diols; Pavlova; Diacronema; chemotaxonomy; fatty acids. Microalgae in the class Haptophyceae are com- mon in the marine environment and are significant.
J. Phyrol. 33, 10161023 (1997)
UNUSUAL DIHYDROXYSTEROLS AS CHEMOTAXONOMIC MARKERS FOR MICROALGAE FROM THE ORDER PAVLOVALES (HAPTOPHYCEAE) John K. Volkman2 CSIRO Division of Marine Research, GPO Box 1538, Hobart, Tasmania 7001, Australia
Christine L. F ~ m d Institute of Antarctic and Southern Ocean Studies, University of Tasmania, GPO Box 252C, Hobart, Tasmania 7001, Australia
Stephanie M. Barrett CSIRO Division of Marine Research, GPO Box 1538, Hobart, Tasmania 7001, Australia
and Elisabeth L. Sikes Australian Geological Survey Organisation and Antarctic CRC, University of Tasmania, GPO Box 252C, Hobart, Tasmania 7001, Australia ABSTRACT
Recent studies have led to the identification of a n unusual class of dihydroxysterols (steroidal diols t m e d ‘paw lovols’3 in a f m species of microalgaefrom the genus Pavlova vamiij Pavlovaceae, class Haptophyceae = Prymnesiuphyceae). These compounds haue a n additional hydro31 group at G 4 in the sterol A ring, which appears to be v q rare in sterol biosyntheticpathways. The sterol compositions of many other haptophytes )om different orders have been analyzed, but to date all have lacked pavlovols. We now rt=pmtthe occurrence of these compounds in Diacronema vlkianum Prauser and two strains of Pavlova pinguis Green. This is the$rst report of the lipid composition of thse species. Both microalgae contained ‘24methylpavb vol” (4a,24-dimethy1-5a-cholestan-3p, 4p-diol), P. pinguis also contained “24-ethylpavlovol” (4a-methyl-24ethyl-5a-cholestan-3P,4p&ol), and D. vlkianum contained a diol identified from its mass spectrum as 4a,24/3dimethyl-5a-cholest-22E-en-3~,4~-diol. Both species contained structurally analogous 4-desmethyl sterols and 4-methyl sterols, although there were major differences in the proportions in each series. The major 4-desmethyl sterol in both species was 24-ethylcholesta-5,22E-dien-3~-ol and the major 4-methyl sterol was 4~~-rnethyl-24-ethyl-5a-cholest22E-en-3p-01. The presence of pavlovols in P. pinguis, combined with earlier data, suggests that all Pavlova species might have this distinguishing lipid feature. However, their identtjication in D. vlkianum extends the occurrence of these compounds to another genus and shows that they are not unique to the genus Pavlova. However, thq are probably restricted to species )om the order Pavlovales. The modes of biosynthesis and functions of pavlovols remain unknown. Received 1 April 1997. Accepted 15 August 1997. Author for reprint requests; e-mail: john.volkman@marine. csiro.au. ’ Present address: Major Projects Tasmania, GPO Box 646, Hobart, Tasmania 7001, Australia.
Key index words: sterols; pavlovols; Pavlovales; steroidal diols; Pavlova; Diacronema; chemotaxonomy;fatty acids Microalgae in the class Haptophyceae are common in the marine environment and are significant sources of organic matter in marine sediments. Most haptophytes are marine species and often make up a major part of the marine phytoplankton. Species which are both widespread and seasonally abundant include Emiliania huxleyi, Gephyrocapsa oceanica, and Phaeocystis pouchetzi. This class is also referred to as the Prymnesiophyceae, although this name was not formally described. For a discussion of the history of the taxonomy of this class the reader is referred to Green and Jordan (1994). Below the class level, there is a considerable diversity of views on the taxonomy, in terms of the number of orders and families as well as assignment of species. According to Chretiennot-Dinet et al. (1993), the class Haptophyceae contains the orders Isochrysidales, Coccolithophorales, Prymnesiales, and Pavlovales. Most authors recognize the Pavlovales as a separate, highly distinct order which in some schemes is elevated to subclass level (see Green and Jordan 1994). Microalgae often contain compounds whose structures are sufficiently distinctive that they can be used as biomarkers in support of taxonomic classification (e.g. Conte et al. 1994). They can be of particular value for the classification of the nanoplankton, whose taxonomy is particularly difficult because of their small size and lack of distinctive morphologies. Thus, the recognition of novel compounds may be used as a tool to aid in the classification of a particular species. The lipids of haptophyte algae have been intensively studied after it was recognized that a few species synthesize large amounts of unusual, very long-chain, unsaturated ketones and alkenes (Volkman et al. 1980). Haptophytes display a wide variety of lipid compositions. In a recent re-
1016
DIHYDROXYSTEROL MARKERS IN MICROALGAE R
1017
one of which is not from the genus PuvZova, and provide information about the chemical structure of the sterols present. Such data can be used to address questions of taxonomy in the Haptophyceae and are also of interest to those studying the sources of organic matter in marine ecosystems and sediments. MATERIALS AND METHODS
I
R
IV
I11
R=
+
b
a
C
d
e
FIG. 1. Structures of 4desmethylsterols (I and II), 4methyl sterols (III), and 3,4-dihydroxy-4methylsterols ( N ) identified in Pavloua pinguis and Diacronema vlkianum. Quantitative data and full names are shown in Table 1.
