Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol31.Issue6.2000:

A comparative study of lipids in

Sphagnum

species

Marianne Baas

a

_{, Rich Pancost}

a

_{, Bas van Geel}

b

_{, Jaap S. Sinninghe DamsteÂ}

a,

_*

a_{Netherlands Institute for Sea Research (NIOZ), Department of Marine Biogeochemistry and Toxicology, PO Box 59, 1790 AB}

Den Burg, The Netherlands

b_{The Netherlands Centre for Geo-ecological Research, Department of Palynology and Paleo/Actuo-ecology, University of Amsterdam,}

Kruislaan 318, 1098 SM Amsterdam, The Netherlands

Received 21 December 1999; accepted 3 March 2000 (returned to author for revision 24 February 2000)

Abstract

The free lipid compositions of twelve species ofSphagnumwere determined by capillary gas chromatography/mass spectrometry as part of a study to identify characteristic lipids forSphagnumin peat bogs. Complex mixtures of lipids, comprised of C28±C29 sterols, C30 triterpenoids, C16±C30 fatty acids, C22±C30fatty alcohols, C21±C33 n-alkanes and

isoprenoid and straight-chain wax esters, were identi®ed and quanti®ed. Sterols are dominated by the C29sterols,

24-ethylcholesta-5,22-dien-3b-ol and 24-ethylcholest-5-en-3b-ol, whilst in some species C28 sterols are also abundant.

Summed concentrations of triterpenoids varied widely (20±3500mg/g dry weight), with only small concentrations pre-sent in the mesotrophic species,S. ®mbriatumandS. palustre. Ursolic acid is always the major triterpenoid detected. Although absolute concentrations vary signi®cantly, the carbon number distributions of fatty acids, fatty alcohols and

n-alkanes are similar in all examined species. Thus, the distributions of these compounds, and especially the dominance of C23 and C25 n-alkanes, are a useful chemotaxonomic ®ngerprint for Sphagnumspecies and can thus be used in

compound-speci®c13_{C and}14_{C studies of peat bogs.#2000 Elsevier Science Ltd. All rights reserved.} Keywords: Sphagnum; Sterols; Triterpenoids; Fatty acids; Fatty alcohols;n-Alkanes; Wax esters; Peat

1. Introduction

Sphagnumspecies (Bryophyta) can comprise a large fraction of peat bog deposits. The composition of such peat deposits records climatic information and can serve as a useful tool in palaeoclimate reconstructions. One way to obtain such information is to determine the pollen and macrofossil record (e.g. Grosse-Brauckmann, 1972, 1974; van Geel, 1978; Moore et al., 1991); however, such records are not always preserved. An alternative method is to analyse bog deposits for lipids derived from plants contributing to the peat (e.g. Dehmer, 1993; Lehtonen and Ketola, 1993; Farrimond and Flanagan, 1995; Ficken et al., 1998; Nott et al., 2000). However, chemotaxonomic

data for lipids from Sphagnum species are scarce and largely limited to alkyl compounds.

Sever et al. (1972) and Marseli et al. (1972) reported that the n-alkane distributions ofSphagnum aneand

S. teresare dominated by the C31member. The majorn

-alkane in four other species ofSphagnawas C23or C25

(Corrigan et al., 1973). This characteristic pattern was also reported recently for three out of six species of

Sphagna(Nott et al., 2000). Early studies have identi®ed sterols, triterpenoids and fatty acids in a fewSphagnum

species (Black et al., 1955; Ives and O'Neill, 1958a,b; Marseli et al., 1972). Karunen and co-workers reported the lipid composition of fresh and decaying S. fuscum

(Karunen et al., 1979; 1983; Karunen and Salin, 1980; Karunen and Ekman, 1981). Ficken et al. (1998) reported then-alkane, fatty acid and fatty alcohol dis-tributions for S. capillifolium andS. fuscum. Here we present data on the lipid composition of twelve species ofSphagna.

