Fucoxanthin Pigment Markers of Marine Phytoplankton Analysed by HPLC and HPTLC

MARINE ECOLOGY - PROGRESS SERIES Vol. 38: 259-266, 1987 1 Published July 13 Mar. Ecol. Prog. Ser. l

Fucoxanthin pigment markers of marine phytoplankton analysed by HPLC and HPTLC

Simon W. wrightl & S. W. ~effrey~

Antarctic Division, Department of Science, Channel Highway, Kingston, Tasmania 7150. Australia L CSIRO Division of Fisheries Research, Marine Laboratories, GPO Box 1538, Hobart, Tasmania 7001, Australia

ABSTRACT: High-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC) were used to separate fucoxanthin, 19'-hexanoyloxyfucoxanthin and a 19'- butanoyloxyfucoxanthin-like pigment from algal cultures. Pavlova luthen (Prymnesiophyceae) con- tained fucoxanthin only, whereas Emiliania huxleyi, Phaeocystis pouchetii (Prymnesiophyceae) and Pelagococcus subviridis (Chrysophyceae) contained all 3 fucoxanthin pigments in widely different proportions. The 3 pigments, which were also found in phytoplankton samples from Antarctic waters, may provide useful markers of phytoplankton in field samples. The HPTLC method provides a rapid screening technique for fucoxanthin pigments, complementing the more powerful but time-consuming HPLC

INTRODUCTION Fucoxanthin (Fig. 1) is confined in the phytoplankton to diatoms, prymnesiophytes, raphidophytes and chry- Knowledge of the range of photosynthetic pigments sophytes (Jeffrey & Vesk 1981, Stauber & Jeffrey in a phytoplankton sample can indicate the algal unpubl.). 19'-hexanoyloxyfucoxanthin is the major classes present in phytoplankton populations, informa- carotenoid of the coccolithophorid Emiliania tion not available from simple chlorophyll and (Coccolithus) huxleyi (Arpin et al. 1976) and was also 'pheopigment' analyses (Jeffrey 1974). For example, ascribed to natural blooms of the colonial prym- thin-layer chromatography (TLC) unexpectedly nesiophyte Crymbellus aureus (Gieskes & Kraay, revealed significant quantities of chlorophyll b (green 1986a). Several dinoflagellates (e.g. Gyrodinium algae) in the North Pacific Ocean (Jeffrey 1976), and aureolum; Bjerrnland & Tangen 1979, Tangen & Bj~rn- fucoxanthin (diatoms, prymnesiophytes) was found to land 1981) which contain fucoxanthin and 19'-hex- be the dominant carotenoid compared to the insigni- anoyloxyfucoxanthin may contain prymnesiophycean ficant peridinin (photosynthetic dinoflagellates) in endosymbionts (Bjerrnland et al. 1984). Recently, a new Australian coastal and oceanic phytoplankton (Jeffrey fucoxanthin derivative from a Norwegian isolate of & Hallegraeff 1980, Hallegraeff 1981, Hallegraeff & Pelagococcus (Coc. min., Chrysophyceae) was une- Jeffrey 1984, Jeffrey & Hallegraeff 1987). TLC (Jeffrey quivocally characterized as 19'-butanoyloxyfucoxan- 1981) has also allowed separation and identification of thin (Bjrarnland et al. 1985). The full paper on this many types of chlorophyll degradation products in phy- pigment has yet to be published. A similar pigment was toplankton field samples (Hallegraeff & Jeffrey 1985). The application of high-performance liquid chroma- tography (HPLC) in phytoplankton studies (e.g. Man- toura & Llewellyn 1983, Wright & Shearer 1984) has

extended the range of pigment separations, making fucoxanthin available more 'marker' pigments for finger-printing algal types in the water column. Recent additions include zeaxanthin, ascribed as a proposed marker for cyanobacteria (Guillard et al. 1985), alloxanthin, a marker for cryptomonads (Gieskes & Kraay 1983), and prasinoxanthin, a marker for particular prasinophytes Fig. 1. Chemical structures of fucoxanthin, 19'-butanoyloxy- (Foss et al. 1984). fucoxanthin and 19'-hexanoyloxyfucoxanthin