view, Conte et al. (1994) recognized five groups of species based on fatty acid distributions and four groups based on sterol distributions. The sterol composition of the Pavlovales appears to be unique (see Fig. 1 for structures). The common sterols cholesterol (Ia) and 24methylcholesta5,22Edien-3P-ol (Ib), observed in species from other orders, are either minor constituents or not present at all. Instead, 24methylcholest-5-en-3P-01(Ic), 24ethylcholesta-5,22E-dien-3P-o1 (Id), and 24ethylcholest-5-en-3P-01 (Ie) predominate, together with 5a-stanols (11) , 4a-methylsterols (111) , and unusual 3,4-dihydroxy-4-methylsterols(IV), termed pavlovols, which have not been found in other microalgae. In P. Zutheri, the latter occur exclusively as glycoside conjugates (Vkron et al. 1996). In this paper, we present further data on the occurrence of pavlovols in two other species from the Pavlovales,
Paulova pinguis Green belongs to the order Pavlovales, family Pavlovaceae. Puvlova pinguis ((3286) was isolated and identified by J. C. Green from seawater collected in the northeastern Atlantic Ocean at 3Y58.6' N 16O54.8' W. The other strain of P. pinguis examined (CS375) was isolated from Pipeclay Lagoon in southeastern Tasmania, Australia, in 1992 by J.-M. LeRoi. The 5-8 by 3-4 pm, yellow-green cells are ovate and slightly compressed. The cells have three appendages: two flagella and a haptonema. The two flagella are of unequal length with the longer (10-12 pn) easily seen by microscopy beating with a sinuous wave action characteristic of members of the Pavlovales.The long flagellum carries a garniture of fine hairs and knob scales (Green 1980). Diacronema vlkianum Prauser (CS266) was isolated near Ryde on the Isle of Wight, U.K. by M. Parke. The cells are dorsoventrally flattened and heart shaped in outline, with a length of 3-6 km and a width of 3.5-7.5 pm (Fournier 1969, Green and Hibberd 1977). The three appendages, consisting of two unequal flagella and a haptonema, arise from the center of the ventral side. The two flagella possess a terminal hair point. Green and Hibberd (1977) described the haptonema as being just over 1 p n in length, noncoiling and distally tapered. Algal culture. Cultures were obtained from the CSIRO culture collection. Stock 75-mL cultures of P. pinguis (CS286) and D. vlkianum (Cs'266) were aseptically transferred to 500-mL culture flasks containing 300 mL of GSe medium. This medium has a reduced salinity (ca. 24%0) compared with ocean waters and was prepared by mixing 80% seawater, 17% sterile distilled water, 1.9%nutrient solution, and 1% soil extract solution. The seawater was collected from the ocean side of Maria Island on the east coast of Tasmania and was purified using activated charcoal and filtered through a 0.2-pm mesh before use. The cultures were maintained in a constant environment room at 12-13' C and illuminated from beneath with 80 pmol.m-2.s-' (measured using a Biospherical Instruments light meter QSL-100) white fluorescent light (Philips daylight tubes) on 12:12 h LD cycles. The haptophyte cultures were harvested after growth for 4-9 days when cells were in the exponential growth phase. At harvest, 5 mL was transferred into 75 mZ, of media such that back up cultures could be grown, and 2 mL was reserved for cell counts. The remaining volume was filtered through 0.4pm, precombusted (24 h at 450" C) Whatman GF/C filters. Cells were counted microscopically using a Neubauer hemocytometer after immobilizing motile cells by the addition of 2-3 drops of 2% Lugols solution. Diacronema vlkianum was harvested at a cell count of 2.25 2 0.32 X lo6 mL-' and P. pinguis at a cell count of 1.82 2 0.13 X lo6 mL-'. The second strain of P. pinguis (CS37.5) was cultured on a larger scale to provide more material for analysis. A starter culture (200 mL in midlogarithmic phase) was inoculated into 1.4 L seawater enriched with f/2 culture medium (Guillard and Ryther 1962) in a 2-L Erlenmeyer flask. The flask was bubbled with air and placed on a glass shelf at 22" ? 2" C and illuminated at 100 pmol.m-2.s-' as described previously. When the culture had reached mid- to late logarithmic growth phase the contents were transferred to a sterilized polyethylene bag containing 80 L 0.2pm filtered seawater (3.5% salinity from Pipeclay Lagoon) enriched with f/2 nutrients. The bag mass culture was also grown at 22 2 2" C with 100 pmol.rn-*.s-' white fluorescent light with no dark period. The culture was mixed by bubbling with air enriched with CO, at 20 L-min-', which maintained the pH of the culture between 7.4 and 7.8. Sixty liters of the culture was harvested in stationary phase by centrifugation using an Alfa Lava1 (Lausanne, Switzerland) 106AE cream separator at a flow rate of
1018
JOHN K. VOLKMAN ET AL.
