designated U~'7which does not include the concentration of the tetra-unsaturated alkenone should be used since the Ca7:4 alkenone was not abundant in many sediment or seawater samples. That is, U~'7 = [37:2]/([37:2] + [37:3]). U'37 has proven to be a useful indicator of past sea surface temperature (SST) in low to mid latitudes. It has been applied to a wide variety of paleoceanographic topics from glacial scale SST changes to estimates of upwelling intensity and frequency of El Niflo events (see Brassell, 1993, for a review; Sikes and Keigwin, 1994). 513
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J.K. Volkman ei al.
k e n o n e s and alkenoates. From this, they c o n c l u d e d that species from the G e p h y r o c a p s a c e a e have produced alkenones for at least the last 45 million years. The family G e p h y r o c a p saceae extends back at least to the Cretaceous (Tappan, 1981)) and is characterized by a diversity o f species and subspecies many o f relatively short duration. The genus Gephyrocapsd is k n o w n from the Pliocene onwards, but relatively few species are extant. Species which are c o m m o n today include Gephyrocapsa oceanica, G. ericsonii. G. muelleri, and G. caribbeanica (e.g., Winter et al,, 1994; Okada and Mclnlyre, 1977). W e report here a study o f the lipids in the widely distributed haptophyte G. oceanica Kampter. This alga is c o m m o n in tropical to temperate waters, but it only rarely occurs in m o n o specific b l o o m s (Blackburn and Cresswell, 1993; W i n t e r et al., 1994). Its range is more restricted than E. huxleyi and it is usually found in warm marginal seas ( O k a d a and Honjo, 1975) and boundary regions in low latitudes ( B l a c k w e l d e r , 1984), although it is occasionally found in upwelling areas ( M i t c h e l l - I n n e s and Winter, 1987). In waters around Australia, it is the d o m i n a n t coccolithophorid in the Gulf o f Carpentaria, on the North W e s t S h e l f and in the Coral Sea (Hallegraeff, 1984); in s u m m e r higher concentrations occur at higher latitudes (Hallegraeff, 1984; Blackburn and Cresswell, 1993). Both E. huxleyi and G. oceanica o c c u p y similar biogeographic and ecological niches ( M c l n t y r e , 1967). Their coccolith structures are distinctive and so they are readily distinguished in seawater samples, but in s e d i m e n t s the effects o f dissolution can make coccolith identification quite difficult. The n e w data presented here show that G. oceanica contains alkenones and alkenoates and thus caution is n e e d e d w h e n alkenone distributions are used to infer absolute paleo-SST values.
METHODS Algal Samples Gephyrocapsa oceanica bloomed in Jervis Bay, New South Wales, eastern Australia (34°S) between December 16, 1992 and January 22, 1993. The bloom was clearly visible from aircraft and detectable in the visible band of the NOAA-11 satellite. Water temperatures were 18°C or higher and cell counts were up to 1.8 x 107 cells L The coccospheres were remarkably uniform in size with a diameter of 7-8/~m, and the coccoliths showed a characteristic bridge of two elements arching across a single pore. This size uniformity and oceanographic data suggest that the bloom was seeded from a small genetically similar stock derived from oceanic waters outside the bay. Full details of the bloom and description of the alga can be found in Blackburn and Cresswell (1993). Cultures were established from cells isolated from water samples collected on December 31, 1992. A clonal culture designated strain JB02 was lodged with the CSIRO Algal Culture Collection at the CSIRO Division of Fisheries in Hobart and used in these studies. Insufficient cells were available from the natural samples for detailed lipid analysis since these were only provided for species identification. Culture Conditions Cultures were grown at selected temperatures between I I and 29°C in 200 mL Erlenmeyer flasks containing 150 mL GPM medium ( Loeblich, 1975) with selenium added as H2SeO~ at I x 10 8 M. Initial inocula were temperature-adjusted log phase cultures. The cultures were maintained under cool-white fluorescent lighting at 80 #E m 2 s-~ under a 12:12 light:dark cycle; light levels were measured with a QSL-100 (Biospherical Instruments) light meter. Temperatures
were measured with a thermometer calibrated to _+0. I °C. Subsamples (2 mL) were fixed in Lugol's iodine solution and cell counts were measured with a Neubauer haemocytometer on a Zeiss Axioplan microscope (errors +_6%). The alga was cultured on three occasions. In a preliminary study, one culture was grown at 20°C and analysed for the presence of alkenones. When the results proved positive, two experiments were carried out to examine how the biochemical composition changed with growth temperature. In Experiment 1, algae from a single starter culture were grown at 11.1, 15.7, 20, 24, and 29°C, whereas in Experiment 2 cultures were grown at II .9, 15.9, 18.1, and 20°C.
