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Fatty acid and sterol composition of twoevolutionarily closely related dinoflagellatemorphospecies from cold Scandinavian brackish andfreshwatersJeffrey D. Leblond a , Brian Anderson a , David Kofink a , Ramiro Logares b , KarinRengefors b & Anke Kremp ca Department of Biology , Middle Tennessee State University , P.O. Box 60, Murfreesboro,TN 37132, USAb Department of Ecology , Lund University , Lund, SE-22362, Swedenc Tvärminne Zoological Station, University of Helsinki , 10900 Hanko, FinlandPublished online: 20 Feb 2007.

To cite this article: Jeffrey D. Leblond , Brian Anderson , David Kofink , Ramiro Logares , Karin Rengefors & Anke Kremp(2006) Fatty acid and sterol composition of two evolutionarily closely related dinoflagellate morphospecies from coldScandinavian brackish and freshwaters, European Journal of Phycology, 41:3, 303-311, DOI: 10.1080/09670260600804843

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Eur. J. Phycol., (2006), 41(3): 303–311

Fatty acid and sterol composition of two evolutionarily closely

related dinoflagellate morphospecies from cold Scandinavian

brackish and freshwaters

JEFFREY D. LEBLOND1, BRIAN ANDERSON1, DAVID KOFINK1, RAMIRO LOGARES2,

KARIN RENGEFORS2 AND ANKE KREMP3

1Department of Biology, Middle Tennessee State University, P.O. Box 60, Murfreesboro, TN 37132, USA2Department of Ecology, Lund University, Lund, SE-22362, Sweden3Tvarminne Zoological Station, University of Helsinki, 10900 Hanko, Finland

(Received 10 November 2005; accepted 10 May 2006)

Little is known about the lipid composition of cold-adapted dinoflagellates. Here, we investigate Peridinium aciculiferum and

Scrippsiella hangoei, two cold-adapted dinoflagellate morphospecies that present significant differences in general

morphology, physiology and habitat, but which share identical ribosomal DNA (rDNA) sequences, indicating a recent

evolutionary divergence between them. Despite their present taxonomic names, it is now known that these two genetically

isolated morphospecies are evolutionarily related to the so-called Pfiesteria-like species. We have found that chloroplast-

associated glycolipids from P. aciculiferum and S. hangoei contained a higher relative percentage of two polyunsaturated

C18 fatty acids (octadecapentaenoic [18:5(n-3)] and octadecatetraenoic [18:4(n-3)] acids), and a lower relative percentage of

hexadecanoic acid (16:0) than typically observed in previously examined warm-adapted dinoflagellate species. This suggests

that these two fatty acids provided the requisite membrane fluidity for chloroplasts under a cold growth condition in these two

organisms. A similar increase in 18:5(n-3) and 18:4(n-3) over warm-adapted species was also found in the phospholipids and

triglycerides. P. aciculiferum and S. hangoei were also examined for the production of unique sterols that may serve as

chemical biomarkers. Both were found to have a very similar sterol composition, consisting of two common dinoflagellate

sterols, cholestanol and dinostanol, as dominant sterols, along with a number of other common dinoflagellate sterols as minor

sterols. This demonstrated the absence of a specific steroidal biomarker but, more importantly, showed that these two

morphospecies still produce very similar sterols despite having diverged in several other traits.

Key words: Algae, cold-tolerance, dinoflagellate, Dinophyceae, fatty acid, lipid, Peridinium, Scrippsiella, sterol

Introduction

Polar and cold-temperate marine environmentssupport rich assemblages of microalgae that serveas the nutritional base for higher trophic levels(Garrison et al., 1987; Treguer & Jaques, 1991;Kirst & Wiencke, 1995; Longhurst, 1995; Smithet al., 1999). Dinoflagellates can be among thepredominant types of algae in these marineenvironments, with the most well-studied examplebeing Polarella glacialis Montresor, a speciescommonly found in both Arctic and Antarcticmarine waters (reviewed by Montresor et al.,2003). Likewise, under-ice communities in cold-temperate and Alpine lakes can reach highbiomasses (e.g. Rodhe, 1955), and dinoflagellates

