13
Terpenoid composition of three fossil resins from Cretaceous and Tertiary conifers Angelika Otto a; , Bernd R.T. Simoneit a , Volker Wilde b , Lutz Kunzmann c , Wilhelm Pu « ttmann d a Environmental and Petroleum Geochemistry Group, College of Oceanic and Atmospheric Sciences, Oregon State University, Corvallis, OR 97331, USA b Forschungsinstitut Senckenberg, Sektion Pala «obotanik, D-60325 Frankfurt/Main, Germany c Staatliches Museum fu «r Mineralogie und Geologie zu Dresden, D-01109 Dresden, Germany d Institut fu «r Mineralogie^Umweltanalytik, J.W. Goethe-Universita «t Frankfurt/Main, D-60054 Frankfurt/Main, Germany Received 14 March 2001; received in revised form 18 September 2001; accepted 11 December 2001 Abstract The terpenoid composition of three fossil resins from macrofossils of Cretaceous and Tertiary conifers has been analyzed by gas chromatography^mass spectrometry (GC^MS). The mono-, sesqui- and diterpenoids which have been identified in the resin extracts are derived from precursors produced by the respective source plants and may be used as chemosystematic markers when compared with terpenoids in extant conifers. Sesquiterpenoids (cedrene, cuparene, cadinanes) and phenolic diterpenoids (ferruginol and derivatives) are the major components in Cupressospermum saxonicum Mai (Miocene). The terpenoid characteristics strongly support a relationship to the Cupressaceae s. str. The resin of Doliostrobus taxiformis (Sternberg) Kvac ›ek (Eocene) consists of abietane and pimarane type resin acids accompanied by minor amounts of phenolic diterpenoids (ferruginol, hinokiol). According to morphological and anatomical characteristics, D. taxiformis was previously compared to both, extant Araucariaceae and Cupressaceae s.l., but the terpenoid pattern of the resin now supports a relationship to the Cupressaceae s.l. rather than to Araucariaceae. Degraded diterpenoids of the abietane type are the major compounds in the extract of Tritaenia linkii (Roemer) Ma «gdefrau et Rudolf (Lower Cretaceous) indicating considerable oxidative alteration of the resin. Since the terpenoids in the resin of T. linkii are highly degraded or belong to the common abietane class, the leaves cannot be assigned or compared to any modern family based on their terpenoid composition. The presence of ferruginol probably excludes pinaceous affinities. Terpenoids proved to be valuable chemosystematic markers for fossil conifers once they are adequately preserved. The analysis of resin extracts by GC^MS is a suitable tool for the investigation of soluble compounds in fossil plants. ȣ 2002 Elsevier Science B.V. All rights reserved. Keywords: conifers; Cupressaceae; fossil resin; gas chromatography^mass spectrometry; terpenoids 0034-6667 / 02 / $ ^ see front matter ȣ 2002 Elsevier Science B.V. All rights reserved. PII:S0034-6667(02)00072-6 * Corresponding author. Present address: Institut fu «r Mineralogie^Umweltanalytik, J.W. Goethe-Universita «t Frankfurt/Main, Georg-Voigt-str. 14, D-60054 Frankfurt/Main, Germany. Fax +49-69-79828702. E-mail addresses: [email protected] (A. Otto), [email protected] (V. Wilde), [email protected] (L. Kunzmann), [email protected] (W. Pu « ttmann). Review of Palaeobotany and Palynology 120 (2002) 203^215 www.elsevier.com/locate/revpalbo

Terpenoid composition of three fossil resins from Cretaceous and Tertiary conifers

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Page 1: Terpenoid composition of three fossil resins from Cretaceous and Tertiary conifers

Terpenoid composition of three fossil resins fromCretaceous and Tertiary conifers

Angelika Otto a;�, Bernd R.T. Simoneit a, Volker Wilde b, Lutz Kunzmann c,Wilhelm Pu«ttmann d

a Environmental and Petroleum Geochemistry Group, College of Oceanic and Atmospheric Sciences, Oregon State University,Corvallis, OR 97331, USA

b Forschungsinstitut Senckenberg, Sektion Pala«obotanik, D-60325 Frankfurt/Main, Germanyc Staatliches Museum fu«r Mineralogie und Geologie zu Dresden, D-01109 Dresden, Germany

d Institut fu«r Mineralogie^Umweltanalytik, J.W. Goethe-Universita«t Frankfurt/Main, D-60054 Frankfurt/Main, Germany

Received 14 March 2001; received in revised form 18 September 2001; accepted 11 December 2001