Q Inter-Research/Printed in F. R. Germany 260 Mar. Ecol. Prog. Ser. 38: 259-266, 1987

reported from other strains of Pelagococcus subviridis General Oceanics rosette sampler equipped with (Lewin et al. 1977, Vesk & Jeffrey 1987), and is referred twelve 5 1 Nishn bottles was used to collect water to here as a 19'-butanoyloxyfucoxanthin-like pigment. samples from 0, 10, 25, 35, 50, 75, 100 and 200 m depth. Sucrose TLC was originally used to separate fuco- Between 2 and 4 1 of each sample were filtered for xanthin and the 19'-butanoyloxyfucoxanthin-like pig- pigment analysis. ment from Pelagococcus subviridis (Lewin et al. 1977). Pigments: algal cultures. Cells were harvested at the In the present work, cellulose TLC (Jeffrey 1981) was end of log phase by centrifuging either in a plankton used to separate mixtures of fucoxanthin (from Pavlova centrifuge at 8000 X g (large volumes) or in a bench luthen] and 19'-hexanoyloxyfucoxanthin (from centrifuge at 2000 X g for 3 to 5 min (small volumes). Emiliania huxleyi]. However, neither TLC system Pigments were extracted from the cell pellet in 100 % resolved the 19'-hexanoyloxyfucoxanthin from the 19'- acetone (except Pelagococcus subvin'dis, which butanoyloxyfucoxanthin-like pigment. Therefore an required freezing in ether/dry ice mixtures before HPTLC (high-performance thin-layer chromatography) acetone extraction; Vesk & Jeffrey 1987). method (Vesk & Jeffrey 1987) and the HPLC technique For determination of chlorophylls a and c, spec- of Wright & Shearer (1984) were both used to separate trophotometric analyses of acetone extracts were all 3 fucoxanthin pigments. The present work reports carried out using the appropriate equations of Jeffrey & the use of both methods in cultured microalgae and Humphrey (1975). Cell counts of the original cultures phytoplankton field samples from Antarctic waters. were made using a haemocytometer. For TLC, pigments were transferred from acetone to peroxide-free diethyl ether by adding an equal volume of ether to the acetone extract and gently mixing with MATERIALS AND METHODS 20 volumes of cold (5 "C) 5 % NaCl solution. The pig- ments migrated to the ether hyperphase, which was Algal cultures. The cultures used were from the collected, concentrated under a stream of NZ, dried CSIRO Algal Culture Collection (Jeffrey 1980). Pavlova with a little solid NaC1, and stored at - 15 'C until use. lutheri (CSIRO Culture Code CS-23) and Emiliania Pigments: phytoplankton field samples. Samples huxleyi (CS-57; Clone BT6 from Dr R. R. L. Guillard) were filtered under slight vacuum onto Whatman GF/F were used as source material for standard fucoxanthin filters (47 mm diameter). As magnesium carbonate can (Berger et al. 1977) and standard 19'-hexanoyloxy- bind pheophytins and chlorophyllides (Daley et al. fucoxanthin (Arpin et al. 1976) respectively. Pelago- 1973), it was not used as a filter aid. The filters were coccus subw.ridis (East Australian Current strain; CS- immediately frozen in liquid nitrogen until ready for 99) was used as the source of the 19'-butanoyloxy- analysis. fucoxanthin-like pigment (Vesk & Jeffrey 1987). These The frozen filters were cut into pieces of approxi- cultures were grown in 125 m1 Erlenmeyer flasks at mately 5 mm diameter, sonicated in 4 m1 methanol 17.5 "C in 40 ,uE m-' S-' white fluorescent light (Phillips (30 S at 50 W) using a Braun Labsonic 1510 sonicator Daylight tubes) on 12: 12 h light: dark cycles in medium equipped with a 4 mm diameter needle probe and f2 (Guillard & Ryther 1962). Light intensities were filtered using the centrifugal system described in measured with a Biospherical Optics light meter. Three Wright & Shearer (1984). Methanol was used as an strains of Phaeocystis pouchetii (one isolated by J. L. extraction solvent for HPLC because acetone caused Stauber in 1981 from the East Australian Current [CS- peak spreading. The filter debris was washed with 1651, one isolated by P. Thomas from Prydz Bay, 0.5 m1 methanol and recentrifuged. The combined Antarctica in 1985. and one isolated by S. I. Blackbum extract was filtered through a Millex-SR filter unit from Antarctic waters) were grown in G5 medium (0.5 pm pore-size, Millipore Corp.), and chromato- (Loeblich & Smith 1968) under the same conditions. graphed immediately. No significant allomerization of Both strains on Antarctic P. pouchetii were grown at chlorophylls occurred in the 10 min required for extrac- 5 "C. For larger volumes, 1 to l0 l cultures were either tion. aerated, or shaken on an orbital shaker at 100 cycles High-performance thin-layer chromatography. min-l, in Erlenmeyer flasks. None of the cultures used Because the cellulose TLC plate (Jeffrey 1981) was in this study were completely axenic. unable to separate all the fucoxanthin pigments, a 1- Collection of phytoplankton field samples. Samples dimensional reverse-phase HPTLC plate was used, were collected from the MV Nella Dan every 1" latitude which successfully separated the 3 pigments. The sys- from 60"s to the Antarctic coast, along 7 north-south tem comprised a Merck RP-8 bonded silica plate, transects between 58" and 93"E longitude made during developed for 30 min with methano1:water = 9:1 (v/v) January 1985 as part of the Second International BIO- (Vesk & Jeffrey 1987). MASS Experiment, Phase 11 (SIBEX 11; Wright 1987). A High-performance liquid chromatography. Samples Wright & Jeffrey: Fucoxanthin pigments of phytoplankton 261