are important components of the winter phyto-plankton community in cold-temperate lakes(Rodhe et al., 1966; Steinberg, 1981; Spauldinget al., 1993; Rengefors, 1998) and brackish waters(Niemi, 1975); their importance as primary produ-cers is becoming increasingly apparent (Lignellet al., 1993; Stoecker et al., 2000). For example,Peridinium aciculiferum Lemmermann regularlyforms dense blooms in ice-covered lakes inSweden (Rengefors, 1998) where it can be foundalong with Woloszynskia ordinata (Skuja)Thompson (Nauwerck, 1963) and Peridiniumeuryceps Rengefors & Meyer, two other cold-adapted forms (Rengefors & Meyer, 1998). Inthe Baltic Sea, cold-adapted dinoflagellates, suchas Scrippsiella hangoei (Schiller) Larsen,Woloszynskia halophila (Biecheler) Elbrachter& Kremp and Peridiniella catenata Levander,regularly occur during winter and spring in

Correspondence to: J. D. Leblond. e-mail: jleblond@mtsu.edu

ISSN 0967-0262 print/ISSN 1469-4433 online/06/030303–9 � 2006 British Phycological Society

DOI: 10.1080/09670260600804843

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sea ice and coastal open waters (Larsen et al., 1995;Kremp et al., 2005) where they may form highbiomass blooms.The extreme environmental conditions in polar

and cold-temperate aquatic habitats during winterare characterized by very low temperatures as wellas low and/or variable irradiance. Consequently,the physiological, ecological, and biochemicaladaptation of cryophilic phytoplankton to theseextreme habitats are of great interest. Because oftheir environmental importance as bloom formers,dinoflagellates have had a rich history of examina-tion of their lipid biochemistry, in order tounderstand their cellular physiology and, morecommonly, to search for steroidal biomarkers thatcan be used to trace these organisms in theenvironment. To date, most studies of dinoflagel-late lipids have centered upon those organisms thatare isolated and grown under warm-temperateconditions around 20�C. Thus, with respect todinoflagellates found only under polar or wintercold-temperate conditions, two key questions arise.Firstly, in order to survive in cold waters (0–4�C),do cold-adapted dinoflagellates possess higherquantities of polyunsaturated fatty acids(PUFAs) than warm-adapted dinoflagellatesto maintain sufficient membrane fluidity? Andsecondly, do cold-adapted dinoflagellates produceunique sterols that can be used as chemicalbiomarkers to track them in the environment?Production of large quantities of PUFAs is

considered an adaptation to cold environments(Thompson, 1989; Suutari et al., 1997), and isthought to be essential for maintaining photo-synthesis at low temperatures (Routaboul et al.,2000). In a recent study of the lipids of Polarellaglacialis, Thomson et al. (2004) found thatapproximately 75% of the fatty acids derivedfrom a total lipid extract were polyunsaturated,with octadecapentaenoic acid [18:5(n-3)] anddocosahexaenoic acid [22:6(n-3)] being thedominant forms. However, dinoflagellates grownunder warmer conditions have also been found toproduce large quantities of these same PUFAs. Forexample, five dinoflagellates grown at 18.5�C werefound to produce between 51.3 and 83.0% PUFAs(average 65.4%), with the dominant formsgenerally being 18:5(n-3) and 22:6(n-3) (Mansouret al., 1999).Similar observations were made in a survey of

sixteen warm-adapted dinoflagellates by Leblond& Chapman (2000). This is not surprising sincePUFAs are often considered necessary for photo-synthesis in eukaryotes, such as Arabidopsis, evenunder a warm temperature (22�C; McConn &Browse, 1998). Yet, because only one cold-adapteddinoflagellate was examined by Thomson et al.(2004), it is not clear whether these particular

