Chemical Composition of Paleozoic and Mesozoic Fossil Invertebrate

The cuticles of 15 fossil invertebrates ranging in age from Silurian to Cretaceous, and including both marine and terrestrial organisms, have been ana...
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Energy & Fuels 1997, 11, 515-521

515

Chemical Composition of Paleozoic and Mesozoic Fossil Invertebrate Cuticles As Revealed by Pyrolysis-Gas Chromatography/Mass Spectrometry B. Artur Stankiewicz,*,†,‡ Derek E. G. Briggs,† and Richard P. Evershed‡ Biogeochemistry Research Centre, Department of Geology, University of Bristol, Queen’s Road, Bristol BS8 1RJ, U.K., and Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, U.K. Received October 14, 1996. Revised Manuscript Received January 2, 1997X

The cuticles of 15 fossil invertebrates ranging in age from Silurian to Cretaceous, and including both marine and terrestrial organisms, have been analyzed using pyrolysis-gas chromatography/ mass spectrometry (py-GC/MS). Modern invertebrate cuticles were analyzed in the same way as a basis for comparison. The modern cuticles yielded pyrolysis products derived from chitin and proteins, but none of these components was detected in the pyrolysates of the fossil cuticles. The fossil cuticles fall into two, chemically distinct groups: aliphatic, yielding pairs of n-alk-1enes and n-alkanes upon pyrolysis, and aromatic, producing pyrolysates dominated by alkylbenzenes and alkylindenes. Aliphatic pyrolysates may derive through polymerization of lipids, e.g., epicuticular waxes, during diagenesis. Alternatively the aliphatic moieties found in algae (algaenan) or in plants (e.g., cutan, suberan) may have been incorporated into the animal cuticles by unknown diagenetic processes. Alkylindenes are major pyrolysis products of the fossil cuticles that generate predominantly aromatic components. This association may resolve the enigma of the frequent occurrence of alkylindenes as minor components in the pyrolysates of most types of kerogen. The abundant thiophenes in the same pyrolysates may reflect sulfur incorporation during diagenesis of the original amino sugar (glucosamine) moieties that comprise the chitin biopolymer.

Introduction Organic geochemical studies of sedimentary organic matter (SOM) have investigated materials ranging from peat and coal to kerogen. Many have focused on the investigation of identifiable organic matter such as algal biomass1-5 or parts of terrestrial vegetation.6-10 These investigations led to the wide acceptance of a model of kerogen formation through the selective preservation * Corresponding author. † Department of Geology. ‡ School of Chemistry. X Abstract published in Advance ACS Abstracts, March 1, 1997. (1) Largeau, C.; Derenne, S.; Casadevall, E.; Kadouri, A.; Sellier, N. Org. Geochem. 1986, 10, 1023-1032. (2) Largeau, C.; Derenne, S.; Casadevall, E.; Berkaloff, C.; Corolleur, M.; Lugardon, B.; Raynaud, J. F.; Connan, J. Org. Geochem. 1990, 16, 889-895. (3) Derenne, S.; Largeau, C.; Casadevall, E. Org. Geochem. 1991, 17, 597-602. (4) Derenne, S.; Largeau, C.; Casadevall, E.; Raynaud, J. F.; Berkaloff, C.; Rousseau; B. Geochim. Cosmochim. Acta 1991, 55, 10411050. (5) Douglas, A. G.; Sinninghe Damste´, J. S.; Fowler, M. G.; Eglinton, T. I.; de Leeuw, J. W. Geochim. Cosmochim. Acta 1991, 55, 275-291. (6) Nip, M.; Tegelaar, E.; de Leeuw, J. W.; Schenck, P. A.; Holloway, P. J. W. Naturwissenschaften 1986, 73, 579-585. (7) Tegelaar, E. W.; de Leeuw, J. W.; Largeau, C.; Derenne, S.; Schulten, H.-R.; Muller, R.; Boon, J. J.; Nip, M.; Sprenkels, J. C. M. J. Anal. Appl. Pyrolysis 1989, 15, 29-54. (8) van Aarssen, B. G. K.; Cox, H. C.; Hoogendoorn, P.; de Leeuw, J. W. Geochim. Cosmochim. Acta 1990, 54, 3021-3031. (9) Collinson, M. E.; van Bergen, P. F.; Scott, A. C.; de Leeuw, J. W. In Coal and Coal-bearing Strata as Oil-prone Source Rocks?; Scott, A. C., Fleet, A. J., Eds.; Geol. Soc. Spec. Publ.: London, 1994; pp 3170. (10) van Bergen, P. F.; Collinson, M. E.; Briggs, D. E. G.; de Leeuw, J. W.; Scott, A. C.; Evershed, R. P.; Finch, P. Acta Bot. Neerl. 1995, 44, 319-342.

