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Molecular Taphonomy of Graptolites

February 14, 2007

By Gupta, Neal S; Briggs, Derek E G; Pancost, Richard D

Graptolites are important fossils in Early Palaeozoic assemblages. Preserved graptolite periderm consists dominantly of an aliphatic polymer, immune to base hydrolysis. It contains no protein even though its structure, and chemical analyses of the periderm of the living relative Rhabdopleura, indicate that it was originally collagen. This anomaly was previously interpreted as the result of replacement by macromolecular material from the surrounding sediment. New analyses suggest that the aliphatic composition of graptolite periderm reflects direct incorporation of lipids from the organism itself by in situ polymerization. A similar process may account for the preservation of most organic fossils.

Graptolites are the dominant component within many Early Palaeozoic fossil planktonic assemblages, and may be enormously abundant within organic-rich hemipelagic mudstone facies. This, combined with their widespread distribution and rapid evolution, accounts for their importance in biostratigraphy. Graptolites lacked a biomineralized skeleton. Similarities to the periderm of closely related living pterobranchs such as Rhabdopleura (Briggs et al. 1995, and references therein) and Cephalodiscus indicate that the periderm of graptolites was composed originally of collagen. This confirms interpretations of its composition based on the structure of the periderm as revealed by transmission electron microscopy (Towe & Urbanek 1972; Crowther & Rickards 1977; Crowther 1981).

The skeleton of graptolites often provided a locus for the precipitation of authigenic minerals such as clays, which may be altered to chlorite (Underwood 1992). Pyrite may preserve the 3D morphology by growing as an infill (Underwood & Bottrell 1994). In many cases, however, the graptolite periderm is preserved as organic material (Bustin et al. 1989; Briggs et al. 1995), and graptolites can be released from carbonates by dissolving the matrix. Carbonaceous traces of the stolon of graptoloid graptolites have been reported (Loydell et al. 2004) but evidence of the morphology of the zooids rarely survives and relies on authigenic mineralization (Rickards et al. 1991).

Analysis of Monograptus from the Wenlock Cape Phillips Formation of Cornwallis Island revealed an aromatic structure with an aliphatic component (Bustin et al. 1989). Briggs et al. (1995) analysed Monograptus instrenuus from the same locality, and Amphigraptus sp. from the Caradoc Viola Limestone Formation of Oklahoma (Table 1), and concluded that the aliphatic material that they discovered in the periderm could not have been derived from an original proteinaceous material such as collagen. They argued that the material in the fossils must have been introduced from an external source that included a decayresistant macromolecule. The aliphatic material might have been derived by diagenetic replacement from algal cell walls, for example, which were a component of the surrounding matrix (Briggs et al. 1995). This was proposed in the light of the prevailing paradigm of organic matter preservation in sediments as a result of the selective preservation of decay- resistant components (Tegelaar et al. 1989; De Leeuw & Largeau 1993; Baas et al. 1995).

Recent research has shown that selective preservation is not an adequate explanation for the preservation of a number of fossil materials, including leaves (Gupta et al. 2006a-c) and arthropod cuticle (Briggs 1999; Stankiewicz et al. 2000; Gupta et al. 2006d). Nor can the preservation of fossil arthropod cuticle be explained by the introduction of material from external sources: thermochemolysis of co-occurring insect and plant fossils, and the associated organic- rich matrix, from the Oligocene Enspel Formation, Germany, revealed differences in the distribution of the constituent fatty acyl components, indicating that the aliphatic component of the fossil is at least partly derived endogenously (Gupta et al. 20066).

Here we test the hypothesis that the organic preservation of graptolite periderm, like that of the cuticle of arthropods and leaves (Gupta et al. 2006e), is a result of in situ polymerization of the lipid and other labile constituents rather than selective preservation of decay-resistant components or the introduction of material from an external source such as the matrix. This study also provides a further test of the model of in situ polymerization, because the original composition of graptolite periderm differs from that of arthropod cuticles and leaves, but fossils of all three are composed of dominantly aliphatic macromolecular material.

Methods. The graptolites investigated were selected from the Yale Peabody Museum collections to provide stratigraphie coverage from the Early Ordovician to the Silurian and a variety of genera from different localities (Table 1). They were either on the rock (e.g. Paleodiclyota anastomotica), in which case samples were scraped from the surface, or they had been released from the matrix by acid digestion and stored in glass vials in glycerin (e.g. Dictyonema peltatum). Samples were extracted ultrasonically three times, 15min each, with 2:1 CH^sub 2^Cl^sub 2^ (dichloromethane)-CH^sub 3^OH (methanol), to yield an insoluble residue. This residue was analysed by pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) to reveal the molecular distribution of compounds. Samples of selected graptolites were subjected to thermochemolysis (De Leeuw & Baas 1993) to permit further structural analysis of the macromolecule. Graptolites were hydrolysed in IM 95% methanoic NaOH (saponification) for 1 h at 70 C to yield a non-hydrolysable residue, to test the resistance of the macromolecule to base hydrolysis. Py-GC/MS was carried out on the periderm of modern Rhabdopleura before and after solvent extraction to evaluate the molecular distribution in the presence and absence of lipids. Further structural analysis of Rhabdopleura was carried out by subjecting samples to thermochemolysis without solvent extraction to reveal the complete molecular distribution including lipids.