PUFAs are indeed more abundant in coldtemperature-grown dinoflagellates. In addition,because only total lipid extracts were examined,it is uncertain whether certain lipid classesbecome more enriched in PUFAs thanothers (e.g. chloroplast-associated glycolipids v.cytoplasmic membrane-associated phospholipids).With respect to the second question regarding

sterol biomarkers, there is evidence from numerousstudies on warm-adapted dinoflagellates for theproduction of dinosterol, a 4�-methyl sterol rarelyfound in other classes of algae (Volkman et al.,1998; Volkman, 2003). Hence, dinosterol is oftenconsidered indicative of the class Dinophyceae asa whole. Dinosterol has been used as a biomarkerto track both recent and ancient dinoflagellateblooms (Volkman, 1986; Volkman et al., 1998;Volkman, 2003). Some groups of dinoflagellates,such as the closely related harmful species, Kareniabrevis (Davis) Hansen & Moestrup, K. mikimotoi(Miyake & Kominami ex Oda) Hansen &Moestrup, and Karlodinium micrum (Leadbeater& Dodge) Larsen, produce more unusual sterolsthat may provide a level of specification beyondthat of dinosterol (Giner et al., 2003; Leblond &Chapman, 2002). With respect to cold-adapteddinoflagellates, only P. glacialis has been examined(Thomson et al., 2004), and has been observed toproduce 27-nor-24-methylcholest-5,22E-dien-3�-olas its principal sterol. This sterol has a limiteddistribution within the class Dinophyceae, beingfound in a small number of other gymnodinoidssuch as Gymnodinium simplex (Lohmann) Kofoid& Swezy and Symbiodinium microadriaticumFreudenthal, and may thus serve as a chemicalbiomarker for these organisms. To date, nothing isknown about the potential presence of sterolbiomarkers in other bloom-forming cold-adapteddinoflagellates.The objectives of this study were threefold.

Firstly, to characterize PUFAs in two cold-adapted dinoflagellates, Peridinium aciculiferum(freshwater) and Scrippsiella hangoei (marine-brackish), with the aim of determining whethercertain lipid classes are more enriched in PUFAsthan those of dinoflagellates grown under warmertemperature conditions. Secondly, to ascertainwhether these two organisms produce uniquesterol biomarkers. And thirdly, since these twodinoflagellates are morphospecies that divergedrecently (perhaps within the past 10,000 years) andshare identical rDNA sequences but are presentlygenetically isolated (as evidenced by AmplifiedFragment Length Polymorphism, AFLP), andhave differences in morphology, physiologyand habitat (freshwater v. marine-brackish;Logares et al., 2006), to assess the evolution ofsterol composition in comparison to other traits.

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Completion of these objectives has advancedour understanding of the lipids of cold-adapteddinoflagellates, and has given new insight into theevolution of sterol composition in organisms thathave diverged recently.

Materials and methods

Cultures and growth conditions

A culture of Peridinium aciculiferum strain PAER-1isolated from a single cyst (Rengefors & Legrand, 2001)was grown in duplicate as cultures A and B in 2 litres ofmodified Woods Hole medium (MWC; Guillard &Lorenzen, 1972) in Fernback flasks. Scrippsiella hangoeiclonal strain SHTV-6 (Kremp et al., 2005) was grown induplicate in 2 litres of f/2 medium (Guillard & Ryther,1962; Guillard, 1975) prepared in 1/6 strength seawater.Cultures were grown and harvested in latelogarithmic phase according to the methodology ofLeblond & Chapman (2000) but at 4�C rather than21�C. Cell concentrations at harvest were approximately7� 104 cellsml�1 for each culture.

Lipid extraction and fractionation

Extraction of lipids from glass fibre filters wasperformed according to a modified Bligh and Dyerextraction (Guckert et al., 1985). The total lipidextracts were separated into five component lipidfractions on columns of activated Unisil silica (1.0 g,100–200 mesh, activated at 120�C, ClarksonChromatography, South Williamsport, PA, USA).The following solvent regime was used to separatelipids according to polarity, with fraction 5 being themost polar, and probably charged, lipids (Leblond &Chapman, 2000): (i) 12ml methylene chloride (sterolesters); (ii) 15ml 5% acetone in methylene chloridewith 0.05% acetic acid (free sterols, tri- anddiacylglycerols, and free fatty acids); (iii) 10ml 20%acetone in methylene chloride (monoacylglycerols);(iv) 45ml acetone (glycolipids, including monogalac-tosyldiacylglycerol, digalactosyldiacylglycerol, and sul-phoquinovosyldiacylglycerol); and (v) 15ml methanolwith 0.1% acetic acid (phospholipids). The roles ofglycolipids, and potentially the phospholipid phos-phatidylglycerol, as components of chloroplastmembranes, and of more polar lipids, includingphospholipids, as components of other cellular mem-branes are discussed by Thompson (1996) andHarwood (1998).

