Biochemical Systematics and Ecology 31 (2003) 1125–1145 www.elsevier.com/locate/biochemsyseco Variability of the fatty acids of the marine green algae belonging to the genus Codium V.M. Dembitsky a,∗, H. Řezanková b, T. Řezanka c, L.O. Hanuš a a Department of Medicinal Chemistry and Natural Products, School of Pharmacy, P.O. Box 12065, Hebrew University of Jerusalem, Jerusalem 91120, Israel b Department of Statistics and Probability, University of Economics, W. Churchill Sq. 4, 13067, Prague, Czech Republic c Institute of Microbiology, Videnska 1083, 14220, Prague, Czech Republic Received 10 July 2002; accepted 3 January 2003 Abstract Analysis of fatty acids by gas chromatography-mass spectrometry (GC-MS) using serially coupled capillary columns with different polarity of stationary phases is reported. More than 40 volatile compounds, including, low molecular, dioic and fatty acids were determined of two marine green algae Codium dwarkense and C. taylorii. There are large variations in individual fatty acid contents according to species, season and location of the genus Codium. Statistical analysis of variability of fatty acids belonging to the genus Codium is reported. Published by Elsevier Science Ltd. Keywords: Fatty acids; Codium; GC-MS; Statistical analysis 1. Introduction The scientific investigation of fatty acids of marine oils began in the first decade of the 20th century (Ackman, 1989). In the field of lipid chemistry, a great deal of investigation of fatty acids of marine plants has been carried out since the 1950s, when gas-liquid chromatography was developed. The taxonomic classification of many plants is assisted by chemical investigation of some secondary metabolites. ∗ Corresponding author. Tel.: +972-2-675-7549; fax: +972-2-675-8201. E-mail address: dvalery@cc.huji.ac.il (V.M. Dembitsky). 0305-1978/03/$ - see front matter. Published by Elsevier Science Ltd. doi:10.1016/S0305-1978(03)00043-7 1126 V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 Plant lipids, fatty acids, and also other volatile compounds provide interesting information for assignment of the taxonomic position, particularly of marine, but also of freshwater algal species (Dembitsky et al., 1990, 1991, 1992; Dembitsky and Rozentsvet, 1996; Rozentsvet et al., 1995). Fatty acids, sterols and hydrocarbons are also perfect biogeochemical markers for studies of fossil structures of ancient organisms. Marine algae are potential suppliers of fatty acids, sterols and hydrocarbons, which are found in marine sediments (Peters and Moldowan, 1993; Volkman et al., 1998). Analysis by gas-chromatography-mass spectrometry is essential for identification of natural fatty acids, sterols and alkanes isolated from biological samples, including marine and freshwater algae, sediments and crude oil (Volkman et al., 1998; Christie, 1998a, Christie, 1998b; McClosky, 1971). Some papers have been published recently on the subject of optimising capillary GC separations through the use of serially coupled columns (Repka et al., 1989; Benicka et al., 1990; Engewald and Maurer, 1990; Maurer et al., 1990; Williams and Mitchell, 1991; Lou et al., 1993; Rastogi, 1993). A number of different variables have been manipulated to change the selectivity of the coupled system. However, the use of these approaches has not been fully investigated, for instance, in the separation of fatty, dioic, and carboxylic acids and other metabolites from marine algae. In this paper we describe the application of serially coupled capillary columns with consecutive nonpolar and semipolar stationary phase coating for separation of fatty, dioic and low molecular acids isolated from two marine green algae Codium dwarkense and C. taylorii. The results obtained are discussed, together with literature data, to verify previously proposed fatty acid distributions for marine green algae belonging to the genus Codium. 