0146-6380/00/$ - see front matter#2000 Elsevier Science Ltd. All rights reserved. P I I : S 0 1 4 6 - 6 3 8 0 ( 0 0 ) 0 0 0 3 7 - 1

www.elsevier.nl/locate/orggeochem

* Corresponding author. Tel.: 222-369550; fax: +31-222-319674.

2. Experimental

2.1. Plant selection and identi®cation

Field specimens ofSphagnum species were obtained from the ``Bargerveen'' peat bog reserve (Zwartemeer, SE-Drenthe, the Netherlands) except forS. imbricatum, which was obtained from the Irish Curraghmore Bog, near Galway. After collection the plants were immedi-ately washed repeatedly with water and any non-indi-genous material was removed. Species identi®cation was con®rmed by observation with light microscopy (Smith, 1978). Aliquots of the samples for chemical studies were stored atÿ20C.

2.2. Extraction and derivatisation

The total lipid extract of the freeze-driedSphagnum

species (ca. 100±200 mg dry weight) were obtained by sonication with a progressively less polar mix of solvents (MeOH/CH2Cl21/0, 3/1, 1/1, 1/3 and 0/1, respectively).

After addition of a standard (10±40mg of 2,3-dimethyl-5-(1,1-d2-hexadecyl)thiophene), the total extract was

methylated with diazomethane, chromatographed over silica 60 (Merck 60, 0.063±0.2 mm, 70±230 mesh) with ethyl acetate as an eluent to remove very polar com-pounds and silylated with a 1/1 mixture of BSTFA (1% TMCS) and pyridine at 60_{C for 20 min.}

2.3. Gas chromatography (GC) and gas chromatography±mass spectrometry (GC±MS)

GC was performed using a Hewlett-Packard 6890 instrument, equipped with an on-column injector. A fused silica capillary column (25 m0.32 mm) coated

with CP-Sil 5 (®lm thickness 0.12 mm) was used with helium as carrier gas. The e‚uent was monitored with a ¯ame ionization detector (FID). The samples were injected at 75_{C and the oven was programmed to}

130_{C at 20C/min and then at 4C/min to 320C, at}

which it was held for 15 min. GC±MS analyses used a Hewlett-Packard 5890 gas chromatograph interfaced with a VG Autospec Ultima mass spectrometer oper-ated at 70 eV with a mass range ofm/z 40±800 and a cycle time of 1.7 s (resolution 1000). The gas chromato-graph was equipped with a fused silica capillary column of the same dimensions as described for GC. The carrier gas was helium. The same temperature program as for GC was used.

2.4. Quantitation

Components were quanti®ed by integration of peak areas in the FID chromatograms, in some cases assisted by mass chromatography of speci®c ions from GC±MS analysis of the same fraction.

3. Results

Twelve species of Sphagna, eleven from a peat bog reserve in the Netherlands and one from a peat bog in Ire-land, were analysed for free lipids using GC and GC±MS. A chromatogram of a typical total lipid fraction is shown in Fig. 1. Numbers refer to compounds listed in Table 1.

3.1. Sterols

Sterols are the most abundant free lipids in many of the investigated Sphagnum species. Summed concentrations vary from 450 to 1600mg/g dry weight (Table 1). In all cases, C29sterols (I±II; see Appendix for structures) as a

group dominate over C28 sterols, although

24-methyl-cholest-5-en-3b-ol (III) is sometimes the second most abundant sterol present. 24-Ethylcholesta-5,22-dien-3b -ol (I) is typically the most abundant sterol, with 24-ethylcholest-5-en-3b-ol (II) usually present in somewhat lower concentrations. In three cases (S. ®mbriatum, S. recurvum, and S. magellanicum), 24-ethylcholest-5-en-3b-ol (II) is the most abundant sterol. The less abundant C28 sterols comprise of 24-methylcholest-5-en-3b-ol

(III), 24-methylcholesta-5,22-dien-3b-ol (IV) and 24-methylcholesta-5,7,22-trien-3b-ol (V). These results are in good agreement with studies of sterols in an unidenti®ed

Sphagnum species (Ives and O'Neill, 1958a), S. teres

(Marsili et al., 1972) and S. fuscum (Karunen and Ekman, 1981; Karunen et al. 1983).