of 250 p1 of extract were injected directly into a Waters 0.48) - from extracts of cultures of Pavlova luthei Associates liquid chromatograph. Two RCM-100 radial Emiliania huxleyi and Pelagococcus subviridis and compression modules containing Rad-Pak A cartridges mixtures of these (Fig. 2A, B, C, D). All 3 pigments were (5 pm particle size, octadecyl silicae) were used in found in the East Australian Current strain and 2 series, protected by a precolumn filter and an RCSS Antarctic strains of Phaecystis pouchetii, although the Guard-Pak. The pigments were eluted using a linear proportion of the fucoxanthin pigments was variable gradient from 90:10 acetonitri1e:water (v/v) to ethyl (Fig. 2E, F, G). The Antarctic concentrate also con- acetate over 20 min at 2 m1 min-l. They were detected tained the 3 fucoxanthin pigments (Fig. 2H). by the sum of absorbances at 405 and 436 nm (Wright & Shearer 1984), and integrated using a Waters Data Module. For small peaks, where the signal-to-noise ratio was low, the data module was found to be inaccu- rate, so these peaks were measured manually and converted to pigment abundance using individual calibration curves based on extinction coefficients of 160 l g-' cm-' (at 452 nm; solvent ethanol) for fucoxan- thin and derivatives (Jensen 1966), 250 1 cm-' (at 450 nm; solvent ethanol) for carotene, diadinoxanthin and diatoxanthin (Goodwin 1955), and 88.15 1 g-' cm-' (at 663 nm; solvent acetone for chlorophyll a (Jeffrey & Humphrey 1975). As there was insufficient material in most phytoplankton field extracts to obtain absorption spectra, the pigments could be identified initially only by comparison of retention times with known stan- dards. However, after all field samples were analysed, 0- 0 0 Q Q 0 0 0 @-""gin peak identities were confirmed by pooling selected ABCDEFGH samples into one concentrate (referred to here as Antarctic concentrate), which was rechromatographed. 0 Fucoxanthinder~vatives Absorption spectra for initial identification were then 0 Other carotenoids taken during elution, using a Hewlett-Packard 8450A Fig. 2. Separation of major carotenoids from various algal spectrophotometer. species and the Antarctic concentrate on the reverse-phase Visible absorption spectra. For precise absorption bonded-silica HPTLC plate (Merck. RP-8). (A) Pavlova lutheri; spectra, purified samples from cultures were isolated (B)Emiliania hwdeyi; (C) Pelagococcus subviridis; (D) mixture on TLC systems (reverse-phase RP-8 plate, or washed of A, B & C; (E, F, G) Phaeocystis pouchetii: East Australian Current strain (EAC), and Antarctic strains (S. I. Blackburn cellulose (Jeffrey 1981) and examined either in a Cary [Ant:SIB] and P. Thomas [Ant:PT] respectively); (H) Antarctic Model 14 or a Shimadzu Model RP1 recording spec- concentrate. 1 : peridinin (red); 2: 19'-butanoyloxyfucoxan- trophotometer. thin-like (yellow-orange); 3: fucoxanthin (red-orange); 4: 19'- It should be noted that where only chromatographic hexanoyloxyfucoxanthin (yellow-orange); 5: diadinoxanthn (yellow); 6: diatoxanthin (pale orange) and visible absorption spectroscopy data, and not full chemical characterization, are available, the pigment should be identified by the suffix '-like' (SCOR Work- The visible absorption spectra of these pigments in ing Group 77 on Photosynthetic Pigments, unpublished different solvents are given in Fig. 3 (no quantitative recommendation). Thls has been followed in the pre- relations between the solvents is intended) and absorp- sent paper, e.g. only fucoxanthin from Pavlova luthen tion maxima and band ratios of the 3 fucoxanthin and 19'-hexanoyloxyfucoxanthin from Emiliania hux- pigments from different algal sources are given in leyi have been fully chemically characterized (Arpin et Table 1. The spectra of 19'-hexanoyloxyfucoxanthin al. 1976, Berger et al. 1977). and the 19'-butanoyloxyfucoxanthin-like pigment differed from that of fucoxanthin by having a pro- nounced minimum between Peaks I1 and I11 (sensu Ke RESULTS et al. 1970), whereas in fucoxanthin this minimum was absent, Peak 111 being a shoulder. The 1II:II band ratios The reverse-phase RP-8 HPTLC plate separated the for the 19'-hexanoyloxyfucoxanthin and 19'-butanoy- 3 fucoxanthin pigments - fucoxanthin (RF value 0.43), loxyfucoxanthin-like pigment differed depending on 19'-hexanoyloxyfucoxanthin (RFvalue 0.40) and the algal source. We ascribe these variations to solvent 19'-butanoyloxyfucoxanthin-like pigment (RF value effects caused by slight differences in the concentration 262 Mar. Ecol. Prog. Ser. 38: 259-266, 1987