Lipid Extraction and Fractionation Cultures were harvested at late logarithmic phase 9 - 1 2 days after inoculation by filtering through a 47 mm diameter glass fibre filter (Whatman GFF, nominal pore size 0.7 ffm) and extracted immediately with chloroform-methanol-water (1:2:0.8, 5 X 5 mL) using ultrasonication. Chloroform and purified water (Milli-Q ® system) were added to the combined extracts to give a final chloroform-methanol-water ratio of 1:1:0.9, to initiate phase separation. The upper aqueous phase containing salts and water-soluble material was discarded and the total solvent-extractable lipids (TSE) were recovered in the lower chloroform phase. The solvents were then removed under vacuum, and the lipids stored under nitrogen at -20°C. In Experiments I and 2 the TSE was transesterified directly with methanol-chloroform-HCl (10:1:1, v/v/v; 3 mL) at 80°C for 2 h. After addition of 1 mL of Milli-Q® water, the lipids were extracted with hexane-chloroform (4:1, v/v; 3 x 3 mL). The samples were treated with bis(trimethylsilyl)trifluoroacetamide (BSTFA) immediately before GC analysis to convert compounds containing free hydroxyl groups (e.g., sterols) to their trimethylsilyl-ether derivatives. This extract contained fatty acid methyl esters as well as the alkenones. Note that any ethyl esters of the 36:2 fatty acid are converted to the methyl ester by this procedure. Samples were stored under nitrogen at -20°(?.
Analysis by Capillary Gas Chromatography (GC) All samples were analysed with a Shimadzu 9A gas chromatograph equipped with a flame ionisation detector and cooled OCI-3 on-column injector (SGE, Australia). Samples were dissolved in chloroform to which a known amount of 23:0 fatty acid methyl ester was added as internal standard. The sample was injected at 50°C onto a nonpolar methyl silicone fused-silica capillary column (50 m × 0.32 mm i.d., Hewlett Packard). After one minute, the oven temperature was raised to 120°C at 30°C per min and then to 320°C at 3°C per rain. This final temperature was then maintained for 20 rain. Hydrogen was used as the carrier gas and the detector temperature was 330°C. These analysis conditions provided excellent separation of all of the very long-chain compounds present (Fig, 1 ). Peak areas were quantified with DAPA software (DAPA Scientific Pty. Ltd., Kalamunda, Western Australia) and converted to concentrations using appropriate response and recovery factors.
Analysis by Gas Chromatography-Mass Spectrometry (GCMS) GC-MS analyses were performed with an HP 5890 Series II GC and 5790 MSD fitted with a cooled on-column injector. The nonpolar column and chromatography conditions were the same as those described above except that the oven temperature was raised to 315°C and helium was used as the carrier gas, Electron impact mass spectra were acquired and processed with an HP 59970A Computer Workstation. Typical MSD operating conditions were: electron multiplier 2000 volts; transfer line 315°C; electron impact energy of 70 eV; 0.8 scans per second; mass range 40-600 Da. Compounds were identified by comparison with compounds previously isolated from E, hux-
leyi. RESULTS AND DISCUSSION The transesterified T S E fractions contained long-chain alkenones, alkenoates, and a simple distribution o f sterols con-
Use of alkenones in paleoclimate studies
38:2 • 38:3
515
The major sterol in each culture was 24-methylcholesta5,22E-dien-3/~-ol. This is the predominant sterol in many haptophytes, including closely related E. huxleyi (Volkman et al., 1980b), but it is also abundant in some other microalgae such as diatoms (Volkman, 1986). The concentration of this sterol per cell varied from 59 to 155 fg/cell (Table 1 ), with a trend towards highest cellular content at either extreme of the temperature range (Fig. 2a). Other unidentified sterols represented less than 2% of the total sterols at all temperatures.
