LIPID BIOMARKER DIVERSITY IN THE COCCOLITHOPHORID EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) AND THE...

J. P ~ Y C O ~ . 31, 272-282 (1995)

LIPID BIOMARKER DIVERSITY IN THE COCCOLITHOPHORID EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) AND THE

RELATED SPECIES GEPHYROCAPSA OCEANICA'

Maureen H . Conte,= Anthony Thompson, Geofrey Eglinton Biogeochemistry Centre, University of Bristol, Bristol BS8 1 TS United Kingdom

and

John C. Green Plymouth Marine Laboratory, Citadel Hill, Plymouth PL1 2PB United Kingdom

ABSTRACT

Twentyfour strains of Emiliania huxleyi and two strains of Gephyrocapsa oceanica were grown at 15OC under identical culture conditions to assess genetic variability in key lipid biomarker profiles (C3,-C39 alkenones, C,, and Cj7 alkyl alkenoates, and C,-C?,alkenes). Under our culture conditions, little divergence an biomarker composition was detected between E. huxleyi strains f rom different oceanic regions or between E. huxleyi and G. oceanica even though the strains originated f rom biogeo- graphical regions as diverse as the subpolar North At- lantic and subtropical Western Pacific. The major daxer- ences observed were i n tetraunsaturated alkenone abundance and alkene projles, which tended to separate neritic from open ocean strains. Dafferent strains from the same locality were as diferent as strains originating f rom widely separated ocean basins, indicating extreme genotypic di- zlersity within a population. Replicate cultures of the same strain showed signijicant variability in their biomarker profiles even though the culture temperature varied by only k0.3" C, indicating that their synthesis ratios are injluenced by environmental and/or physiological variable@), as yet unidentijied, i n addition to temperature. Strong covariance in C,, and C,, methyl alkenone unsaturation ratios (V17 and U38M#, respectively) and, in coastal strains, C3, alkene and alkenone unsaturation ratios indicates that these compounds are biochemically linked. Key index words: alkenes; alkenoates; alkenones; biomarkers; Emiliania huxleyi; Gephyrocapsa oceanica; haptophytes; lipids; Prymnesiophyceae; strain variability

The coccolithophorid Emiliania huxleyi (Lohm.) Hay and Mohler (Prymnesiophyceae) occurs throughout the world's oceans, from polar regions of high productivity to oligotrophic subtropical regions (reviewed in Westbroek et al. 1993). This species often accounts for a major fraction of the total coccolithophorid community and is noted for

Received 1 1 August 1994. Accepted 12 January 1995. * Present address and author for reprint requests: Department

of Marine Chemistry and Geochemistry, Woods Hole Oceano- graphic Institution, Woods Hole, Massachusetts 02543.

its propensity to form large blooms, easily visualized by satellite, in coastal and mid- to high-latitude areas. These blooms, which cover areas of 10-105 km*, have a major impact on the biological carbon cycle in the ocean and on ocean/atmosphere fluxes of carbon dioxide and volatile sulfur compounds (Westbroek et al. 1993).

In addition to its importance as a major producer of CaCO,, E. huxleyi synthesizes a unique suite of long-chain alkenone and alkyl alkenoates whose sedimentary distribution provides an important proxy record of paleo-sea surface temperature (paleo-SST; reviewed in Brassell 1993). Early culture studies (Marlowe 1984, Prahl et al. 1988) showed that the synthesis ratio of the individual alkenones and alkyl alkenoate compounds in E. huxleyi, in particular the unsaturation ratio of the CS7 methyl alkenones (UkS7; Table l), is a function of growth temperature. Bras- sell et al. (1 986a, b) extended this work to sediments and demonstrated that alkenone unsaturation in surface sediments was correlated with overlying SST. They also observed that alkenone unsaturation in the sedimentary record was correlated with glacial to interglacial variations in P O and suggested that these compounds preserved a record of past SST variations. Further studies of U',, stratigraphy in ocean sediments have demonstrated the importance of these biomarkers as a paleo-SST proxy (e.g. Poyn- ter et al. 1989, Farrimond et al. 1990, Ten Haven and Kroon 1990, Eglinton et al. 1992).

Most commonly, the sedimentary UkS7 ratio is converted to a paleo-SST using the linear regression calibration of Ws7 versus temperature, which was measured in culture experiments using a NE Pacific strain of E. huxleyi (Prahl et al. 1988). While this temperature calibration is generally appropriate for the open ocean (Prahl and Wakeham 1987, Brassell 1993), recent studies in the North Atlantic (Conte et al. 1992, Conte and Eglinton 1993), Black Sea (Freeman and Wakeham 1992), Southern Ocean (Sikes and Volkman 1993), Norwegian fjords (Conte et al. 1995), and Mediterranean (Ternois et al., unpubl. results) have shown that a single calibration may not be applicable to all geographical regions. Multivariate calibrations derived using the entire suite of alkenone and alkenoate compounds may in

272

273 LIPID BIOMARKERS IN EMILIANIA HUXLEYI AND GEPHYROCAPSA OCEANICA

TABLE 1. Alkenone and alkenoate indices and IPT calibrations used in this study. Shorthand notation is carbon number: number of double bonds. Me = methyl alkenone, Et = ethyl alkenone, FAME = alkyl alkenoate. Complete formulas for compounds are listed in Marlowe et al. (1990).

Index or calibration' Equation Reference

Uk37 37:2Me/(37:2Me + 37:3Me) Prahl and Wakeham 1987 AA56 38:3Et/(38:3Et + 36:2FAME + 36:3FAME) Conte et al. 1992 UkS8Mc 38:2Me/(38:2Me + 38:3Me) Conte and Eglinton 1993 IPTl IPT = (UkS7 + 0.496)/0.056 Conte and Eglinton 1993 IPT2 IPT = (UkSBMe + 0.629)/0.058 Conte and Eglinton 1993 IPT3 Conte and Eglinton 1993 IPT4 IPT = (U'37 + 0.083)/0.037 Brassell 1993 P&W IPT = (U'37 - 0.039)/0.034 Prahl et al. 1988

IPT = 3.73 + 1 1.33(uk58M,) + 10.86(AASs) + 5.21(Uk57)

a The IPT notation was not used in the original reference.

fact be more robust temperature estimators (Conte and Eglinton 1993).