Derivatization and gas chromatography/massspectrometry analysis of fatty acids and sterols

Derivatization of sterols (as their trimethylsilyl ethers)found in the sterol ester and free sterol fractions, and thederivatization of fatty acids (as their fatty acid methylesters, FAMEs) from triglycerides, glycolipids, andphospholipids in fractions 2, 4, and 5, respectively, was

performed according to the methodology utilized byLeblond & Chapman (2000, 2002). Mass spectrometryanalysis was performed on a Finnigan Magnum GC/MS(ThermoFinnigan, Austin, TX, USA) using the condi-tions described in Leblond & Chapman (2000, 2002)with the exception that the final hold temperature was300�C rather than 310�C. Additional separation ofoctadecapentaenoic [18:5(n-3)] and octadecatetraenoic[18:4(n-3)] methyl esters was performed using an Rtx-1MS column rather than an Rtx-5MS column. FAMEdouble bond location was examined by analyzingoxazoline (DMOX) derivatives prepared according tothe procedure of Fay & Richli (1991) under the sameGC/MS conditions as described above. The work of Yuet al. (1989) was also especially useful in interpretationof DMOX data.

Suggested structures for sterols were arrived at bycomparison of the mass spectra of their trimethylsilylether derivatives with those of commercially availablestandards, relative retention times, library entries, and/or published GC/MS values. The works of Jones et al.(1994) and Mansour et al. (1999) were found to beparticularly useful sources of GC/MS values and relativeretention times.

Results

Fatty acid composition of glycolipids andphospholipids

Within the glycolipid fractions of Peridiniumaciculiferum and Scrippsiella hangoei, the vastmajority of the fatty acid content was found inthe form of octadecapentaenoic [18:5(n-3)] andoctadecatetraenoic [18:4(n-3)] acids (Table 1). Bothorganisms possessed 18:5(n-3) at an average ofslightly greater than 50%, and 18:4(n-3) atapproximately 40%.By contrast, the glycolipid fractions in the

sixteen warm-adapted dinoflagellates studied byLeblond & Chapman (2000) possessed 18:5(n-3)and 18:4(n-3) at averages of 33.1� 12.8% and24.0� 15.5%, respectively. Although it isdifficult to assign statistical significance to thiscomparison because of the paucity of differentcold-adapted dinoflagellate genera availablefor lipid characterization (compared to the multi-tude of warm-adapted dinoflagellates available),the apparent increases in 18:5(n-3) and 18:4(n-3)were mirrored by a decrease in the relativepercentage of hexadecanoic acid (16:0, Table 1)relative to warm-adapted dinoflagellates. Theaverage relative percentage of 16:0 in the twocold-adapted dinoflagellates was approximately3%, whereas warm-adapted dinoflagellates hadan average of 16.1� 7.7%. Conversely, there wasevidence for a lower relative percentage of the C22

PUFA, docosahexaenoic acid [22:6(n-3)], in thecold-adapted dinoflagellates. Its average withinthe glycolipid fraction of the sixteen warm-adapted

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dinoflagellates was 5.4� 2.8% (Leblond &Chapman, 2000), whereas in the cold-adapteddinoflagellates it was approximately 1%.The phospholipid fraction of these cold-adapted

dinoflagellates also appeared to be more enrichedin 18:5(n-3) than the corresponding fraction inwarm-adapted dinoflagellates. 18:5(n-3) was foundin P. aciculiferum and S. hangoei at averages of15.7 and 9.6% (Table 2), respectively, whereas inthe warm-adapted dinoflagellates examined byLeblond & Chapman (2000) it was found at2.1� 3.2%. In P. aciculiferum and S. hangoei,18:4(n-3) was found at averages of 2.5 and 5.8%,respectively (Table 2), whereas in warm-adapteddinoflagellates it was found at 4.3� 3.0%. Withinthe triglyceride fraction, the most abundant fatty

acids were 18:5(n-3) and 18:4(n-3), with 18:4(n-3)being slightly more abundant (Table 3).Although not reported by Leblond & Chapman(2000), 18:5(n-3) and 18:4(n-3) were found inwarm-adapted dinoflagellates at lower averagesof 6.9� 6.4 and 6.7� 5.7%, respectively.The relative percentages of other fatty acidsassociated with phospholipids and triglycerides,and comparisons to their presence in warm-adapted dinoflagellates, are shown in Tables 2and 3, respectively.