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of decay resistant biomacromolecules,11 thus providing an alternative pathway to that originally proposed by Tissot and Welte (1984)12 involving random repolymerization and recondensation of lipids, sugars, etc. Recent investigations have also revealed the importance of socalled amorphous organic matter (AOM) as a major component of marine Type II kerogens.13-17 It has been shown that AOM from different geographical areas can be characterized by its optical and chemical properties.2,16,17 However, the exact composition and origin of much AOM remains unclear. While algal and bacterial biomass is generally accepted as the major contributor to the formation of marine organic matter, invertebrates have been largely neglected as a possible source. The primary analytical tool used to elucidate the chemical structure of resistant biomacromolecules found in coal and kerogen is analytical pyrolysis (py) in combination with gas chromatography (GC), mass spectrometry (MS), or gas chromatography/mass spectrom(11) Tegelaar, E. W.; de Leeuw, J. W.; Derenne, S.; Largeau, C. Geochim. Cosmochim. Acta 1989, 53, 3103-3106. (12) Tissot, B.; Welte, D. H. Petroleum Formation and Occurrence, 2nd ed.; Springer-Verlag: Berlin, 1984. (13) Stach, E.; Taylor, G. H.; Mackowsky, M. T.; Chandra, D.; Teichmu¨ller, M.; Teichmu¨ller, R. Stach’s Textbook of Coal Petrology, 3rd ed.; Gebru¨der Borntraeger: Berlin, 1982. (14) Mukhopadhyay, P. K. Org. Geochem. 1989, 14, 269-284. (15) Senftle, J. T.; Brown, J. H.; Larter, S. R. Int. J. Coal Geol. 1987, 7, 105-117. (16) Boussafir, M.; Gelin, F.; Lallier-Verge´s, E.; Derenne, S.; Bertrand, P.; Largeau, C. Geochim. Cosmochim. Acta 1995, 59, 37313747. (17) Stankiewicz, B. A.; Kruge, M. A.; Mastalerz, M.; Salmon, G. L. Org. Geochem. 1996, 24, 495-509.