19'-he~an0ylo~yf~~0~anIhin-like-- 19'-butanoyloxyfucoxanthin-like - lsolvent:D diethyl ether

Fig. 3.Visible absorption spectra of fucoxanthin pigments in acetone, ethanol and ðyl ether. (A) Fucoxanthin from Pavlova lutheri; (B) 19'-hexanoyloxyfucoxanthin from Emiliania huxleyi;(C) 19'-butanoyloxyfucoxanthin-like pigment from Pelagococcus subviridjs; (D) the 19'-hexanoyloxyfucoxanthn-likeand 19'-butanoyloxyfucoxanthin-like pigments from Phaeocystispouchetii in diethyl ether. (Band I1 is the 450 nm peak and Band I11 is the 470 nm peak referred to in Table 1; Ke et al. 1970). of water and other cell components extracted with the presence of minor components, including the cis-isom- pigments. ers of fucoxanthin and 19'-hexanoyloxyfucoxanthin, The separation of the 3 fucoxanthin pigments on the and an unknown carotenoid pigment, which eluted just HPLC system of Wright & Shearer (1984) is shown for before fucoxanthin. the first time in Fig. 4. The algal extracts and Antarctic Quantitative analyses of the major pigments in Pav- concentrate were the same as those used on the HPTLC lova luthen, Emiliania huxleyi, Phaeocystis pouchetii system (Fig. 2). HPLC confirmed the resolution of the and the Antarctic samples are shown in Table 2, and major fucoxanthin derivatives obtained with the the ratios of the 3 fucoxanthin pigments are given in HPTLC plate, but the extra sensitivity also revealed the Table 3.