Abstract

The terpenoid composition of three fossil resins from macrofossils of Cretaceous and Tertiary conifers has beenanalyzed by gas chromatography^mass spectrometry (GC^MS). The mono-, sesqui- and diterpenoids which have beenidentified in the resin extracts are derived from precursors produced by the respective source plants and may be usedas chemosystematic markers when compared with terpenoids in extant conifers. Sesquiterpenoids (cedrene, cuparene,cadinanes) and phenolic diterpenoids (ferruginol and derivatives) are the major components in Cupressospermumsaxonicum Mai (Miocene). The terpenoid characteristics strongly support a relationship to the Cupressaceae s. str.The resin of Doliostrobus taxiformis (Sternberg) Kvac›ek (Eocene) consists of abietane and pimarane type resin acidsaccompanied by minor amounts of phenolic diterpenoids (ferruginol, hinokiol). According to morphological andanatomical characteristics, D. taxiformis was previously compared to both, extant Araucariaceae and Cupressaceaes.l., but the terpenoid pattern of the resin now supports a relationship to the Cupressaceae s.l. rather than toAraucariaceae. Degraded diterpenoids of the abietane type are the major compounds in the extract of Tritaenia linkii(Roemer) Ma«gdefrau et Rudolf (Lower Cretaceous) indicating considerable oxidative alteration of the resin. Since theterpenoids in the resin of T. linkii are highly degraded or belong to the common abietane class, the leaves cannot beassigned or compared to any modern family based on their terpenoid composition. The presence of ferruginolprobably excludes pinaceous affinities. Terpenoids proved to be valuable chemosystematic markers for fossil conifersonce they are adequately preserved. The analysis of resin extracts by GC^MS is a suitable tool for the investigation ofsoluble compounds in fossil plants. 9 2002 Elsevier Science B.V. All rights reserved.

Keywords: conifers; Cupressaceae; fossil resin; gas chromatography^mass spectrometry; terpenoids

0034-6667 / 02 / $ ^ see front matter 9 2002 Elsevier Science B.V. All rights reserved.PII: S 0 0 3 4 - 6 6 6 7 ( 0 2 ) 0 0 0 7 2 - 6

* Corresponding author. Present address: Institut fu«r Mineralogie^Umweltanalytik, J.W. Goethe-Universita«t Frankfurt/Main,Georg-Voigt-str. 14, D-60054 Frankfurt/Main, Germany. Fax +49-69-79828702.

E-mail addresses: [email protected] (A. Otto), [email protected] (V. Wilde), [email protected](L. Kunzmann), [email protected] (W. Pu«ttmann).

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1. Introduction

Terpenoids are common constituents of theresins of higher plants and they are useful che-mosystematic characteristics of extant plants,especially conifers (Hegnauer, 1962, 1992;Langenheim, 1969; Erdtman and Norin, 1966;Thomas, 1986; Otto and Wilde, 2001). The chem-ical composition of many fossil resins and ambershas been analyzed and the identi¢ed compoundswere used for a chemosystematic evaluation of thesource plants (e.g. Langenheim, 1969; Gough andMills, 1972; Mills et al., 1984; Simoneit et al.,1986; Grimalt et al., 1988; Czechowski et al.,1996; Alonso et al., 2000). Since many ambersare preserved separately from remains of theirsource plant, it is di⁄cult to determine the botan-ical source based on the chemical characteristicsof the amber alone. However, the wood, shoots,and cones of fossil conifers may still contain res-ins which are preserved in situ or intimately asso-ciated with the plant material thus providingchemical and morphological characteristics ofthe fossil plant at the same time. Most of thecompounds identi¢ed in the fossil resins are thediagenetic products (biomarkers) of terpenoidswhich were synthesized by living organisms (Si-moneit, 1986). Despite various chemical altera-tions during diagenesis, the biomarkers still havethe characteristic basic skeletal structures of theirprecursors and can thus be used as chemosys-tematic markers.Langenheim (1969) stated that resinous materi-

al potentially preserved in macroscopic remains ofconifers may have been overlooked before. Untilnow, only a few resins preserved in or closely

associated with macrofossils have been analyzedat all (e.g. Va¤vra and Walther, 1993; Andersonand LePage, 1995). Va¤vra and Walther (1993) de-tected mono- and sesquiterpenoids in the extractsof the resin of Cunninghamia miocenica Ettings-hausen (Cupressaceae s.l.) from Miocene sedi-ments in Germany using gas chromatography^mass spectrometry (GC^MS). The identi¢ed ter-penoids were unaltered terpenoids and diageneticproducts of natural precursors which are knownfrom related extant species of Cupressaceae s.l.Resinites associated with fossil cones of Metase-quoia Miki (Cupressaceae s.l.), Pinus L. and Pseu-dolarix Gordon (both Pinaceae) from the Eoceneof Axel Heiberg Island, Canada, have been ana-lyzed by pyrolysis^GC^MS (Py^GC^MS), and itwas speculated that Pseudolarix and the succiniteproducing species share a common ancestor (An-derson and LePage, 1995).For the present paper the terpenoid composi-

tions of the resins of three selected conifers ofCretaceous to Tertiary age were studied (Table1). The systematic of conifers as applied in thepresent study follows the recent concept of theCupressaceae (Cupressaceae s.l. ; Gadek et al.,2000), including a monophyletic alliance compris-ing the former Cupressaceae ( =Cupressaceae s.str.) and a number of isolated clades which wereunited before under the Taxodiaceae. The rankand position of the Geinitziaceae, as introducedby Kunzmann (1999) in the sense of a morpho-logically and anatomically intermediate extincttaxon between former Cupressaceae and Taxodia-ceae, are in need of clari¢cation by more compre-hensive studies which are beyond the scope of thepresent paper.