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etry (GC/MS).6,9,18-23 Although this approach is destructive and does not yield precise quantitative information, it has proved to be the most appropriate in studies of insoluble macromolecules where the amounts of material available for study are limited.10,22 Geological samples, particularly fossil remains, are often available in only milligram quantities, so limiting or completely precluding the application of wet-chemical methods or other techniques such as solid state 13C NMR. Py-GC/MS has notable advantages in requiring submilligram samples and yielding reliable structural information from polycondensed materials. Perhaps the most important feature of py-GC/MS is its capacity to generate biomarker information from otherwise intractable materials, thus allowing specific inputs to be “fingerprinted” and/or traced to a particular source.10,22 For example, pyrolysates with abundant n-alk-1-enes, n-alkanes, R,ω-dienes, and/or ketones can be related to a specific algal precursor3,4,24 or cutan polymer,6,7 whereas those rich in methoxyphenols indicate an origin in a lignin biopolymer.25-27 Invertebrate cuticles are composed of two main biopolymers, chitin and proteins,28 cross-linked via catecholamine and histidyl (aspartic) moieties. Our understanding of the chemical structure of cuticle has been significantly advanced by utilizing solid state 13C and 15N NMR.29 Although py-GC/MS has been used to study chitin and protein/amino acids,30-35 these investigations considered the biopolymers separately rather than linked in cuticles, and the emphasis was on biochemistry rather than their occurrence in the geosphere. Py-GC/MS has been applied only recently to modern and fossil cuticles,36 revealing the preferential preservation of chitin over the proteinaceous compo(18) Larter, S. R.; Douglas, A. G. In Advances in Organic Geochemistry 1979; Douglas, A. G., Maxwell, J. R., Eds.; Pergamon Press, 1980; pp 579-583. (19) Philp, R. P.; Russel, N. J.; Gilbert, T. D.; Friedrich, J. M. J. Anal. Appl. Pyrolysis 1982, 4, 143-161. (20) Larter, S. R. In Analytical Pyrolysis-Methods and Applications; Voorhees, K., Ed.; Butterworth: London, 1984; pp 212-275. (21) Horsfield, B. Geochim. Cosmochim. Acta 1989, 53, 891-901. (22) de Leeuw, J. W.; van Bergen, P. F.; van Aarssen, B. G. K.; Gatellier, J.-P. L. A.; Sinninghe Damste´, J. S.; Collinson, M. E. Philos. Trans. R. Soc. London 1991, B 333, 329-337. (23) Boon, J. J. Int. J. Mass Spectrom. Ion Processes 1992, 118/119, 755-787. (24) Gelin, F.; Boogers, I.; Noordeloos, A. A. M.; Sinninghe Damste´, J. S.; Hatcher, P. G., de Leeuw, J. W. Geochim. Cosmochim. Acta 1996, 60, 1275-1280. (25) Saiz-Jimenez, C.; de Leeuw, J. W. Org. Geochem. 1984, 6, 417422. (26) Saiz-Jimenez, C.; de Leeuw, J. W. Org. Geochem. 1986, 10, 869876. (27) Hatcher, P. G.; Lerch, H. E., III; Kotra, R. K.; Verheyen, T. V. Fuel 1988, 67, 1069-1075. (28) Muzzarelli, R. A. A. Chitin; Pergamon Press: Oxford, U.K., 1977. (29) Schaefer, J.; Kramer, K. J.; Garbow, J. R.; Jacob, G. S.; Stejskal, E. O.; Hopkins, T. L.; Speirs, R. Science 1987, 235, 1200-1204. (30) van der Kaaden, A.; Boon, J. J.; de Leeuw, J. W.; de Lange, F.; Schuyl, P. J. W.; Schulten, H.-R.; Bahr, U. Anal. Chem. 1984, 56, 21602165. (31) Franich, R. A.; Goodin, S. J.; Wilkins, A. L. J. Anal. Appl. Pyrolysis 1984, 7, 91-100. (32) Davies, D. H.; Hayes, E. R.; Lal, G. S. In Chitin in Nature and Technology; Muzzarelli, R., Jeuniaux, Ch., Gooday, G. W., Eds.; Plenum Press: New York and London, 1985; pp 365-370. (33) Tsuge, S.; Matsubara, H. J. Anal. Appl. Pyrolysis 1985, 8, 4964. (34) Munson, T. O.; Fetterolf, D. D. J. Anal. Appl. Pyrolysis 1987, 11, 15-24. (35) Boon, J. J.; de Leeuw, J. W. J. Anal. Appl. Pyrolysis 1987, 11, 313-327. (36) Baas; M., Briggs, D. E. G.; van Heemst, J. D. H.; Kear, A. J.; de Leeuw, J. W. Geochim. Cosmochim. Acta 1995, 59, 945-951.