Table 1. Absorption maxima (nm) and band ratios of fucoxanthin pigments in various solvents

Alga CSIRO Culture Plgrnent Solvent Maxima Band ratios Code No. 1II:II (O/O) Band I1 Band I11 I

Pavlova lutheri CS-23 Fucoxanthin Ethanol 448 -470 0 Acetone 446 470 Diethyl ether 446 470 3.3 Emiliania h uxleyi 19'-hexanoyloxy- Ethanol 447 fucoxanthin Acetone 445 Diethyl ether 444 Pelagococcus subvindis' 19'-butanoyloxy- Ethanol 446 fucoxanthin-like. ' Acetone 445 Diethyl ether 444 Phaeocystispouchetli 19'-hexanoyloxy- Ethanol 446 fucoxanthin-like' ' Acetone 444 Diethyl ether 444 19'-butanoyloxy- Ethanol 447 fucoxanthin-like " Acetone 445 Diethyl ether 444

For a full analysis of P. subviridis pigments (2 strains), see Vesk & Jeffrey (1987) ' ' Where only chromatographic and spectral data are available, and not full chemical characterization, the suffix-'like' is added (SCOR Worlung Group 77, unpubl. recommendation) Wnght & Jeffrey: Fucoxanthin pigments of phytoplankton 263

completely swamped the tiny amount of the 19'- butanoyloxyfucoxanthin-like pigment available from the field samples, for which no infrared spectra were obtained at this time. Other minor and trace pigments were also present in the cultures and the Antarctic concentrate (Wright & Shearer 1984, Vesk & Jeffrey 1987), but these will be discussed fully elsewhere (Wright 1987).

DISCUSSION

Algal cultures 6 8 Pelagococcus The present work confirms the presence of fucoxan- a thn alone (free from traces of other fucoxanthin pig- ments) in Pavlova lutheri, previously examined by Berger et al. (1977). Emiliania huxleyi, in adhtion to containing the dominant 19'-hexanoyloxyfucoxanthin (Arpin et al. 1976), also contained small amounts of fucoxanthn and the 19'-butanoyloxyfucoxanthin-like

3 8 pigment (Tables 2 & 3). Pelagococcus subviridis con- Phaeocystis (Ant.PT) tained fucoxanthin and the 19'-butanoyloxyfucoxan- 6 thin-like pigment as the major carotenoids (Vesk & 1 h 7 Jeffrey 1987), but HPLC also revealed the presence of small amounts of 19'-hexanoyloxyfucoxanthin (Fig. 2). The identity of the fucoxanthin and 19'-hexanoyl- oxyfucoxanthln was confirmed by using pigment stan- dards from well-characterized sources, but the identity of the 19'-butanoyloxyfucoxanthin-like pigment from Pelagococcus subviridis is stdl unclear. True 19'- butanoyloxyfucoxanthn was chemically identified together with fucoxanthin in the Norwegian isolate of a I'"'I'"'I""IJ~"I' 0 5 10 15 20 Pelagococcus-like strain (Coc. min.) by Bj~lrnlandet al. Retent~ontime (m~nutes) (1984), although the full data, including visible spectra