The differences in the alkenone/alkenoate versus temperature relationship observed for different ocean regions suggests that there are genetic differences among E. huxleyi and/or other alkenone- synthesizing populations in these regions. Gephyro- capsa oceanica (Volkman et al. 1995) and presumably other members of the genus Gephyrocapsa also syn- thesize alkenones and alkyl alkenoates. These species coexist with E. huxleyi in mid- to low-latitude regions and may contribute a sizable fraction of the overall production (Winter et al. 1994). In addition to genetic differences among alkenone/alkenoate- synthesizing populations, environmental variables other than temperature may also influence biomarker production ratios.

The aim of the present laboratory investigations was to assess genetic differences in lipid biomarker synthesis among E. huxleyi strains and between E. huxleyi and the closely related species Gephyrocapsa oceanica. In this paper, we report long-chain alkenone, alkenoate, and related alkene biomarker profiles for 24 clonal isolates of E. huxleyi and two isolates of Gephyrocapsa oceanica grown at 15" C under identical culture conditions. Results of molecular genetic and pigment studies on many of the same cultures are reported in Barker et al. (1995), Medlin et al. (1994), and Mantoura et al. (unpubl. results).

MATERIALS A N D METHODS

The E. huxleyi and G. oceanica strains examined in this study are listed in Table 2. The North Atlantic strains of E. huxleyi were collected during 1989-199 1 UK-JGOFS Biogeochemical Ocean Flux Study cruises. On these cruises, cells were concentrated from approximately 500 mL of surface water obtained from Conductivity-Temperature-Density bottles or the nontox- ic seawater supply (4-m depth) by gravity filtration through a 3-am Nucleopore filter. A 1-mL aliquot of the concentrated seawater was added to 30 mL of enriched seawater culture medium (Keller et al. 1987). The rough cultures were maintained at 10"- 15" C (depending on the ambient water temperature) under a 12:12 h LD regime until return to the laboratory. Emiliania hux- leji cells were then isolated using single-cell isolation methods to establish clones. The Norwegian fjord strains were obtained during the 1992 Norwegian mesocosm experiment (Conte et al. 1995, Heimdal et al. 1995). Strains were established from single-cell

isolates taken during different bloom stages in the mesocosm enclosures and from the surrounding fjord waters. Single-cell isolations were made directly from concentrated seawater (Barker et al. 1995). The subsequent culturing history of the Norwegian strains was identical to that of the North Atlantic strains at the same maintenance temperature. The strains from other geographical regions (Sargasso Sea, Gulf of Maine, English Channel, North Sea, Indian Ocean, and NE and SW Pacific Ocean) were obtained from culture collections. The full culture history of ' many of these strains is not known; however, after arrival at the Plymouth Marine Laboratory culture facility, they were treated in a manner identical to that used for the North Atlantic and Norwegian strains.

Culturing methods. Stock cultures were maintained in 1 00-mL Erlenmeyer flasks or 250- x -1 5-mm culture tubes. Innocula were transferred to 250-mL Erlenmeyer flasks and grown at 15' C for approximately 10 days before bulking up into the 500-mL flasks used for the experimental cultures. Prahl et al. (1988) previously showed that the alkenone and alkenoate distributions within liv- ing E. huxleyi cells adjusts within 2 days to a 5' C shift in growth temperature, so we believe this 10-day period was sufficient for acclimation to the experimental temperature.

Strains for the experiments were batch-cultured (nonaxenic) in 500 mL of enriched natural seawater (Keller et al. 1987) (initial NO,-concentration 8.83 x lo-' M). From three to seven different strains were cultured in each run group. All strains were cultured at 15" C (+0.3" C variation) under a 12:12 h LD regime in incubators. The growth of the cultures was monitored by de- termining cell counts with a Coulter multisizer. The mean stan- dard deviation of replicated cell counts for 90 samples was 3.4%. There was little or no lag phase after transfer so the cells were actively growing. All cultures were harvested during logarithmic growth after about 2-3 days growth in the experimental culture flasks. Preliminary biomarker analyses identified a significant degree of variation in lipid distributions between replicate cultures of the same strain. Therefore, two or three replicates of each strain were cultured for biomarker analyses in different experimental run groups.

Each batch culture was subsampled upon harvest for morpho- metric studies, genetic analyses (Medlin et al. 1994, Barker et al. 1995), bulk inorganic and organic carbon, lipids, and pigments (Mantoura et al., unpubl. results). Samples for particulate organic carbon (POC) and particulate organic nitrogen (PON) analyses and particulate calcium were filtered onto precombusted (375' C for > 12 h) 25-mm GFF filters (Whatman) until analysis. The GFF filters for C/N and calcium analyses were then rinsed with clean culture media and stored at less than -20" C until analysis. Samples for lipids were filtered onto precombusted 47-mm GFF filters using an all-glass Millipore filtration unit. The filters were placed in precleaned ("Nochromix," Godax Laboratories, New York) Pyrex test tubes fitted with Teflon liners in Teflon caps, and the tubes were filled with the extraction solvent (2:l chlo-

274 MAUREEN H. CONTE ET AL.

TABLE 2. E. huxleyi and G. oceanica strains analyzed. (B) indicates T@e B coccolith morphology. Symbols used infigures aregivm in parmtkses.