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nents in shrimps decayed in laboratory conditions. This study36 showed that none of the pyrolysis products of the cuticle of a Carboniferous shrimp could be related directly to chitin or proteins. The highly aliphatic signatures obtained resembled the cutan polymer characteristic of terrestrial plants rather than the chitinous cuticle of a modern arthropod! A similar aliphatic signature was obtained from the periderm of Ordovician and Silurian graptolites, even though the periderm of pterobranchs, their closest living relatives, is proteinaceous.37 In this paper, we present data obtained from py-GC/ MS of several invertebrate cuticles ranging from Cambrian to Cretaceous in age. The fossil organisms represent both marine and terrestrial environments of deposition. The chemical characteristics of the fossil cuticles are compared with those of their modern relatives to allow the chemical changes that take place during diagenesis to be inferred. Methods Sample Description and Preparation. Fossil cuticles of a wide range of geological ages and paleoenvironments were analyzed. Specimens were selected on the basis of brown color and organic appearance by examination under the light microscope. The cuticles were separated from the rock matrix mechanically to avoid any possibility of chemical alteration or contamination (Table 1). Modern arthropod specimens were purchased from retailers and freeze-dried (Table 1). Cuticle and other parts for analysis (Table 1) were separated from the bodies mechanically and immediately solvent-extracted to minimize the growth of microorganisms. All samples, both fossil and modern, were extracted ultrasonically with CH2Cl2 (4 × 10 min) to remove solvent soluble constituents and possible contaminants originating from handling. The resulting insoluble residues were dried under a stream of nitrogen before analysis. Chitin separated from crab shells (Aldrich Chemical Co., Inc.), commercially produced amino acids (histidine, aspartic acid, valine, alanine, proline, tyrosine, tryptophan, cysteine, serine, glycine, lysine, arginine), and polypeptides were analyzed to provide reference data for the identification of chitinous and proteinaceous constituents of the cuticles (for details, see Stankiewicz et al. (1996)).38 Pyrolysis-Gas Chromatography/Mass Spectrometry (Py-GC/MS). Analyses were performed using a CDS (Chemical Data System) 1000 pyroprobe, connected to a Carlo Erba 4130 gas chromatograph (GC) coupled to a Finnigan 4500 mass spectrometer (MS). Compounds were separated using a 50 m CP Sil-5 CB column (0.32 mm i.d., film thickness 0.4 µm). Each sample was loaded into the quartz tubes, weighed (up to 1 mg), and pyrolyzed in a flow of helium (0.65 kg cm2) for 10 s. The pyrolysis interface was held at 250 °C and the GC injector maintained at 250 °C. The temperature of the GC/MS transfer line was set at 310 °C. The GC oven was operated under the following program: isothermal for 5 min at 35 °C; temperature programmed at 4 °C min-1 to 310 °C; and then isothermal for 15 min. The MS was operated in full scan mode (40-650 Da, 1 scan s-1, 70 eV electron energy, 300 mA emission current, and ionization source temperature of 170 °C). Peaks were identified on the basis of their mass spectral characteristics and GC retention times and on comparison with the NIST mass spectral library and published GC and MS data.33,38 Additional identifications were based on a comparison with the mass spectra and retention times of compounds in the (37) Briggs, D. E. G.; Kear, A. J.; Baas, M.; de Leeuw, J. W.; Rigby, S. Lethaia 1995, 28, 15-23. (38) Stankiewicz, B. A.; van Bergen, P. F.; Duncan, I. J.; Carter, J. F.; Briggs, D. E. G.; Evershed, R. P. Rapid Commun. Mass Spectrom. 1996, 10, 1747-1757.

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Table 1. List of Fossil and Modern Specimens Analyzed by Py-GC/MS in This Study specimena

locality

age

(1) stylonurid eurypterid (1) eurypterid (1) shrimpb Anthracophausia (1) scorpion (1) decapod shrimp (1) squidc Plesioteuthis (1) squidc Mastigophora (1) decapod shrimp (1) decapod shrimp (1) shrimpd Pseudastacus (3) shrimpd Delclosia (3) water bug Iberonepa (2) nymphb (2) mantis shrimpd (2) shrimpd Crangon crangon cockroachb Periplaneta americana squidc Loligo

Wiarton, Ontario, Canada Wiarton, Ontario, Canada Fort Erie, Ontario, Canada Bearsden, Scotland Ballycastle, N. Ireland Solnhofen, Germany Solnhofen, Germany Oxford Clay, England Osteno, Italy La Voulte, France Las Hoyas, Spain Las Hoyas, Spain Las Hoyas, Spain Las Hoyas, Spain Lebanon U.K. U.K. U.K.

Silurian Silurian Silurian Carboniferous Carboniferous Jurassic Jurassic Jurassic Jurassic Jurassic Cretaceous Cretaceous Cretaceous Cretaceous Cretaceous modern modern modern

scorpionb

chemical character aliphatic aliphatic aliphatic aliphatic aliphatic aliphatic aromatic aromatic aromatic aliphatic aromatic aromatic aliphatic aliphatic aromatic chitin + proteins chitin + proteins chitin + proteins

a Indicates number of specimens analyzed. b Pyrograms shown in Figure 1. c Pyrograms shown in Figure 2. Figure 3.