sponding compounds isolated from E. huxleyi and they had identical mass spectra so we assume that the double bonds have the same positions and trans-geometry previously established for some of the alkenones by De Leeuw et al. (1980) and Reckha and Maxwell (1988). The concentrations of the major alkenones showed different responses to increasing growth temperature (Fig. 2). The concentration of the triunsaturated 37:3 methyl alkenone and 38:3 ethyl alkenone showed a quasi-linear decrease over the entire temperature range (Fig. 2c,e). In contrast, both the 37:2 methyl alkenone and 38:2 ethyl alkenone concentrations showed almost no change from 11 to 20°(2 followed by a dramatic increase above 20°C (Fig. 2d,f ). The abundances of the C38 methyl alkenones (Fig. 2g,h ) followed the same trends as the corresponding C37 alkenones. The mean ratio of triunsaturated C37 methyl alkenone to C38 ethyl alkenone was 1.08 and the range was only 0.90 to 1.34, apart from the culture at 24°C which had an inexplicably high ratio of 2.35 (Table 2). The ratio of diunsaturated C37 methyl alkenone to C38 ethyl alkenone was remarkably constant from 11 to 20°(2 (mean 0.41 ; range 0.35 to 0.48), but almost doubled at temperatures higher than this (Table 2). Prahl et al. (1988) observed that the ratio of total C37 methyl alkenones to C38 methyl plus ethyl alkenones in Emiliania changed little with growth temperature suggesting that this may be a characteristic feature of this alga. The ratio in strain JB-02 of Gephyrocapsa was also remarkably constant (mean: 0.70; range: 0.59-0.81; Table 2), but significantly different from that found by Prahl et al. (1988) for E. huxleyi ( mean: 1.46; range: 1.18-1.71; Table 2) over a similar range of temperatures. Data presented by Conte et al. (1994) for twelve strains of E. huxleyi grown at 15°(2 suggest that significant differences can also occur between strains of the same species (mean: 1.16; range 0.86-2.16), but these data still do not overlap with those for this strain of G. oceanica. In all strains of E. huxleyi analysed to date the C37 methyl alkenones are more abundant than the C3s ethyl alkenones whereas the reverse is true for the strain of G. oceanica analysed here (mean: 0.81; range 0.69-0.95; Table 2). However, the data presented by Conte et al. (1994) for twelve strains grown at 15°C shows that the ratio varies between strains (mean: 1.7; range 1.3-2.8; Table 2) and further research on Gephyrocapsa strains may reveal a similar degree of variability. If so, this could limit the use of apparent differences in these distributions between species to identify different sources of alkenones.