Table 1Samples of fossil conifer resins

Species Organ Stratigraphy Locality

Cupressospermum saxonicum Mai shoot Neogene, Lower Miocene(Spremberg Member)

Kleinsaubernitz near Bautzen,Sachsen

Doliostrobus taxiformis Sternberg(Kvac›ek)

cone Paleogene, Middle Eocene(Messel Formation)

Messel near Darmstadt, Hessen

Tritaenia linkii (Roemer) Ma«gdefrau etRudolf

leaves, resin Lower Cretaceous, Berriasian(Bu«ckeberg Formation)

Hils south of Hannover,Niedersachsen

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2. Materials and methods

2.1. Samples and sample preparation

Needle-like leaves with a length of up to 8 cmhave repeatedly been described from Wealden-fa-cies sediments (Bu«ckeberg Formation, Berriasian,Lower Cretaceous; Pelzer and Wilde, 1987) of theHils, Deister and Osterwald south of Hannover(Niedersachsen, Germany). They may be assignedto Tritaenia linkii (Roemer) Ma«gdefrau et Rudolf,the type species of a form genus for needle-likeleaves with more than two abaxial stomatal bands(Wilde, 1991; Manum et al., 2000). Sometimesthese leaves are found isolated, sometimes theyare almost exclusively accumulated in layers ofimpure coal up to more than 10 cm in thickness(Pelzer et al., 1992; Pelzer, 1998). No distinct res-in canals have been proven for the species, but theinternal tissues of the leaves were obviously richin resinous cell ¢llings (Manum et al., 2000).There are reddish^brown resin bodies embeddedin the matrix of the leaf coals which have beeninterpreted to be derived from the T. linkii plant.Although T. linkii obviously represents the foliageof a conifer, it could not be assigned or comparedto any of the modern families (Manum et al.,2000).The Middle Eocene oilshale of Messel near

Darmstadt (Hessen, Germany) ¢lls an isolatedstructure of tectonic or volcanic origin. It wasmined for about a century before the pit wasabandoned in 1972. Commercial mining was fol-lowed by scienti¢c exploration which turned thesite into a famous locality for extremely well pre-served fossils, especially vertebrates, insects, andplants (Schaal and Schneider, 1995). This ¢nallylead to it being declared as a World Heritage Siteby UNESCO (Schaal, 1996). The plant taphocoe-nosis of Messel comprises numerous taxa ofleaves, fruits and seeds, pollen and spores, andcomplete £owers (Schaarschmidt, 1988). Remainsof conifers are comparatively rare, includinga number of leafy shoots and cone scales ofDoliostrobus taxiformis (Sternberg) Kvac›ek( =D. cf. certus Bu'zek, Holy¤ et Kvac›ek: Wilde1989). They show the resin canals still ¢lled by

visible amounts of light yellow resin (Wilde,1989).

Cupressospermum saxonicum Mai represents anextinct genus. It is commonly found in terrestrialto marginally marine coal basins of Oligocene toMiocene age in Europe (Kunzmann, 1999), andfrequently dominated the initial peat formingplant associations of Miocene coals in easternGermany which represent eutrophic swamps (K-facies sensu Schneider, 1992). It has also beendiscussed as a possible source of amber whichmay have been produced by the respective plantsas a reaction to the in£uence of brackish water(Mai and Schneider, 1988). The resinous shoots ofC. saxonicum which were analyzed in the presentstudy were sampled from core KS 1/70 drilled atKleinsaubernitz near Bautzen (Sachsen, Ger-many). The material was taken from the fourthLusatian browncoal seam (Spremberg Member,Lower Miocene; local £oral zone II) which over-lies an Oligocene diatomite famous for a rich £ora(Walther, 1999). The specimen analyzed is thecounterpart to LA/KS 409 as illustrated by Kunz-mann (1999: pl. 21, ¢g. 2) and was de¢ned bycuticular analysis.The resins of Cupressospermum saxonicum and

Doliostrobus taxiformis were picked from theshoots and cone scales, respectively, with a scalpelpreviously cleaned with organic solvent. Tritaenialinkii leaves and the associated resin bodies weresampled during mechanical disintegration of theleaf coal by careful splitting with a scalpel.

2.2. Extraction and derivatization

The resins and Tritaenia linkii leaves were pul-verized and sonicated three times with dichloro-methane:methanol (1:1; v/v) for 5 min. The totalextracts were ¢ltered, dried under nitrogen blow-down and weighed. The Cupressospermum resinyielded 100%, the Doliostrobus resin 91%, the Tri-taenia resin 84% and the Tritaenia leaves 1.2%extract, respectively. Aliquots of the total extractswere converted to trimethylsilyl derivatives by re-action with N,O-bis-(trimethylsilyl)tri£uoroaceta-mide and pyridine for 3 h at 70‡C to deactivatepolar functional groups.

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Table 2Terpenoids identi¢ed in the resins of Cupressospermum saxonicum (Cup) and Doliostrobus taxiformis (Dol), and in the resin andleaves of Tritaenia linkii (Tri)