pyrolysates of the reference materials (chitin, polypeptide, and amino acids).38

Results and Discussion Pyrolysis of the modern invertebrate cuticles yielded components that can be assigned to either chitin or a protein biopolymer.38 Detailed investigation of the pyrolysates of the fossil samples, on the other hand, failed to reveal any components that could be identified positively as deriving from chitin or proteins. A similar result was obtained by Schimmelmann and others (1988)39 using wet chemical methods (even their Tertiary specimens did not yield identifiable chitin). The fossil cuticles fell into two categories based on their pyrolysis products, each with a distinctive chemical signature: (1) aliphatic, yielding pyrolysates characterized by pairs of n-alk-1-enes and n-alkanes (Figure 1) and (2) aromatic, with relatively abundant C0-C3 (alkyl)benzenes and C0-C2 (alkyl)indenes (Figures 2 and 3). The chemical character of the fossil cuticles does not appear to reflect taxonomic affinity, geological age, sedimentary environment, or geographical location. “Aliphatic” Cuticles. All the Paleozoic samples, and the insects from the Cretaceous of Las Hoyas, Spain, were characterized by aliphatic components and yielded a series of n-alk-1-enes and n-alkanes upon pyrolysis. The carbon number of the alkyl chain varied from sample to sample, encompassing the range from C5 to C30 and reaching an upper limit in the Carboniferous shrimp from Bearsden, Scotland (Figure 1C). Aromatic components were minor constituents of the total pyrolysate, the most prominent being C0-C3 (alkyl)benzenes. In view of the close evolutionary relationship between many of the fossil arthropods analyzed and their modern relatives, it is very unlikely that the original composition of the cuticle of the Paleozoic and Mesozoic taxa differed fundamentally from that of arthropods living today. Thus, the absence of any components other than highly aliphatic in the pyrolysates of the fossil cuticles is anomalous. The only aliphatic substances known in modern arthropods are cuticular waxes,40 but these are not part of the insoluble (39) Schimmelmann, A.; Krause, R. G. F.; DeNiro, M. J. Org. Geochem. 1988, 12, 1-5.

d

Pyrograms shown in

macromolecular structure and are easily extracted with organic solvents. It is possible, however, that cuticular waxes and/or lipids from internal tissues were polymerized during diagenesis and chemically “replaced” the chitin and proteins (Figure 4), which are rapidly degraded in most modern environments41,42 (the relationship between the morphological and chemical preservation of these cuticles is the subject of a parallel investigation). An alternative mechanism was suggested by Baas et al. (1995),36 who proposed that the original cuticle was replaced by highly aliphatic organic matter from an external source, resulting in pyrolysates typical of algaenan or cutan (Figure 4). A hypothesis involving mobilization and migration of aliphatic moieties from aliphatic-rich substances (liptinites) to typically nonaliphatic ones (huminite/vitrinite) with increasing diagenesis has been used to explain chemical changes in composition of coal macerals derived from chemically aliphatic-poor macromolecules such as lignin.43-45 However, microscale migration between macerals in an organic-rich coaly matrix is supported by the proximity of specific source material,43-45 whereas the evidence for migration of aliphatics from some unknown source into animal cuticles, often through an organic-poor matrix, is purely circumstantial. In the biotas analyzed, for example, arthropods are often preserved at different sedimentary levels to plant cuticles (Las Hoyas, Spain). Equally early Paleozoic cuticles (e.g., Silurian eurypterids) have been transformed to an aliphatic composition even though terrestrial plants had yet to diversify. However, in these cases, algae remain a possible source of aliphatics. An important factor in the diagenesis of all cuticles is the maturation level, which is influenced by temperature, (40) Jackson, L. L.; Blomquist, G. J. In Chemistry and Biochemistry of Natural Waxes; Kolattukudy, P. E., Ed.; Elsevier: Amsterdam, 1976; pp 201-233. (41) Gooday, G. W. In Advances in Microbial Ecology; Marshall, K. C., Ed.; Plenum Press: New York and London, 1990, pp 387-430. (42) Poulicek, M.; Jeuniaux, C. Biochem. System Ecol. 1991, 19, 385-394. (43) Stout, S. A. In Coal and Coal-bearing Strata as Oil-prone Source Rocks?; Scott, A. C., Fleet, A. J., Eds.; Geol. Soc. Spec. Publ.: London, 1994; pp 93-106. (44) Zhang, E.; Hatcher, P. G.; Davis, A. Org. Geochem. 1993, 20, 721-734. (45) Stankiewicz, B. A.; Kruge, M. A.; Mastalerz, M. Org. Geochem. 1996, 24, 531-545.