1 I,.miii-l. The harvested cells were freeze-dried and stored under liquid nitrogen prior to analysis. Lipid extraction. A modification of the Bligh and Dyer (1959) technique was used. The filters with the collected algal cells of CS286 and CS266 and 15 mg of the freeze-dried CS-375 reconstituted in deionized water (Milli-Q System, Millipore Co.) were extracted with chloroform : methanol :water (1:2:0.8v/v/v, 5 X 5 mL) using ultrasonication. Chloroform and deionized water were added to the combined extracts to give a final chloroform : methanol :water ratio of 1:l:O.g (v/v/v) to initiate phase separation. The upper aqueous phase containing salts and water-soluble material was discarded, and the lipids were recovered in the lower chloroform phase. The solvents were then removed under vacuum, and the lipids were stored under nitrogen at -20" C . Saponij5cation of Extract to yield nonsaponifiabk lipid fraction. An aliquot of the total solvent extract was evaporated to near dryness under N, and saponified in 5% KOH in methanol :water (80:20 v/v) at 80" C for 2 h. The nonsaponifiable lipids (NSLs) were extracted with hexane: chloroform (4:l v/v) and stored under nitrogen at -20" C . This fraction was treated with bis(trimethy1siIyl) trifluoroacetamide (BSTFA; Alltech Associates Inc.) immediately before gas chromatography (GC) analysis to convert compounds containing free hydroxyl groups to their trimethylsilylether (TMSi-ether) derivatives. Dihydroxysterolswere derivatized by the use of Power Sil-Prep (Alltech Associates, Deerfield, Illinois), because BSTFA only derivatized one of the hydroxyl groups. Fatty ucid methyl estm. Fatty acids were extracted from the saponified sample after acidification to pH 2. The fatty acids were converted to fatty acid methyl esters (FAME) using methanol: hydrochloric acid:chloroforin (1Ol:l v/v/v) at 80" C for 2 h under a N, atmosphere. After cooling, water was added and the FAME were extracted with hexane :chloroform (4:lv/v). Analysis of lipid class composition of the total lipid extract. Thin layer chromatography combined with flame ionization detection (TLCFID) was used to identify and quantify the nonpolar lipids and total polar lipids in algal samples (Volkman and Nichols 1991). The instrument used was an latroscan MkIII TH-10 TLGFID analyzer with silica gel SIII Chrornarods. The peak areas obtained from TLC-FID were converted to amounts of particular compounds using calibration cumes for each compound class. Analysis of lapids by gas chromatocqaphy and gas chromatographymass spectrometly. The TMSi-derivatized NSL fractions of CS286 and CS266 were arialysed with a Shimadzu 9A gas chromatograph equipped with an FID and cooled OCI-3 oncolumn injector (SGE, Ringwood, Victoria, Australia). Samples in chloroform were injected at 45' C onto a nonpolar methyl-silicone fused-silica capillary column (HPI, 50 m X 0.32 rnm i.d., Hewlett Packard, Avondale, Perinsylvania).After 1 min, the oven temperature was raised to 120" C at 30" Gniin-' and then to 320" C at 4" Csmin-'. This final temperature was then maintained for 20 min. The detector temperature was 330" C . FAME were analyzed siniilarly except that the second temperature ramp was at 3" Gmin-'. The P. p i n p i s CS-375 NSL fraction was analyzed with a Variari (Walnut Creek, California) High Temperature Series 5410 gas chi-omatograph with a Series 8100 autosampler and a septumequipped programmable injector (SPI). The column type and temperature program were the same as above except the first segment end temperature was 140" C, and the second temperature ramp was 3" C.mi1i-l to 310" c to improve the resolution. The initial temperature of the SPI was 50" C which was held for 0.15 min, then raised to 310" C at a rate of 200" C.niin-' and inairitdined for 40 min. The detector temperature was 31.5" C . Hydrogen was used as the carrier gas for both gas chromatographs. Peak areas were quantified with DAPA software (DAPA Scientific Pty. Ltd., Kalamunda, Western Australia). Individual lipids were identified from their relative retention time, coinjection with previously identified compounds and authentic standards, and from interpretation of mass spectra obtained using gas chromatography-mass spectrometly (GC-MS). Fractions from CS286 and CS266 were analysed by GGMS using a Hewlett Packard 5790 MSD connected by direct capillary inlet to an HP 5890 GC fitted with a split/splitless injector used in the