No. Name Composition MW IDa Relative abundanceb

Cup Dol Tri

resin leaves

1 Borneol C10H18O 154 S 14.4 ^ ^ ^2 1,6,8-Trimethyl-1,2,3,4-tetrahydronaphthalene C13H18 174 L ^ ^ 2.6 ^3 1,1,6-Trimethyl-1,2,3,4-tetrahydronaphthalene C13H18 174 L ^ ^ ^ 4.04 1,2-Dihydro-1,1,2,6-tetramethylnaphthalene C14H18 186 I ^ ^ ^ 5.85 Pentamethylindane C14H20 188 S ^ ^ 36.3 ^6 Methylionene C14H20 188 I ^ ^ 59.6 100.07 Tetramethylbutylbenzene C14H22 190 I ^ ^ 8.4 ^8 L-Ionone= Irisone C13H20O 192 L ^ ^ 26.8 ^9 Cadalene C15H18 198 L ^ ^ 18.9 36.610 Calamenene isomer C15H22 202 I ^ ^ 100.0 ^11 Calamenene C15H22 202 S, L 25.0 0.5 87.8 ^12 Calamenene isomer C15H22 202 I ^ ^ 87.8 ^13 5,6,7,8-Tetrahydrocadalene C15H22 202 L 6.0 0.3 21.9 13.414 5,6,7,8-Tetrahydrocadalene isomer C15H22 202 I ^ ^ 21.9 ^15 K-Cedrene C15H24 204 S 9.1 ^ ^ ^16 Dihydro-ar-curcumene C15H24 204 S 19.1 0.8 ^ ^17 Unknown sesquiterpenol C15H26O 222 ^ 4.1 ^ ^ ^18 Unknown sesquiterpenol C15H26O 222 ^ 13.2 ^ ^ ^19 Unknown sesquiterpenol C15H26O 222 ^ 11.1 ^ ^ ^20 Unknown sesquiterpenol C15H26O 222 ^ 9.2 ^ ^ ^21 Unknown sesquiterpenol C15H26O 222 ^ 9.6 ^ ^ ^22 Unknown sesquiterpenol C15H26O 222 ^ 16.7 ^ ^ ^23 18,19-Bisnorsimonellite C17H20 224 I ^ ^ ^ 10.424 15,16,17-Trisnorabieta-8,11,13-triene C17H24 228 I ^ ^ 11.0 ^25 16,17,19-Trisnorabieta-8,11,13-triene C17H24 228 I ^ ^ 76.8 ^26 16,17,18-Trisnorabieta-8,11,13-triene C17H24 228 I ^ ^ 26.7 ^27 1,2,3,4-Tetrahydroretene C18H22 238 L ^ ^ 3.1 7.028 16,17-Bisnordehydroabietane C18H26 242 I ^ ^ 26.8 9.629 7-Oxo-16,17,18-trisnorabieta-8,11,13-triene C17H22O 242 I ^ ^ 13.5 ^30 Simonellite C19H24 252 S ^ ^ 0.9 1.631 5L(H)-18-Norabieta-8,11,13-triene C19H28 256 L ^ ^ 2.7 ^32 12-Hydroxysimonellite C19H24O 268 I 46.8 ^ 4.3 ^33 16,17-Bisnordehydroabietic acid C18H24O2 272 I ^ ^ 34.8 ^34 6,7-Dehydroferruginol C20H28O 284 I 5.7 ^ 0.6 ^35 Ferruginol C20H30O 286 S 100.0 2.6 6.0 ^36 Dehydroabietic acid C20H28O2 300 S ^ 100.0 ^ ^37 Sugiol (7-ketoferruginol) C20H28O2 300 S 3.1 ^ ^ ^38 10K-9,10-Secodehydroabietic acid C20H30O2 302 I ^ 0.6 ^ ^39 10L-9,10-Secodehydroabietic acid C20H30O2 302 I ^ 1.0 ^ ^40 Isopimara-8,15-dien-18-oic acid C20H30O2 302 I ^ 1.3 ^ ^41 Isopimaric acid C20H30O2 302 S ^ 0.4 ^ ^42 Abietic acid C20H30O2 302 S ^ 1.4 ^ ^43 1- or 2-Hydroxyferruginol C20H30O2 302 I 9.1 ^ ^ ^44 Hinokiol (3-hydroxyferruginol) C20H30O2 302 S 5.8 0.6 ^ ^45 Isopimar-8-en-18-oic acid C20H32O2 304 I ^ 0.2 ^ ^46 Pimar-8-en-18-oic acid C20H32O2 304 I ^ 5.1 ^ ^47 13K(H)-Abiet-8(14)-en-18-oic acid C20H32O2 304 I ^ 0.3 ^ ^48 13L(H)-Abiet-8-en-18-oic acid C20H32O2 304 I ^ 3.4 ^ ^49 Isopimar-7-en-18-oic acid C20H32O2 304 I ^ 5.0 ^ ^50 13L(H)-Abiet-7-en-18-oic acid C20H32O2 304 I ^ 0.4 ^ ^

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2.3. GC^MS

GC^MS analyses of both the underivatized andderivatized total extracts were performed on aHewlett-Packard model 6890 GC coupled to aHewlett-Packard model 5973 quadrupole MSD.Separation was achieved on a fused silica capillarycolumn coated with DB5 (30 mU0.25 mm i.d.,0.25 Wm ¢lm thickness). The GC operating con-ditions were as follows: temperature hold at 65‡Cfor 2 min, increase from 65 to 300‡C at a rate of6‡C min31 with ¢nal isothermal hold at 300‡C for20 min. Helium was used as carrier gas. The sam-ple was injected splitless with the injector temper-ature at 300‡C. The mass spectrometer was oper-ated in the electron impact mode at 70 eVionization energy and scanned from 50 to 650Da. Data were acquired and processed with theChemstation software. Individual compoundswere identi¢ed by comparison of mass spectrawith literature and library data, comparison withauthentic standards and interpretation of mass

spectrometric fragmentation patterns and GC re-tention indices. Due to clipping of the GC columnthe retention times of compounds changed andwere re-calculated using standard reference com-pounds.

