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Preservation of Chitin in 25-Million-Year-Old Fossils

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Science  06 Jun 1997:
Vol. 276, Issue 5318, pp. 1541-1543
DOI: 10.1126/science.276.5318.1541

Abstract

Chitin is present in fossil insects from the Oligocene (24.7 million years ago) lacustrine shales of Enspel, Germany. This result, which was obtained by analytical pyrolysis, extends by nearly 25 million years the length of time that chemically detectable remains of this biomolecule are known to survive. The embedding sediment is dominated by diatoms, which reflect high productivity in the paleolake. The primary control on the preservation of chitin is thus not time; it may persist in older sediments where suitable paleoenvironmental conditions prevailed.

Chitin, which is one of the most abundant macromolecules on Earth, is also one of the most enigmatic of molecular fossils. An estimated 1011 tons of chitin is produced annually in the biosphere, most of it in the oceans (1). It occurs in a range of organisms but is particularly important as a constituent of arthropod cuticles (1-4), where it is cross-linked with proteins via catechol and histidyl moieties (5). Although biodegradation in the water column and sediment normally removes almost all of the chitin produced in the oceans (3), experiments have demonstrated that chitin is the component of shrimps that is most resistant to degradation (6) and organic remains of arthropod cuticle are abundant in the fossil record, in some cases preserving remarkable morphological detail (7). Chitin has been detected in insects in terrestrial deposits of Pleistocene age (∼130,000 years ago) (8) and in asphalt deposits from California (9). However, analyses have failed to provide evidence for its presence in older fossils except (6, 10-12) for traces of amino sugars in the calcified skeletons of one Cretaceous and three Tertiary decapod crustaceans (13). Even where the morphology of the cuticle appears well preserved, the original chemical composition may be completely altered.

Pyrolysis–gas chromatography–mass spectrometry (py-GC-MS) (6, 14-16) provides a powerful tool for the chemical characterization of invertebrate cuticles (9, 15), particularly in cases in which the sample size is limited to a few micrograms. Investigation of the cuticles of fossil arthropods from 15 sites ranging in age from Silurian to Cretaceous revealed no trace of the original chemical components of chitin (12). The chemical signature either (i) was dominated by the n -alk-1-ene and n -alkane doublets characteristic of highly aliphatic biopolymers or (ii) contained a substantial aromatic component including alkylbenzenes and alkylindenes and relatively abundant sulfur-containing compounds such as thiophenes (12).

Here we demonstrate that chitin is preserved in the late Oligocene [24.7 million years ago (Ma)] Enspel Fossillagerstätte (17) near Bad Marienberg, in the Westerwald, Rheinland-Pfalz, Germany, where maar-lake deposits are interbedded with tuff (18). This deposit yields abundant remarkably preserved plants and animals (18), complete with soft body outline, and insects, some of which retain the original iridescent color. We used py-GC-MS (19) to study beetles [eight specimens of Coleoptera: Curculionoidea (Fig. 1A)] and flies (two specimens of Diptera: Bibionidae), which represent the dominant insect orders from Enspel (20), from the 14-cm-thick layer 12 in the bituminous volcanoclastic shales (21).

Figure 1

Photomicrographs of (A) the ventral view of a beetle (Coleoptera: Curculionoidea) from level 12 of the Oligocene (24.7 Ma) of Enspel, western Germany; (B) an SEM image of the cuticle of a modern mealworm beetle (Tenebrio molitor) that reveals the layers of cuticle overlapping at different angles; (C an SEM image of a fractured edge of cuticle of the specimen illustrated in (A) that shows chitinous fibers; and D) an SEM image of the shale matrix that contains the fossil insects and reveals that pennate diatoms are the dominant constituent. (Images were made with a Cambridge Stereoscan 250 Mk3 SEM at 7 to 12 kV after specimens were coated with gold.)