splitless mode with an injector temperature of 310" C. GC-MS analysis of the P. pinguis CS375 NSL fraction was performed with a Fisons Instruments (Rydalmere, NSW, California) MD800 and Fisons AS800 autosampler equipped with a Carlo Erba (Milan, Italy) oncolumn injector. The nonpolar column and chromatography conditions were the same as those described for the Varian GC above except that the second temperature ramp was 4" C.min-l and helium was used as the carrier gas. Electron impact mass spectra were acquired and processed with an HP 59970A Computer Workstation or Fisons Masslab software on a personal computer. Typical mass spectrometer operating conditions were: transfer line, 310" C; electron impact energy, 70 eV; 0.8 scans.s-[; mass range, 40-650 daltons. RESULTS
The total lipid content of the two species was reasonably similar (2.0 pg cell-' for P. pinguis and 1.2 pg-cell-' for D. vlkianum). Other studies of Pavlova species have reported higher total lipid contents of 4.5-8.0 pgcell-' when analyzed by TLC-FID (Volkman et al. 1991). In both species, polar lipids were the dominant group of lipids detected. Sterols r e p resented less than 8% of the total lipids in both species. Neither P. pinguis nor D. vlkianum contained the unusual long-chain unsaturated ketones (alkenones) produced by some haptophyte algae (Volkman et al. 1980, Marlowe et al. 1984) 4-Desmethylsterols. Pavlova pinguis contained two major 4desmethylsterols (55-65% of total sterols) and four minor ones, whereas D. vlkianum contained two major sterols plus another minor component (Table 1). All three sterols found in D. vlkianum were present in P, pinguis, but the latter also contained small amounts of cholesterol (Ia; structures shown in Fig. 1) and 5a-cholestanol (IIa) which were not found in D. vlkianum. The major 4 desmethyl sterol in both species was 24ethylcholesta-5,22E-dien-3P-ol (Id). Substantial amounts of 2 4 ethyl-5a-cholest-22E-en-3P-1 (IId) (25.8 and 20.2% of the total steroidal compounds) were also detected in P. pinguis but not in D. vlkianum. Indeed, the latter species contained no Mesmethyl sterols lacking a A5 double bond (i.e. 5a-stanols), but both species contained 4methyl sterols lacking a A5 double bond. 4-Methylsterols. Both species contained sterols containing a methyl group at C-4, although these comprised a much higher percentage of the total sterols in D. vlkianum (27.2%) than in P. pinguis (9.9 and 1 4.8% ) . Both 4a, 24-dime thyl-5a-cholestan-3 f3-01 (IIIc) and 4a-methyl-24ethyl-5a-cholest-22E-en-3P01 (IIId) were observed in D.vlkianum, but only the latter was detected in P. pinguis. Dihydroxysterols. Three compounds eluting after the main sterols were identified as belonging to an unusual class of dihydroxysterols. These represented 11.4 and 12.8% of the total sterols in the two strains of P. pinguis, but they were much more abundant in D. vlkianum where they comprised over 30% of the total sterols. A C,, saturated diol was detected in both species, whereas P. pinguis also contained a C3" saturated diol and D. vlkianum also contained a Cg9
1019
DIHMROXYSTEROL MARKERS IN MICROALGAE
TABLE 1. Perrentage composition of 4-desmethylsterols, 4-methyLsterols,and 3,4-dihydroxysterols in Pavlova pinguis and Diacronema vlkianum. Strain
Structure’
Sterol
4-desmethylsterols Ia cholest-5-en-3P-ol IIa 5a-cholestan-SP-0I Ic 24-methylcholest-5-en-3P-ol Id 24ethylcholesta-5,22Edien-3~-ol IId 24ethyl-5acholest-22E-en-3P-ol Ie 24ethylcholest-5-en-3~-ol 4methylsterols IIIc 4a,24dimethyl-5arholestan-3~-0l IIId 4a-methyl-24ethyl-5a-choIest-22E-en-3P-ol 3,4dihydroxy-4methylsterols IVC 4a,24-dimethyl-5a-cholestan-3P,4P-diol
20.2
3.7
3.3
2.7 35.3
2.5
24.9 15.0
-
-
2.3
9.9
14.8
24.9
3.5 9.3 5.5
7.2 23.3
rn
4a,24-dimethyl-5a-cholest-22E-eri-3P,4P-diol
2.5 -
IVe
4a-methyl-24-ethyl-5a-cholestan-3P,4P-diol
8.9
Othersh
-
a
4.7 0.7
6.4 1.1 1.4 39.6 25.8
-
Structure numbers refer to Figure 1.
’’ Gas chromatograms of the NSL from strain CS375 showed a significant peak eluting after 4a-methyl-24ethyl-5acholest-22E-en-3P-ol in the middle of the sterol region. However, when this sample was reanalyzed by GC a few days later this peak had disappeared and was replaced by a broad lump. Further attempts to derivatize this compound were uiisuccessful and its identity remains unknown.