Collection temperature E. huxlcyi Culture Colldon Collection

urain collection' location date c C)

NE Atlantic, Iceland Basin (0)

G1779Ga PML 60'0'N, 20"o'W Jun 89 9.8 G1779Ge PML 60'0'N. 20OO'W Jun 89 9.8 DW60/3/8 PML 61"74'N, 22'49'W Jun 91 12.5 DW6 1/3/16 PML 5 1'4 1 'N, 1 5'53'W JuI 91 12.5 DW6 1 / 12/6 PML 61'05'N, 20'00'W JuI 91 12.4 DW61/67/5 PML 60°41'N, 20'16'W Ju191 12.9 DW61/87/17 PML 56"14'N, 19'58'W Ju191 13.8

5/90/25g PML 48'20'N, 17'33'W May 90 14.3 NE Atlantic, temperate (0)

NE Atlantic, subtropical (0)

Sargasso Sea (0)

DW53/74/6 PML 2427'N, 20'24'W Oct 90 21.8

M181b PML 32'N, 62OW Jun 87 ndc MCH-1 (B) BIG "Off Bermuda" Jan 67 nd

CH25/90 (B) NIOZ 57'26'N, 06'13'E Ju190 nd 92D (B) PML 50'02'N, 04'22'W Jun 75 nd 92E PML. 49"52", 06'12'W Jun 92 nd

88COCCO BIG Gulf of Maine JuI 88 nd 88E BIG Gulf of Maine Ju188 nd

English Channel/North Sea (A)

Gulf of Maine (0)

SW Norwegian fjords (m) B92/ 1 1 PML MESOSd: prebloom Apr 92 7.8

B92/131 PML MESOSd: midbloom May 92 10.2

B92/2 1 PML 60'26'N, 5'38'E Apr 92 nd (Trengereid)

B92/317 PML MESOSd: midbloom May 92 13.4

S-AFRICA WITS 29'49'S, 31'01'E Mar 83 nd

VAN55 NEPCC 50'1 l'N, 145'57'W nd nd VAN556 NEPCC 49'40'N, 144'40'W nd nd

EH2 TSUKUBA Great Barrier Reef, Dec 90 nd

Indian Ocean, subtropical ( X )

NE Pacific, temperate (+)

SW Pacific, subtropical (A)

Australia G. oceanica strains (0)

EH 1 TSUKUBA Great Barrier Reef, Dec 90 nd

GO 1 TSUKUBA 41'N, 141'E Oct 91 nd Australia

* PML, Plymouth Marine Laboratory (UK); BIG, Bigelow Marine Laboratory (USA); WITS, University of Witwatersrand, Johan- nesburg (South Africa); NIOZ, Netherlands Institute of Sea Research (NIOZ), Texel (The Netherlands): NEPCC, Northeast Pacific Cultur; Collection, University of British Columbia (Canada); TSUKUBA, University of Tsukuba (Japan).'

Bigelow strain designation 1 A 1. nd, no details. MESO isolated from mesocosm enclosures; see text.

roform: methanol). The lipid samples were stored at less than -20" C until analysis. Filter blanks (CFF filters rinsed with clean culture media only) were made for each run group.

Sample filters for POC and PON determinations were dried at 60' C and preacidified using high-purity sulphurous acid (Al- drich) to remove carbonates. POC and PON were analyzed on a Carlo Erba CN analyzer (combustion temperature 1000' C). Par- ticulate Ca was analyzed by atomic adsorption spectroscopy. Ca measurements also include detached coccoliths suspended in the medium. Values for all analyses were blank-corrected using media + GFF blanks taken at harvest for each run group.

Lifiid analyses. Lipid analytical methods are detailed in Conte

et al. (1992, 1995). Briefly, lipids were extracted in 2:1 chloro- form :methanol using ultrasonication and separated from non- lipid components by washing with 0.88% aqueous KCI (Folch et al. 1957). The lipid extract was dried by passing it through com- busted Na,SO, and evaporated to just dryness. Lipids were transesterified using 5% methanolic HCI in toluene (55" C for 12 h; Christie 1982). The transesterified products were extracted into hexane. Just prior to gas chromatography, the sample was trans- methylsilylated under N, using BSTFA in pyridine to derivatize free sterols (and other compounds containing OH groups). The reagents were evaporated, and the sample was taken up in CH,CI, for gas chromatography. Samples were analyzed on a 50-m-x-

LIPID BIOMARKERS IN EMILIANIA HUXLEYI AND GEPHYROCAPSA OCEANICA 275

0.32-pm Chrompack CPSil5CB (0.25-pm film) column using a Carlo Erba 5160 gas chromatograph fitted with on-column in- jection. The gas chromatograph was programmed from 50" to 150°C at lO".min-I and from 15" to 310°C at 4".min-'. H, was used as the carrier gas. Compounds were quantified using Min- ichrom (VG Scientific) data acquisition/chromatography soft- ware. Compounds were identified by gas chromatography-mass spectrometry (GC-MS) using a Finnigan 4500 quadrupole mass spectrometer in electron impact mode (70 eV, emission current 350 mA, 1 scan.s-'). Gas chromatography conditions for GC-MS were identical except that He was used as the carrier gas.

a /

RESULTS

We observed a large amount of variability among replicate cultures of the same strains. Variations in cellular concentrations of carbon and lipid biomarkers were proportional to cell density at harvest, suggesting that cultures in which cell density was low had larger mean cell size. However, variations in alkenone, alkenoate, and alkene biomarker composition could not be attributed to measured differences in growth temperature or other culture variables. The variability among replicate cultures in alkenone ratios especially was unexpected, as it is believed that alkenone and alkenoate distributions are primarily controlled by growth temperature.