Alkenone Distributions
Alkenoates
Partial gas chromatograms showing the distributions of alkenones at the two extreme growth temperatures clearly show a trend from dominance of triunsaturated alkenones at temperatures around 11°C to an almost complete predominance of diunsaturated alkenones at 29°C (Fig. 1 ). The major methyl alkenones had 37 and 38 carbon atoms, whereas ethyl alkenones had 38 and 39 carbon atoms, with the former in much greater abundance in both cases (Table 1). Small amounts of C39 methyl alkenones were also detected by GC (Fig. 1) and GC-MS, but their abundance was too small to quantify accurately. The alkenones co-eluted with the corre-
G. oceanica also contained high concentrations of esters of a diunsaturated C36 fatty acid, particularly at the lower growth temperatures where the abundance of 36:2 methyl ester (after transesterification) was almost as high as the 2 major alkenones (Fig. 1 ). Small amounts of the 36:3 esters were also found (Table 1 ). Concentrations of the total 36:2 methyl plus ethyl esters ranged from 183 fg/cell at l l . I ° C to 85 fg/cell at 18.1°(2 (Table 1 ), and showed a slight decline with increasing growth temperature (Fig. 2b). Since samples were analysed after direct transmethylation, information on the possible occurrence of ethyl esters in these samples was not ob-
• 37:3
• 38:3 j
/e 37:: • 38:3\
•38:2 •39:3
\
• //•39:2 ~ , ~ . ~ • 39:2
• 37:2
;8:2
o38:3
• 38:2\ , / • 37:3~ ~J
•
38:2
/ .38:3~• 74
76 Time(mins)
/~,/•jj39:2• 39:2 78
FIG. I. Partial capillary gas chromatograms showing the distribution of long-chain lipids (alkenones and alkenoates) in G. oceanica grown at low (I 1.1"(2) and high (29°(2) temperatures. Concentration data are shown in Table !. • methyl alkenones; • ethyl alkenones; & methyl esters (but note that these samples were transesterifiedso that ethyl esters have been converted to methyl esters). The tri-unsaturated alkenones are abundant at the low temperature, but are almost undetectable at the high growth temperature. sisting almost entirely of a single sterol (Table 1 ). Long-chain alkenes of the type found in some strains of E. huxleyi (Volkman et al., 1980a) were not detected at any temperature. Details of each distribution are given below. Sterois
516
J. K. V o l k m a n et al.
Table 1. Cellular concentrations ofalkenones, esters and sterols in Gephyrocapsa oceanica (fg/cell)
Growth temperature (°C) Experiment number
11.1 1
11.9 2
15.7 1
15.9 2
18.1 2
20 1
20 2
24 1
29 1
131
109
111
73
59
79
71
113
1~;5
206 25 62 6 4 303
203 34 55 9 5 306
118 28 30 4 2 182
77 25 4 2 130
57 25 14 3 1 100
79 33 16 4 1 133
69 37 18 5 1 130
47 131 9 14 201
9 377 2 46 434
213 71 39 12 335
169 80 29 11 289
131 78 19 10 238
85 62 13 8 168
53 53 7 7 120
59 82 6 7 154
56 77 8 9 150
20 183 2 14 219
9 420 30 459
183 12 195
114 6 120
158 6 164
107 4 111
85 2 87
100 2 102
100 3 103
89 tr 89
103 103
833
715
584
409
307
389
383
509
996
Sterols: 24-mcthylcholesta-5,22E-dien-3~-ol
Methyl alkenones : 37:3 37:2 38:3 38:2 39:3 subtotal:
22
Ethyl alkenones: 38:3 38:2 39:3 39:2 subtotal
Fatty acid esters*: 36:2 36:3 subtotal: Total alkenones and alkenoates
*determined as the methyl ester after transesterification,
but likely to be mainly ethyl esters. Trace amounts of C39 methyl alkenones were
found in some cultures (Fig. 1)
tained. However, direct GC analysis of the total extract of the 20°C sample from the preliminary experiment showed that the fatty acid occurred almost entirely as the ethyl ester with less than 5% as the methyl ester. Thus, the data in Table 1 for the 36:2 "methyl ester" represents the sum of the methyl and ethyl esters, with the latter likely to be the major form. The ratio of tri- to di-unsaturated esters changes systematically with temperature (Table 1 ), but the 36:2 esters always greatly predominate over the 36:3 esters even at the lowest growth temperatures. The ratio of the abundances of these esters to the alkenones is considerably higher in Gephyrocapsa than in Emiliania at all growth temperatures (Prahl et al., 1988). Others have constructed temperature calibrations based on alkenoate and alkenone abundances in Emiliania ( e . g . , AA36, see Conte et al., 1992; Conte and Eglinton, 1993), but from our limited data for Gephyrocapsa it seems likely that different temperature relationships will be found between the two genera of algae.