monounsaturated diol. The mass spectra of the C,, and Cs0 saturated compounds (as the mono TMSi ethers) were very similar (Figs. 2a and 2b). The base peak in each mass spectrum represents the loss of 147 daltons (C,H,O,TMSi) due to fragmentation of the A ring. These compounds were identified as 4a,24P-dimethyl-5a-cholestan-3P,4P-diol (IVc) and 4a-methyl,24~-ethyl-5a-cholestan-3f3,4~-diol (IVe) by comparison with compounds previously isolated from other Pavlova species (Volkman et al. 1990; Patterson et al. 1992, 1993). The mass spectrum of the C,, monounsaturated diol did not closely resemble that of the saturated dihydroxysterols (contrast Fig. 2a and 2b with 2c). The highest ion observed was m/z 484, which we interpret as loss of H,O from a molecular ion of m/z 502, indicating the presence of a single double bond. Further loss of 90 daltons (OTMSi group) from this ion gives an ion at m/z 394, which in turn loses a methyl group to give the base peak at m/z 379. The most likely position for the double bond is at C22-23 in the side chain, as found in the 4 desmethyl and 4methylsterols (Table 1). In previous studies of pavlovols it was noted that the hindered 4hydroxy group could not be derivatized either to the TMSi-ether (Volkman et al. 1990) or to the acetate form (Gladu et al. 1991, V6ron et al. 1996). In our study we tried a number of derivatizing agents and were finally able to derivatize the dihydroxysterols to form the di-TMSi-ethers using the reagent “Power Sil-Prep” following the same method as used for the BSTFA derivatization. The mass spectra of the 2 pavlovols (as di-TMSi-ethers) in D. vlkianum are shown in Figure 3. In the region below m/z 300 these spectra differ greatly from
those produced from the mono-TMSi-ethers and show a strong ion at m/z 117 (CH,OTMSi) from cleavage and loss of the OTMSi group in ring A. Both show an ion at m/z 147 characteristic of TMSiethers containing two hydroxy groups (Eglinton et al. 1969). The similarities in the distributions of the low mass ions in the mass spectra (which are due to ring A cleavage) of both the saturated and monounsaturated C,, diols (Fig. 3) confirm that the double bond is not associated with the AB ring system and hence it is associated with the side chain. The mass spectrum of the monounsaturated C,, compound gave a molecular ion at m/z 574, thus confirming the presence of two hydroxy groups and a single double bond. The base peak of m/z 379 and relative proportions of ions above m/z 300 were remarkably similar in mass spectra of both the mono- and diOTMSi-ether derivatives (compare Fig. 2c with Fig. 3b). Although final structural elucidation requires NMR data, the most likely structure consistent with these mass spectral and GC retention data is 4a,24 dimethyl-5au-cholest-22E-en-3~,4~diol (IVb) . This is also the structure assigned by Wren et al. (1996) to this compound based on interpretation of the mass spectrum of the monoacetate derivative. Fatty acids. Data for total fatty acid compositions are shown in Table 2. Both species had relatively simple fatty acid distributions with four compounds predominating, viz. 20:5(n-3), 16:l (n-7), and 14:O and 18:4(n-3). These distributions are very similar to those previously published for other species of Pavlova (Volkman et al. 1991), but differ significantly from those found in many other haptophytes from other genera (Conte et al. 1994). For example, the unusual 18:5(n-3) polyunsaturated fatty acid
1020
JOHN K. VOLKMAN ET AI,.
100, ’
357
43
117
95 445 156
I
I
147
0
II
MassiCharge
100
200
130
I
I00
400
300
i
95
300
200
576 561
500
600
MassiCharge
371
3
0
I
486
100
%
357 191
379
400
500 %
Mass/Charge
I379 0 100
200
400
300
500
600
Madcharge
0
100
200
300
400
500
FIG. 3. Mass spectra of (a) the C , saturated diol 4a,24pdimethyl-5a-cholestan-3~,4~diol ( N c )as the di-TMSietheI-and (b) the C,, monounsaturated diol4a,24P-dimethyl-5a-cholest-22Eun3P,4pdiol (IVd) as the di-TMSi-ether;both compounds from Diacronema vlkianum.
MassiCharge
FIG. 2. Mass spectra of (a) the CpQsaturated diol 4a,24P-dimethyl-5a-cholestan-3P,4~diol (IVc) as the mono-TMSi-ether from Dincrnnmn iilkinnum (also found in Pavlma pinguis), (b) the C,, saturated diol 4a-methyl-24~-ethyl-5a-cholestan-3P,4P-diol (IVe) as the mono-TMSi-etherfrom Pniiloiia pinguis (CS286),and (c) the C,, monounsaturated diol 4a,24P-dimethyl-5a-cholestTABLE 2. Composition .f major fatty acids in Pavolva pinguis and YLE-eri-3P,4Pdiol (IVd) as the mono-TMSiether from Dincronema Diacronema vlkianum. iilkianum.
Fatty acid’
found in E~ilianzahuxleyi, I m a ~ t o n ~rotunda, u Dicrutoiu inornutu,and other haptophytes was not detected at all in P. pinguis or D. vlkianum. DISCUSSION
Taxonomy. Haptophytes are usually flagellates or have flagellate stages in their life history. The basic cell commonly has two flagella and is characterized by the presence of a haptonema between the flagella (Hibberd 19’16).In most species, elliptical organic scales cover the outside of the cells and in some organisms a layer of inorganic coccoliths is found outside the scales. Christensen (1962) distinguished the Prymnesiophyceae from the Chrysophyceae (see Lee 1980), and Hibberd (1976) suggested
14:0 16:0 16:l (n-7) 1 61(n-5) 18:1(n-9) 18:2(n-6) 18:3(11-3) 18:4(11-3) 2O:5(n-3) 22:5 (n-3) 22:5 (n-6) 223311-3) Others
P. ping-uir
P. pinpis
CS286
cs575
22.3
14.6 14.2 12.6 4.1 0.2 0.8 1.0 8.0 20.9 0.1 11.1 7.8 4.6
5.3 10.3 6.8 0.8 1.1 0.6 11.3 26.1 0.2 2.4
9.7 3.1
D. vlkianum CS266
14.4 8.9 14.9 tr. 1.8 1.1 tr. 13.2 27.1 2.3 2.4
7.7 6.2
”Fatty acids are designated as “total number of carbon atoms: number of double bonds (n-x)” where “x” is the position of the ultimate double-bond from the terminal methyl group. Double bonds in polyunsaturated fatty acids (PUFA) are separated by a methylene group.