Particulate organic and inorganic carbon. Cellular concentrations of organic carbon and calcium showed a strong relationship with the cell density measured at the time of harvest (Fig. 1). Cellular concentrations in cultures having cell densities of <2 x lo6 cells-mL-I rose sharply. Modal cell di- ameter measured on the multisizer was variable but generally higher in cultures having cell densities < 1 x lo6 cells.mL-', indicating larger cells in these cultures, which had lower growth rates. Organic carbon averaged 6-7 pg C.cel1-l for those cultures in which the cell density was > 1.5 X lo6 cells.mL-' but increased to 10-30 pg C-cell-l in more dilute cultures (Fig. la). No difference in cellular organic carbon between calcifying and noncalcifying strains was apparent. Cellular calcium concentrations in calcifying cultures were more variable, ranging from 3 to 18 pg Ca-cell-l (Fig. 1 b). Part of this variability may be attributed to the presence of free liths in the filtrate. Particulate calcium averaged 10-12 pg Ca-cell-' and also increased in dilute cultures. Cel- lular organic carbon for both strains of Gephyrocapsa oceanica was similar to that measured for the E. hux- 1eji strains sampled at the same cell density, but the cellular Ca ratios were about 2-3 times higher. Par- ticulate organic to inorganic carbon (POC/PIC) ratios were > 1 in most cultures (Fig. lc) except for a few that contained cells having multiple layers of coccoliths (supercalcifying). There was no correlation of POC/PIC ratios with cell density. POC/PIC ratios averaged 2.9 for cultures in which all cells were calcifying. It is important to note here that these POC/PIC ratios exclude dissolved or remi- neralized organic carbon and thus underestimate

101 . . . ' . ' . ' .

c l Cell density at time of harvert

(cells x 10-6 ml-1)

FIG. 1 . Cellular concentrations of particulate organic carbon and calcium and the POC/PIC ratio for each culture plotted versus cell density at harvest. All strains were cultured at 15" C under conditions described in the text. The POC/PIC ratio is plotted only for cultures in which most cells were calcifying. The symbol designation for the geographical origin of the strains is listed in Table 2.

the true organic to inorganic carbon synthesis (C/ P) ratio.

Lipid biomarkers. Both E. huxleyi and G. oceanica contain a suite of di-, tri-, and tetraunsaturated CS7- Cs9 methyl and ethyl alkenones and corresponding c36 and CS7 di- and triunsaturated alkenonates (reviewed in Conte et al. 1994). The double-bond configuration of the CS7 alkenones is in the biologically rare trans configuration (Recka and Maxwell 1988); the double-bond configuration of the other alkenones and alkyl alkenoates is assumed to be analo- gous. Species containing long-chain alkenones and alkyl alkenoates also contain one or more C,,-C,, alkenes. An exception is G. oceanica strain JB-02, in which alkenes were not detected (Volkman et al. 1995). The presence of long-chain (C,,, Css, Cs7, Cs8) alkenes in alkenone-synthesizing species and their absence in other species suggests that the alkenes are biochemically linked to the alkenones and alkenoates.

Cellular concentrations of alkenones and alkyl al-

2'76 MAUREEN H. CONTE ET AL.

0

8

0 1 2 3 4 6 6

(cellr x 10-6 ml-1) Cell dmelty at tlmr of harveat

0

A 0 .

m 00

Q 0 0

0

C '

0 ;

0 ,

0 '

1

"0 1 2 3 4 5 6

" L 1 A 2

A - 92D

1 1 3

4 , o o , : / 5 6

Coll drnelty at tlmr of harvrrt (cells x 10-8 ml-1)

FIG. 2. C17-CIp alkenones and Cs,, and C,7 alkyl alkenoates for individual culture replicates versus cell density at harvest. The symbol designation for the geographical origin of the strains is listed in Table 2. a) Cellular concentrations of total longchain alkenones and alkyl alkenoates. b) Total long-chain alkenones and alkyl alkenoates as a percentage of cellular organic carbon. c) Alkyl alkenoates as a percentage of total longchain alkenones and alkyl alkenoates. d) Tetraunsaturated C17 and Cm alkenones as a percentage of total long-chain alkenones and alkyl alkenoates. Error bars are 1 SD of gas chromatography replicate measurements.

kenoates in E. huxleyi cultures ranged from 0.2 to 1.5 pgvcell-l (Fig. 2a). The higher cellular concentrations are comparable to those observed in E. hux- leqi blooms in the North Atlantic (Conte and Eglin- ton 1993, Conte et al., unpubl. results), but they are slightly lower than those observed in induced E. huxleyi blooms in mesocosm studies in a Norwegian fjord (Conte et al. 1995). As with organic carbon, replicate cultures of the same strain showed large differences in cellular concentrations. Cellular concentrations of alkenones and alkenoates increased in cultures having cell densities at harvest of <2 x 1 O6 cells-mL-I, presumably due to the larger mean cell size in these cultures. Total alkenones and alkenoates as a percentage of cellular organic carbon was quite variable and ranged from 2.5 to 15% (Fig. 2b). In cultures containing >1.5 x lo6 cells.mL-l, alkenones and alkenoates averaged about 5% of organic carbon. These values are about 60% of the cellular levels of alkenones reported by Prahl et al. ( 1 988) for their cultures.

Due to the high variability among replicate cultures, no significant difference in total alkenone and alkenoate concentrations, on either a per cell or percentage of carbon basis could be detected among E. huxleyi strains isolated from the different geographical regions. Cultures grown at more extreme temperatures or tighter control of culturing conditions and timing of harvesting may yet reveal genetic differences among the strains.

Alkyl alkenoates comprised from 6 to 22% of the total (Fig. 2c). Both G. oceanica strains tended to have lower alkenoate abundances than the E. huxleyi strains. T h e lower relative abundance of alkenoates in the G. oceanica strains examined here contrasts with the higher alkenoate abundances found for G. oceanica strain JB-02 from eastern Australia waters (Volkman et al. 1995).

Significant differences among the strains were found for the relative abundances of tetraunsaturated alkenones. Oceanic strains of E. huxleyi and G. oceanica contained low levels of tetraunsaturated alkenones, whereas some coastal strains from the Gulf of Maine and Norwegian fjord and the English Channel strain 92D contained tetraunsaturated alkenone abundances 2-5 times higher (Fig. 2d). As with the di- and triunsaturated alkenones and alkenoates, abundances of tetraunsaturated alkenones varied among different culture replicates (e.g. strains B92/131, B92/317, and 92D). This indicates that tetraunsaturated alkenone abundance is controlled by environmental factors in addition to genetic makeup.