2. Experimental 2.1. Algal material Codium dwarkense Børgesen and C. taylorii P. Silva were collected from littoral rocks in the Mediterranean Sea near Ashdod and Ashkelon (Israel) during June–July 2000 at 2–5 m depth. Freshly collected algae were carefully cleaned of extraneous matter and only clean tissue was used for homogenisation in a high-speed unit (Dembitsky et al., 1990; Dembitsky and Rozentsvet, 1996). 2.2. Extracted procedure Fresh material of Codium dwarkense and C. taylorii was treated with MeOH and heated for 6 hrs at 60 °C. After cooling the extract to room temperature, a cold mixture of n-C5H12–H2O (3:6, v/v) was added. The mixture was shaken for 15 min, and then cooled under ice. The C5H12 and methanol-water phases were separated. The methanol-water phase was extracted with CH2CI2 three times, and the phases were separated, and used for preparation of fatty acid methyl esters. The pentane and dichloromethane extracts were washed with 0.9% KCl solution three times. Then V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 1127 the pentane and dichloromethane extracts were combined and evaporated under nitrogen at 10 °C. The remaining organic matter was kept at ⫺20 °C for analysis by GC-MS. 2.3. Preparation of fatty acid derivatives The fatty acids were converted to methyl esters by reaction with 5% methanolic HCl (overnight, 50 °C (Christie, 1989). After methylation, the mixture was cooled to room temperature, and the pentane-extract was washed three times with 0.9% KCl, and kept at -20 °C before analysis by GC-MS. 2.4. Apparatus and chromatographic conditions Separation of hydrocarbons and fatty acid methyl esters was carried out with a Hewlett-Packard 5890 (series II) gas chromatograph (Palo Alto, CA), equipped with a 5971B MSD mass selective detector. Low molecular carboxylic, dioic and fatty acids were analyzed by gas chromatography using two coupled capillary columns with different stationary phases; a RTX-1 column 30 m, ID = 0.32 mm, film thickness 0.25 µm, (Restek, PA) coupled with a second capillary column RTX-1701 30 m, ID = 0.32 mm × 0.25 µm film thickness (Restek, PA), as described previously (Dembitsky et al., 1999). The GC oven program had an initial temperature of 40 °C for 2 min, 2 °C/min run to 300 °C and final hold at 300 °C (20 min). Injector temperature was kept at 180 °C (splitless), and carrier gas (helium) flow rate was 25 cm/sec. The MS detector was operated at 194 °C, scan range was from 30 to 650 m/z at 0.9 scan/sec scan rate. Solvent delay was 10 min. Fatty acids and other volatile compounds were identified by mass spectral library search (Wiley 7 Edition), followed by matching of MS data and comparison of some components with commercial standard fatty acids. 2.5. Statistical analysis Statistical package STATISTICA 6.0 (Stat Soft, Inc, Tulsa, OK) was used for this purpose. 