DIHYDROXYSTEROL MARKERS IN MICROALGAE
they should be recognized as a separate class. The primary feature distinguishing haptophytes from chrysophytes is that haptophytes generally lack flagellar hairs, although species from the Pavlovales are an exception because fine nontubular hairs are present (Hibberd 1980). The Chrysophyceae have a fairly characteristic pattern of carotenoids, but even so, the distributions are not always sufficiently distinctive to separate the two algal classes (Withers et al. 1981). The taxonomy of the Pavlovales has often been revised (Green and Manton 1970). The genus PavZova was established with the description by Butcher (1952) of the type species Pavlova gyrans. They were named after the Russian ballerina Anna Pavlova because of their gyrating motion. Green (1976) erected the order Pavlovales and the family Pavlovaceae to accomodate related species within the class Prymnesiophyceae (Haptophyta) . Three genera were recognized: Diacronema Prauser, Exarthemachlysis Lepailleur, and Pavlova Butcher. Pavlova luthm‘ was originally named Monochlysis luthm’ (Droop) and was included in the Chrysophyceae, but later electron microscopy studies showed the presence of a haptonema and flagellar organization characteristic of the Prymnesiophyceae (Green 1975). The Pavlovales is a very successful group with r e p resentatives in oceanic, coastal, brackish, and freshwater habitats (Green 1980). Individual species appear to be widely distributed geographically. Pavlova species are small (3-10 pm) flagellates that lack the calcium carbonate coccoliths and unmineralized scales found in many other species of the Haptophyceae. Most Pavlova species thrive in brackish water environments, and only one species is known from a freshwater lake (Green 1980); several species of Pavlova are common in Australian coastal waters (Hallegraeff 1983). Pavlova Zutheri is widely used as an algal food for larval animals in mariculture hatcheries. Lipids as taxonomic markers. The complex mixture of 4desmethylsterols, 4methylsterols, and dihydroxysterols in species of the order Pavlovales (Ballantine et al. 1979, Volkman et al. 1990, Gladu et al. 1991, Patterson et al. 1992, 1993, Vkron et al. 1996) distinguishes the sterol composition of these microalgae from other haptophytes and, indeed, all other classes of microalgae. To date, no species from other orders have been found that contain these unusual dihydroxysterols. Indeed, the occurrence of 3P,4p-dihydroxy steroid systems seems to be exceedingly rare although a trio1 24-methylcholesta-5,24 dien-3P,4P,20Striol containing the same 3P,4P-diol systems was recently reported in the leaves of the plant Dysoxylum malabancum (Govindachari et al. 1997). The function and biosynthetic pathways of such sterols are not known, but the presence of the 4P-hydroxy group may be associated with the removal of one of the original two methyl groups from this position.
1021
4-Desmethyl sterols. The sterol distribution in P. pinp i s is very similar to other Pavlova species and consistent with the observation that the major 4desmethyl sterol in Pavlova species is 24ethylcholesta5,22E-dien-3P-ol (Id) (Volkman et al. 1990, Gladu et al. 1991, Vkron et al. 1996). The stereochemistry of the methyl group at C-24 in this sterol has been assigned as 24p (i.e. poriferasterol) by Gladu et al. (1991) based on melting point and nuclear magnetic resonance (NMR) data. Vkron et al. (1996) have shown that this sterol occurs predominantly as a free sterol and as a steryl glycoside. P. luthm’, P. gyrans, P. salina, and two unidentified Pavlova species also contain 24ethylcholest-5-en-3P-01(Ie) and varying amounts of 24methylcholest-5-en-3P-01(Ic), 24methyl-5a-cholest-22E-en-3P-01 (IIb), and other minor constituents. The predominance of 24ethyl5a-cholest-22E-en-3P-01 (IId) in P. pinguis corresponds to that found in two unnamed Pavlova species analyzed by Volkman et al. (1990). The high abundance of 4desmethylsterols lacking a double bond in the A ring (i.e. 5a-stanols; 11) in P. pinguis and other Pavlova species was also observed in dinoflagellates (Withers 1983), but it is only rarely encountered in other microalgae. Many haptophytes have very simple sterol distributions in which 24methylcholesta-5,22E-dien-3P-ol (Ib) is the major or even sole component (Conte et al. 1994). However, this sterol was not detected in either D. vlkianum or P. pinguis, further highlighting the distinctive sterol composition of these algae. 4-Methyl sterols. Previous studies have identified four different 4methyl sterols in Pavlova species (Volkman et al. 1990). Only two 4methyl sterols were fully identified in this study, although very small amounts of additional minor 4methyl sterols were also present. Volkman et a]. (1990) and Gladu et al. (1991) reported 4cy-methyl-24ethyl-5cy-cholest22E-en-3P-01 (IIId) as the major 4methyl sterol in P. lutheri, P. salina, P. gyrans, and two Pavlova species. The other 4methyl sterols detected in these species of Pavlova are 4a,24dimethyl-5a-cholest-22E-en-3P01 (IIIb), 4a,24dimethyl-5a-cholestan-3P-ol (IIIc), and 4a-methyl-24-ethyl-5cy-cholestan-3P-01 (IIIe) (Volkman et al. 1990, Patterson et al. 1992). Dihydroxysterok. Dihydroxysterols have previously only been detected in haptophyte species of the genus Pavlova. The unusual dihydroxysterols of the Pavlovales were first detected in P. lutheri (as Monochlysis luthen‘) by Ballantine et al. (1979) from a stationary phase culture, but they were not found when the cells were harvested in the exponential growth phase. Two different compounds were observed (labeled as sterols “M-15” and “M-16”). Their exact structure was not deduced, but it was postulated that M-15 was a C,, saturated stanol with an extra sterically hindered hydroxyl group in the nucleus, and that M-16 was a C,,, dihydroxysterol. Volkman et al. (1990) detected three dihydroxysterols, labeled as compounds 13, 14, and 15. Com-
1022
JOHN K. VOLKMAN ET AI..
