Phycoiogicai Research 1998; 46: 21-28

Chemotaxonomy of planktonic cyanobacteria based onnon-polar and 3-hydroxy fatty acid composition

Renhui Li,' Akira Yokota,' Junta Sugiyama, Masayuki Watanabe/ Mikiya Hiroki' and Makoto M. Watanabe-"^'Institute of Biological Sciences, University of Tsukuba, Tennondai 2-1-1, Tsukuba, Ibaraki 305, Japan, Institute ofMolecular and Cellular Biology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan, 'Department of Botany,National Science Museum, 4-1-1 Amokubo, Tsukuba, Ibaraki 305, Japan and "National Institute for EnvironmentalStudies, 16-2 Onogawa, Tsukuba, Ibaraki 305, Japan

SUMMARY

Twenty-eight axenio planktonic cyanobacterial strains(10 Microcystis, three Osciiiatoria, one Spiruiina, oneAphanizomenon, 13 Anabaena) were investigated fortheir fatty acid composition by measurement of non-polar and hydroxy fatty acids. No 2-hydroxy fatty acidswere detected in any strain, but 3-hydroxy fatty acidswere detected in minor quantities in 24 strains. Thehighest portion of total fatty acids were non-polar fattyacids. Qualitative and quantitative analyses of 3-hy-droxy fatty acids showed no taxonomic value in thesestrains, while the type of non-polar fatty acid compo-sition was shown to be consistent within Microcystisand Anabaena strains, distinguishing them as type 4,characterized by the presence of 18:4, and type 2,characterized by 18;3 (a) of the Kenyon-Murata sys-tem. Two Osciiiatoria agardhii Gomont strains were alsoincluded in the type 2 group due to the presence of 18:3 (a), but the difference in characteristics of 16:2 and16:3 between 0. agardhii and Anabaena turther dmdetitype 2 into two subgroups: type 2A for Anabaena andtype 2B for 0. agardhii. A simplified unweighted pairgroup method with arithmetic averages (UPGMA) den-drogram demonstrated that the classification of 28strains {Microcystis spp., Anabaena spp,, Aphanizo-menon fios-aquae (Lemmermann) Ralfs f. graciie (Lem-mermann) Elenkin, 0. agardhii and SpiruiinasubsaisaOersted ex Gomont based on numerical analysis of non-polar fatty acids corresponded to morphological speciescriteria, suggesting that non-polar fatty acid composi-tion is a valuable chemical marker in the taxonomy ofplanktonic cyanobacteria. However, the fatty acid com-position in Csciiiatoria raciborskii is similar to that ofMicrocystis and very different from that of 0. agardhii,suggesting its special position in Osciiiatoria and thechemical diversity in the genus Osciiiatoria.

Key words: 3-hycJroxy fatty acids, chemotaxonomy,fatty acid, non-polar fatty acids, planktonic cyano-bacteria.

INTRODUCTION

Freshwater planktonic cyanobacteria such as Anabae-na, Aphanizomenon, Microcystis, Osciiiatoria and Spi-ruiina, occur abundantly and sometimes form waterblooms in eutrophic lakes, reservoirs and ponds. Mostof them possess gas vacuoies, and some species pro-duce a toxic substance such as microcystin, anatoxinand saxitoxin (Carmichael 1980; Skulberg etal. 1993),while some excrete geosmin-like odor substances thatcause environmentai problems (Juttner 1987). Howev-er, the taxonomy of these groups is still in an unsatis-factory state. For example, in the genus Microcystis,which is the most common genus in water blooms, col-ony form, sheath and cell size are mainly used as tax-onomic criteria (Komarek 1958), These characters,however, show marked variation such as disappearanceof the colonial form and variation in cell size due toculture conditions (Stainer ef a/. 1971; Eloff and Wes-thuizen 1981; Kruger etai. 1981; Scott 1986), In thefilamentous genus Anabaena, typical in water blooms,the taxonomic criteria of the trichome form and char-acteristics of the vegetative cell, heterocyst and akineteare also open to doubt due to their occasional absenceor phenotypic changes under different environmentalconditions (Li et ai. 1997), Since cyanobacteria sharethe same prokaryotic properties as bacteria and a com-prehensive set of characteristics have been used in thetaxonomy of bacteria, a modern approach to taxonomyof cyanobacteria strongiy suggests that not only mor-phological characters, but also physiological, chemicaland genetic features should be investigated (Castenholzand Waterbury 1989).

Fatty acid composition is a useful analytical tool inbacterial taxonomy (Welch 1991; Embley and Wait1994). In the cyanobacteria, Holton etai. (1968) dem-onstrated a significant correlation between the morpho-logicai complexity of species and their fatty acid com-position. Kenyon (1972) and Kenyon et ai. (1972)

'To whom correspondence should be addressed.Communicating editor: S. Hino.Received 10 June 1997; accepted 23 October 1997.