3.2. Triterpenoids

The concentrations of triterpenoids are much more variable than those of sterols, ranging from 20 to 3500

mg/g dry weight (Table 1). In two species,S. ®mbriatum

andS. palustre, they are almost completely absent. The distribution is always dominated by ursolic acid (VI) with oleanoic acid (VII) and another triterpenoid, ten-tatively identi®ed as ursolic acid containing an addi-tional double bond, also typically abundant. The latter compound's mass spectrum [m/z201 (100%), 73 (85%), 260 (55%), 247 (30%), 190 (45%) 540 (25%)] is quite similar to that of ursolic acid but a number of the frag-ment ions have shifted by two daltons. Apart from these three triterpenoids, other triterpenoids [i.e. a- and b -amyrin (VIII andIX) and lupeol (X)] occur in several

Sphagnumspecies in varying relative abundances. InS. papillosumandS. pulchrum relatively high amounts of lupeol (X) occur. Lupenone (XI) was only encountered in S. molle. These ®ndings are in agreement with the presence of ursolic acid inS. teres(Marsili et al., 1972).

3.3. Fatty acids

debris was vigorously washed prior to extraction, it is possible that some bacterial biomass was not removed; consequently, a bacterial contribution to the C16±C18

fatty acids (especially the highly unsaturated compo-nents) seems plausible. Summed concentrations of C20+

fatty acids vary much less (140±420 mg/g dry weight). These fatty acids are almost exclusively composed of even carbon numbered components with distributions dominated by the C24or C26members. This is in good

agreement with earlier studies reporting a C24

dom-inance in the fatty acid distribution of two di€erent

Sphagnumspecies (Karunen and Salin, 1980; Ficken et al., 1998). Marsili et al. (1972) reported the dominance of C16±C18fatty acids (including polyunsaturated

com-ponents) inS. teres.

3.4. Fatty alcohols

Total concentrations of fatty alcohols vary from 80 to 340mg/g dry weight (Table 1).n-Alcohols occur in the range C22±C30 and are predominantly composed of

even-carbon numbered components. The distributions are somewhat more variable than those of the fatty acids and are dominated by the C24, C26 and, in some

cases, the C28alcohols. Ficken et al. (1998) also reported

a dominance of the C24and C26alcohols in two di€erent

species ofSphagna.

3.5. Hydrocarbons

Straight chain alkanes occur in relatively small con-centrations (100±290 mg/g dry weight; Table 1). Their distributions are dominated by C21+ odd-carbon

num-bered homologues, and in all but one species (S. rubel-lum) their distributions maximize at either C23or C25. In

about half of the studied species (S. compactum, S. molle,S. papillosum,S. recurvumandS. tenellum) there is a clear second maximum at C31and inS. rubellumthe

C31n-alkane is the dominant hydrocarbon.

These results are generally in agreement with literature data. InS. anethen-alkane distribution is dominated by the C31 homologue (Sever et al., 1972). Corrigan et al.

(1973) reported that the dominant n-alkane in S. fus-cum,S. magellanicumandS. rubellumis C25, whereas in S. recurvumit is C23. Ficken et al. (1998) reported that the n-alkane distributions in S. capillifoliumand S. fuscum

maximize at the C23, C25and C31, and C25homologues,

respectively. Nott et al. (2000) recently reportedn-alkane

Table 1

Concentrations (mg/g dry weight of various lipids in twelve di€erentSphagnumspecies

Compoundb _{Sphagnumspecies}

(a)a _{(b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l)}

Sterols

1 24-Methylcholest-5,7,22-trien-3b-ol (V) 62 15c ₁₉c ₇₀c ₅₈c ₉₂c ₄₁c _{93 11}c ₂₃c ₈₀c ₂₄c