Sterol composition

In Peridinium aciculiferum, approximately 97% ofthe sterols were found as free sterols in fraction 2,

Table 1. Relative abundance (in % of total peak area) of selected fatty acid methyl esters (FAMEs) derived from glycolipids

(fraction 4) of cold-adapted dinoflagellates.

Peridinium aciculiferum Scrippsiella hangoei

Warm-adapted

FAME RT PAER-1-A PAER-1-B SHTV-6-A SHTV-6-B dinoflagellatesb

14:0 19:15 0.6 0.3 1.4 3.3 2.5� 3.4

16:0 26:31 2.9 2.1 2.4 4.3 16.1� 7.7

18:5(n-3) 34:26a 55.5 46.9 58.6 45.1 33.1� 12.8

18:4(n-3) 34:26a 35.0 48.5 35.4 44.1 24.0� 15.5

18:0 35:50 ND ND Tr Tr 1.5� 1.8

20:5(n-3) 41:53 3.3 Tr Tr ND 6.3� 9.0

22:6(n-3) 47:38 0.8 1.2 0.8 1.9 5.4� 2.8

Others 1.5 1.4 0.7

aRelative percentage of 18:5(n-3) v. 18:4(n-3) was determined by injection on an Rtx-1MS column; these two FAMEs coeluted on an Rtx-5MS

column used to separate all other FAMEs.bAverage values from warm-adapted dinoflagellates examined by Leblond & Chapman (2000).

Abbreviations: RT: retention time in minutes; Tr: Trace FAMEs (less than 0.1% of the total peak area); ND: Not detected.

Table 2. Relative abundance (in % of total peak area) of selected fatty acid methyl esters (FAMEs) derived from

phospholipids (fraction 5) of cold-adapted dinoflagellates.

Peridinium aciculiferum Scrippsiella hangoei

Warm-adapted

FAME RT PAER-1-A PAER-1-B SHTV-6-A SHTV-6-B dinoflagellatesb

14:0 19:15 14.5 5.8 21.8 11.9 1.82� 1.83

16:1(n-7) 25:31 1.9 1.4 1.2 ND 1.4� 1.4

16:1(undetermined) 26:05 3.1 1.8 3.9 2.1 0.6� 0.4

16:0 26:31 8.6 5.9 20.0 23.1 30.5� 6.7

18:5(n-3) 34:26a 10.6 20.7 12.3 6.9 2.1� 3.2

18:4(n-3) 34:26a 5.0 ND 2.9 8.6 4.3� 3.0

18:2(n-6) 34:08 1.4 1.2 2.1 1.6 1.4� 1.7

18:1(n-9) 34:30 ND 2.9 5.5 5.2 3.9� 1.7

18:1(n-8) 34:45 ND 0.6 2.3 6.1 ND

18:1(n-3) 35:22 4.5 ND 3.5 6.0 0

18:0 35:50 Tr ND Tr ND 3.7� 1.8

20:5(n-3) 41:53 5.0 7.8 2.2 3.6 1.5� 1.1

22:6(n-3) 47:38 44.8 51.9 20.4 25.1 27.2� 13.5

Others 0.6 1.9

a,b See Table 1. Abbreviations as Table 1.