3. Results and discussion

3.1. Cupressospermum saxonicum Mai(Lower Miocene)

The total extract of the resin from the shoots ofCupressospermum saxonicum contains severalmono-, sesqui- and diterpenoids (Fig. 1). The rel-ative abundances of the identi¢ed compounds aregiven in Table 2. Mass spectrometric data of thecompounds identi¢ed by interpretation of theirfragmentation patterns are listed in Table 3.The monoterpenoid borneol (1) has been de-

scribed from several ambers (e.g. Mills et al.,1984; Czechowski et al., 1996). Borneol occurs

Fig. 1. Derivatized total extract of the resin from Cupressospermum saxonicum, Kleinsaubernitz, Miocene. Numbers refer toTable 2.

a ID= Identi¢cation of compound: S= standard, I = interpretation of mass spectral patterns, L=published data in literature(Simoneit and Mazurek, 1982; Philp, 1985) or Wiley MS library.

b Relative abundance normalized to major compound=100.

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Table 3Mass spectrometric data of compounds identi¢ed by interpretation of their fragmentation patterns

No. Compound name Composition MW Characteristic fragmentsm/z (%)

4 1,2-Dihydro-1,1,2,6-tetramethylnaphthalene C14H18 186 171 (100), 156 (50), 186 (29), 141 (16), 155(16), 172 (14), 153 (9), 157 (9)

6 Methylionene C14H20 188 173 (100), 188 (17), 174 (15), 128 (9), 158(9), 143 (8), 145 (7), 115 (6)

7 Tetramethylbutylbenzene C14H22 190 147 (100), 190 (15), 133 (11), 148 (11), 91(6), 105 (5), 115 (5)

10 Calamenene isomer C15H22 202 159 (100), 128 (26), 129 (26), 115 (20), 160(14), 144 (13), 202 (9), 105 (7)

12 Calamenene isomer C15H22 202 159 (100), 129 (36), 128 (33), 160 (23), 115(18), 144 (17), 202 (12), 105 (9)

14 5,6,7,8-Tetrahydrocadalene isomer C15H22 202 187 (100), 202 (18), 188 (14), 172 (10), 157(9), 142 (7), 128 (6), 145 (6)

23 18,19-Bisnorsimonellite C17H20 224 209 (100), 224 (34), 210 (21), 165 (21), 179(20), 169 (18)

24 15,16,17-Trisnorabieta-8,11,13-triene C17H24 228 131 (100), 213 (91), 157 (42), 228 (27), 128(22), 143 (22), 185 (22), 214 (18)

25 16,17,19-Trisnorabieta-8,11,13-triene C17H24 228 131 (100), 213 (72), 157 (37), 228 (25), 143(18), 128 (16), 141 (13), 142 (12)

26 16,17,18-Trisnorabieta-8,11,13-triene C17H24 228 131 (100), 213 (72), 157 (45), 228 (24), 159(20), 143 (18), 128 (16), 185 (13)

28 16,17-Bisnordehydroabietane C18H26 242 227 (100), 145 (76), 131 (66), 157 (57), 242(40), 143 (36), 69 (36), 171 (27)

29 7-Oxo-16,17,18-trisnorabieta-8,11,13-triene C17H22O 242 145 (100), 227 (90), 171 (41), 242 (29), 228(22), 128 (16), 141 (16), 199 (14)

32 12-Hydroxysimonellite C19H24O 268 253 (100), 268 (54), 211 (21), 254 (18), 165(14), 209 (12), 178 (10), 152 (8)

12-Hydroxysimonellite-TMS C22H32OSi 340 325 (100), 340 (84), 73 (42), 326 (28), 341(25), 283 (10), 237 (9), 251 (6)

33 16,17-Bisnordehydroabietic acid-TMS C21H32O2Si 344 211 (100), 212 (18), 73 (15), 145 (14), 329(14), 157 (13), 344 (11), 227 (8)

34 6,7-Dehydroferruginol C20H28O 284 202 (100), 284 (92), 199 (67), 213 (57), 200(44), 185 (40), 159 (31), 269 (31)

6,7-Dehydroferruginol-TMS C23H36O2Si 356 356 (100), 73 (61), 341 (51), 285 (27), 300(18), 221 (17), 313 (13), 257 (11)

38 10K-9,10-Secodehydroabietic acid-TMS C23H38O2Si 374 73 (100), 146 (74), 133 (48), 117 (39), 263(39), 284 (32), 359 (28), 374 (20)

39 10L-9,10-Secodehydroabietic acid-TMS C23H38O2Si 374 73 (100), 146 (62), 263 (60), 133 (52), 117(39), 374 (37), 173 (31), 91 (27),

40 Isopimara-8,15-dien-18-oic acid-TMS C23H38O2Si 374 241 (100), 73 (60), 256 (28), 359 (24), 257(24), 242 (21), 91 (18), 374 (14)

43 1- or 2-Hydroxyferruginol-di-TMS C26H46O2Si2 302 446 (100), 73 (53), 447 (40), 431 (30), 341(27), 261 (18), 299 (18), 103 (13)

45 Isopimar-8-en-18-oic acid-TMS C23H40O2Si 376 73 (100), 243 (64), 361 (58), 376 (35), 145(27), 105 (24), 131 (24), 175 (22)

46 Pimar-8-en-18-oic acid-TMS C23H40O2Si 376 243 (100), 73 (45), 258 (23), 244 (220), 361(20), 259 (18), 376 (13), 91 (11)