Fig. 4. HPLC traces of pigment separations of the algae shown and chromatographic systems, have yet to be pub- In Fiq. 2 and the Antarchc concentrate Piqment fractions are. lished. The fucoxanthin derivative present in both our 1 19'-butanoyloxyfucoxanthin-like pigment; 2 fucoxanthn; 3 strains of Pelagococcus (Vesk & Jeffrey 1987) was 19'-hexanovlox~fucoxanthin;. . 4 cis-fucoxanthln; 5 as 19'-he- chromatographically different from 19'-hexanoyloxy- xanoyloxyfucoxanthn; 6 diadinoxanthin: 7 dlatoxanthin; 8 chlorophyll a. Retention times as shown. It should be noted fucoxanthn, yet spectrally similar (Fig. 3), and we ini- that Peak 3 in both Phaeocystis pouchetri isolated should be tially assumed it to be the 19'-butanoyloxyfucoxanthin referred to as '19'-hexanoyloxyfucoxanth~n-like',untd chemi- of Bj~lrnlandet al. (1984). However, in both our HPLC cal characterization has been carried out and HPTLC systems, where the chromatographic mobdity of an homologous series is related to the That the pigments in the Antarctic concentrate were length of the hydrophobic substituent, the sequence of identical with the fucoxanthin derivatives in the vari- the 3 fucoxanthin pigments was different from that we ous algal cultures was confirmed by absorption spec- would expect if this pigment were 'true' 19'-butanoy- troscopy and by CO-chromatography in the HPLC and loxyfucoxanthin. On the basis of chemical structures HPTLC systems. In addition, allene groups, which are (Fig. l), 19'-butanoyloxyfucoxanthin should be inter- present in fucoxanthin and its derivatives, were mediate in polarity between fucoxanthn and 19'-he- detected in the fucoxanthin and 19'-hexanoyloxyfuco- xanoyloxyfucoxanthin, but in both our chromato- xanthin fractions isolated from the Antarctic samples graphic systems (HPLC and HPTLC) it appeared to be by an absorption peak at 1929 nm, using infrared spec- more polar than both (Fig. 2 & 3). The possibility troscopy. Colourless contaminants (presumably lipids) remains that our pigment either has an addtional polar prevented full characterization of these pigments and group (e.g. hydroxyl) or has lost the methyl ester of the 264 Mar. Ecol. Prog. Ser. 38: 259-266, 1987

Table 2. Quantitative analyses of major pigments in Pavlova lutheri, Emiliania huxleyi, Phaeocystis pouchetii, Pelagococcus sub~disand the Antarctic concentrate, from HPLC analysis for carotenoids and spectrophotometric analysis (Jeffrey & Humphrey 1975) for chlorophylls a and c. Minor carotenoids (Fig. 4) are not included

Pigment Algal sample P. lutheri E. huxleyi P, pouchetii P. subviridis Antarctic pg (10' cells)-' pg (10' cells)-' pg (10' cells)-' ~ig(10' cells)-' concentrate &gI-'

Chlorophyll a 1 31.0 Chlorophyll c 8.0 p,p-carotene 0.39 Fucoxanthin 0.41 19'-hexanoyloxyfucoxanthin 31.3 19'-butanoyloxyfucoxanthin-like 0.27 Diadinoxanthm 4.3 Diatoxanthin 2.4

Table 3. Ratios of fucoxanthin pigments from HPLC analysis of algal cultures and Antarctic water samples

I Alga or phytoplankton sample Pigment ratios I Fucoxanthm 19'-hexanoyloxy- 19'-butanoyloxy- fucoxanthin fucoxanthin-like pigment

Pavlova lutheri Emiliania huxleyi Phaeocystis pouchetii (EA Current) Phaeocystis pouchetii (Antarctic) ' Phaeocystis pouchetii (Antarctic) ' ' Phaeocystis pouchetii (Antarctic) ' (senescent culture) Antarctic concentrate Average of 40 Antarctic water samples