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Figure 1. Reconstructed ion chromatograms (pyrolysis at 610 °C for 10 s) of cuticles of (A) modern cockroach, (B) scorpion from Silurian, Ontario, Canada, (C) shrimp Anthracophausia from Carboniferous of Bearsden, Scotland, and (D) Plecoptera nymph from Cretaceous, Las Hoyas, Spain. Numbers below pyrograms indicate carbon number for alkene/alkane pairs: +, n-alk-1-enes; ×, n-alkanes; b, pyrolysis products directly related to chitin polymer; 0, pyrolysis products of protein moieties; OX, oxazoline derivatives;30 DKP, 2,5-diketopiperazines.38 Chemical structures are given for the most important pyrolysis products derived directly from amino acid moieties (A) and aromatic components in fossil specimens (B-D).

pressure, and time, and may induce chemical changes, such as degree of aromatization, in organic matter.12 “Aromatic” Cuticles. Squid pens from the Jurassic Solnhofen Limestone, Germany, and Oxford Clay, England (Figure 2), and shrimps from the Cretaceous of Las Hoyas and the Lebanon (Figure 3) yielded pyrolysates dominated by aromatic compounds. The major products were a series of C0-C3 alkylated benzenes and, more interestingly, alkylated indenes (Figures 2 and 3). Naphthalene and its C1 and C2 homologues, and fluorene, were minor aromatic components. Sulfur compounds, represented by thiophene, its C1-C3 alkylated

homologues, and benzothiophene, were abundant in all samples. Series of C5 up to C18 n-alk-1-enes and n-alkanes were minor components. Here, as in the aliphatic fossil cuticles, pyrolysis products of chitin or proteins were not detected. The origin of the aromatic pyrolysis products is unknown. C0-C3 alkylbenzenes are common pyrolysis products of almost all organic materials46 and therefore have little potential as markers. The relatively high (46) Hartgers, W. A.; Sinninghe Damste´, J. S.; de Leeuw, J. W. Geochim. Cosmochim. Acta 1994, 58, 1759-1775.

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Figure 2. Reconstructed ion chromatograms (pyrolysis at 610 °C for 10 s) of (A) commercial chitin (Aldrich), (B) modern squid Loligo pen, (C) squid pen from Jurassic Oxford Clay, Swindon, England, and (D) squid pen from Jurassic, Solnhofen, Germany: +, n-alk-1-enes; ×, n-alkanes; b, pyrolysis products directly related to chitin polymer; 0, pyrolysis products of protein moieties; OX, oxazoline derivatives;30 DKP, 2,5-diketopiperazines.38 Numbers below pyrograms indicate carbon number for alkene/alkane pairs. Chemical structures are given for the most important pyrolysis products derived directly from chitin (A), amino acid moieties (B), and aromatics and sulfur compounds in fossil specimens (C-D).

abundance of indene-type compounds, on the other hand, is more diagnostic. Indenes are minor components of the total pyrolysate of various types of organic matter.17,45,47,48 There is no known chemical explanation for the transformation of chitin or protein into indene moieties. Nevertheless, the abundance of indene and its alkylated homologues in the pyrolysates of invertebrate cuticles points to them as a possible source of these substances in kerogens (Figure 4). This may also apply to AOM, which is often structureless and difficult to characterize optically. This hypothesis re(47) Nip, M.; de Leeuw, J. W.; Schenck, P. A. Geochim. Cosmochim. Acta 1988, 52, 637-648. (48) Nip, M.; de Leeuw, J. W.; Crelling, J. C. Energy Fuels 1992, 6, 125-136.

quires testing through an investigation of the pyrolysates of AOM, which might reveal the presence of a relatively high abundance of alkylated indenes. The possibility that these indene-type compounds might be pyrolytic artifacts, however, cannot be ruled out. The vulcanization of aliphatic moieties49,50 is not a ready explanation for the presence of relatively abundant alkylated thiophenes in fossil cuticles, as the aliphatics are absent from the polymeric structure of (49) Sinninghe Damste´, J. S.; Rijpstra, W. I. C.; Kock-Van Dalen, A. C.; de Leeuw, J. W.; Schenck, P. A. Geochim. Cosmochim. Acta 1989, 53, 1343-1355. (50) Kohnen, M. E. L.; Sinninghe Damste´, J. S.; Rijpstra, W. I. C.; de Leeuw, J. W. Geochim. Cosmochim. Acta 1993, 57, 2515-2528.