Large differences were observed between replicate cultures in biomarker indices for CS7 and C,, methyl alkenone unsaturation (Uk',7 and UkSBMe, respectively; Table 1) and for AA,, (Table l) , an index of alkenoate abundance (Fig. 3). Given this variability, we could not detect strain-related differences. T h e variability in biomarker indices within a

LIPID BIOMARKERS IN EMILIANIA HUXLEYI AND GEPHYROCAPSA OCEANICA 277

0.6 1 I

1 . I-. . , . . : : : : : : : : : : : : : : : .........................

0.9

02 " ' ~ ' ~ ' " ~ "

FIG. 3. Biomarker indices in strains of E. huxleyi and G. oceanica (U",?, Uk,aHL, AA,,). Formulae for biomarker indices are given in Table 1. Data points shown are average values for each strain, with 1 SD of strain replicates (either two or three replicates per strain). Mean values are Uk',? = 0.34 k 0.05; Uk,8He = 0.34 k 0.10; AA,, = 0.52 k 0.09.

single culture was not random. A strong covariance was observed between Uk'37 and UkSBMc, indicating that the observed variability was in response to a presently unidentified cofactor that also affects both CS7 and C,, alkenone unsaturation similarly (Fig. 4). This covariation indicates that the biosynthesis of the Cs, and Cs8 alkenones is closely coupled.

We have not yet identified the factor@) influenc- ing alkenone unsaturation. The variation in biomarker ratios was not correlated with very high or low cell density at harvest (an indicator of growth rate) or cellular Ca content. This suggests that differences in growth conditions or calcification do not seem to be major factors. Light is an obvious pos- sibility, but cell shading can be ruled out, as there was no correlation in biomarker ratios with cell density. Furthermore, pigments do not indicate any large differences in light conditions among cultures (Man- toura et al., unpubl. results).

The integrated production temperature (IPT; Conte et al. 1992) is the cumulative growth temperature signal recorded by the alkenone/alkenoates. Several different calibration algorithms have been derived using culture and field data to estimate IPT from alkenone and alkenoate distributions (Ta- ble 1). The most commonly used IPT calibration is a simple linear regression of Uk'37 versus temperature, which was measured by Prahl and Wakeham ( 1 987) in a culture of a NE Pacific strain of E. huxleyi

0.6 .

0.5 -

i 3 0.4 . Y 3

0.3 .

0 2 .

0.2 0.3 0.4 0.6 0.6 0.7

Uk.37

FIG. 4. UVs7 versus Uk,a,, for individual culture replicates. Formulae for biomarker indices are given in Table 1. Error bars are 1 SD of gas chromatography measurements. The fitted regression is UkSllMe = -0.031 + 0.964Uk,7 (n = 53, + = 0.915).

(revised calibration given in Prahl et al. 1988). More recently, we derived empirical simple and multivariate IPT calibrations from field studies of alkenone/ alkenoate distributions in >0.8-pm surface water particles in the North Atlantic (Conte et al. 1992, Conte and Eglinton 1993). Our North Atlantic strains (Table 2) were collected from the same waters as the particulate matter samples that provided these calibration data. T h e Brassell (1993) revision of the simple Uk',, temperature calibration includes field data from all studies published up to 1993.

We used these five calibrations (Table 1) to estimate IPT in each culture replicate. Differences among IPT estimates in culture replicates ranged over a 7"- 12" C temperature span, although the actual deviation in the incubation temperature was C0.3" C. This range in I P T estimates resulted from the large variation in biomarker ratios in the cultures due to the influence of unidentified cofactor(s) on alkenone synthesis ratios. As for the individual biomarker ratios, there was no correlation of IPT with culture density or with geographical origin of the strain. Average IPT estimated using the three algorithms derived from the North Atlantic data set ( IPTl , IPT2, and IPT3) were not significantly different and agreed well with the actual culture temperature of 15.0" C (Table 3). Even so, individual estimates in the cultures ranged from 10" to 20" C. In contrast, IPT estimates using the Prahl et al. ( 1 988) calibration were consistently lower than the culture temperature, averaging only 9.4" C. IPT estimates using the revised field calibration of Brassell (1993) were also lower than the culture temperature, averaging only 12.0" C.

All strains of E. huxleyi and G. oceanica contained multiple isomers of a 3 1:2 alkene and 33:3 and 33:4 alkenes (Table 4). In addition, some oceanic E. hux- 1eqi strains also contained sizable amounts of 37:3 and 38:3 triunsaturated alkenes. Some of the Ice- land Basin strains also contained small amounts of a second 37:3 alkene, which has not been reported


TABLE 3. IPT estimates for culture experiments. IPT algorithms are listed in Table 1. The measured temperature in the incubator during the experiments was 15.0' f 0.3" C. The variability in IPT estimates indicates the injluence of a secondary cofactor that, in addition to temperature, controlled alkenone unsaturation ratios in these cultures.

Calibration Range Mean SD

IPT 1 12.5-19.8 15.3 1.5 IPT2 1 1.6-20.6 16.2 1.6 IPT3 10.6-19.1 14.9 1.5 IPT4 7.8-1 8.8 12.0 2.3 P&W 4.9-16.9 9.4 2.5

in previous studies (e.g. Volkman et al. 1980). Small amounts of 37:3 and 38:3 alkenes were detected in one but not both cultures of G. oceanica strain GO 1, suggesting that the synthesis of these compounds is also, in part, physiologically controlled.