2 24-Methylcholest-5,22-dien-3b-ol (IV) 58 230 21c ₂₀c ₉c ₁₇₅c ₅₇c _{84 166 29}c ₈₈c ₅₅c

3 24-Methylcholest-5-en-3b-ol (III) 120 300 405 63 98 82 130 160 115 205 175 120 4 24-Ethylcholest-5,22-dien-3b-ol (I) 370 640 280 145 78 870 280 476 346 160 350 255 5 24-Ethylcholest-5-en-3û-ol (II) 200 180 450 195 215 350 150 160 94 345 155 135 Summed sterols 810 1370 1180 500 460 1570 660 970 730 760 850 590

Triterpenoids

6 a-Amyrin (VIII) 215 56c _{0 0 0 225 0 100 35 25 0 17}d

7 b-Amyrin (IX) 78 9 0 0 12 54 0 57 68 26 0 26d

8 Lupeol (X) 290 19 0 0 25 200 0 250 230 47 70 99 9 Oleanoic acid (VII) 485 66 0 39 65 405 0 145 110 125 110 145 10 Ursolic acid (VI) 1785 280 27 73 270 1640 20 615 465 430 350 590 11 Unsaturated ursolic acid 665 100 0 29 65 555 0 205 155 175 105 170 Summed triterpenoids 3520 530 27 140 440 3080 20 1370 1060 830 640 1050

Fatty acids

12 C16:0 35 15 44 7 175 11 21 33 40 13 15 26

13 C18:0 12 3 13 6 10 2 5 14 7 2 8 8

14 C18:5 39 88 47 3 60 17 56 100 62 36 62 67

15 C18:2+3 26 41 48 23 590 34 12 86 26 13 53 20

16 C22:0 21 16 13 23 14 15 13 22 13 21 16 21

17 C24:0 100 68 94 115 70 143 84 97 52 62 79 61

18 C26:0 75 45 75 155 105 225 100 110 39 39 63 54 19 C28:0 15 14 14 30 9c _{22 20}c ₁₈c ₆c ₁₂c ₁₈c ₂₀c

20 C30:0 14c ₁₄c ₉c ₃c _{<1 24}c ₁₅c ₈c ₅c ₆c ₁₆c ₁₈c

Summed fatty acids 340 300 360 370 1030 490 330 490 250 200 330 300

Fatty alcohols

21 C22:0 21 14 21 9 9 12 7 8 13 14 34 12

22 C24:0 53 27 56 49 29 76 44 38 18 32 88 41

23 C26:0 66 30 49 110 47 155 70 45 27 28 59 40

24 C28:0 105 27 49c ₅₉c _{13 95 88}c _{30 13 18}c ₄₈c ₇₂

25 C30:0 15c ₅c ₅c ₅c ₃c _{0 6}c _{5 4 3}c ₇c ₈c

Summed fatty alcohols 260 100 180 230 100 340 210 130 76 95 240 170

n-Alkanes

26 C21:0 11 15 7 52 12 27 19 30 15 33 27 15

27 C23:0 30 58 31 74 17 42 51 72 41 48 35 61

28 C25:0 55 17 64 40 42 71 40 44 22 21 41 27

29 C27:0 9 5 36 8 7 15 10 9 9 11 11 8

30 C29:0 15 8 13 1c _{6 24 16 10 10 28 44}c ₁₅

31 C31:0 53 22 19c _{10 6 57 16}d _{31 14 42 87 24}

32 C33:0 0 0 0 0 14c _<1c _{0 0 0 18 46 0}

Summed n-alkanes 170 130 170 180 100 240 150 200 110 180 250 150

Wax esters

33 i-C20:1 FA- i-C20:1 alc (XII) 0 180 195 0 14 0 18 18 21 24 65 0 34 C16:0 FA- C22:0 alc 53 18 52 0 14 0 13 18 24 38 100 0 35 C16:0 FA- C24:0 alc 140 80 110 37 43 165 71 110 43 51 225 74 36 C16:0 FA- C26:0 alc 150 100 56 80 44 265 86 150 49 22 94 74 Summed wax esters 340 380 410 120 120 430 190 300 140 140 480 150