22 R. Li etai

showed four types of fatty acid composition based onthe analysis of 66 axenic strains of cyanobacteria.These findings \Nere later confirmed by Murata et ai.(1992) in a study of glycolipid metabolism and theproperties of unsaturated fatty acids in cyanobacteria.Cohen e^ ai. (1995) examined the fatty acid composi-tion of many strains of Spiruiina for biotechnologicalexploitation, and showed the existence of a fifth type offatty acid composition in cyanobacteria, suggestingmodification of the Kenyon-Murata classification sys-tem. Caudales and Wells (1992) investigated the cel-lular fatty acids of free-living, nitrogen-fixing cyanobac-teria belonging to Anabaena and Ncstoc. They showeda significant difference between these two genera andconcluded that the fatty acid composition and relativepercentages of individual components in each genuscould reflect evolutionary changes and metaboiic andbiochemical processes in cyanobacteria. Kruger et ai.(1995) evaluated the taxonomic importance of fattyacid composition at the genus and subgenus level byanalysing the fatty acid composition of different Micro-cystis isolates and other members of the order Chroo-coccales. However, information about the fatty acidcomposition of other planktonic cyanobacteria such asplanktonic Anabaena, Osciiiatoria and Aphanizomenonis very limited. Moreover, the above studies were basedon total cellular fatty acid composition. In bacterialstudies, some minor fatty acid components such as the2-hydroxy and 3-hydroxy ones have been noted for theirchemotaxonomic value (Oyaizu and Komagata 1983;Yokota 1989). In this paper, we examined 28 strains ofplanktonic cyanobacteria including Microcystis, Oscii-iatoria, Spiruiina, Anabaena and Aphanizomenon by an-alysing their non-polar and 3-hydroxy fatty acid com-position. The value of these two kinds of fatty acids incyanobacterial taxonomy shown by relating fatty acidcomposition to morphological classification is dis-cussed.

MATERIALS AND METHODS

Organisms and culture conditionsAxenic strains of 28 planktonic cyanobacteria, includ-ing 10 Microcystis and 13 Anabaena strains from dif-ferent culture collections (NIES, TAO, CCAP), wereused in the present experiments {Table 1). Some Ana-baena strains have been re-identified after the akineteinduction according to the method developed by Li efai. (1997), Microcystis strains were cultivated in MAmedium, the others in CT medium (Watanabe and Hi-roki 1997). The cultures were grown in 3 L medium in5 L flasks equipped with aeration using a 0,22 [j.m fil-ter. All cultures were grown at 20X under a 12:12 L:Dh cycle with a photon irradiance of 40 ^jimol-m-^-sec-'provided by daylight fluorescent lamps. When all cul-tures had grown for 4 weeks, cells were harvested by

centrifugation (1000 g, 10 min), washed twice with dis-tilled water and subsequently freeze-dried for 5 days.

Fatty acids analysisApproximately 50 mg of lyophilized cells was trans-methylated with 5% HCI-Methanol Reagent 10 (TCI-SU, Japan) at 100°0 for 3 h. The methylated acids wereextracted with 1.5 mL hexane, washed with 0.5 mL dis-tilled water, and then concentrated to a volume of 100|i.L by nitrogen gas or by vacuum evaporation. The con-centrated methylated acids were fractioned by thin-lay-er chromatography on a silica gel plate (25 TLC plates20 X 20 cm Silica Gel 60 F^r^^, Merck, Germany) withhexane/diethyl ether (50:50 by volume) (Fig. 1). Non-polar fatty acid methyl esters and hydroxy fatty acidmethyl esters were separated and stained under UV byspraying with 0.02% dichlorofluorescein (Sigma, USA)in ethanol solution. Individual stained spots were re-moved, resolved in 2 mL hexane and concentrated to50 (xL, A 1 |i.L portion of concentrated sample was in-jected into a Shimadzu GC-17A gas chromatographequipped with a hydrogen flame ionization detector anda capillary column (25 m x 0,25 mm x 0.25 |i.m)coated with methyl silicone as a polar stationary phase(007-FFAP, Tokyo Kasei). The fiow of helium gas was40 mL min-^ and the column temperature was pro-grammed to increase from 130X to 220°C at 5°C min '.The methyl esters were identified by cochromatographyof authentic standards (Sigma, USA) and by mass spec-troscopy. The relative concentrations of fatty acids werecalculated by comparing the areas under the chromato-graphic peaks using a data processor (0-R 6 A, Shi-madzu). The quantitative fatty acid data were analysednumerically using the NTSYS-pc program package (Ap-plied Biostatistics Inc., Setanket, NY, USA), and resultspresented as dendrograms drawn using the unweightedpair group method with the arithmetic averages(UPGMA) clustering method.

RESULTS

3-Hydroxy fatty acid compositionNo 2-hydroxy fatty acid was detected in any strain, but3-hydroxy fatty acids were detected in 24 strains whichshowed bright fluorescence in TLC after spraying. Table2 summarizes the 3-hydroxy fatty acid composition ofthese strains, which included 12:0, 14:0,15:0, 16:0,18:0 (saturated) and 15:1, 16:1, 16:3 and 18:1 (un-saturated). The most widely distributed componentswere 14:0, 16:0 and 16:3 which existed in all 24strains with 3-hydroxy fatty acids. However, other com-ponents such as 12:0, 18:0 and 18:1 differed with re-spect to presence or absence within these strains, evenwithin the same genus (e.g. in Microcystis and Anabae-na). The values for all components of 3-hydroxy fattyacids varied largely with different strains. A simplified