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whereas the sterols in Scrippsiella hangoei wereapproximately evenly distributed between freesterols in fraction 2 and sterol esters in fraction 1.Overall, there were 11 sterols and 2 steroidalketones produced among these two strains, withthe largest number of compounds being C28 andC30 compounds (Table 4).The free sterol compositions of Peridinium

aciculiferum and Scrippsiella hangoei were foundto be very similar, with little to distinguish thesestrains from each other. In general, they weredominated by high relative percentages of thecommon dinoflagellate sterols, 5�-cholestan-3�-ol(cholestanol, compound 1), 4�,23,24-trimethyl-5�-cholest-7-en-3�-ol (compound 12), and 4�,23,24-trimethyl-5�-cholestan-3�-ol (dinostanol, com-pound 13) (Table 5). Both organisms also containedslightly lower relative percentages of the common

dinoflagellate sterols, 24-methyl-5�-cholestan-3�-ol (compound 4), 4�,24-dimethyl-5�-cholestan-3�-ol (compound 7), and 4�,23,24-trimethyl-5�-chol-est-22E-en-3�-ol (dinosterol, compound 9).The sterol ester compositions generally mirrored

the free sterols (Table 5). Small amounts of thesteroidal ketones, 4�,24-dimethyl-5�-cholestan-3-one (compound 6) and an unidentified C30:1

steroidal ketone (compound 11) were found in asingle replicate of SHTV-6.

Discussion

Our study shows that the cold-adapted dinoflagel-lates tested contained higher relative percentages ofcertain PUFAs, particularly polyunsaturated C18

forms in glycolipids, than the warm-adapteddinoflagellates examined by Leblond & Chapman

Table 3. Relative abundance (in % of total peak area) of selected fatty acid methyl esters (FAMEs) derived from triglycerides

(fraction 2) of cold-adapted dinoflagellates.

Peridinium aciculiferum Scrippsiella hangoei

Warm-adapted

FAME RT PAER-1-A PAER-1-B SHTV-6-A SHTV-6-B dinoflagellatesb

14:0 19:15 11.8 0.6 26.4 6.1 4.3� 3.1

16:1(n-7) 25:31 0.9 0.5 ND ND ND

16:0 26:31 13.7 8.4 20.1 16.5 28.2� 11.8

18:5(n-3) 34:26a 12.2 11.3 18.3 21.8 6.9� 6.4

18:4(n-3) 34:26a 13.3 23.5 15.4 26.6 6.7� 5.7

18:2(n-6) 34:08 13.6 15.4 14.7 20.7 4.0� 3.3

18:1(n-3) 35:22 11.1 14.7 ND ND 0.7� 1.2

18:3(n-6) 35:35 14.5 11.0 ND ND ND

18:0 35:50 2.9 1.3 5.2 8.4 3.8� 2.4

20:5(n-3) 41:53 ND 0.6 ND ND 2.5� 3.1

22:6(n-3) 47:38 6 12.7 ND ND 12.3� 7.0

Others

a,b See Table 1. Abbreviations as Table 1.

Table 4. Sterols and steroidal ketones found in this survey of cold-adapted dinoflagellate lipids.

Carbon no. Compound no. Suggested structure MWa RT RRTb

C27 1 5�-Cholestan-3�-ol (cholestanol) 460 32.98 1.03

C28 2 4�-Methyl-5�-cholest-7-en-3�-ol 472 34.77 1.25

3 4�-Methyl-5�-cholestan-3�-ol 474 35.10 1.29

4 24-Methyl-5�-cholestan-3�-ol 474 35.45 1.33

5 24-Methyl-5�-cholest-7-en-3�-ol 472 36.87 1.50

6 4�,24-Dimethyl-5�-cholestan-3-one 414c 37.83 1.62

C29 7 4�,24-Dimethyl-5�-cholestan-3�-ol 488 38.30 1.68

C30 8 Unidentified C30:2 sterol 498 38.88 1.75

9 4�,23,24-Trimethyl-5�-cholest-22E-en-3�-ol (dinosterol) 500 39.22 1.79

10 Unidentified C30:1 sterol 500 39.85 1.87

11 Unidentified C30:1 steroidal ketone 426c 40.20 1.91

12 4�,23,24-Trimethyl-5�-cholest-7-en-3�-ol 500 40.92 2.00

13 4�,23,24-Trimethyl-5�-cholestan-3�-ol (dinostanol) 502 41.80 2.11

aMW of sterols as their TMS ether derivatives.bRelative retention time with respect to cholesterol calculated according to Jones et al. (1994) on an Rtx-5MS capillary column.cMolecular weight of underivatized steroidal ketone.