As with the alkenones and alkenoates, the cellular abundances of the total alkenes varied inversely with culture density (Fig. 5a). Cellular abundances averaged 0.005-0.015 pg-cell-' for most cultures and were significantly higher in strains that contained C37 and CS8 alkenes. Alkenes comprised from 0.25 to 1 .O% of cellular organic carbon in strains lacking CS7 and Cs8 alkenes but up to 2-3% of organic carbon in strains that contained C3, and CS8 alkenes (Fig. 5b). Gephyrocapsa oceanica contained alkenes at

TABLE 4. Long-chain alkene distributions. Distribution data are shown as the percentage of total a l h e s . The compound designation is carbon number: number of double bonds. Values shown are meansfor each strain. + indicates that the compound comprises <0.5% of total a l h e s . The a l h e s are listed in order of elution on a CPSilSCB column. Locations of double bonds were not determined. No analyses were made for strains G1779Ga, MCH-I, or B92149.

Akene E. h u x y Z ALK

strain 31:P 31:P 31:P 31:P 33:4 33:s 33:4 33:s 33:4 37:s 3 7 3 383 @g.cell-') % POC

NE Atlantic, Iceland Basin G1779Ge 6 -b DW60/3/8 11 3 DW6 1/12/6 2 1 DW61/3/16 3 2 DW61/67/5 2 3 DW61/87/17 14 2

5/90/25g 6 5 NE Atlantic, temperate

NE Atlantic, subtropical

Sargasso Sea

English Channel/North Sea

DW53/74/6 18 -

Ml8l 15 +

C H 2 5/90 2 4 92 D 16 - 92E 8 5

88COCCO 6 3 88E 5 4

B92/11 10 1 B92/131 7 4 B92/21 7 2 B92/317 9 6

%AFRICA 18 1

VAN55 1 1 VAN556 1 +

EH2 5

EH 1 11 5 GO 1 12 4

Gulf of Maine

Norwegian fjords

Indian Ocean, subtropical

NE Pacific, temperate

NW Pacific, subtropical

G. oceanica strains

48 41 10 10 9

28

36

53

51

21 44 22

17 27

30 32 31 35

60

4 5

44

34 25

2 4 1 2 2 1

2

3

1

3

3 -

+ 2

- 2

3 -

-

1 +

3

4 25

- 3 1 + 3 2

2

2

2

+ - -

13 6

6 6

19 5

3

+ + -

- -

5 - - - 0.02 0.34 11 4 25 14 9 13 2 - - - 0.05 0.76 5 4 6 1 48 5 16 0.38 1.52 6 3 6 1 47 1 18 0.15 0.84 5 1 9 2 46 1 18 0.11 0.81

10 3 6 1 15 4 14 0.55 1.48

12 17 17 3 - - - 0.02 0.27

10 4 6 4 - - - 0.02 0.27

11 5 9 7 - - - 0.01 0.22

4 5 11 2 39 - 10 0.04 0.30 15 15 8 1 - - - 0.01 0.07 5 10 5 1 31 - 11 0.05 1.16

26 16 18 2 - - - 0.04 0.33 13 16 24 3 - - - 0.02 0.25

20 10 19 5 - - - 0.04 0.27 12 17 15 3 - - - 0.04 0.70 16 8 17 1 - - - 0.01 0.21 12 15 13 2 - - - 0.02 0.25

7 3 5 3 - - - 0.07 0.80

3 2 5 1 68 - 18 0.21 2.08 2 1 5 1 67 - 18 0.18 2.43

12 6 7 6 - - - 0.03 0.47

17 6 17 6 - - - 0.13 0.64 10 4 14 3 3 - 1 0.10 0.52

a Includes a small amount of an unidentified coelutant. Not present or trace component.

279 LIPID BIOMARKERS IN EMILIANIA HUXLEYI AND GEPHYROCAPSA OCEANICA

:= "/" A , , , , , , ~ , , , . ol L . A . e m 0

0 1 2 3 4 5 6 0.0

t b l

2 3 4 5 8 1 -

0 1 Cell denrlty mt Ilma of hiwas1

(eella I 104 ml-1)

FIG. 5. Long-chain C,,, C,,, C,,, and C,, alkenes. The symbol designation for the geographical origin of the strains is listed in Table 2. a) Cellular concentrations (pg.cell-l) of total alkenes in individual cultures plotted against cell concentration at the time of harvest. b) Total alkenes as the percentage of organic carbon.

cellular concentrations comparable to E. huxleyi and had similar alkene distributions to other oceanic strains, providing further evidence that these genera are closely related.

Alkene distributions showed significant differences among the strains. Again, differences primarily separated neritic versus open ocean strains. The coastal Gulf of Maine and Norwegian fjord strains all lacked Cs7 and C,, alkenes. Furthermore, these strains had consistently lower CSI alkenes relative to the Css alkenes [C,,/(C,, + Css)] than other strains (Fig. 6a). In contrast, oceanic strains containing Cs7 and C,, alkenes had the highest C,,/(Csl + CSs) alkene ratios, and this ratio was strongly correlated with Cs7 and C,, alkene abundance (Fig. 6b). Rep- licate cultures of strains CH25/90 (North Sea) and 92E (Isles of Scilly, NE Atlantic) showed large differences in abundance of CS7 and C,, alkenes, indicating that alkene distributions also varied, in part, in response to environmental or physiological variables.

The relative ratio of the tri- to tetraunsaturated C,, alkenes [2(33:3)/X(33:4)] also showed small but significant differences between neritic and oceanic strains (Fig. 6c). The 2(33:3)/2(33:4) ratio was consistently lower in coastal Gulf of Maine and Nor- wegian strains, ranging from about 0.25 to 1.0. The 2(33:3)/2(33:4) ratio in coastal Gulf of Maine and Norwegian fjord strains, but not in the oceanic strains, was also correlated with Uk's7. The correlation of this ratio with Uk',, suggests that alkene unsaturation in these strains is also controlled in part by the same factors that control alkenone unsaturation.