P. Thomas isolate I' ' S. Blackburn isolate native fucoxanthin. On the basis of chromatographic cultured Phaeocystis pouchetii (pers. comm.). Our properties, this pigment is probably identical to the results are in apparent contradiction to those of Gieskes unknown fucoxanthin derivative found by Gieskes & & Kraay (1986a), who stated that field populations of Kraay (1986b) in the tropical Atlantic. The chemical Phaeocystis pouchetii do not contain 19'-hexanoyloxy- structure of this 19'-butanoyloxyfucoxanthin-like pig- fucoxanthin, but without further details it cannot be ment from our strains of Pelagococcus subviridis is determined whether culture, extractions methods or currently under investigation. strain differences would explain the conflicting obser- Cultured strains of Phaeocystis pouchetii examined vations. in the present work contained all 3 fucoxanthin pig- ments, with the pigment chromatographically and spectrally identical to 19'-hexanoyloxyfucoxanthin Antarctic water samples dominant in each strain. The 19'-butanoyloxyfucoxan- thin-like pigment was CO-dominant in the East Austra- Extensive use of the HPLC method during the SIBEX lian Current strain, but was present as a minor compo- I1 experiment in Antarctic waters allowed distribution nent with fucoxanthin in both Antarctic strains. Signifi- of the 3 fucoxanthin pigments (integrated for the top cant differences in the proportions of these pigments 100 m) to be determined over a wide area (Wright were observed between strains and age of culture 1987). Significant differences in the abundances of the (Table 3). These results are in agreement with those of 3 pigments were noted. The range of values for fuco- Dr S. Laaen-Jensen, who found both 19'-hexanoyl- xanthin was 2.2 to 247 mg m-2, compared to 5.1 to oxyfucoxanthin and 19'-butanoyloxyfucoxanthin in 323 mg m-2 for chlorophyll a, 2.4 to 83 mg m-2 for 19'- Wright & Jeffrey: Fucoxanthin pigments of phytoplankton 265