pounds 13 and 15 were present in P. luthm', P. salinu, and two unnamed Pavlova sp. Compound 14 was only present in significant amounts in P. salina. Comparison of the mass spectral data showed that compounds 13 and 15 were the same as M-15 and M-16 detected by Ballantine et al. (1979). Volkman et al. (1990) showed that these compounds were based on a 3p,4p-dihydroxy-4aL-methyl sterol structure by converting the C,, pavlovol to the known 4 methyl steroid ketone. The P. lutheri strain studied by Volkman et al. (1990) was harvested in exponential growth phase. This species had also been studied by Lin et al. (1982), but these authors did not observe the dihydroxysterols. However, Gladu et al. (1991) detected three dihydroxysterols in P. lutheri and four in P. gyrans and termed them pavlovols. Ethylpavlovol (the C,, saturated compound) was reported as the major dihydroxysterol of P. gyrans, and methylpavlovol (the C,, saturated compound) was the dominant dihydroxysterol of P. lutheri. Patterson et al. (1993) used 400 MHz NMR to complete the structural characterization of the two most abundant saturated pavlovols in P. gyrans and P. lutheri and defined the positioning of the second hydroxyl moiety and stereochemistry of the alkyl group at C-24. Methylpavlovol was shown to be 4a,24~-dimethyl-5a-cholestan-3(3,4~-diol (IVc), and ethylpavlovol was 4a-methyl-24p-ethyl-5a-cholestan3P,4p-diol (Ive), These are structurally similar to the 4methyl sterols, 4a,24dimethylda-cholestan-3~01 (IIIc) and 4a-methyl-24ethylda-cholestan-3p-01 (IIIe), present in these algae. Compound 13 (Volkman et al. 1990) and M-15 (Ballantine et al. 1979) were thus identified as methylpavlovol and compound 15 and M-16 as ethylpavlovol. The identification of the stereochemistry of the methyl group as 4a is as expected because this is the usual stereochemistry found in the 4methyl sterols of microalgae (Withers 1983). C-24 epimers are not separated on the nonpolar columns using gas chromatographic conditions commonly used for sterol analysis, nor are chiral phases of sufficient thermal stability available for sterol analysis, so we were not able to confirm the C-24 stereochemistry in our study. However, the assignment by Patterson et al. (1993) of the stereochemistry of the alkyl group at C-24 as 24p is of interest. This stereochemistry is found in the sterols of many algal groups (Volkman 1986, Gladu et al. 1990, Patterson et al. 1992), although it has been noted that the C,, sterols of centric diatoms have the 24p configuration, and the sterols of pennate diatoms are 24a (Rubinstein and Goad 1974, Gladu et al. 1991). The C-24 stereochemistry of higher plants seems to be mostly 24a, particularly for C,, sterols (Goad and Goodwin 1972). This is also the stereochemistry found in 24amethylcholesta-5,22E-dien-3~-01 (Ib) synthesized by Emiliania huxleyi (Maxwell et al. 1980, Goad et al. 1983) which is also from the Haptophyceae. The oc-
currence of both stereochemistries in microalgae from the same class is unusual and, if confirmed, further distinguishes the unusual sterol pattern of the Pavlovales. Comparison between strains. The availability of two strains of P. pinguis isolated from opposite sides of the earth provided an opportunity to compare their lipid compositions (Tables 1 and 2). The two sterol distributions were remarkably similar despite the fact that different culturing methods had been used. Both species contained the same suite of sterols and the relative abundances were almost identical both within and between each class of sterol (4desmethyl, Cmethyl, and pavlovols) . The fatty acid distributions (Table 2) are also broadly similar apart from enhanced proportions of 16:O and 22:5(n-6) and lower proportion of 20:5(n-3) in strain CS375. Such differences are often noted because the abundance of individual fatty acids can be significantly affected by environmental conditions and growth stage. Geochemical applications. Dihydroxysterols have been detected in the sediments of a saline Antarctic lake, Ace Lake (Volkman et al. 1990), but as yet no alga from the lake has been shown to produce these compounds. This is the only report of these compounds in sediments of which we are aware. This is somewhat surprising since haptophyte algae in general are significant contributors of organic matter to marine sediments. This implies either that species from the Pavlovales are not major sources of organic matter, that the pavlovols have been overlooked in earlier geochemical studies, or that the pavlovols rapidly degrade in sediments. The latter possibility seems quite likely since treatment of the pavlovols with a Lewis acid converts them readily to the steroid ketone (Volkman et al. 1990). Taxonomic signijicance. Previous studies indicated that dihydroxysterols were probably limited to haptophytes belonging to the order Pavlovales. Twelve named species are included in this order (Green 1980), but sterol compositional data are only available for two undescribed species and five named species: P. luthm', P. salina, P. gyrans, P. pinguis, and D. vlkianum. Similar distributions of dihydroxysterols have been detected in all species of the Pavlovales studied to date. Only three steroidal diols were observed in this study despite the greater number of 4methyl and 4desmethylsterols present. Diamonema vlkianum is the first species in the Pavlovales not of the genus Pavlova that has been studied for its sterol content and composition. Although it is impossible to study all species, it appears from our data that pavlovols may only be synthesized by species of the order Pavlovales and hence could be useful chemotaxonomic markers for this order. Furthermore, each species contains a distinctive and restricted distribution of 4a-methyl sterols and 5a-stanols, which is rarely encountered in other microalgae.