a _{(a)S. compactum, (b)S. cuspidatum, (c)S. ®mbriatum, (d)S. imbricatum, (e)S. magellanicum, (f)S. molle, (g)S. palustre, (h)S.}

papillosum, (i)S. pulchrum, (j)S. recurvum, (k)S. rubellum, (l)S. tenellum.

distributions in six species ofSphagnaand noted for all species an increased relative abundance of the C23and,

sometimes, C25 homologue in comparison to

distribu-tions of other modern bog plants.

3.6. Wax esters

Substantial amounts of wax esters (100±500mg/g dry weight; Table 1) were encountered in all investigated species. They are predominantly comprised of C16:0

fatty acids esteri®ed with C22:0, C24:0 and C26:0 fatty

alcohols. In addition, an isoprenoid wax ester (XII), comprising phytol esteri®ed to phytenic acid, was ten-tatively identi®ed. This component was detected only in some species and is especially abundant inS. cuspidatum

andS. ®mbriatum.

3.7. Other compounds

a-Tocopherol (XIII) was encountered in all species. All species also contain relatively small amounts of C25

and C27 methyl ketones. In a number of species (S. imbricatum,S. compactum,S. magellanicum,S. palustre,

S. papillosum, andS. pulchrum) relatively high amounts of free sugars (detected as their TMS derivatives) were encountered in the total lipid extract.

4. Discussion

The distributions of free lipids in the twelve Sphag-numspecies are rather similar. Exceptions to this general observation include relatively high abundances of the isoprenoid wax ester XIIin S. cuspidatumandS. ®m-briatumand the very low abundances of triterpenoids in

S. ®mbriatumandS. palustre. Remarkably, these latter two species are mesotrophic species as opposed to the other tenSphagna. For the rest, no clear relationships between chemotaxonomy and biological classi®cation or habitat could be made.

The distribution within the various lipid classes var-ied, but not to a large extent. Steroids are typically dominated by C29members and triterpenoids by ursolic

acid (VI). Fatty acids, fatty alcohols and n-alkanes all show rather characteristic carbon number distributions, with only small variations. These data are generally in good agreement with data reported in the literature for other Sphagnum species (Ives and O'Neill, 1958a,b; Marsili et al., 1972; Sever et al., 1972; Corrigan et al., 1973; Karunen et al., 1979, 1983; Karunen and Salin, 1980; Karunen and Ekman, 1981; Ficken et al., 1998; Nott et al., 2000). Then-alkane distributions of all but one species maximize at C23or C25(cf. Corrigan et al., 1973). In

contrast, higher plants often containn-alkanes but their distribution is typically dominated byn-C29,n-C31orn-C33

and is characterized by only small contributions ofn-C23

and n-C25 (e.g. Eglinton and Hamilton, 1967). Peats

with a large contribution fromSphagnashow the same characteristic n-alkane pattern (Lehtonen and Ketola, 1993; Nott et al., 2000; Pancost et al., unpublished results), although this is not always the case (Ficken et al., 1998). Consequently, the C23or C25n-alkanes have strong

potential as source-speci®c indicators forSphagnum con-tributions to ancient peat deposits. Moreover, the 14_C

and13_{C contents of such diagnostic compounds should}

be useful in the age determination of peats and in the reconstruction of their environmental depositional con-ditions, respectively. The fatty acid and alcohol dis-tributions also seem to be rather characteristic but their distribution is not distinct enough from those of other higher plants to be of use in tracingSphagnuminputs.

Acknowledgements

We thank Dr. M. O'Connell for supplying S. imbri-catumand J. de Vries (SBB Zwartemeer) for identifying theSphagnumspecies in the ®eld.

Appendix

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