Abbreviations: MW: molecular weight; RT: retention time in minutes; RRT: relative retention time.

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(2000). These results suggest that there is a generaldifference in the fatty acid composition betweencold- and warm-adapted species, as we hypothe-sized. Although warm-adapted species producelarge quantities of PUFAs, the production ofPUFAs by P. aciculiferum and S. hangoei appearsto be even higher. It should also be recognizedthat these are general trends and do not excludeplasticity within other dinoflagellate species whichcan grow under wider temperature ranges. Forexample, Parrish et al. (1993) observed thatGymnodinium (formerly Gyrodinium) aureolum(Hulburt) Hansen grown at 18�C produced more18:5(n-3) and less 22:6(n-3) than when grownat 13�C.Our description of the fatty acid compositions of

Peridinium aciculiferum and Scrippsiella hangoeiconfirm the initial findings of high levels of PUFAsin Polarella glacialis, the only cold-adapted dino-flagellate to have its lipid composition described todate (Thomson et al., 2004). Incidentally, Polarellaglacialis (grown in full-strength f/2 medium),Peridinium aciculiferum (grown in a freshwatermedium) and S. hangoei (grown in reduced salinityf/2 medium) all had similar high concentrationsof PUFAs. Since Polarella glacialis is distantlyrelated to the other two species, our results suggestthat temperature, rather than genetic similarityappeared to be the driving factor in PUFAproduction. A similar cold- v. warm-adaptedalgae comparison was conducted by De Angeliset al. (2005), who found that PUFAs were moreprevalent under cold conditions. However, ourresults also extend the findings of Thomson et al.(2004) by detailing how the PUFA composition ofglycolipid, phospholipid, and triglyceride classes

compares to dinoflagellates grown in warmerwater. In particular, for the lipid classes examined,Peridinium aciculiferum and S. hangoei had ahigher percentage of C18 PUFAs than thewarm-water species, but not of 22:6(n-3). Thisincrease in C18 PUFAs appeared to come at theexpense of the C16 saturated fatty acid. Basedon these results, it appears that C18 PUFAsprovide the most optimal membrane fluidity atthe colder growth temperature of 4�C. Productionof these PUFAs can thus be considered to be amembrane fluidity adaptation.Further studies are needed to understand better

the enzymology and genetics behind fatty acidsynthesis as a function of growth temperature andother environmental variables. Currently, this typeof understanding in the field of dinoflagellatelipid biochemistry lags behind that of plants andcyanobacteria (e.g. studies by Sakamoto et al.,1997; Saruwatari et al., 1999).Future studies on the production of PUFAs

should also begin to migrate into the realm oflipidomics, a new approach to lipid analysis usingliquid chromatograph/mass spectrometry/massspectrometry (LC/MS/MS) that will potentiallyallow assessments of which glycolipid and phos-pholipid species are associated with particular fattyacids (Welti et al., 2002; Han & Gross, 2003). Forexample, one may determine which particularglycolipid is most abundant in 18:5(n-3). Suchstudies will represent a great leap beyond currentmethodology in dinoflagellate lipid biochemistry,and will significantly advance our understanding ofdinoflagellate lipids.With respect to sterol composition, both

Peridinium aciculiferum and Scrippsiella hangoei

Table 5. Relative percentage distribution of sterols and steroidal ketones found in this survey of cold-adapted

dinoflagellate lipidsa.

Compound no.

Peridinium aciculiferum Scrippsiella hangoei

PAER-1-A PAER-1-B SHTV-6-A SHTV-6-B

1 14.7 (37.2) 33.2(25.4) 18.4 (9.6) 31.1 (9.4)

2 ND ND Tr ND

3 ND ND Tr ND

4 Tr 1.1 6.9 7.5 (5.3)

5 ND ND 2.2 (5.3) 3.0

6 ND ND (Tr) ND

7 2.3 1.9 4.3 (9.6) 4.4 (6.4)

8 2.3 2.3 ND ND

9 15.4 8.6 (10.3) 6.7 (11.0) 5.5 (11.6)

10 Tr Tr Tr Tr

11 ND ND (1.8) ND

12 10.8 9.0 (18.2) 24.4 (15.9) 18.1 (22.6)

13 53.0 (62.8) 44.0 (46.1) 35.4 (46.9) 29.6 (44.6)

a Relative percentage of sterols found as free sterols in fraction 2 are shown without parentheses.