3. Results and discussion The problem of the simultaneous analysis of lipid metabolites is a difficult one because of the large number of different simple lipids, low molecular acids (from C2 to C10), dioic acids, and fatty acids that may be present. As a rule, for the separation of such a complex mixture of lipid components, it is necessary to resort to preliminary separation of low molecular carboxylic and fatty acids and other products, using preparative TLC (thin-layer chromatography), or some other manipulations. This, however, results in the loss of a number of components, or their oxidation and other undesirables processes may happen. Low molecular acids (C2–C10), 1128 V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 dioic acids, and also other metabolites are practically detectable by GC analysis. Use of coupled columns with different degrees of polarity of stationary phases gives a satisfactory separation capability for all the above mentioned components without their preliminary separation. The genus Codium attracts attention because these seaweeds contain unusual structures of some branched fatty acids (Aliya et al., 1992; Aliya and Shameel, 1993), rare and novel sterols (Sheu et al., 1995; Ahmad et al., 1993; Aknin et al., 1992a; Pollesello et al., 1992; Romeo and Toscano, 1983; Rubinstein and Goad, 1974), carotenoids (Pollesello et al., 1992; Egeland et al., 1997; Matsuno and Hirao, 1989), sulphated polysaccharides (Siddhanta et al., 1999), sulphated galactans (Matsubara et al., 2001), and halogenated and other bioactive metabolites (Dembitsky and Srebnik, 2002; Tringalli, 1997; Siddhanta and Shanmugam, 1999). Representatives of the genus Codium collected in various regions of the world have been investigated regarding their content of various fatty acids (Aliya et al., 1991, 1992; Aliya and Shameel, 1993; Herbreteau et al., 1997; Vaskovsky et al., 1996; Aknin et al., 1992b; Shameel and Khan, 1991); the members of the genus investigated are presented in Table 1. Data of the quantitative analysis of fatty acids are shown in Tables 2–4, and in essence do not differ from published data. Special attention was given to dioic acids. The analysis of published data on Codium has shown that only Aliya and Shameel (1993) have found propane-1,3-dioic acid in C. iyegarii. With the usual columns, detection of dioic acids is rather problematic. However, in our experience use of coupled columns solves the problem perfectly. Doubtlessly, the greatest interest was taken in the composition of the saturated fatty acids (Table 2). The system used allows the separation and identification of the highly polar fatty acids. The composition of monoenoic acids is shown in Table 3. Again, we came to the conclusion that our system enables the identification of considerably more monoenoic acids in the total lipid extract. It is possible to separate trans- and cis-isomers of various fatty acids. Polyenoic fatty acids (Table 4) present in various species of Codium are represented by a large spectrum of acids. It is difficult to interpret the absence of polyenoic acid in some species indicated by numbers 6, 10, 15. It is possible, that these are artefacts. Special attention was paid to C. fragile (Table 5). This species is widespread in marine ecosystems from the shores of Australia up to northern Europe, Africa and the Black Sea. As shown in Table 5, C. fragile from different locations contains acid 18:4, as also do other species of this genus. The obtained results are of interest from the viewpoint of biochemistry and chemotaxonomy and also provide more complete data for the pharmaceutical industry (Ackman, 1981, 1989; Dembitsky, 1996a; Pohl and Zurheide, 1979). Previously we described using package STATISTICA 6.0 for statistical analysis of fatty acids and triacylglycerols in vegetable and molluscs (Rezanka and Rezankova, 1999; Go et al., 2002), and showed that this procedure is effective for chemotaxonomic and related investigations. We applied this package to calculate the relationship between algal species inside the genus Codium grown in different marine locations from Australian, European and America coasts (Table 6). Table 1 Codium