Chemotaxonomy of planktonic cyanobacteria 23

Table 1. Strains of cyanobacteria used in the present study

Species Strain Source

Microcystis viridls{(\. Brown) Lemmermann

Microcystis aeruginosa (Kutzing) Lemmermann f. aeruginosa Elenkin

M. aeruginosa t. aeruginosa

M. aeruginosa t. aeruginosa

M. aeruginosa \. aeruginosa

Microcystis aeruginosa {Ku^z'mg) Lemmermann f. flos-aquae {W\nrocW) Elenkin

Microcystis wesenbergii Komarek

M. wesenbergii

M. wesenbergii

M. wesenbergii

Anabaena affinis Lemmermann

A. crassa (Lemmermann) Komarkova-Legenerova et Cronberg

A. crassa

A. crassa

A. tycra/f7/ca (Schkorb) M. Watanabe

A. ucrainica

A. soiitaria Klebahan

A. soiitaria

A. sm/f/7/7 (Komarek) M, Vi/atanabe

A. planktonica Brunnthaher

A. planktonica

A. flos-aquae Brebtsson ex Bornet et Flahault

A. flos-aquae

Aphanizomenon flos-aquae (Lemmermann) Ralfs f. graciie (Lemmermann)

Osciiiatoria agardhii Gomont

0. agardhii

0. raciborskii V '̂olosynska

Spiruiina subsalsa Oersted ex Gomont

NIES-102NIES-44

NIES-87

NIES-89

NIES-298

NIES-98

NIES-104

NIES-Ul

NIES-112

NIES-604

NIES-40

NIES-76

NIES-79

TAC436

TAC448

TAC449

NIES-78

NIES-80

TAC432

TAC421

INBA-5

CCAP

CCAP

NIES^81

NIES-204

NIES-205

NIES-207

NIES-27

NIESNIES

NIES

NIES

NIES

NIES

NIES

NIES

NIES

NIES

NiES

NIES

NIES

NSM

NSM

NSM

NIES

NIES

NSM

NSM

NIES

CCAP

CCAP

NiES

NIES

NiES

NiES

NIES

NIES, Microbia Culture Collection, National Institute for Environmentai Studies, Tsukuba, Japan; NSM, National Science Museum,

Tsukuba, Japan; CCAP, Culture Collection of Algae and Protozoa, Windermere, tJK.

UPGMA dendrogram showed that 3-hydroxy fatty acidcomposition did not correspond with morphological cri-teria at the taxonomic levels of order, family, genus orspecies (Fig. 2).

Non-polar fatty acid compositionAll 28 strains showed very bright fluorescence blots inTLC which represented a high concentration of non-po-lar fatty acids (Fig. 1). Non-polar fatty acids occupied95% to 98% of total fatty acids. Their composition andrelative concentration are summarized in Table 3. Allstrains included 14:0, 16:0, 16:1 (cis), 18:0, 18:1,18:2 and 18:3 (a), apart from the absence of the lastfrom Spiruiina subsaisa Oersted ex Gomont, Coccoidcyanobacteria [Microcystis spp.) contained 18:3 (7)and 18:4, but lack 16:2 and 16:3. All strains of Ana-baena, Aphanizomenon and Osciiiatoria agardhii Go-mont contained 16:2 and lacked 18:3 (7) and 18:4.However, Ariabaena and Aphanizomenon strains con-tained 16:1 (cis-), 16:2 and 16:3, while 0. agardhiilacked 16:3. Spiruiina subsaisa contained 1.8% 18:3(•y) instead of 18:3 (a). Unexpectedly, the fatty acid

composition of Osciiiatoria raciborskii Woloszynska(NIFS-207) was similar to that of Microcystis, whichcontained 18:3 (7) and 18:4 but neither 16:2 nor 16:3, a situation markedly different from the two strains of0. agardhii (NIES-204, NIES-205). Quantitatively, thelargest single component was 16:0; the values of whichwere higher for Microcystis spp., 0. raciborskii and S.subsaisa (51.7-60,2%), and lower for Anabaena,Aphanizomenon and 0. agardhii strains (30,5% to41.9). Conversely, the former contained less 18:3 (a)(0-19.3%) than the latter (24.0-45.8%).

A simplified UPGMA dendrogram demonstrated twomajor clusters: cluster 1 (unicellular) and cluster 2 (fil-amentous) (Fig, 3). The classification of 13 Anabaenastrains and one Aphanizomenon strain based on the nu-merical analysis of non-polar fatty acids correspondedto morphological species criteria. This is also true forthe Microcystis strains except for Microcystis aerugi-nosa {Kutzing) Lemmermann f, aeruginosa (Wittrock)Elenkin (NIES-44). Osciiiatoria agardhii and S. subsai-sa formed groups different from each other and fromthe heterocystous cyanobacteria, Anabaena ar\6 Aphan-

24 R. Li etal.

iNonpolar

3-OH

Nonpolar 2-OH 3-OH Sample

Standard

Fig. 1. Sketch of separation of non-polar fatty acids and hydroxy

fatty acids. Non-polar, 3-OH and 2-OH represent non-polar, 3-

hydroxy and 2-hydroxy fatty acids.

izomenon and from the coccoid cyanobacteria, Micro-cystis. However, 0. raciborsk 11 was included in the clus-ter for Microcystis.