47 13K(H)-Abiet-8(14)-en-18-oic acid-TMS C23H40O2Si 376 73 (100), 121 (66), 376 (60), 107 (56), 215(45), 259 (44), 361 (44), 120 (36)

48 13L(H)-Abiet-8-en-18-oic acid-TMS C23H40O2Si 376 243 (100), 73 (62), 258 (54), 361 (47), 259(35), 376 (25), 244 (22), 105 (18)

49 Isopimar-7-en-18-oic acid-TMS C23H40O2Si 376 243 (100), 73 (93), 258 (71), 361 (51), 259(49), 376 (32), 91 (26), 244 (25)

50 13L(H)-Abiet-7-en-18-oic acid-TMS C23H40O2Si 376 73 (100), 237 (46), 376 (44), 75 (26), 361(24), 143 (19), 93 (18), 259 (18)

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in modern conifers (Sukh Dev, 1989) and hasbeen preserved unchanged in the fossil resin.The distribution of K-cedrene (15) in modern

conifers seems to be restricted to species of theCupressaceae s. str. (Hegnauer, 1992; Otto andWilde, 2001). The occurrence of this sesquiterpe-noid in the resin thus suggests a relationship ofCupressospermum saxonicum to the Cupressaceaes. str. Dihydro-ar-curcumene (16) is an aromaticsesquiterpenoid which probably derived from bi-sabolane type precursors. Calamenene (11) and5,6,7,8-tetrahydrocadalene (13) of the cadinanetype also occur in the resin extract. Bisabolanesand cadinanes are common constituents of higherplants and thus non-speci¢c markers (Karrer etal., 1977; Hu«rlimann and Cherbuliez, 1981;Otto and Wilde, 2001). A series of unknown com-pounds (17^22) which were interpreted as sesqui-terpenols according to their mass spectrometricfragmentation patterns and molecular weights(MW) have also been observed in the resin ex-tract.Phenolic abietanes are the major diterpenoids

in the Cupressospermum saxonicum resin andcomprise 6,7-dehydroferruginol (34), ferruginol(35), 12-hydroxysimonellite (32), 1- or 2-hydroxy-ferruginol (43), sugiol (7-ketoferruginol, 37), andhinokiol (3-hydroxyferruginol, 44). Ferruginol,hydroxyferruginols, hinokiol and sugiol are natu-ral products known from modern conifers, espe-cially from the Cupressaceae s.l. and Podocarpa-ceae (Karrer, 1958; Erdtman and Norin, 1966;Thomas, 1970; Hegnauer, 1962, 1992). 6,7-Dehy-droferruginol (34) and 12-hydroxysimonellite (32)are interpreted to be diagenetic products fromferruginol and its derivatives.

Cupressospermum saxonicum was initiallyplaced in the Cupressaceae s. str. by Mai (1960).Cupressaceous a⁄nities were later denied by Maiand Schneider (1988) in comparing the species toextinct Mesozoic conifers of unknown or uncleara⁄nities. Kunzmann (1999) recently assigned thespecies to the Geinitziaceae, a new extinct taxonwhich he regarded as morphologically and ana-tomically intermediate between former Cupressa-ceae and Taxodiaceae. The chemical characteris-tics of the resin are in accordance with a positionof C. saxonicum near to (or even among) the Cu-

pressaceae s. str. Since the terpenoids identi¢ed inour material are known from several genera of theCupressaceae s. str., it can chemically not be com-pared to a modern genus of the respective clade.The chemical results would thus support a posi-tion of the Geinitziaceae sensu Kunzmann (1999)near to the extant Cupressaceae s. str.

3.2. Doliostrobus taxiformis (Sternberg) Kvac›ek(Middle Eocene)

The terpenoids identi¢ed in the total extract ofthe Doliostrobus taxiformis resin are listed in Ta-ble 2 and shown in Fig. 2. Diterpenoids of theabietane and pimarane classes are the predomi-nant compounds in the resin.The non-speci¢c sesquiterpenoids dihydro-ar-

curcumene (16), calamenene (11), and 5,6,7,8-tet-rahydrocadalene (13), present in Cupressosper-mum saxonicum, have also been identi¢ed in theDoliostrobus taxiformis resin.The diterpenoids are comprised mainly of un-

changed resin acids of the abietane, pimarane andisopimarane classes and their diagenetic products(Fig. 2a). Dehydroabietic acid (36) is the domi-nant terpenoid of the Doliostrobus taxiformis resinand comprises approximately 80% of the totalextract. Dehydroabietic acid is the alterationproduct of the biological precursor abietic acid(42) which is also preserved in the resin. The re-maining abietane type resin acids present in theextract are also interpreted as diagenetic productsfrom abietic acid. 13K(H)-Abiet-8(14)-en-18-oicacid (47), 13L(H)-abiet-8-en-18-oic acid (48) and13L(H)-abiet-7-en-18-oic acid (50) were probablygenerated by the reduction of one double bond inabietic acid followed by further isomerization pro-cesses. In 10K-9,10-secodehydroabietic acid (38)and its isomer 10L-9,10-secodehydroabietic acid(39) ring B of dehydroabietic acid was openedby diagenetic processes. The resin acids of theisopimarane series comprise isopimaric acid (41)as the biological precursor and its presumed dia-genetic products isopimara-8,15-dien-18-oic acid(40), isopimar-8-en-18-oic acid (45) and isopi-mar-7-en-18-oic acid (49). The pimarane type di-terpenoids are represented only by pimar-8-en-18-oic acid (46). Resin acids of the abietane, isopi-