species investigated for their content of fatty acids 1. decorticatum 2. dichotomum 3. duthiae 4. duthiae 5. dwarkense 6. dwarkense 7. elongatum 8. elongatum 9. flabellatum 10. flabellatum 11. galeatum 12. harveyi 13. harveyi 14. intractum 15. iyengarii 16. muelleri 17. muelleri 18. pomoides 19. taylorii 20. fragile CHLOROPHYCOTA Chlorophyceae Codiales Bryopsidaceae Codium Collected place Collected date Reference Indian Ocean, Karachi, Pakistan Senegalese coast, Dakar, Senegal Pacific Ocean, Victoria, Australia Pacific Ocean, South Australia Mediterranean Sea, Israel Buleji Bay, Karachi, Pakistan Senegalese coast, Dakar, Senegal North Sea, Germany Buleji Bay, Karachi, Pakistan Buleji Bay, Karachi, Pakistan Pacific Ocean, Victoria, Australia Pacific Ocean, Victoria, Australia Pacific Ocean, South Australia Sea of Japan, Japan Indian Ocean, Karachi, Pakistan Pacific Ocean, South Australia Pacific Ocean, Tasmania, Australia Pacific Ocean, Victoria, Australia Mediterranean Sea, Israel North Sea, Germany November 1988 July 1990 July 1995 August 1995 June 2000 November 1989 July 1990 September 1966 November, 1988 February 1990 February 1995 July 1995 August 1995 July 1980 November 1988 August 1995 October 1995 July 1995 July 2000 August 1962 Aliya and Shameel (1993) Aknin et al. (1992) Xu et al. (1998) Xu et al. (1998) Present study Shameel and Khan (1991) Aknin et al. (1992) Pohl and Zurheide (1979) Aliya and Shameel (1993) Shameel and Khan (1991) Xu et al. (1998) Xu et al. (1998) Xu et al. (1998) Kato and Ariga (1982) Aliya and Shameel (1993) Xu et al. (1998) Xu et al. (1998) Xu et al. (1998) Present study Klenk et al. (1963) (continued on next page) V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 DIVISION Class Order Family Genus Species 1129 1130 DIVISION Class Order Family Genus Species 21. 22. 23. 24. 25. 26. 27. 28. 29. fragile fragile fragile fragile fragile fragile fragile fragile fragile CHLOROPHYCOTA Chlorophyceae Codiales Bryopsidaceae Codium Collected place Collected date Reference Amur Bay, Sea of Japan, Russia Sea of Japan, Japan Sillon Bay, Cotes d’Amor, France Feodosiya Bay, Black Sea, Russia Yellow Sea, China Sea of Japan, Yamaguchi, Japan Pacific Ocean, Victoria, Australia Sea of Japan, Japan Amur Bay, Sea of Japan, Russia August 1991 June 1974 June 1993 July 1987 October 1995 May 1996 July 1994 July 1980 August 1991 Khotimchenko (1993) Sato (1975) Herbreteau et al. (1997) Dembitsky (1996) Vaskovsky et al. (1996) Kaneniwa et al. (1998) Xu et al. (1998) Kato and Ariga (1982) Khotimchenko (1993) V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 Table 1 (continued) Table 2 Comparison of the proportion of saturated and branched fatty acids obtained in the present study with data reported in literature for Codium species 1a Ethane-1,2-dioic Propane-1,3-dioic Hexane-1,6-dioic Octane-1,8-dioic Nonane-1,9-dioic Total Dioic acids C 5:0 2,3-Dimethyl-3:0 0.39 C 4:0 Butanoic C 6:0 Hexanoic C 7:0 Heptanoic C 8:0 Octanoic C10:0 4,6-Dimethyl-8:0 C 9:0 Nonanoic C10:0 Decanoic C11:0 8-Methyl-10:0 C11:0 9-Methyl-10:0 C11:0 Undecanoic C12:0 10-Methyl-11:0 C12:0 Dodecanoic C14:0 Tetradecanoic C15:0 9-Methyl-14:0 C15:0 13-Methyl-14:0 C15:0 Pentadecanoic 4.95 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0.17 1.20 0.77 0.18 0.32 1.44 1.20 16 17 18 19 0.11 0.56 0.21 0.38 0.31 1.57 0.45 0.16 0.18 0.23 0.27 2.73 1.12 0.48 0.49 2.07 6.23 2.80 2.70 1.40 0.10 0.10 0.10 0.25 0.24 0.21 0.20 0.19 0.54 5.33 0.22 0.21 0.64 0.98 0.47 1.50 0.20 2.40 4.40 0.87 0.45 3.10 3.50 2.80 0.10 0.10 2.40 0.73 0.11 0.21 0.33 0.27 0.24 0.22 0.31 0.18 0.26 0.19 0.17 0.59 2.90 1.00 7.80 5.38 0.29 0.20 0.10 0.10 0.58 (continued on next page) V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 Acid type Systematic name 1131 1132 Acid type Systematic name 1a 2 C16:0 C17:0 C17:0 C17:0 C18:0 C19:0 C20:0 C21:0 C22:0 C23:0 C24:0 C27:0 C29:0 Total 5.09 32.90 26.60 36.90 26.03 83.79 28.40 10.90 5.85 83.59 41.20 26.80 44.80 21.20 8.98 0.29 0.25 0.10 0.10 0.23 0.30 9.10 0.40 7.66 0.70 1.10 1.20 1.67 2.53 1.10 2.00 2.67 2.50 0.90 1.70 3.53 4.40 0.20 0.20 0.20 0.30 15.18 0.30 0.30 0.30 0.23 4.08 1.46 0.90 2.30 0.70 3.57 2.40 0.80 2.20 4.74 4.75 2.07 0.40 0.20 0.31 0.80 0.30 0.80 5.30 a Hexadecanoic 2-Methyl-16:0 14-Methyl-16:0 Heptadecanoic Octadecanoic Nonadecanoic Eicosanoic Heneicosanoic Docosanoic Tricosanoic Tetracosanoic Heptacosanoic Nonacosanoic 2.58 4.60 9.59 3.75 3.64 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 38.80 25.60 25.00 22.90 0.42 0.31 0.20 0.26 1.10 0.40 1.00 1.19 0.24 0.20 0.10 0.40 0.22 0.29 1.80 0.90 0.15 0.18 0.20 0.30 0.30 0.36 2.18 36.77 36.70 32.10 42.40 39.43 88.26 31.50 24.40 53.06 88.06 50.70 32.70 52.70 23.60 42.14 45.10 28.40 34.70 37.41 Species and references indicated in Table 1. V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 Table 2 (continued) Table 3 Comparison of the proportion of monoenoic fatty acids obtained in the present study with data reported in literature for Codium species C 9:1 C10:1 C11:1 C12:1 C13:1 C14:1 C14:1 C15:1 C16:1 C16:1 C16:1 C16:1 C16:1 C16:1 C16:1 C16:1 C17:1 (E)-2-Nonenoic 9-Decenoic 1D-Undecenoic 9-Dodecenoic Tridecenoic 9Tetradecenoic 5Tetradecenoic Pentadecenoic All isomers 5Hexadecenoic 7Hexadecenoic 9Hexadecenoic (Z)-9Hexadecenoic 11Hexadecenoic (E)-13Hexadecenoic transHexadecenoic Heptadecenoic 1a 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 0.24 1.17 2.95 1.35 1.81 8.40 0.16 2.77 2.72 1.98 0.40 0.30 2.07 0.09 3.13 10.20 0.64 1.75 0.10 0.16 3.20 0.20 0.10 2.00 0.66 0.90 0.50 0.40 3.00 0.63 1.10 0.50 2.15 3.62 4.83 1.32 1.07 1.30 1.90 3.19 5.61 6.64 10.02 3.88 2.50 2.30 2.60 4.41 2.30 4.00 0.50 0.50 0.91 V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 Acid Systematic type name (continued on next page) 1133 1134 Table 3 (continued) C18:1 C18:1 C18:1 C18:1 C18:1 1a All isomers 7-Octadecenoic 8-Octadecenoic 9-Octadecenoic 4.85 (Z)-9Octadecenoic C18:1 (E)-9Octadecenoic C18:1 11Octadecenoic C19:1 Nonadecenoic C20:1 7-Eicosenoic C20:1 9-Eicosenoic 3.55 C20:1 11-Eicosenoic C21:1 Heneicosenoic C22:1 11-Docosenoic C23:1 Tricosenoic 4.20 Total Monoenoic 33.52 acids C13:1 Tridecynoic C16:1 12-Methoxy-4Hexadecynoic Total Monoynoic acids a 2 3 4 5 1.00 1.70 1.20 2.83 2.12 14.50 13.60 9.50 6 7 8 9 8.92 10 11 12 13 7.43 14 15 16 17 18 19 1.40 2.91 2.21 22.90 0.90 2.80 2.70 2.80 11.30 18.30 2.58 12.70 13.10 15.80 2.40 6.83 19.60 18.30 15.40 6.07 5.01 0.71 0.49 1.41 1.95 2.10 0.20 0.50 20.00 0.10 0.20 0.20 6.20 2.84 0.70 1.12 4.74 0.30 0.10 0.10 0.20 0.20 0.10 0.30 0.30 0.50 4.57 17.60 13.40 19.07 11.69 17.90 36.40 22.90 11.90 18.40 18.60 21.20 26.10 41.84 26.00 19.70 18.50 25.20 Species and references indicated in Table 1. 0.65 9.22 9.87 V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 Acid Systematic type name Table 4 Comparison of the proportion of polyenoic fatty acids obtained in the present study with data reported in literature for Codium species C11:2 3,8-Dimethyl-2,7Nonadienoic C14:2 7-Ethyl-3-MethylUndecadienoic C16:2 All isomers C16:2 6,9Hexadecadienoic C16:2 7,10Hexadecadienoic C16:2 9,12Hexadecadienoic C17:2 Heptadecadienoic C18:2 All isomers C18:2 8,11Octadecadienoic C18:2 9,12Octadecadienoic C18:2 10,13Octadecadienoic C18:2 11,14Octadecadienoic C20:2 11,13-Eicosadienoic Total dienoic fatty acids 1a 