DISCUSSION

Analysis of cellular fatty acids including non-polar, 2-hydroxy and 3-hydroxy acids has proved to be very use-ful in identification of bacteria, particularly gram-neg-ative bacteria (Dees ef al. 1981; Moss 1981). Somereports on the use of fatty acid analysis for classificationof cyanobacteria have been published (Caudales andWells 1992; Krijger ef al. 1995). However, almost allresults of fatty acid composition in cyanobacteria byformer investigators neglected the composition of hy-droxy fatty acids because of their minor contents. Forinstance, hydroxy fatty acids in relation to total fattyacids in Anabaena and Nostoc were about 0.29% and0.27%, respectively (Caudales and Wells 1992). How-ever, it has been suggested that extensive taxonomicinformation may be obtained from abundance and vari-ations in some minor biochemical components of mi-croorganisms (Jones ef al. 1994). So, in this study, thefatty acid aniaysis was based on separate measure-ments of non-polar, 2-hydroxy and 3-hydroxy acids. Ourresults showed no trace of 2-hydroxy fatty acids, butclear evidence of 3-hydroxy fatty acids, although theircontent were very lovi/ in relation to the total fatty acids

Table 2. 3-Hydroxy fatty acid composition of pianktonic cyanobacteria

Species and strains 12:0 14:0 15:0 15:1 16:0 16:1 16:3 18:0 18:1

Microcystis viridis{NIES-102)

M. aeruginosa f. aeruginosa (NIES-44)

M. aeruginosa f. aeruginosa [NIES-87)

M. aeruginosa f, aeruginosa (NIES-89)

M. aeruginosa f. aeruginosa (NIES-298)

M. aeruginosa f. flos-aquae {H\ES'98)

M. wesenbergiiiN\ES-104)

M. wesenbergii{N\ES-\1\)

M. wesenbergii W\IS-112)

M. wesenbergii(n\ES-60A)

Anabaena affinis (NIES-40)

A. crassa (NIES-76)

A. crassa(NIES-79)

A. ucrainica {TAC449)

A. solitaria {H\ES-78)

A. sotitaria iWES-SO)

A. smithii {JAC432)

A. flos-aquae (CCAP1403/13B)

A. flos-aquae (CCAP1403/13G)

Aphanizomenon flos-aquae f, gracile {NIES-81)

Oscillatoria agardhii (NIES-204)

0. agardhii {WES-205)

0. raciborskii mES-207)

Spirulina subsaisa (NIES-27)

6.9

12.56,3

—

—

—

13,1—

10,89.7

—

13.8

11.55.6_

12.84.2

4.3

4,1

—

—

10.16,5

14.2

19,621.527,324,029.929,928,432,023,224.2

3,4

1.4

2,1

4.2

4.2

6,9

1,1

20,512,226,119,34.4

14.21.6

18,714.514.9

5.3

24.116.818,933.214,114,913,711.016,918,313.112,715.914.8

5.2

1,8

27,112.7—

5,9

15,112,67.9

9,9

10.116.915,411.06,9

3,86.8

15.46,5

4,0

4.3

2.7

5,9

5.2

2.5

0,9

2,5

21.8—

15,1

12

12.39.0

33,25,4

5.5

1,9

12.61,1

14,947,83231,843,148.432.536,8

8.1

14.948.428,419.527.840.3

5.5—

12,4—

—

9.5

3.1

—

7.4

—

—

—

—

—

—

—

—

4.6

7.4

—

—

4.6

—_

9.87,9

4,8

15.323,110,113.111,118.318,826.915.416.419,029,820,220,426,934,620.812.1

3,2

42,914,8

12.19,8

8.7

—

7,4

11,36.1—

7.9

6,3

—

11,11.9

—

—

5,7

—

2,4

—

2.2

—

10.58,6

5.0

—8,8

8,4

12,2—

—

—

—

10.47,1

—

—

12.65,2

—

6,2

15,313,219,1—

10.713,2—

2.2

—, not detected.

Chemotaxonomy of planktonic cyanobacteria 25

M. aeruginosa f. aerugfinosa{NIES-89 )0 .agardhi i{NIES-2 04)M.wesenbergii(NIES-111)M.aeruginosa f.aeruginosa(NIES-44)M.aeruginosa f.aeruginosa(NIES-87)W.weseni)ergii(NlES~104)M.vesenjbergii(NIES-112 )O.agardhii(NIES-205)M.aeruginosa f.fIos-aguae(NIES-9 8)M.viridis(NIES-102)A.affinis(NIES-40)A.soIitaria(NIES-7 8)A.ucrainica(TAC449)A.soiitaria{NIES-80)A.crassa(NIES-79)S.sujbsaIsa(NIES-27)A.smithii{TAC432)A.crassa(NIES-76)A.fIos-aguae(CCAP1403/13B)Aph.flos-aquae f.gracile(NIES-81)M.iv'esenfc)ergii(NIES-604)

M-aeruginosa f.aeruginosa{NIES-29 8)A.fIos-aguae(CCAP1403/13G)O,racit)orsJk:ii{NIES-207)

3.0 2.0 1,0

Euclidian distance

Fig. 2. Dendrogram of planktonic cyanobacteria on the basis of their 3-hydroxy fatty acid composition.