DIHYDROXYSTEROL MARKERS IN MICROALGAE We thank Dr. David Lyons (IASOS), Dr. S. W. Jeffrey (CSIRO Division of Marine Research), and colleagues for many useful discussions. Dr. Susan Blackburn (CSIRO Division of Marine Research) is thanked for her advice on algal cultures. The assistance of Suzanne Norwood,Jeannie-Marie LeRoi, and others in the CSIRO algal culture collection laboratory is greatly appreciated. We thank Dr. Malcolm Brown, Malcolm McCausland (culturing),and Richard Knuckey (all CSIRO Division of Marine Research) for provision of the mass culture of CS375 and use of the Alpha Lava1 centrifuge. Dr. Andrew Revill and Graeme Dunstan provided helpful comments on an earlier draft of the paper. Ballantine, J. A,, Lavis, A. & Morris, R. J. 1979. Stelols of the phytoplankton-effects of illumination and growth stage. Phytochemistly 1 81459-66. Bligh, E. G. & Dyer, W. J. 1959. A rapid method of total lipid extraction and put-ification.Can. J. Bzocha. Physiol. 37:911-7. Butcher, R. W. 1952. Contributions to our knowledge of the smaller marine algae. J. Mar. Bid. Assoc. IJ.K. 31:175-91. Chritiennot-Diriet, M.J., Sournia, A,, Ricard, M. & Billard, C. 1993. A classification of the marine phytoplankton of the world from class to genus. Phycologia 32:159-179. Christensen, T. 1962. Alger. I n Biicher, T. W., Lange, M. & Sewensen, T. [Eds.] Botanik, Vol. 2, No. 2. Munksgaard, Copenhagen, pp. 1-178. Conte. M. H.. Volkman. I. K. & Eelinton. G. 1994. Lioid biomarkers of the Haptiphyta. I n Ereen, J. C. & Leadbkater, B. [Eds.] The Haptuphyte Algur. Clarendon Press, Oxford, pp. 351-77. Eglinton, G., Hunneman, D. H. & McCormick, A. 1968. Gas chromatographic-mass spectrometric studies of longchain hydroxy acids-111. The mass spectra of aliphatic hydroxy acids. A facile method of double bond location. Or@ Mass Spectrom. 13595-611. Fournier, R. 0. 1969. Observations on the flagellate Diauonema vlkianum Prauser (Haptophyceae) . Br. Phycol. J. 4:185-90. Gladu, P. K., Patterson, G . W., Wikfors, G. H., Chitwood, D. J. & Lusby, W. R. 1990. The occurrence of brassicasterol and epibrassicasterol in the Chromophycota. Comp. Biochem. Physiol. Y7B491-4. Gladu, P. K., Patterson, G. W., Wikfors, G. H. & Lusby, W. R. 1991. Free and combined sterols of Pavloua gyrans. Lzpids 2 6 6569. Goad, L. J. & Goodwin, T. W. 1972. The biosynthesis of plant sterols. Pmg. Phytochm. 3:113-98. Goad, L. J., Holz, G . G. & Beach, D. H. 1983. Identification of (24S)-24methylcholesta-5,22-dien-3P-ol as the major sterol of a marine cryptophyte and a marine haptophyte. Phytochemistly 22475-6. Govindachari, T. R., Krishna Kumari, G. N. & Suresh, G. 1997. Ergosta-5,24(24’)diene-3P,4P,20Striol, an ergostane steroid from Dysoxylum malaban‘cum. Phytochemistly 44: 153-5. Green, J. C. 1975. The finestructure and taxonomy of the haptophycean flagellate Paulova lutheri (Droop) comb. nov. (= Monochrysis lutheri).J. Mar. Biol. Assoc. U.K. 55:785-93. 1976. Notes on the flagellar apparatus and taxonomy of Paulova mesolychnon Van der Veer, and on the status of Pavlova Butcher and related genera within the Haptophyceae.J. Mar. Biol. Assoc. U.K. 56:595-602. 1980. The fine structure of Pavlova pinguis Green and a preliminary survey of the order Pavlovales (Prymnesiophyceae). Br. Phycol. J. 15:151-91. Green, J. C. & Hibberd, D. J . 1977. The ultrastructure and taxonomy of Diacronema ulkianum (Prymnesiophyceae) with special reference to the haptonema and flagella apparatus. J. Mar. Biol. ASSOC. U.K 57: 1125-36. I
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