1.01. . - . . . . , . . . , . . . . . . . . . . . I

0 10 20 30 4 0 5 0 6 0 7 0 8 0 9 0 100

c37 + C38 alkanes (W of total rlkenes)

3.51 - . . . . I . , ,

0.0' . . ' . ' . I -

0.20 0.30 0.40 0.50 0.60

u k'37

FIG. 6. Alkene ratios. The symbol designation for the geographical origin of the strains is listed in Table 2. a) Ratio of sum total C,, alkenes to total C,, + C,, alkenes [ q 3 1)/E(3 1 + 33)] in individual cultures plotted against cell density at harvest. The range encompassing the coastal Gulf of Maine and Norwegian fjord strains is circled. b) Ratio of C,, alkenes to total C,, + C,, alkenes versus total C,, + C,, alkenes as a percentage of total alkenes. The line shown is the best fit line for strains containing significant concentration of C,, and C,, alkenes (y = 0.67 + O.O029x, n = 13, I* = 0.89). c) Ratio of sum total 33:3 to total 33:4 [2(33:3)/2(33:4)] versus U",,. The regression line shown is for coastal Gulf of Maine and Norwegian fjord strains, shown here by solid squares (y = -1.14 + 4.86x, n = 12, r* = 0.63).

DISCUSSION

The POC/PIC ratio, which in our cultures presumably provided a lower limit on the photosynthesis to calcification ratio, was >1 in most cultures, indicating that net photosynthesis exceeded calcification under these conditions. This implies that all intracellular CO, generated by calcification (Paasche 1964, Sikes et al. 1980, Nimer and Merrett 1992) was used to meet photosynthetic demands and that no excess C 0 2 was released into the surrounding medium. If these ratios are representative of those in natural waters, E. huxleyi production would not generally constitute a CO, source in surface waters, although calcification, which provides an alternative intracellular CO, source for photosynthesis, will reduce the amount of C 0 2 drawdown.


Overall, only small differences were found in biomarker profiles among the E. huxleyi and G. oceanica strains examined here, in spite of the fact that the strains originated from oceanic regions as diverse as the Iceland Basin and subtropical, oligotrophic Pa- cific. Strain differences were most apparent in the relative abundances of tetraunsaturated alkenones and in alkene profiles, which tended to separate neritic strains from oceanic strains. This division between neritic and oceanic strains was also observed in pigment profiles, with neritic strains having a higher fucoxanthin to 19’-hexanoylfucoxanthin ratio (Mantoura et al., unpubl. results). Divergence between neritic versus oceanic strains has previously been reported in reproductive rates and isozyme banding patterns in the marine diatom Thalassiosira pseudonana (Brand et al. 1981).

N o significant differences in lipid profiles were apparent between calcifying and noncalcifying cells, in agreement with the results of Volkman et al. (1980), or between cells having Type A or B morphology. These results are in agreement with molecular genetic evidence (Medlin et al. 1994) that suggests that Type A and B morphotypes are not distinct subspecies (cf. van Bleijswijk et al. 1991).

Differences in biomarker profiles among strains isolated from the same area were often as large as those between biogeographically separated strains. For example, large differences were found among Norwegian fjord strains in tetraunsaturated alkenone abundance and among Iceland Basin strains in alkene profiles. This diversity is consistent with RAPD analyses indicating genetically diverse populations within these North Atlantic regions (Medlin et al. 1994, Barker et al. 1995) and the results of Brand (1 982), who demonstrated genetic differences in reproductive rates among E. huxleyi clones isolated from the same Sargasso Sea population. Similarly, Brand et al. (1981) also found significant genetic differences in reproductive rates and in elec- trophoretic mobility of isozymes within a population of the diatom Thalassiosira pseudonana isolated from a single water bottle. Our results provide additional evidence for genetic diversity within oceanic phy- toplankton populations.

We observed only small differences in lipid biomarker profiles between E. huxleyi and G. oceanica. This similarity between G. oceanica and E. huxleyi biomarker distributions is consistent with morpho- logical and paleontological inferences (reviewed in Marlowe et al. 1990) and recent molecular genetic evidence (Fujiwara et al. 1994) that the two genera are very closely related. Further studies of other Gephyrocapsa species and strains are clearly needed to define the alkenone/alkenoate versus temperature relationship(s) for this genus. The four G. oceanica strains examined to date (this study, Volkman et al. 1995, M. H. Conte, unpubl. results) indicate significant genetic diversity in G. oceanica, as found for E. huxleyi.

The differences we observed in tetraunsaturated abundances among the strains in culture are consistent with field observations from the collection locations. Low levels of tetraunsaturated alkenones were found in >0.8-pm surface water particles in subarctic waters of the eastern North Atlantic (Conte and Eglinton 1993), whereas tetraunsaturated alkenones were abundant in Norwegian fjord particles collected at similar water temperatures (Conte et al. 1995). The low abundances of tetraunsaturated alkenones in the oceanic strains examined is also consistent with the near absence of these compounds in the underlying oceanic sediments (Marlowe et al. 1990).

The anomalously high abundances of tetraunsaturated alkenones in strain 92D relative to oceanic strains provides an explanation for earlier discrep- ancies between the temperature calibration work of Marlowe (1984) and Prahl et al. (1988). In his initial culture studies, Marlowe (1984) used the English Channel strain 92D whereas Prahl et al. (1988) used an unidentified NE Pacific strain. Extending Mar- lowe’s work, Brassell et al. (1 986b) included a term for 37:4Me concentration in the original index for CS7 alkenone unsaturation (Uks,; Table 1). Prahl et al. (1988) found that the correlation with temperature in their cultures was improved using a modi- fied UkS7 ratio (VS7; Table 1) that omitted the 37:4Me term. In contrast, the correlation with temperature in Norwegian fjords is best when the original UkS7 ratio, which includes the 37:4 term, is used (Conte et al. 1995). The difference in the goodness of fit seen between Uk’S7 and UkS7 in these two studies is consistent with our culture findings: both 92D and Norwegian fjord strains contain significant abundances of the tetraunsaturated alkenones, whereas the NE Pacific and other oceanic strains do not. These culture results indicate that in strains in which tetraunsaturated alkenones are relatively abundant the biochemical response to temperature involves major adjustment of the levels of tetraunsaturated as well as di- and triunsaturated alkenones.