Solvent-extracted graptolite and Rhabdopleura periderm was analysed using Py-GC/MS (see Gupta & Pancost 2004, for parameters), and compounds were identified using spectra reported in the literature (Stankiewicz et al. 1997a; Reeves & Francis 1998). For thermochemolysis, the extracted residues were transferred to a fresh vial and 1 ml TMAH (tetramethylammonium hydroxide) solution was added. They were soaked in TMAH solution for 3-4 h prior to analysis to ensure that sufficient TMAH was available during on-line pyrolysis. Thermochemolysis cleaves ester bonds to release constituent fatty acyl moieties (Challinor 1991a,b; De Leeuw & Baas 1993). A discussion of the behaviour of protein compounds under TMAH conditions has been given by Zhang et al. (2001).

Results. The major pyrolysis products of Rhabdopleura periderm (without lipid or solvent extraction) included phenols, indoles, pyrimidine, diketodipyrrole and diketopiperazine derivatives (Fig. 1a, Table 1). The abundance of diketodipyrrole, a marker for hydroxyproline, is characteristic of collagen (Stankiewicz et al. 1997a). Pyrimidine is also derived from collagen, although it cannot be ascribed to any particular amino acid.

The m/z 85 + 83 (Fig. 1a) mass chromatogram revealed a series of n-alkanes and n-alkenes. However, analysis of Rhabdopleura periderm after lipid extraction did not reveal the presence of these n- alkanes and n-alkenes, indicating that they were derived from a soluble, probably lipid, component.

Thermochemolysis of Rhabdopleura (Fig. 1b) revealed a distribution of fatty acyl moieties ranging in carbon number from C^sub 7^ to C^sub 18^. C^sub 16^ and C^sub 18^ saturated and unsaturated components were the most abundant (see Fig. 1b: m/z 74 + 87). Homologues greater than C^sub 10^ showed an even over odd predominance.

Samples of the graptolites Paleodictyota anastomotica, Dictyonema peltatum and Diplograptus sp. were analysed after solvent extraction. No moieties diagnostic of collagen were detected (Fig. 2). The graptolites comprised a largely aliphatic polymer with n- alkyl components in the pyrolysate extending at least from C^sub 9^ to C^sub 21^; C^sub 10^ to C^sub 15^ alkane/alkene homologues were the most abundant (Fig. 2, Table 1). A very similar distribution of chain lengths was observed in Monograplus and Amphigraptus (Briggs et al. 1995; Table 1). The aromatic component detected in all these consisted of benzene derivatives, phenols and naphthalene. Some glycerin was detected in the analysis of Dictyonema peltatum (Fig. 2), presumably as a result of contamination from the storage medium, which could not be completely removed during extraction (no glycerin was detected in the samples obtained directly from rock). Fatty acyl moieties released by thermochemolysis of the same graptolite samples (Fig. 3) ranged from C^sub 7^ to C^sub 18^ with an even-over-odd predominance (especially for those >C^sub 10^). The most abundant of these were C^sub 16^ and C^sub 18^ fatty acyl moieties, both saturated and unsaturated. These fatty acyl moieties were dominant when compared with the alkane/alkene homologues (see mass chromatogram m/z 74 + 87 + 85 + 83). The pred\ominance of fatty acyl moieties emphasizes the importance of ester functional groups in cross-linking the aliphatic polymer in these fossils (Versteegh et al. 2004; Gupta et al. 2006b). To determine the nature of these bonds, the fossils were hydrolysed in basic conditions and a residue was recovered. The weight of residue after hydrolysis was similar to that after extraction, and thermochemolysis of this residue yielded fatty acids in distribution similar to that after extraction. Thus, the aliphatic polymer in the fossils is nonextractable and largely non-hydrolysable, attesting to its recalcitrant nature. Pyrolysis failed to detect any ketones (Fig. 2), suggesting low amounts of ether linkages in the graptolite periderm. The primary oxygen- containing functional group in the graptolites may be as esters. The n-alkyl chains may protect the ester functional group in three dimensions by steric hindrance, accounting for their immunity to base hydrolysis (McKinney et al. 1996; Gupta et al. 2006a,b).