(2-5%). Nine kinds of 3-hydroxy fatty acid have beendetermined by GC-MS. However, our qualitative andquantitative results of 3-hydroxy fatty acid compositiondid not shov^ much taxonomic value (Table 3). A den-drogram based on the results of 3-hydroxy fatty acidcomposition in 24 strains did not correspond to mor-phological criteria of taxa of planktonic cyanobacteriaat the order, family, genus or species level.

Non-polar fatty acids represented a high proportionof the total fatty acid content (95-98% of total) in thisstudy. All results for cyanobacterial total fatty acid com-position in previous studies were actually based on thenon-polar fatty acid composition. Hence, our results fornon-polar fatty acid composition can be compared tothose existing in the literature. Based on differences inthe fatty acid composition, Kenyon (1972) and Kenyonef al. (1972) suggested that fatty acid composition incyanobacteria can be classified into four types. The firsttype contains saturated and mono-unsaturated fatty ac-ids without polyunsaturated fatty acids (PUFA). Thesecond contains 18;3 (a), the third type 18:3 (7), andthe fourth type contains 18:4. This classification waslater supported by Murata ef ai (1992). Kenyon ef al.(1972) claimed that unicellular strains belong to thefirst or third groups, while filamentous ones were dis-tributed among the second, third and fourth types. Mu-rata ef al. (1992) found that the filamentous and uni-

cellular cyanobacteria examined were distributed in allfour types.

In this study, unicellular Microcystis had a uniformnon-polar fatty acid composition pattern in which 18:2, 18:3 (a), 18:3 iy) and 18:4 was contained as PUFA,being classified to type 4 according to the Kenyon-Mur-ata system. This result is similar to that of Ahlgren efal. (1992), but differs from those of Kenyon (1972) inwhich one isolate of Microcystis aeruginosa containedonly 18:2 and 18:3 (7) as PUFA, but neither 18:3 (a)nor 18:4, and therefore was classified into type 3, andthose of Kruger et al. (1995) in which PUFA of Micro-cystis also only contained 18:2 and 18:3 (which hadnot been divided into 18:3 (a) and 18:3 (7)) but no 18:4. Quantitatively, the relative amounts of 16:0 in thetotal non-polar fatty acid content of M/crocysf/s strainsin this study range from 50% to 60%, vwhich were high-er than those of one isolate of Microcystis (45%) stud-ied by Kenyon (1972) and those (31-45%) by Krugeret al. (1995). There has been no previous report on thefatty acids of filamentous planktonic cyanobacteriasuch as Anabaena and Aphanizomenon, although somestudies on fatty acids of filamentous benthic cyanobac-teria have been carried out. In this study, filamentousplanktonic Anabaena and Aphanizomenon strains con-tained 16:2, 16:3, 18:2 and 18:3 (a) as PUFA and,therefore, were classified to type 2 according to the

26 R. Li ef al.

Table 3. Nonpolar fatty acid composition of planktonic cyanobacteria

Species and strains

Microcystis viridis {NIES-102)

M. aeruginosa t aeruginosa (NIES-44)

M. aeruginosa t aeruginosa {H\ES-87)

M. aeruginosa t aeruginosa {N\E.S-89)

M. aeruginosa t aeruginosa iN\ES-298)

M. aeruginosa f. flos-aguae {N\ES-98)

M. wesenbergii {N\ES-104)

M. wesenbergii{N\ES-ll\)

M. wesenbergii W\ES-l 12)

M. wesenbergii W\ES-eQ4)

Anabaena affinis (N-40)

A. crassa {mES-76)

A. crassa (NIES-79)

A. ucrainica {TAC448)

A. ucrainica {TAC449)

A. crassa {TAC436)

A. solitaria {H\ES-78)

A. solitaria (mES-80)

A. smithii (TAC432)

A. planktonica {'\AC42l)

A. planktonica imBA-e)

A. flos-aquae {CCAP1403n38)

A. flos-aquae (CCAP1403n3G)

Aphanizomenon flos-aquae f. gracile (NIES-81)

Oscillatoria agardhiHN\ES-204)

0. agardhii {H\ES-205)

0. raciborskii {H\ES-2Q7)

Spirulina subsaisa (NlES-27)

14:0

2,9

2,0

2.9

2.4

2.42.5

2,4

2,7

2.0

2,2

4,6

2.8

2,6

3,2

3,4

2,6

5,3

6,1

3.75.3

4.4

5.9

5,3

2,1

2.22.2

1,6

2,8

16:0

60.2

51.7

55,3

55,8

55,1

57,6

54.2

58,6

54.1

56,3

30,5

37.1

41,3

36,7

34.9

39,2

37,4

41.9

40.8

39,5

34,8

33.9

31,3

33.9

33.1

31,0

56.8

50.7

16:1*

tr

0,9

1,3

0.7

0,5

tr

0,8

0.5

1,2

tr

2.1

1.7

1,5

1.8

1,8

1.6

2,5

2.4

2,6

2,2

3.6

0,5

0.9

1.2—

—

—

15,3

16:l(cis-)16:2*

1,1

1,7

2.0

1,7

2.3

1.3

1,8

1,6

3.0

1,2

3,6

3,8

3.2

2.9

5,1

4,2

4.8

5,1

2,5

4.1

3,7

1.9

2,5

2.2

24

23.2

1,2

6,2

—

—

—.