Calibration of U~ against Culture Temperature In Fig. 3, we have plotted U~'7values against temperature for the G. oceanica culture data. The data can be fitted by a linear relationship of the form U~'7 = 0.049T - 0,520 (r: = 0.89). This relationship is statistically different at the 95% confidence interval by 2-way t test from that established for E. huxleyi clone 55a in culture where U~'7 = 0.033T + 0.043 (Prahl and Wakeham, 1987). In particular, U~'7values for any given temperature are higher for the E. huxleyi clone than for G. oceanica (by 0.1 at 28°C and 0.4 at 11°C). The slope of the line is significantly different and the error envelopes of the two relationships do not overlap. Note that these changes in U~'7 with temperature in G. oceanica are not due to a simple conversion of one alkenone to another, but reflect complicated changes in cellular abundance. From l 1 to 20°C, the change is mainly due to a decrease in the abundance of the 37:3 alkenone, but at higher
temperatures the change is mainly due to a dramatic increase in the cellular content of the 37:2 alkenone (Fig. 2). For this reason, it is perhaps not surprising that a residuals analysis indicates nonlinearity in the Gephyrocapsa dataset. This can be seen visually in Fig. 3 where all the cold and warm water data points fall above the linear relationship and all the points at intermediate temperatures fall below. A better fit to the data can be achieved by applying a polynomial such as a Richard's growth curve to the dataset. This function is mathematically bounded by 0 and l - - a s is U~'v--and reproduces observations from field data that the change in alkenone ratios is much less at extremes of the temperature range (Sikes and Volkman, 1993). The relationship U~'7 = [21 + e -3°3~r-2522)] 0o5 reduces the least mean squared residuals from 0.007 for the linear regression to 0.002. However, we stress that other polynomials may be more applicable and a choice between them must await better information about the biophysical function of alkenones in these algae. For practical purposes, the data are reasonably represented by a linear regression at temperatures above 1I°C and for paleoclimate studies a linear function is certainly more suitable given the uncertainties involved. M. Conte (pets. commun., 1994) also found that U~'7changes in an approximately linear fashion for a different strain of Gephyrocapsa isolated from the subtropical western Pacific although the intercept differs from that found here. In Fig. 4 we have plotted U~'7values for seawater samples published by Prahl and Wakeham (1987), Prahl et al. ( 1988 ), Conte and Eglinton (1993), and Sikes and Volkman (1993) against surface seawater mixed layer (SML) temperatures. The combined dataset represents approximately the entire range of SML temperatures in the open ocean and includes seawater samples from the eastern Pacific, Caribbean, North Atlantic, equatorial Pacific, and Southern oceans. It is clear from these data that U~'7changes systematically with temperature although there is variability where data from different geographical regions having similar SML temperatures are
Use of alkenones in paleoclimate studies
36:2 m d ~ d estmr |aftur
tnmmmWffoellonl
~
j
200 150
1°°i 5o i
..,
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15 25 T*mlNmnur* I°C)
|
200
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50
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.
m
0
35
15
5
Temperature
25 (°C)
35
517
water or sediment samples (e.g., Conte and Eglinton, 1993; Rostek et al., 1993), and in such cases it might be possible to estimate the relative contributions of the two algae from linear addition of the different alkenone profiles making use of the fact that the C37:C38 alkenone ratios or alkenone:alkenoate ratios appear to be different in these species (Table 2). This requires that the whole distribution of C37C39 alkenones and alkenoates must be quantified, not just the C37 components as done in most studies at present.
d 37:2methyldkenone
37:3methylalkenone
250 200 i ,(
Caveats on the use of U~'7 calibrations in different water masses and sediments o f varied geological ages
400. ~ 350 300 250 200~
150 100
°
!