The extreme variability observed here in biomarker ratios among replicate cultures of the same strain provides conclusive evidence that unsaturation ratios of both CS7 and CS8 methyl alkenones are controlled by variable(s) in addition to temperature. Two possible factors are suggested by our study. We used seawater from an offshore coastal location to make the culture media. The dissolved organic carbon and trace nutrient composition of this seawater, which would be expected to vary over the course of the experiments, could have had an influence on biomarker synthesis. However, we did not observe any systematic differences between culture run batches, suggesting that the difference in seawater composition was not a factor. Alternatively, the stage of the growth cycle or growth conditions could have varied among cultures. Dunstan et al. (1 993) found that cellular alkenone concentrations in the hapto-

LIPID BIOMARKERS IN EMILIANIA HUXLEYI AND GEPHYROCAPSA OCEANICA 28 1

phyte Zsochrysis galbana were sensitive to culture age and growth conditions. We tried to ensure that the cells were harvested in the log phase of growth; nevertheless, some of the cultures may have been later in the growth cycle than others. We did not observe, however, any consistent difference in biomarker ratios between replicate cultures with high or low final cell densities, suggesting that growth rate per se, as reflected by final cell density, had not strongly affected the biomarker profiles in our cultures.

The influence of additional factor(s) controlling alkenone unsaturation ratios may account in part for the variation in Uks7 and UkSBMe seen in natural waters, especially at lower temperatures (Conte and Eglinton 1993, Sikes and Volkman 1993, Conte et al. 1995). It is important to note that the variability in biomarker ratios observed in these cultures great- ly exceeds that observed in surface water particles, suggesting that this variation is largely averaged out in the natural environment.

While this variability obscured more subtle strain- related differences in alkenone/alkenoate profiles, it allowed us to examine the biochemical adjust- ments of biomarker distributions in response to the forcing variable(s). The nearly perfect covariance observed between Uk’s7 and UksEMe (Fig. 4) indicates that CS7. and Cs8 methyl alkenone unsaturation is biochemically linked and that the biochemical response to this cofactor is essentially identical in all E. huxleyi and G. oceanica strains. Similar covariation in degree of unsaturation has also been observed in >0.8-pm surface water particles from the North At- lantic (Conte and Eglinton 1993) and Norwegian fjords (Conte et al. 1995) and in sediments from the NE Atlantic (Madureira et al. 1995).

Alkene distributions also varied in a systematic fashion in the cultures. In Gulf of Maine and Nor- wegian fjord strains, the unsaturation ratio of the C,, alkenes was positively correlated with UkS7, suggesting that the alkene and alkenone unsaturation in these neritic strains is controlled by similar factors. In contrast, oceanic strains did not show any correlation of Css unsaturation with Uk’s7. Instead, those strains that contained Cs7 + Cs8 alkenes showed a close correlation in the relative abundance of Csl versus C,, alkenes and total Cs7 + Cs8 alkene abundance. These observations, as well as the greater significance of tetraunsaturated alkenones in some neritic strains, suggest that the oceanic and neritic populations have genetically diverged to a greater extent than strains from different ocean basins.

Our results have several implications for the re- finement of alkenone/alkenoate temperature calibrations for use in paleoclimatic studies. First, field studies should be used to calibrate the alkenone/ alkenoate versus temperature response, as culture calibrations of a single strain may not be representative of the alkenone-synthesizing population as a whole and additional cofactor(s) that also influence

alkenone unsaturation patterns may not be representative of the natural environment. In this regard, it is remarkable that the culture calibration of Prahl et al. (1988) agrees so closely with field data. Second, the strong covariance between UkS7 and UkSBMe provides further support for the use of multivariate temperature calibrations (Table 1) to reduce mea- surement errors. The redundancy of the two inde- pendent measurements is advantageous for calibration as estimation error associated with measure- ment of a single variable are minimized. Routine analysis of both ratios in cores, as well as the alkenoate abundance ratio (AASB), could easily identify outliers and coelution problems resulting in erro- neous biomarker indices and reduce analytical scat- ter in IPT estimates.

Our study provides strong evidence that the alkenones, alkenoates, and alkenes are biochemically linked and vary in a coordinated manner in response to unknown environmental variables in addition to temperature. Only small differences in alkenone/ alkenoate distributions within the E. huxleyi strains and between G. oceanica and E. huxleyi were detected under the culture conditions used here. Differences observed between clones isolated from the same water mass were as great as those among biogeographically separated strains. The lack of strong divergence among oceanic strains suggests that E. huxleyi has the genetic plasticity to respond physiologically over the range of environmental conditions found in open ocean waters. In contrast, both lipid and pigment (Mantoura et al., unpubl. results) profiles suggest that E. huxleyi populations have diverged in response to differences between the coastal and oceanic environment. We thank L. A. S. Madureira, R. Jordan, R. Harris, and R. Head for assistance with collections on BOFS cruises and S. Brown and R. Green for assistance with laboratory analyses. We also thank R. L. A. Andersen (Bigelow Laboratory), R. N. Pienaar (Uni- versity of Witwaterstrand), F. J. R. Taylor (University of British Columbia), and I . lnouye (University of Tsukuba), who kindly made cultures available to us. This work was supported by NERC grants GST/02/386, GR3/8139, and GR9/787 and by EC fund- ing to the MAST I1 EHUX project, contract No. MASGCTS- 0038. Support for the mass spectrometry facilities by NERC grant GR3/3758 is also acknowledged. BOFS contribution No. 248 and EHUX (MAST) contribution No. 22.

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