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marane and pimarane classes are abundant con-stituents of conifer resins (Thomas, 1970; Heg-nauer, 1962, 1992; Otto and Wilde, 2001). Dueto their wide distribution among the conifer spe-cies they are non-speci¢c chemosystematicmarkers.Minor amounts of the phenolic diterpenoids

ferruginol (35) and hinokiol (44) are also foundin the resin (Fig. 2b). Ferruginol is common inmodern species of Cupressaceae s. l. and Podocar-paceae (Karrer, 1958; Thomas, 1970; Hegnauer,1962, 1992). Hinokiol (3-hydroxyferruginol) hashitherto been described only from Cupressaceaes.l. and Torreya Arn. (Taxaceae) (Erdtman and

Fig. 2. Derivatized total extract of the resin from Doliostrobus taxiformis, Messel, Eocene: (a) total ion current trace, and (b) en-larged section of the total ion current trace. Numbers refer to Table 2.

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Norin, 1966; Harrison and Akasawa, 1987). Fer-ruginol derivatives are abundant in Cupressaceaes.l. and Podocarpaceae, but were only rarely re-ported from species of Pinaceae (Hegnauer, 1962,1992; Otto and Wilde, 2001).The anatomical and morphological characteris-

tics of Doliostrobus taxiformis led to comparisonswith modern Araucariaceae (e.g. Bu'zek et al.,1968) and the cunninghamioid alliance of formerTaxodiaceae (e.g. Mai, 1976). According to someterpenoids (ferruginol, hinokiol) identi¢ed in theresin, D. taxiformis shows a closer relationship toCupressaceae s.l. than to Araucariaceae. Ferrugi-nol derivatives were hitherto reported from onlyone species of Araucariaceae, and tetracyclic di-terpenoids (e.g. kauranes, phyllocladanes), whichare common in the Araucariaceae (Otto andWilde, 2001), were not detected in Doliostrobus.A similar composition of abietane, isopimaraneand pimarane type diterpenoids accompanied byferruginol and hinokiol has been observed in res-inous cones of Cunninghamia chaneyi from theMiocene Clarkia formation in Idaho and extantCunninghamia lanceolata (Lambert) Hooker (Otto

et al., in preparation). This supports recent resultsfrom morphological and anatomical studies whichplace D. taxiformis again nearer Cunninghamia R.Brown (Kunzmann, 1999), an extant genus form-ing a monogeneric alliance within the Cupressa-ceae s.l. (Gadek et al., 2000; Kusumi et al., 2000).Interestingly, Cunninghamia appears repeatedlynext to Araucaria Jussien in a recent analysiscombining morphological and anatomical datafrom a number of extant and extinct conifers(Miller, 1999).

3.3. Tritaenia linkii (Roemer) Ma«gdefrau etRudolf (Lower Cretaceous)

Sesquiterpenoids of the cadalane type andmonoaromatic diterpenoids are the major constit-uents of the leaf and resin extracts accompaniedby substituted hydronaphthalenes (Figs. 3 and 4;Table 2).

L-Ionone (8), tetramethylbutylbenzene (7),1,6,8-trimethyl-1,2,3,4-tetrahydronaphthalene (2),pentamethylindane (5), and methylionene (6) arehighly degraded diagenetic products of various

Fig. 3. Derivatized total extract of the resin from Tritaenia linkii, Hils, Lower Cretaceous. Numbers refer to Table 2.

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sesqui- and diterpenoids (Bendoraitis, 1974).These degradation products thus cannot be as-signed to certain terpenoid classes, because thebasic structure of their parent molecules hasbeen severely altered by oxidation during diagen-esis. The predominant sesquiterpenoids in the res-in extract belong to the non-speci¢c cadinane se-ries and are comprised of calamenene (11), twocalamenene isomers (10 and 12), 5,6,7,8-tetrahy-drocadalene (13) and its isomer (14), and cadalene(9). The calamenene and tetrahydrocadalene iso-mers di¡er in the stereochemical position of hy-drogen and were generated by isomerization pro-cesses during diagenesis (Alexander et al., 1994;Barstow et al., 1997).A series of mono- and diaromatic abietane di-

terpenoids have been identi¢ed in the Tritaenialinkii resin. 16,17-Bisnordehydroabietic acid (33),15,16,17-trisnorabieta-8,11,13-triene (24), 16,17,19-trisnorabieta-8,11,13-triene (25), 16,17,18-tris-norabieta-8,11,13-triene (26), 16,17-bisnordehy-droabietane (28), 7-oxo-16,17,18-trisnorabieta-8,11,13-triene (29), 5L(H)-18-norabieta-8,11,13-

triene (31), 1,2,3,4-tetrahydroretene (27), and si-monellite (30) are interpreted as successively de-graded abietane type diterpenoids (e.g. Simoneit,1986). Pimaranes and isopimaranes may also beprecursors for the tris- and bisnorabietanes. Abie-tanes, pimaranes, and isopimaranes are widelydistributed among extant conifers and thereforenon-speci¢c terpenoid classes (Hegnauer, 1962,1992; Otto and Wilde, 2001). The resin extractalso contains small amounts of 6,7-dehydroferru-ginol (34), ferruginol (35), and 12-hydroxysimo-nellite (32). Since the phenolic abietanes occur inmost of the conifer families, but only rarely inPinaceae, the presence of ferruginol and its deriv-ative may preclude an assignment of T. linkii tothe Pinaceae.The terpenoid pattern of the Tritaenia linkii

leaves (Fig. 4) is similar to the resin extract whichindicates that the respective plants most probablywere the source of the isolated resin bodies. Theleaf extract consists of hydronaphthalene deriva-tives, cadinane type sesquiterpenoids and abietanediterpenoids (Fig. 4). Methylionene (6) is the ma-

Fig. 4. Derivatized total extract of the leaves from Tritaenia linkii, Hils, Lower Cretaceous. Numbers refer to Table 2.