:
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15
25 TemplxltUm IoC|
35
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38:3 ethyl dkenone 250
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500
200 i <
150 100 .i
50.
~
0 5
15
25 Temperature (oC)
100. O~ 5
35
15
25
Temperature
35
(°C)
h
38:2 methyl dkenon4
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-- 50]
70
40 O
10 ¸ 0 5
ill .
15
25 Tenq~rlturl (*C)
35
5
iI
15 Temperlaure
25 (°C)
35
FIG. 2. Cellular concentrations (femtogram per cell) of individual lipids plotted against culture temperature. (a) 24-methylcholesta5,22-dien-3~/-ol;(b) 36:2 fatty acid esters; (c) 37:3 methyl alkenone; (d) 37:2 methyl alkenone; (e) 38:3 ethyl alkenone; (f) 38:2 ethyl alkenone; (g) 38:3 methyl alkenone; and (h) 38:2 methyl alkenone.
combined. It is intriguing that the slope of 0.049 for the linear temperature relationship for G. oceanica is remarkably similar to the value of 0.041 found by Sikes and Volkman (1993) and 0.037 by Brassell (1993) for seawater samples containing mainly E. huxleyi, although it must be pointed out that the intercepts on the temperature axis are quite different. The temperature responses of G. oceanica and the E. huxleyi strain analysed by Prahl et al. (1988) in culture enclose most of the data derived from natural seawater samples. It seems reasonable that while some of the variation is due to differences between strains of Emiliania, some of the scatter must reflect contributions from more than one species. Some authors have noted the presence of Gephyrocapsa in their
Studies by Jasper and Gagosian (1989), McCaffrey et al. (1990), Conte et al. (1992), Freeman and Wakeham (1992), Sikes and Volkman (1993), and others indicate that a single calibration of U~ with SST is not appropriate for all geographic areas, presumably due to the presence of different strains having different biochemical compositions. Indeed, genotypic and phenotypic variations in E. huxleyi have been well documented (Brand, 1982; Van Bleijswijk et al., 1991 ), with at least four genetically distinct morphotypes of E. huxleyi being recognised (Young and Westbroeck, 1991 ), as have differences in lipid compositions between strains (Conte et al., 1994; J. K. Volkman et al., unpubl, data). Calibrations based on field data are the most appropriate for studies of ancient sediments. For sediments in which E. huxleyi is the dominant coccolithophorid, the choice of calibration will probably be determined by the likely SST temperature range present and type of oceanic regime. For example, calibrations derived from Southern Ocean data (Sikes and Volkman, 1993 ) are clearly quite inappropriate for the Black Sea (Freeman and Wakeham, 1992). For sediments where Gephyrocapsa is dominant the choice is not so clear, and the difficulty is compounded in sediments which predate the first appearance of E. huxleyi since calibrations for E. huxleyi are probably not appropriate. Sediments older than 85000 years can also be expected to yield less certain SST estimates since E. huxleyi may be a contributor but not be the dominant source of alkenones. If our data for G. oceanica are typical for the genus Gephyrocapsa then the calibration determined in our work could be used. However, Brand (1982) has documented genetic differences in G. oceanica, and recent studies of other strains of Gephyrocapsa indicate that a range of responses is likely as with E. huxleyi (M. Conte, pers. commun., 1994). Fortunately, the differences in inferred SST values above 25°C may not be very large irrespective of which calibration is used, but at temperatures from 10 to 25°C the difference may be 6°(2 or more (Fig. 4). G. oceanica is most common in waters of temperatures above 20°C whereas other species may predominate at lower temperatures. Clearly a degree of caution is needed, and field calibrations are essential, when using U~'7to infer absolute paleo-SST values. Fortunately the slopes of the various calibrations are not greatly different so that estimated changes in paleo-SST are similar despite large differences in inferred absolute values. One must also consider what temperature is actually being measured using the alkenone calibrations. It is obvious that the alkenone composition reflects the temperature at which
518
J . K . Volkman et al. Table 2. Ratios of the abundances of selected alkenones in Gephyrocapsa oceanica and EmUiania huxleyi
Grov~ Temp. *C
37:3 methyl 37:2methyl + 38:3 ethyl 4-38:2 ethyl
Z37 methyl + 238 ethyl
237 methyl + Y38 alkenones
k' U37
Ref.