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Figure 3. Reconstructed ion chromatograms (pyrolysis at 610 °C for 10 s) of cuticles of (A) modern shrimp Crangon, (B) shrimp Pseudastacus from Cretaceous Las Hoyas, Spain, (C) shrimp Delclosia from Cretaceous Las Hoyas, Spain, and (D) mantis shrimp from Cretaceous, Lebanon: +, n-alk-1-enes; ×, n-alkanes; b, pyrolysis products directly related to chitin polymer; 0, pyrolysis products of protein moieties; OX, oxazoline derivatives;30 DKP, 2,5-diketopiperazines.38 Numbers below pyrograms indicate carbon number for alkene/alkane pairs. Chemical structures are given for the most important pyrolysis products derived directly from chitin and amino acid moieties (A) and aromatics and sulfur compounds in fossil specimens (B-D).

the equivalent modern cuticles (Figures 1-3). Thus the incorporation of sulfur must have involved other macromolecules (e.g., chitin and proteins). Kok et al. (1996)51 recently showed that the sulfurization of the polysaccharide component of nonhydrolyzable algal biomass results in an increase in the proportion of sulfurcontaining pyrolysis products. Sulfurization of chitin, an amino sugar biopolymer, would have a similar effect. (51) Kok, M. D.; Osinga, R.; Schouten, S.; Sinninghe Damste´, J. S. In Geochemistry Newsletter, Fall 1996; American Chemical Society: Washington, DC, 1996; p 88.

This hypothesis is currently being tested through laboratory experiments on the sulfurization of chitin and cuticles. Conclusions This investigation represents a novel application of analytical pyrolysis-gas chromatography/mass spectrometry in the characterization of fossil invertebrate cuticles. Analyses of a variety of fossil animal cuticles using py-GC/MS revealed the presence of products that could not be directly related to either chitin or protein

Composition of Fossil Invertebrate Cuticles

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Figure 4. Proposed mechanisms for the chemical changes induced by diagenesis during the fossilization of invertebrate cuticles.

biopolymers. Thus a selection of well-preserved fossils more than 60 My old failed to show the preservation of these macromolecules. The fossil cuticles revealed two distinctive chemical signatures upon pyrolysis, highly aliphatic and aromatic, but the origin of these signatures is currently difficult to explain. The polymerization of external waxes and/or lipids from internal tissues is a possible mechanism for generating the aliphatic composition. There is no known process that could modify chitin or protein into indene and its alkylated homologues, as derived from the aromatic cuticles (Figure 4). Nonetheless the abundance of alkylated indenes in the pyrolysates of sedimentary organic matter may indicate an invertebrate cuticle source. Artificial maturation experiments are underway to explore the chemical changes that occur in invertebrate cuticles during diagenesis and to facilitate an assessment of their contribution to sedimentary organic matter. Acknowledgment. This investigation was made possible by a large number of individuals who kindly provided samples: J. Waddington (Royal Ontario Museum), eurypterids and scorpions from Ontario; N. D. L. Clark (Glasgow), shrimp from Bearsden; A. J. Jeram (Belfast), scorpion from Northern Ireland; G. Viohl

(Eichstatt), squid from Solnhofen; P. R. Wilby (Bristol), squid from Swindon; G. Pinna and G. Teruzzi (Milan), shrimp from Osteno; B. Riou (La Voulte-sur-Rhoˆne), shrimp from La Voulte; F. J. Ortega and J. L. Sanz (Universidad Auto´noma de Madrid) and X. MartinezDelclos (Universidad de Barcelona), shrimps and insects from Las Hoyas; Cees Hof (Amsterdam), mantis shrimp from Lebanon; A. J. Kear, S. Bale, and I. J. Duncan (Bristol), modern squid, shrimp, and insects, respectively. Our work has benefited from advice and discussion with Pim F. van Bergen, Geoffrey Eglinton, and Jan W. de Leeuw. Barbara Mo¨sle is thanked for collecting Las Hoyas specimens during field excavations, and James Carter and Andrew Gledhill, for assistance with GC/MS. Two anonymous reviewers are thanked for their constructive comments and suggestions. This research was funded by a NERC research grant to D.E.G.B. and R.P.E. through the Ancient Biomolecules Initiative (GST/02/1027). NERC also provided financial support for mass spectrometry facilities (F14/6/13). The attendance of B.A.S. at the ACS meeting to present this paper was partially funded by a grant from the Petroleum Research Fund. EF9601778