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 7.70 2.02 2.70 3.00 1.80 0.50 0.30 1.70 0.20 1.40 3.50 0.46 1.50 0.57 0.70 2.20 0.91 3.80 8.90 1.69 4.00 8.52 5.50 7.40 5.00 3.11 2.22 7.30 9.70 3.80 7.00 2.90 3.60 5.10 15.60 2.73 1.49 1.88 1.41 1.46 0.16 0.32 9.20 5.50 8.89 0.00 9.50 12.70 7.70 0.00 4.10 8.70 3.10 8.90 0.00 5.00 8.60 15.60 10.22 (continued on next page) V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 Acid Systematic name type 1135 1136 Table 4 (continued) 1a C11:3 Undecatrienoic C12:3 3,7,10-Trimethyl2,6,10Dodecatrienoic C13:3 Tridecatrienoic C16:3 7,10,13Hexadecatrienoic C17:3 Heptadecatrienoic C18:3 All isomers C18:3 9,12,15Octadecatrienoic C18:3 6,9,12Octadecatrienoic C20:3 5,8,11Eicosatrienoic C20:3 11,14,17Eicosatrienoic C20:3 8,11,14Eicosatrienoic C16:4 4,7,10,13Hexadecatetraenoic C18:4 3,6,9,12Octadecatetraenoic C18:4 6,9,12,15Octadecatetraenoic 1.17 6.42 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2.25 1.57 10.60 5.30 0.76 10.30 0.70 11.50 2.20 6.30 5.70 13.90 3.80 1.06 6.36 21.00 12.00 12.70 12.20 2.65 14.60 16.40 1.50 12.30 4.30 8.00 20.80 3.60 3.41 1.50 5.80 3.10 3.50 2.70 4.90 2.40 2.10 1.90 3.50 0.30 0.52 0.71 0.75 1.07 0.50 0.30 1.10 1.40 0.90 0.30 0.40 0.30 0.20 4.40 5.48 1.40 3.40 0.26 0.10 0.20 0.20 0.30 1.80 1.80 4.90 1.90 1.30 3.30 1.50 1.20 1.30 0.30 8.09 V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 Acid Systematic name type Acid Systematic name type C20:4 5,8,11,14Eicosatetraenoic C20:4 8,11,14,17Eicosatetraenoic C20:5 5,8,11,14,17Eicosapentaenoic C22:4 7,10,13,16Docosatetraenoic C22:5 7,10,13,16,19Docosapentaenoic C22:6 4,7,10,13,16,19Docosahexaenoic C29:3 Nonadecatrienoic Total polyenoic fatty acids Other and non-identified fatty acids a 1a 2 3 4 5 6 7 8 3.90 5.00 4.20 4.90 4.30 4.80 2.60 0.10 0.20 2.70 2.10 1.20 1.90 5.48 1.70 3.60 9 10 11 13 14 10.90 4.00 2.90 4.50 15 16 2.00 1.10 0.60 4.00 0.10 0.20 0.50 0.40 3.20 0.10 0.20 0.50 17 18 19 1.80 3.60 4.50 4.03 0.10 0.30 0.10 1.60 12 0.10 0.20 0.60 1.50 4.50 4.25 0.10 0.10 5.65 21.11 37.10 7.70 37.80 35.10 20.80 0.00 40.30 29.70 16.31 0.00 24.00 35.70 16.20 44.30 0.00 21.40 43.30 20.70 21.55 0.08 0.07 3.30 3.60 Species and references indicated in Table 1. 0.05 0.08 1.80 7.73 0.04 2.80 4.30 6.90 19.30 6.15 2.50 10.50 V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 Table 4 (continued) 1137 1138 V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 Table 5 Comparison of the proportion of fatty acids reported in literature for the C. fragile collected from different locations Acid type Systematic name 20a C10:0 Decanoic C12:0 Dodecanoic C14:0 Tetradecanoic C15:0 Pentadecanoic C16:0 14-Methyl-15:0 C16:0 Hexadecanoic C17:0 Heptadecanoic C18:0 Octadecanoic C20:0 Eicosanoic C22:0 Docosanoic C24:0 Tetracosanoic Total Saturated C16:1 All isomers C16:1 7-Hexadecenoic C16:1 9-Hexadecenoic C16:1 trans-16:1 C18:1 All isomers C18:1 5-Octadecenoic C18:1 7-Octadecenoic C18:1 9-Octadecenoic C20:1 7-Eicosenoic C20:1 9-Eicosenoic C22:1 Docosenoic Total Monoenoic C16:2 All isomers C16:2 7,10Hexadecadienoic C16:3 All isomers C16:3 7,10,13Hexadecatrienoic C18:2 All isomers C18:2 9,12Octadecadienoic C18:3 All isomers C18:3 9,12,15Octadecatrienoic C18:3 6,9,12Octadecatrienoic 21 22 23 24 25 48.60 0.70 1.10 0.80 1.50 3.60 2.50 3.25 1.30 0.10 0.68 2.40 0.32 28.10 26.20 38.00 19.70 19.96 22.40 0.10 0.33 0.90 0.90 1.25 0.90 0.50 1.14 3.20 2.90 0.20 0.29 33.60 34.00 41.60 71.50 28.32 24.80 1.60 7.20 2.30 2.86 1.50 3.30 0.60 0.50 0.70 1.90 14.20 6.20 15.35 1.00 10.80 9.90 1.10 5.20 3.20 15.60 13.60 21.40 8.50 0.90 2.80 12.20 27 28 29 1.60 5.30 0.20 2.30 1.30 34.60 40.50 17.90 17.50 0.30 1.20 0.70 0.20 2.60 1.10 