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jor compound in the leaf extract. 1,1,6-Trimethyl-1,2,3,4-tetrahydronaphthalene (3) and 1,2-dihy-dro-1,1,2,6-tetramethylnaphthalene (4) are furtherhighly degraded diagenetic products which cannotbe assigned to a speci¢c terpenoid source. Thecadinane sesquiterpenoids encompass 5,6,7,8-tet-rahydrocadalene (13) and cadalene (9). Themono- and diaromatic abietane derivatives in-clude 16,17-bisnordehydroabietane (28), 1,2,3,4-tetrahydroretene (27), simonellite (30), and18,19-bisnorsimonellite (23). Cadinane type ses-quiterpenoids and substituted hydronaphthaleneshave also been described as the major compoundsextracted from T. linkii leaves in coals of theNorthwest German Wealden-facies by micro-ther-modesorption (Heppenheimer et al., 1992).The dominance of highly degraded terpenoid

products indicates a major oxidative alterationof both the leaves and resin of Tritaenia linkii atthis locality. Since the material was not heavilya¡ected by thermal alteration (Teichmu«ller andTeichmu«ller, 1950), the organic matter probablywas oxidized by high microbial activity and aera-tion during early diagenesis. This could be ex-plained by the fact that the leaves were obviouslyaccumulated along the shoreline of interchannellakes (Pelzer et al., 1992; Pelzer, 1998; Manumet al., 2000), where they were exposed to atmo-spheric conditions for some time before burial.The terpenoids in the resin and leaves of T. linkiicannot be assigned to certain terpenoid classesdue to their high degradation (substituted hydro-naphthalenes) and the presence of non-speci¢csesquiterpenoids (cadinanes) and diterpenoids(abietanes). According to its terpenoid character-istics, T. linkii thus cannot be assigned to a spe-ci¢c conifer taxon. Since ferruginol derivatives arecommon among all conifers with the exception ofPinaceae, their presence in the T. linkii resin prob-ably precludes pinaceous a⁄nities.

4. Conclusions

The resins of three fossil conifers containmono-, sesqui- and diterpenoids. Sesquiterpenoidsof the cadalane type and diterpenoids of the abie-tane and isopimarane classes dominate in the res-

ins of Miocene Cupressospermum saxonicumshoots and the cone scales of Eocene Doliostrobustaxiformis. The Cretaceous Tritaenia linkii resinconsists mainly of substituted hydronaphthalenes,cadinane type sesquiterpenoids, and degradedabietane derivatives.Terpenoids are useful chemosystematic markers

for the systematic assignment of extant conifers(Hegnauer, 1962, 1992; Erdtman, 1968; Langen-heim, 1969; Thomas, 1986; Otto and Wilde,2001). The chemosystematic relationships of thefossil species analyzed were interpreted by evalu-ating the distribution of the identi¢ed terpenoidsor their biological precursors in extant species.The chemical characteristics of Cupressosper-

mum saxonicum are in accordance with a relation-ship to the Cupressaceae s. str., because the resincontains terpenoids characteristic (phenolic abie-tanes) and in part unique (K-cedrene) for thisclade. The undegraded phenolic diterpenoid ferru-ginol and some of its diagenetic derivatives are thepredominant markers in C. saxonicum.According to morphological and anatomical

characteristics, Doliostrobus taxiformis was com-pared to the Taxodiaceae or Araucariaceae. Theterpenoids identi¢ed in the D. taxiformis resinsupport a closer relationship to the Cupressaceaes.l., because they occur in modern species of thisfamily but have not been reported from Arauca-riaceae. There is even some chemical support for arelationship to the cunninghamioid clade withinthe Cupressaceae s.l.

Tritaenia linkii cannot be related to a distinctconifer taxon according to the terpenoid charac-teristics, because the resin contains only non-spe-ci¢c sesqui- and diterpenoids (cadinanes, abie-tanes) and highly degraded terpenoid derivativeswhich cannot be assigned to their parent terpe-noid classes. The presence of ferruginol deriva-tives weakly excludes pinaceous a⁄nities.In summary, resins preserved in situ or closely

associated with macrofossils are suitable for theanalysis of terpenoids as chemosystematicmarkers. The terpenoids may have been trappedin the resin and thus protected against major deg-radation processes. The analysis of resin extractsby GC^MS is a valuable tool for paleochemosys-tematic studies. It is of advantage compared to

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Py^GC^MS, because the compounds in the ex-tract can be identi¢ed as originally preserved inthe fossil while during pyrolysis the structuresmay be thermally altered.

Acknowledgements

Financial support from the German ResearchFoundation (DFG, Grant Ot 175/1-1), Bonn,and the Max-Kade-Foundation, New York, isgratefully acknowledged. We thank Dr. Ken B.Anderson and Dr. P. van Bergen for their usefulsuggestions to improve this paper.

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