Discussion and conclusion. The ultrastructure of graptolite periderm suggests that its original composition was collagen (Towe & Urbanek 1972; Crowther & Rickards 1977). Collagen, like other proteins (Tegelaar et al. 1989; De Leeuw & Largeau 1993; but see Nguyen & Harvey 1998), is decay-prone, except where it has undergone substantial cross-linking, as in the jaws of polychaetes (Briggs & Kear 1993). Protein and polysaccharide remnants have been shown to survive in archaeological plant remains 1400 years old (Bland et al. 1998), in weevil samples as old as 24.7 Ma (Stankiewicz et al. 1997b; Gupta et al. 2006a) and even in kerogen 140 Ma old (Mongenot et al. 2001), where preservation of labile moieties was facilitated by encapsulation within a resistant aliphatic matrix. The presence of diketodipyrrole and pyrimidine in the periderm of Rhabdopleura is diagnostic of collagen (Stankiewicz et al. 1997a), but these moieities are absent in the periderm of graptolites. Thus, the ultrastructure may reflect the original composition even though the molecular components have been transformed to an aliphatic polymer.

The phenols in the graptolite periderm are probably the product of diagenesis of aromatic structures in the fossil, and not derived from original amino acids. Although encapsulation may promote protein preservation by steric protection of labile compounds (Knicker et al. 2001; Mongenot et al. 2001; Riboulleau et al. 2002), no nitrogen-bearing compounds were detected in the pyrolysates. Thus proteins, including collagen, appear not to have been preserved within the resistant aliphatic matrix that makes up graptolites, and it is likely that protein moieties do not survive in Early Palaeozoic fossils.

Analysis of the periderm of Rhabdopleura confirmed that it is proteinaceous in composition and contains no resistant aliphatic components (Briggs et al. 1995). The graptolites reveal a composition with a dominant aliphatic component similar to Type II kerogen (Bustin et al. (1989) noted that the hydrogen and oxygen indices of graptolite periderm (as determined by RockEval pyrolysis) were similar to Type II kerogen). Although sulphur-bearing compounds were not detected during pyrolysis, such components were detected in the pyrolysate of Monograptus and Amphigraptus in a previous study (Briggs et al. 1995), as a result of diagenetic incorporation of inorganic sulphur species under anoxic conditions (such conditions were probably absent in our samples). This facilitates further cross- linking of the macromolecule, as is often observed in natural vulcanization of kerogen (e.g. Kok et al. 2000).

In the absence of a diagenetically stable aliphatic biopolymer in the living relative, the preservation and aliphatic character of the graptolites cannot be explained by selective preservation. The organic content of the sediment differs from that of the fossils, so migration from an external source can be excluded. Thus, the aliphatic component was probably derived from compounds present in the organism itself. This is supported by the molecular structure. Thermochemolysis of modern Rhabdopleura and the graptolites investigated revealed a very similar saturated fatty acyl distribution ranging from C^sub 7^ to C^sub 18^ with a maximum abundance of C^sub 16^ and C^sub 18^ moieties (Table 1). The unsaturated fatty acyl components (e.g. C^sub 16:1^ and C^sub 18:1^), on the other hand, are more abundant in Rhabdopleura than in the fossils, suggesting that there was loss of unsaturated compounds during diagenesis (Wakeham et al. 1984, 1997).

Preservation of graptolites involves transformation of labile aliphatic components (such as fatty acids) into a recalcitrant cross- linked polymer with a dominant aliphatic component via in situ polymerization. Organic fossils younger than the Tertiary tend to reveal preservation of intact biomolecules (e.g. Gupta et al. 2006b). However, older fossils reveal a dominant aliphatic composition with poor molecular preservation, irrespective of depositional environment and enclosing lithology (for discussion, see Briggs et al. 2000). Evidence for a similar process of lipid incorporation has been reported in fossil leaves (Gupta et al. 2006a,b), dinoclasts (Versteegh et al. 2004), and arthropods (Briggs 1999; Gupta et al. 2006a,d) suggesting that preservation by lipid incorporation is important in the preservation of organic fossils.

We are grateful to C. Underwood, A. Scott and D. McIlroy for useful comments on a previous version of the paper. The Bristol node of the NERC mass spectrometric facility is thanked for analytical help. C. Glastris is thanked for supplying the Rhabdopleura samples.

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Received 18 May 2006; revised typescript accepted 24 July 2006.

Scientific editing by Duncan McIlroy

NEAL S. GUPTA1, DEREK E. G. BRIGGS1 & RICHARD D. PANCOST2

1 Department of Geology and Geophysics, Yale University,

PO Box 208109, New Haven, CT 06520-8109, USA

(e-mail: neal.gupta@yale.edu)

2 The School of Chemistry, University of Bristol,

Bristol BS8 1TS, UK

Copyright Geological Society Publishing House Nov 2006

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