1.40 0.50 41.40 48.80 20.20 18.80 3.20 2.90 2.50 2.30 0.80 22.90 17.70 0.20 2.10 12.50 23.30 0.70 0.90 18.91 11.50 15.00 30.30 26.10 20.60 2.28 1.40 1.20 1.00 10.20 9.40 12.60 4.00 5.50 26 3.60 5.78 8.90 27.20 8.90 5.40 8.50 3.30 5.10 11.00 4.00 12.20 24.90 21.00 22.50 15.10 19.10 15.40 5.60 2.30 2.60 1.90 0.90 (continued on next page) Fig. 1 shows the results of cluster analysis. Euclidean distance and Ward’s method for joining clusters were used for standardized data. Euclidean distance is the geometric distance in a multidimensional space. For two objects i and i⬘ it is computed as: dii’ = 冪Σ p j=1 (xij⫺xi’j)2 Where p is a number of variables. V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 1139 Table 5 (continued) Acid type C20:3 Systematic name 20a 8,11,14Eicosatrienoic C18:4 All isomers C18:4 6,9,12,151.50 Octadecatetraenoic C20:4 All isomers 3.00 C20:4 8,11,14,17Eicosatetraenoic C20:4 5,8,11,14Eicosatetraenoic C20:5 All isomers C20:5 5,8,11,14,171.90 Eicosapentaenoic C22:5 7,10,13,16,19Docosapentaenoic Total Polyenoic 52.20 Other and non-identified fatty acids a 21 22 23 24 25 26 0.40 27 28 29 1.50 1.90 0.20 1.00 1.39 1.40 2.00 1.30 0.30 0.50 0.20 0.10 5.90 2.30 1.50 4.50 8.40 1.80 0.50 4.00 12.47 0.20 4.20 5.20 0.40 3.40 0.40 6.00 10.10 3.49 0.90 0.94 0.30 52.50 36.60 20.00 51.25 66.30 43.60 21.40 53.70 60.60 2.50 12.14 1.52 6.20 13.90 3.30 13.35 9.10 Species of marine algae and references indicated in Table 1. Ward’s method uses the analysis of variance approach to evaluate the distances between clusters. This method attempts to minimize the sum of squares of any two (hypothetical) clusters that can be formed at each step. Two important conclusions may be drawn from this figure. First, the expected taxonomical affinity of the same kinds of Codium sp. (predominantly C. fragile) was confirmed experimentally. The taxonomical affinity was independent of the time and place of sample collection. As an illustrative example, we can use similarities between C. fragile (24) found in the Black Sea and C. fragile (28) collected from the Pacific Ocean near Japan, or two samples of C. fragile (23 and 29) gathered from the coasts of the Atlantic (France) and Pacific Oceans (Far East of Russia). Secondly, the cluster analysis uncovered the bio-geographical affinity of the fatty acid content of Codium sp. An example is the similarity of a closed group of five different strains [C. decorticatum (1), C. dwarkense (5), C. dwarkense (6), C. flabellatum (9), and C. flabellatum (10)] gathered from the north coast of the Indian Ocean in Pakistan compared with distant relative strains of C. fragile (23, 29). Similarly, the strains of C. dwarkense (5) and C. taylorii (19) collected from the Mediterranean Sea show a distinct kinship in fatty acid content. The other isolated cluster constitutes the species to be found along the Atlantic Ocean coasts, such as C. dichotomum (2) and C. elongatum (7) from Senegal and C. elongatum (8) from the North Sea. In order to support our working hypothesis, we identified a close group of similar species that is located in the Pacific Ocean near Australia. The strains of C. fragile (21 and 25) serve as an exception to the rule. The fatty acid content of both strains 1140 Statistical analysis of selected fatty acids isolated from different species of Codium Fatty acids 14:0 15:0 16:0 17:0 18:0 19:0 20:0 22:0 24:0 Saturatedb 16:1a 18:1a 20:1a Mono- 18:2 Dienolcb 16:3 18:3n3 18:3n6 18:4a 20:4a 20:5 Polyenoicb 19 enoicb Number of values 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 Minimum 0.0 0.0 5.10 0.0 0.0 0.0 0.0 0.0 0.0 23.60 0.0 2.58 0.0 11.69 