hexanoyloxyfucoxanthin and 0.06 to 3.2 for the 19'- Acknowledgements. We thank Mrs J. L. Stauber for isolating butanoyloxyfucoxanthin-like pigment. At some sta- the East Australian Current strains of Phaeocystis pouchem. tions 19'-hexanoyloxyfucoxanthin was more abundant and Pelagococcus subvilidis, Mr P. Thomas and Dr S. I. Black- burn for the 2 Antarctic strains of Phaeocystis pouchetii, Dr than fucoxanthin. 19'-hexanoyloxyfucocanthin has S I Blackburn for growing the cultures used in the present recently been reported as present in the Southern work, Mr D. Le for the cell counts, Mr J. D Shearer for Ocean (Gieskes & Elbrachter 1986), in the tropical assistance with the Southern Ocean field sampling, Mrs T. Atlantic (Gieskes & Kraay 1986b) and dominant in the Glaesner for HPLC assistance, and Dr N. Davies (University of Tasmania) for the infrared spectrometry. We are indebted to North Sea during a bloom of the colonial prym- Dr G. M. Hallegraeff and Dr H. Marchant for helpful com- nesiophyte Corymbellus aureus (Gieske & Kraay ments on the manuscricpt. 1986a), during which it reached 7 times the concen- tration of fucoxanthin. The origin of the fucoxanthin derivatives in Antarctic LITERATURE CITED waters is not yet clear, since very little is known of the Arpin, N., Svec, W. A., haaen-Jensen, S. (1976). New fucox- taxonomic distribution of these newly recognized pig- anthin-related carotenoids from Coccolithus husleyi. Phy- ments. Size fractionation of the Antarctic phytoplank- tochemistry 15: 529-532 ton samples showed that both the 19'-hexanoyloxy- Berger, R., Liaaen-Jensen. S.. McAlister, V., Guillard, R. R. L. (1977). Carotenoids of Prymnesiophyceae (Haptophyceae). fucoxanthin and 19'-butanoyloxyfucoxanthin-like pig- Biochem. Syst. Ecol. 5: 71-75 ments were significantly concentrated in the < 5 pm Bj~rnland,T., Pennington, F., Haxo, F. T., Liaaen-Jensen, S. fraction, with most fucoxanthin being found in the (1984). Carotenoids of Crysophyceae and Dinophyceae. > 5 km fraction (Wright 1987), the latter probably Abs. 7th Int IUPAC Symposium on Carotenoids, Munich, originating from diatoms (Stauber & Jeffrey unpubl.). p. 26 Bjernland, T., Tangen, K. (1979). Pigmentation and rnorphol- One source of the fucoxanthin derivatives is Likely to be ogy of a marine Gyrodinium (Dinophyceae) with a major the prymnesiophyte Phaeocystis pouchetii, which was carotenoid different from peridinin and fucoxanthln. J. widespread in the study area during SIBEX 11 (Mar- Phycol. 15- 457-463 chant & Wright unpubl.). Other sources of fucoxanthin Booth, B. C., Marchant, H. J. (1987). Parmales, a new order of marine chrysophytes with descriptions of 3 new genera derivatives are also likely. A significant number of and 7 new species. J. Phycol (in press) nanoplanktonic flagellates (2 to 20 pm) were observed Daley, R.J., Gray, C. B. J., Brown, S. R. (1973). A quantitabve, in Antarctic water samples during this cruise (Mar- semiroutine method for determining algal and sedimen- chant & Wright unpubl.), characterization of which has tary chlorophyll derivatives. J. Fish. Res. Bd Can. 30: not yet been completed. Booth & Marchant (1987) have 345-356 Foss, P,, Guillard, R. R. L., Liaaen-Jensen, S. (1984). Prasinox- described 7 new species from Antarctic water phyto- anthin - a chemosystematic marker for algae. Phytochem. plankton within the newly described order Parmales in 23: 1629-1633 the Chrysophyceae. Elucidation of the pigment com- Gieskes, W. W. C., Elbrachter, M. (1986). Abundance of nano- position of the Parmales and other unidentified forms plankton-size chlorophyll-containing particles caused by diatom disruption in surface waters of the Southern Ocean awaits successful isolation and culture. Pelagococcus (Antarctic Peninsula region). Neth. J. Sea Res. 20: 291-303 subviridis, another minute planktonic chrysophyte (2 to Gieskes, W. W. C.. Kraay, G. W (1983). Dominance of Cryp- 3 pm), is widely distributed in the North Pacific Ocean tophyceae dur~ngthe phytoplankton spnng bloom in the (Lewin et al. 1977),the East Australian Current (Vesk & central North Sea detected by HPLC analysis of pigments. Jeffrey 1987) and Norwegian waters (Throndsen & Mar. Biol. 75: 179-185 Gieskes, W. W. C., Kraay, G. W (1986a). Analysis of phyto- Kristiansen 1985) and can reach concentrations of 106 plankton pigments by HPLC before, during and after mass to 107 cells 1-l. Given the wide range of habitats in occurrence of the microflagellate Corymbellus aureus dur- which it has been found, it is quite possible that P. ing the spring bloom in the open northern North Sea in subviridis and related organisms could be present in 1983. Mar. Biol. 92: 45-52 Gieskes, W. W. C., Kraay, G. W. (198613). Floristic and phy- Antarctic waters as well, and contribute to the 19'- siological differences between the shallow and the deep butanoyloxyfucoxanthin-like distributions. nanophytoplankton community in the euphotic zone of the A few dinoflagellate genera are known to contain open tropical Atlantic revealed by HPLC analysis of pig- combinations of fucoxanthin, 19'-butanoyloxyfucoxan- ments. Mar. Biol. 91: 567-576 thin and 19'-hexanoyloxyfucoxanth~n.However, dino- Goodwin, T. W. (1955). Carotenoids. In: Peach, K., Tracey, M. (ed.) Modern methods in plant analysis, Vol. 3. Springer- flagellates are unlikely to have been a major source of Verlag, Heidelberg, p. 272-31 1 fucoxanthin pigments in these Antarctic water sam- Guillard, R. R. L., Murphy, L. S., Foss, P,, Liaaen-Jensen, S. ples. Although Gymnodinium spp. and other dino- (1985). Synechococcus spp. as likely zeaxanthin-dominant flagellates were present in the study area, they were ultraphytoplankton in the North Atlantic. Lirnnol. Oceanogr. 30: 412414 never abundant (Marchant & Wright unpubl.). Guillard, R. R. L., Ryther, J. H. (1962). Studies of marine plankton diatoms. I. Cyclotella nanaHustedt and Detonula confervacea (Cleve) Gran. Can. J. Microbial. 8: 229-239 266 Mar Ecol. Prog. Ser. 38: 259-266, 1987

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This article was submitted to the editor; it was accepted for printing on April 15, 1987