0.81 0.95 0.70 0.69 0.77 0.79 0.80 0.88 0.90
0.66 0.76 0.60 0.59 0.67 0.70 0.68 0.79 0.81
0.11 0.14 0.19 0.25 0.31 0.29 0.35 0.74 0.98
(1) (1) (1) (1) (1) (1) (1) (1) (1)
1.18 1.34 1.51 2.26 1.22 1.o3 0.91 1.71 1.58
0.29 0,38 0.56 0.25 0.34 0.28 0.35 0.73 0.86
(2) (2)
Gephyrocapsa oceanica 11.1 11.9 15.7 15.9 18.1 20 20 24 29
0.98 1.20 0.90 0.91 1.08 1.34 1.23 2.35 1.00
Emiliania huxleyi 8 10 15 15 15 15 15 20 25
0.35 0.43 0.36 0.40 0.47 0.40 0.48 0.72 0.90
3.76
1.50
2.72
1.61 1.21 1.26
1.86 1.13 0.94
1.69 1.19 1.14
(2)
(3) (4) (4) (4) (2) (2)
(1): This work;(2): PRAHLet al. (1988); (3): clone92d, MARLOWEet al. (1984b); (4): motile,sessileand coccolithformsof a singleclone,VOLKMANel al. (1980a).
the o r g a n i s m grows, w h i c h m a y not necessarily be w h e r e the algae ( o r their r e m a i n s ) are found. C o c c o l i t h o p h o r e s are typically m o s t abundant in well-illuminated surface m i x e d layers w h e r e they can flourish even with e x t r e m e l y low levels o f the m a j o r nutrients ( B r a n d et al., 1983; Balch et al., 1991 ), and thus the appropriate temperature is that o f the surface m i x e d layer ( S M L ) . This is usually e q u a t e d with the sea surface temperature ( S S T ) for practical purposes, although it m a y be inappropriate in those situations w h e r e a subsurface maxim u m in c o c c o l i t h o p h o r e a b u n d a n c e occurs (e.g., Prahl e t a ] . , 1993 ). Such areas are typically found in oligotrophic regions w h e r e c o c c o l i t h o p h o r e s can be important constituents o f the phytoplankton. Seasonal c h a n g e s in SST and their relation-
ship to algal production must also be c o n s i d e r e d (e.g., C o n t e and Eglinton, 1993). A possible c o m p l i c a t i o n arises in the case o f large " m i l k y w a t e r " b l o o m s w h e r e d e t a c h e d coccoliths substantially reflect i n c o m i n g light ( b y up to 2 5 % ) and thus appear as white or grey patches in A V H R R satellite i m a g e s o f the sea surface (e.g., A c k l e s o n et al., 1988; Balch et al., 1991). U n d e r these b l o o m conditions ( a m i s n o m e r since cell a b u n d a n c e s in the water are rarely very h i g h ) a w a r m i n g o f the sea surface temperature by as m u c h as 5°C c o m p a r e d with the surrounding seawater can occur ( A c k l e s o n et al., 1988). In this e x a m p l e , the SST increased at 0.32°C per day during the bloom. Surface w a r m i n g due to near-surface light scattering and absorption
o Gephyrocapsa O: 0,~
- - Richer(Is growth curve I - - iinear regression
/
:
0.9
-
--
05
0.7 0.6
~; /
"
Prahl & W a k e h a m
Gephyrocapsa
O.E
:
All Seawater Data
Gephyrocapsa
~+
O.E
k'
/
U37 o.5
U~7a 0~=
+
0.4
+
0.~
0.2
02
0.1
0.1
1'o"
,;
2;
;~
~o
Temperature (°C) FIG. 3. Plot of U~'7 against temperature for G. oceanica cultures. Two lines of best fit are shown. The linear relationship has the equation U~7 = 0.049T - 0.520 (r2 = 0.89) and the nonlinear curve is described by the Richard's growth curve: U~7 = [21 .+_ e-3.O3(T-25.22)]
+
#C+~t%~
0.3
0.05.