Relative percentage of sterols found as sterol esters, and of steroidal ketones, in fraction 1 are shown in parentheses.

Abbreviations as in Table 1.

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were found to be very similar. Peridinium aciculi-ferum and S. hangoei have diverged recently, as isshown by their identical nuclear ribosomal DNAand very similar cytochrome bmitochondrial DNAsequences (Logares et al., 2006). However, bothspecies are presently genetically isolated (as indi-cated by AFLP), and present clear differences ingeneral morphology, physiology and habitat(Logares et al., 2006). Thus, the lack of geneticinterchange, together with the phenotypic differ-ences, indicate that the morphospecies can beconsidered as two different evolutionary lineagesor true species. Logares et al. (2006) suggest thatthe divergence between P. aciculiferum and S.hangoei was promoted by the different selectivepressures associated with marine/brackish andfreshwater habitats, which started to operate aftera relatively recent marine–freshwater transitionundergone by a common ancestor. Natural selec-tion can cause extremely fast divergences and,when this is the case, a few traits under selectionare expected to change quickly in comparison withseveral others that stay more or less the same (Orr& Smith, 1998; Lee & Bell 1999). Thus, theproduction of similar sterols in P. aciculiferumand S. hangoei suggests that no DNA involved insterol production has been under selectionduring the divergence of both species. AlthoughP. aciculiferum and S. hangoei produced similarfatty acid profiles in the three lipid classesexamined, fatty acid composition is not a reliabletool for assessing dinoflagellate relationshipsbecause most dinoflagellates possess essentiallythe same fatty acids in varying amounts; this is incontrast to a broad diversity of approximately onehundred different sterols that are very unevenlydistributed among dinoflagellates and thus serve asuseful chemical fingerprints (J. Leblond, unpub-lished data). In phylogenetic trees based on SSUrDNA and cytochrome b mitochondrial DNA,P. aciculiferum and S. hangoei cluster with thePfiesteria-like species, being less related to otherScrippsiella species and unrelated to freshwatermonophyletic Peridinium species (Logares et al.,2006).Although compounds 1, 12, and 13 are found in

other dinoflagellates, their presence together as thedominant sterols in P. aciculiferum and S. hangoeiis somewhat unique. The presence of compound 1as a dominant sterol in dinoflagellates is fairly rare(Leblond & Chapman, 2002), although Mansouret al. (1999) found it represented 24% of the sterolsin Scrippsiella sp. CS-295/c. The presence ofcompound 13 as a dominant sterol appears to belimited mainly to a selection of dinoflagellategenera, including Gymnodinium, Gyrodinium,Heterocapsa, Pfiesteria, Prorocentrum, andScrippsiella (Harvey et al., 1988; Piretti et al.,

1997; Mansour et al., 1999; Leblond & Chapman,2002, 2004). Compound 12 has also been found asa dominant sterol among these genera (Nicholset al., 1984; Mansour et al., 1999; Leblond &Chapman, 2002). It thus appears that neitherP. aciculiferum nor S. hangoei possesses candidatesfor specific sterol biomarkers due to their phylo-genetic closeness to the genera Pfiesteria.To conclude, our characterization of the lipids of

P. aciculiferum and S. hangoei showed that bothappear to possess higher relative abundancesof particular PUFAs, primarily 18:5(n-3) and18:4(n-3), than warm-adapted dinoflagellates.In addition, although P. aciculiferum andS. hangoei do not produce unique biomarkersterols individually, they do share an almostidentical and unique sterol composition. This isin agreement with their close evolutionary relation-ship, and indicates that sterol production has notbeen under the same selective pressures that seemto have promoted the divergence between the twomorphospecies in several other traits.

Acknowledgements

Funding to JDL provided by a Middle Tennessee

State University FRCAC 2005 grant is gratefully

acknowledged. The Swedish Research Council

provided a grant to KR. We thank K. Gutseit for

comments on early versions of the manuscript.

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