—

—

—

—

—

—

5.4

4.8

4,0

5,3

5.0

3.9

5,4

4,6

6.4

4.9

5,5

2,6

2,6

1.9

0,5

0,6—

19,0

16:3*

—

—

—

—

—

—

—

8.4

4.3

4,36.1

6,0

4,3

4,7

4.4

3,4

3,8

3,3

9,1

8.0

7,6—

—

—

—

18:0

1.6

1,1

3,2

1,6

0,7

1.9

1,3

1.5

3,2

1.5

0,7

1,1

0.9

1,1

0,7

0,9

0.6

0.7

1.2

0.8

0,9

0.7

0,9

0,6

0,6

1,1

1,2

tr

18:1

0,5

0.6

3.5

3.<:^

2.4

2,4

2,10.8

6,6

1.4

1.6

3.1

1,8

1,7

1,4

2.6

2.1

2,1

2.5

2.5

4,1

1.3

1,1

1,1

1.0

0,7

1,30.7

18:2

3.5

2,3

7,2

15.0

7,6

3,9

4.5

8.4

7,4

3.6

6.2

12,3

10,2

8,9

7,3

11.3

9.5

8.2

13.9

11,4

15.8

3,6

4.4

4.9

8,1

5,2

3,3

4,3

18:3(7)

5.1

2,2

8.3

13,8

23,7

18.1

1,8

9,6

3.2

5,9—

—

—

—-

—-

—

—

—

—

—

—

2,7

1,8

18:3(a)

9,3

18.2

8,8

2,7

1.9

4,8

19,3

7,2

9.7

13,7

36,8

28,9

30,2

32,3

32.3

30.9

27.8

24.5

22.8

25.5

24,0

45.8

42,944,4

31.3

35,2

7.5

—

18:4

15.6

19,4

7.6

3.2

3.2

7.0

11,7

9,2

9,7

14,2

—

—

—

—

—

—

—

24.5

—

"̂ Double band position not determined. —, not detected.

Kenyon-Murata system. Although the only reports ofthe presence of 16:2 and 16:3 in cyanobacteria arethose for some Spirulina isolates by Cohen and Vonshak(1991) and Cohen ef al. (1995), all heterocystous cy-anobacteria examined contained 16:2 (1.9-6.4%) and16:3 (3.3-9.1%) in their fatty acid composition. Tvi'Ostrains of C. agardhii {NIES-204, 205) contained atrace of 16:2 and 18:3 {a) characteristic of type 2.However, these strains had no 16:3, suggesting that itis appropriate to divide the type 2 group into two sub-types: type 2A for Anabaena and Aphanizomenon, andtype 2B for Oscillatoria according to the characteristicsof 16:2 and 16:3. In Spirulina, its PUFA is composedof 16:2 and 18:3 {7) characteristic of type 3. This re-sult is in close agreement with that of Cohen and Von-shak (1991), who analysed the fatty acid compositionof the same strain (NIES-27), although the culture con-ditions differed from the present study. Cohen and Von-shak (1991) also indicated that there were some dif-ferences in fatty acid pattern between freshwater andsalt strains of Spirulina, and proposed that in additionto the existing morphological criteria, the fatty acidcomposition should be used as an additional criterion

for the characterization of Spirulina. Thus, a compre-hensive study combining morphological and chemicalcharacteristics of the genus Spirulina is needed to clar-ify their taxonomy problems.

The simplified UPGMA dendrogram (Fig. 3) demon-strated that the classification of 28 strains of Microcys-tis spp., Anabaena spp., Aphanizomenon flos-aquae f.gracile, 0. agardhii and S. subsaisa based on the nu-merical analysis of non-polar fatty acids correspondedto morphological species criteria with the only excep-tion of M. aeruginosa f. aeruginosa {NIES-44), whichwas positioned in the cluster of Microcystis wesenbergiiKomarek, suggesting that non-polar fatty acid compo-sition is a valuable chemical marker in the taxonomy ofplanktonic cyanobacteria. It is noted that the fatty acidpattern of Aphanizomenon is the same as that of Ana-baena and, quantitatively, it is also similar {0 Anabaenastrains, especially A. flos-aquae Brebisson ex Bornet etFlahault. Horecka and Komarek {1979) pointed outthat no sharp boundary exists between the genera An-abaena and Aphanizomenon based on morphologicalfeatures. The fatty acid composition presented herealso demonstrated the difficulty in separating these two

Chemotaxonomy of planktonic cyanobacteria 27

3.0 2,5 2.0 1,5 1,0Euclidian distance

0.5

S.subsaisa(NIES-27)