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 25% Percentile 0.85 0.0 22.05 0.0 0.95 0.0 0.0 0.0 0.0 32.40 0.0 9.81 0.0 18.15 3.25 4.55 0.0 0.75 0.0 0.15 1.05 0.30 18.51 Median 2.70 0.10 26.60 0.10 1.19 0.0 0.20 0.80 0.20 37.41 0.20 15.30 0.20 19.70 5.00 8.52 0.76 4.30 1.50 1.40 4.10 1.50 21.51 75% Percentile 7.80 0.15 40.00 0.35 2.51 0.0 0.30 2.35 0.38 51.70 4.40 18.30 0.75 26.05 7.99 9.35 8.00 12.50 3.30 4.10 5.05 3.80 37.45 Maximum 7.80 4.98 83.79 9.10 4.60 9.59 15.18 4.74 3.64 88.26 10.20 22.90 6.20 41.84 15.60 15.60 13.90 20.80 5.80 8.09 10.90 5.48 44.30 Mean 2.582 0.374 31.330 1.102 1.573 0.748 1.037 1.274 0.401 43.16 1.912 13.59 0.836 22.04 5.268 6.933 3.464 7.129 1.65 3 2.299 3.681 1.817 24.49 Std. deviation 2.118 1.130 21.640 2.641 1.179 2.366 3.454 1.497 0.824 17.990 2.958 5.612 1.624 7.901 4.026 4.232 4.702 6.632 1.874 2.568 2.875 1.749 14.120 Std. error 0.486 0.259 4.965 0.606 0.270 0.543 0.79 0.343 0.189 4.126 0.678 1.287 0.373 1.813 0.924 0.971 1.079 1.522 0.429 0.589 0.659 0.401 3.240 Lower 95% 1.561 -0.171 20.900 -0.171 1.005 -0.391 -0.628 0.553 0.003 34.500 0.487 10.89 0.053 18.24 3.328 4.893 1.198 3.933 0.749 1.062 2.295 0.974 17.690 3.602 0.919 41.770 2.374 2.141 1.889 2.701 1.996 0.797 51.830 3.338 16.300 1.619 25.850 7.208 8.973 5.730 10.330 2.556 3.537 5.066 2.660 31.300 KS Distance 0.1402 0.4032 0.1855 0.4469 0.2032 0.4799 0.4679 0.2303 0.3424 0.2012 0.3151 0.1460 0.3510 0.1810 0.1152 0.1725 0.2744 0.1915 0.2848 0.2460 0.1529 0.1727 0.1421 P value ⬎0.10 ⬎0.004 ⬎0.10 ⬎0.0185 ⬎0.10 ⬎0.10 ⬎0.10 ⬎0.10 ⬎0.10 ⬎0.10 ⬎0.10 ⬎0.10 ⬎0.10 ⬎0.10 Confidence Intervals Upper 95% Confidence Intervals a All isomers b Total of each types of fatty acids ⬎0.001 ⬎0.10 ⬎0.0003 ⬎0.0005 ⬎0.10 ⬎0.023 ⬎0.10 ⬎0.046 ⬎0.10 V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 Table 6 V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 Fig. 1. 1141 Dendrogram from hierarchical cluster analysis of twenty nine species of Codium. Fig. 2. Two-dimensional graph of the final configuration from a multidimensional scaling of twenty nine species of Codium. matches the “Australian group”. However, they were collected thousands of miles away. The strain C. fragile (21) was gathered from the coast of the Yellow Sea (China) and the other one in Russia (Japan Sea coast). To conclude, the results of cluster analysis revealed a strong relationship between bio-geographical conditions and fatty acid content. Environmental effects such as salinity, temperature, nutrition, solar radiation and taxonomical affinity are very important. 1142 V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 Fig. 3. Two-dimensional graph of a final configuration from the multidimensional scaling of twenty nine species of Codium. Circles correspond to some clusters in Fig. 1 (Some of them correspond to geographical places). Fig. 4. Three-dimensional graph of a final configuration from the multidimensional scaling of twenty nine species of Codium. Similar results were obtained by multidimensional scaling. The same proximity matrix as that for cluster analysis was used on the basis of the Euclidean distance for standardized data (Figs 2–4). Acknowledgements The authors thank Prof. I. Dor (Environmental Division, Hebrew University) for help in the preparation and identification of marine algae. V.M. Dembitsky et al. / Biochemical Systematics and Ecology 31 (2003) 1125–1145 1143 References Ackman, R.G. (Ed.), 1989. Marine Biogenic Lipids, Fats and Oils. Vols. 1 & 2. CRC Press Inc, Boca Raton, FL. Ackman, R.G., 1981. Algae as sources for edible lipids. In: Pryde, E.H., Princen, L.H., Mukheriee, K.D. 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