/
;*+
/
0
+ +
.... 5
~+~
O0
/
~
//
o/"
:
_ o
:/ 0 0
+ +++~.+
+
/
+++
:/
,,"
+ j: + /
+
f
" ,"
+~
p)"
/o /
i
10
15
20
25
30
Temperature (°C) FIG. 4. Plot of U~'7against temperature for the G. oceanica cultures and surface seawater data compiled from the literature (see text). Also shown is the linear fit to the Gephyrocapsa data (U~'7 = 0.1M9T 0.520) and the linear relationship established by Prahl and Wakeham (1987) for clone 55a of E. huxleyi in culture.
Use of alkenones in paleoclimate studies was also observed in an extensive bloom of E. huxleyi which occurred in the northeastern Atlantic Ocean in June 1991 (Holligan et al., 1993). It is not yet clear if such events are important determinants of local SST. However, coccolithophore blooms have certainly been common in the geological past, and thus the temperature recorded by alkenones in sediments may differ from that of seawater outside of the area covered by the bloom. It may thus be useful to measure the total abundance of alkenones per unit mass of sediment as a proxy for algal biomass (e.g., Prahl et al., 1988) in order to identify whether bloom conditions might have prevailed when the sediments were deposited. The difficulty here is that the effects of degradation of alkenones, particularly in oxic surface sediments, must be assessed (Prahl et al., 1989). As with any technique, possible limitations in the alkenone calibrations must be recognized and taken into account when estimates of paieo-SST are made. The great advantage of this approach is that it provides complementary data to techniques based on oxygen isotopes and foraminiferal assemblages (both of which also have restrictions on their use). Further studies in which several of these techniques are used in combination (e.g., Jasper and Gagosian, 1989; Sikes and Keigwin, 1994; Rostek et al., 1993) will undoubtedly lead to more reliable reconstructions of past climate changes. CONCLUSIONS
C37-C39 alkenones and esters of 36:2 and 36:3 fatty acids have been identified in the lipids of a laboratory culture of the coccolithophorid Gephyrocapsa oceanica isolated from a bloom in Jervis Bay, eastern Australia. This confirms earlier predictions based on the fossil record that species of Gephyrocapsa should contain these distinctive biomarkers. The distribution of alkenones in G. oceanica is broadly similar to that in E. huxleyi, but differences in the ratio of C37:C38alkenones or alkenones: alkenoates might make it possible to distinguish between contributions from the two species in seawater and sediments. The ratio of di- to tri-unsaturated alkenones changes systematically with growth temperature, but the relationship is different from those found for strains of E. huxleyi. These data indicate that Emiliania-based alkenone-SST calibrations should be used with caution to infer paleo-SST values from sediments which predate the first appearance of E. huxleyi or in areas where Gephyrocapsa may be important, such as warm marginal seas and subtropical waters. Acknowledgments--We thank Andrew Revill for valuable comments on the manuscript and for his help in preparing the figures. John Wilkin provided very useful assistance with data fitting, Barbara Molfino provided information on the evolution of Emiliania and Tom Truli and Peter Sedwick provided helpful comments on the manuscript. We thank Jervis Bay Sea Sports for providing the G. oceanica bloom sample and Suzanne Norwood for expert assistance with algal culture. Fred Prahl, Manreen Conte, and an anonymous reviewer provided very helpful and detailed comments on an earlier version of the manuscript. M. Conte is also thanked for providing access to unpublished data. This work was supported by the Antarctic Scientific Advisory Committee and the Australian Research Council. Editorial handling: S. Wakeham REFERENCES
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