0.agardhii(NIES-2 04)O.agardhii{tilES-205)Aph.flos-aguae f.graciie{NIES-81)fl.fios-aguae(CCRP1403/13B)A.fios-.aguae(CCaP1403/13G)A.affinis(NIE S-40)A.ucrainica(TAC4 48)A.ucjrainica(TAC449)A.cra3sa(NIES-76)A.crassa(NIES-79),a.erassa(TAC436)fl.soIitaria(NIES-78)A.solitaria(NIES-80)A.planktonica{ThCi2l)A.planktonica(lliBk-6)A.3mitbii{ThCA329M. wesenbergiiO.raciiDorsAii (NIES-111)M.we3&nbergii{i;iES-ll2)

W.aeruginosa f.aeruginosa(KIES-44)M.wesenbergii(NIE S-10 4)«.viridig(NIES-102)M.aeruginoBa f. f io3-agijae(NIES-96 )M.aeruginosa f.aeruginosa(NIES-2 98)«,aeruginosa f.aeruginosa(NIES-87)M. aeruginosa f. aerugi/iO3a( NIES-89 )

Type 3

Type 2B

Type 2ACluster 2

Type 4 Cluster 1

0,0

Fig. 3. Dendrogram showing the relationship among planktonic cyanobacteria on the basis of their non-polar fatty acid composition.

genera. However, as only one strain of Aphanizomenonwas used in this study, further investigations are neededusing more strains.

A surprising result of the present study was that thenon-polar fatty acid composition of 0. raciborskii WIES-207) was markedly different from that of 0. agardhlii(NIES-204, NIES-205), although both species are mor-phologically similar to each other. The non-polar fattyacid composition of 0. raciborskii was very similar tothat of Microcystis (Table 2, Fig. 2). Such a large dif-ference in fatty acid composition suggests the specialposition of 0. raciborskii in genus Oscillatoria, whichshould be further investigated to clarify its taxonomicposition. It is likely that there is much diversity in thegenus Oscillatoria. This suggestion is in agreement withthe result presented by Wilmotte and Golubic (1991),who determined 16S rRNA gene sequences of cyano-bacteria, and also by Neilan (1995), who studied phy-cocyanin gene patterns of water-bloom cyanobacteria byRFLP. Further taxonomic and systematic studies of Os-cillatoria are therefore needed.

The present results indicate that the non-polar fattyacid composition is a valuable chemical characteristicfor the taxonomy of planktonic cyanobacteria as op-posed to 3-hydroxy fatty acids, which are useful for tax-onomy of some gram-negative bacteria. In these iso-lates, particularly in /M/crocysf/s strains and Anabaenastrains, the fatty acid composition may be an importantchemotaxonomic character for classifying axenic strainswhich have changed phenotypically.

ACKNOWLEDGEMENTSWe thank Professors T, Hori and I. Inouye, University ofTsukuba, for their critical advice on this study and help-

ful encouragement. This work Vi'as supported by theJapanese Government's Special Coordination Funds bythe Science and Technology Agency (STA).

REFERENCES

Ahlgren, G., Gustafsson, I. and Boberg. M, 1992, Fatty acid

content and chemical composition of freshwater microal-

gae. J. Phycol. 28: 37-50.

Carmichael, W, W, 1980. Freshwater Blue-green Algae (Cy-

anobacteria) Toxins: A Review, /n Carmichael W. W. (Ed.).

The Water Environment: Algal Toxins and Health. Plenum

Press, New York, pp. 1-14.

Castenholz, R. W. and Waterbury, J, B, 1989. Cyanobacteria.

In Staley, J, T, Bryant, M. P,, Pfennig, IN. and Holt, J. G.

(Eds), Bergey's Manual of Systematic Bacteriology, Vol. 3.

Williams and Wilkins, Baltimore, pp. 1710-27.

Caudales, R. and Wells, J, M, 1992. Differentiation of the

free-living Anabaena and Nostoc cyanobacteria on the ba-

sis of fatty acid composition. Int. J. Syst. Bactenol. 42:

246-51,

Cohen, 2,, Margheri, M, C, and Tomaselli, L, 1995, Chemo-

taxonomy of cyanobacteria. Phytoohem. 40: 1155-8.

Cohen, Z. and Vonshak, A, 1991, Fatty acid composition of

Spirulina and Spirulina-\\ke cyanobacteria in relation to

their chemotaxonomy, Phytochem. 30: 205-5,

Dees, S, B., Hollis, D. G,, Weaver, R. E, and Moss, C. W. 1981,

Cellular fatty acids of Brucella canis and Brucella suis. J.

Clin. Microbiol. 14: 111-2,

Eloff, J. N. andVander Westhuizen,. A. J. 1981, Toxicological

Studies on Microcystis. In Carmichael W, W {Ed,), The Wa-

ter Environment: Algal Toxins and Health. Plenum Press,

New York, pp, 343-64,

Embley, T. M, and Wait, R. 1994, Structural Lipids ot Eubac-

28 R. Li etal.

teria. In Goodfellow M, and O'Donnell (Eds), ChemicalMethods in Prokaryotic Systematics. John Wiley and Sons,Chichester, New York, pp. 121-61,

Holton, R, W,, Blecker, H. H, and Stevens, T S, 1958, Fattyacids in blue-green algae; possible relation to phylogeneticposition. Science 160: 545-7.

Horecka, M. and Komarek, J, 1979, Taxonomy position ofthree planktonic blue-green algae from the genera Aphan-izomenon and Cylindrospermopsis. Preslia. Praha 51 :289-312.

Jones, G. J., Nichols, P D, and Shaw, P M, 1994, Analysisof Microbial Sterols and Hopanoids, In Goodfellow M, andO'Donnell (Eds), Chemical Methods in Prokaryotic System-atics. John Wiley and Sons, Chichester, New York, pp,163-95.

Jiittner, F. 1987. Volatile organic substances. In Fay P andBaalen C. V. (Eds), The Cyanobacteria. Elsevier, Amster-dam, pp. 453-70.

Kenyon, C, N, 1972, Fatty acid composition of unicellularstrains of blue-green algae, J. Bact. 109: 827-34.

Kenyon, C, N,, Rippka, R, and Stanier, R, Y. 1972. Fatty acidcomposition and physiological properties of some filamen-tous blue-green algae. Arch. Mikrobiol. 83: 216-36,

Komarek, J. 1958, Die taxonomische Revision der planktisch-en Blaualgen der Tschechoslowakei. In Komarek, J, andEttI, H. (Eds). Algologischen Studien. Tschech, Akad, Wiss,Frag., Czechoslovakia, pp. 1-206.

Kruger, G, H, J,, Det, W, H,, Kock, J, L, F, and Pieterse, A, J,H, 1995, Fatty acid composition as taxonomic character-istic for /W/crocysf/sand other coccoid cyanobacteria {blue-green alga) isolates. Hydrobiol. 308: 145-51.

Kruger, G. H. J., Eloff, J. N. and Pretorius, J. A. 1981. Mor-phological changes in toxic and non-toxic Microcystis iso-lates at different irradiance levels, J. Phycol. 17: 52-6,

Li, R,, Watanabe, M, and Watanabe, M, M. 1997, Akineteformation of planktonic Anabaena spp, by low temperaturetreatment. J. Phycol. 33: 576-84.

Moss, C, W, 1981, Gas-liquid chromatography as an analyticaltool in microbiology. J. Chromatogr. 203: 337-47,

Murata, N., Wada, H, and Gombos, Z. 1992, Modes of fatty-

acid desaturation in cyanobacteria. Plant Cell Physiol. 33:933-41 .

Neilan, B, A, 1995, Genetic diversity and phylogeny of toxic

cyanobacteria determined by DNA polymorphisms within

the phycocyanin locus. Appl. Environ. Microbiol. 61:3875-83.

Oyaizu, H, and Komagata, K. 1983. Grouping of Pseudomonas

species on the basis of cellular fatty acid composition and

the quinone system with special reference to the existence

of 3-hydroxy fatty acid. J. Gen. Appl. Microbiol. 29: 17-

40.

Scott, W. E, 1986. Examination of toxic and non-toxic Micro-

cystis aeruginosa in the field and laboratory culture. In

Steyn P, S, and VIeggaar R, (Eds). Mycotoxins and Phyco-

toxins. Elsevier Science Publishers, Amsterdam, pp. 4 1 -

50,

Skulberg, 0. M., Carmichael, W. W,, Codd, G, A, and Skulberg,

R. 1993. Taxonomy of Toxic Cyanophyceae (Cyanobacter-

ia). In Falconer I. R. (Ed,), Algal Toxins in Seafood and

Drinking Water Academic Press, London, pp. 145-64.

Stainer, R. Y., Kunisawa, R., Mandel, M. and Cohen-Bazire,

G. 1971, Purification and properties of unicellular blue-

green algae (order Chroccoccales). Bact. Rev. 35: 1 7 1 -

205.

Watanabe, M. M, and Hiroki, M, (Eds), 1997, NIES-Collec-

tion. List of Strains, Algae and Protozoa, 5th ed. National

Institute for Environmental Studies, Environment Agency,

Japan, 140pp,

Welch, D, F, 1991, Application of cellular fatty acid analysis.

Clin. Microbiol. Rev 4: 422-38,Wilmotte, A, and Golubic, S, 1991. Morphological and genetic

criteria in taxonomy of Cyanophyta/Cyanobacteria, Arch.

Hydrobiol. (Suppl. 92), Algol. Studies 64: 1-24,

Yokota, A. 1989 . Taxonomic significance of cellular fatty acid

composition in Rtiizobium, Bradyrhizobium, and Agrobac-

terium species. Inst. Ferment. Osaka Res. Commun. 14:

25-39,

APPENDIX I

Abbreviations

14:0, tedradencanoic acid (myristic acid); 16:0, hexadecanoic acid (palmitic acid); 16:1, hexadecenoic acid (pal-mitoleic acid); 16:1 (cis-), cis- Zl9-hexadecenoic acid; 16:2, hexadecadienoic acid; 16:3, hexadecatrienoic acid;18:0, octadecanoic acid (stearic acid); 18:1, Zl9-octadecenoic acid (oleic acid); 18:2, Zl9,12-octadecadienoic acid(linoleic acid); 18:3 (a), A9, 12,15-octadecatrienoic acid (a-linoienic acid); 18:3 (7), z]6,9,12- octadecatrienoicacid {7 -linolenic acid); 18:4, ZI6,9,12,15-octadecatetraenoic acid.