The distribution of major pyrolysis products of the Enspel beetles is similar to that in elytra of modern beetles (Fig. 2, A and B). The prominent pyrolysis products of chitin, including acetamide, N -acetyl-2-pyridone, acetamidofuran, 3-methyl-5-acetamidofuran, 3-acetamido-4-pyrone, oxazoline derivatives, acetic acid, and pyridines, are present in the fossil samples. Although the distribution of certain components varies among samples, the pyrograms of all the beetle cuticles reveal a similar pattern (Fig. 2). The same diagnostic components are evident in the pyrolysate of chitin isolated from modern arthropods (15). Analyses of the Enspel flies, however, revealed that none of the pyrolysis products were directly related to chitin but instead they composed a series of n -alk-1-enes and n -alkanes characteristic of older fossil arthropod cuticles that have been diagenetically altered (12).

Figure 2

Total ion chromatograms (pyrolysis at 610°C for 10 s) of the (A) elytra of a modern mealworm beetle (Tenebrio molitor), (B) cuticle of the curculionoid beetle from Enspel, and (C) sedimentary matrix from Enspel level 12. The numbers indicate major pyrolysis products derived from chitin: 1, acetic acid; 2, pyridine; 3, acetamide; 4, methylpyridine; 5, 2-pyridinemethanol; 6,N-acetyl-2-pyridone; 7, 3-acetamidofuran; 8, 3-acetamido-5-methylfuran; 9, 3-acetamido-4-pyrone; and 10, 10 and 10′-oxazoline derivatives. Solid squares indicate important components directly related to chitin polymer. Letters indicate products derived from amino acids: A, pyrrole; B, toluene; C, methylpyrroles; D, ethylpyrrole; E, phenol; F, 2-methylphenol; G, 4-methylphenol; H, dimethylphenol and ethylphenol; I, vinylphenol; J, indole; K, methylindole; L, dimethylindole; DKP1, 2,5-diketopiperazine of pro-Ala; and DKP2, 2,5-diketopiperazine of pro-Val. Open squares indicate other important components related to proteins. Δ1-3, cyclic ketones derived from carbohydrate moieties; solid circles, n-alk-1-enes; open circles,n-alkanes; Pr1, prist-1-ene; Pr2, prist-2-ene; asterisks, contamination. Circled numbers indicate the carbon number in the aliphatic chains. Chemical structures are given for most of the important pyrolysis products.

Pyrolysis of the shale matrix that contains the Enspel fossils (Fig.2C) produced a series of C5 to C31n -alk-1-enes, n -alkanes, and α,ω-alkadienes. This trace is characteristic of highly aliphatic resistant macromolecules such as algaenan (derived from algae) and cutan [from the cuticle of vascular plants (22)], both of which are major contributors to Type I kerogens. This pyrolysis trace differs from that of the cuticle of the Enspel flies in that abundant C20 to C34 alkene/alkane pairs are present (Fig. 2C). Scanning electron microscopy (SEM) reveals that the matrix consists overwhelmingly of pennate diatoms (Fig. 1D), although the presence of other algae has been reported (23). Thus, there are a number of possible sources of the highly aliphatic signature of the sedimentary matrix. Such a series of n -alk-1-enes and n -alkanes is also evident among the pyrolysis products of fossils to which the matrix adhered and from which it could not be completely removed (Fig. 2B).

The presence of 2,5-diketopiperazines, which are diagnostic pyrolysis products of proteins (15, 24), indicates that a protein component has survived in the beetle cuticle in addition to the chitin macromolecule. A number of other pyrolysis products are also derived from amino acids, such as tyrosine (phenol and methylphenols), tryptophan (indole and methylindole), phenylalanine (toluene) and proline (pyrrole and methylpyrroles) (Fig. 2A). Although protein moieties have been shown to be more susceptible to decay than chitin in laboratory experiments (6), they appear to be important components of the Enspel fossil samples. The presence of 2-cyclopenten-1-ones, the products of carbohydrate moieties, provides further evidence of the chemical preservation of the beetle cuticle.

SEM of the fossil beetle cuticles from Enspel revealed the overlapping layers of thick fibers in the cuticle (Fig. 1C). The appearance of the curculionid cuticle from Enspel is similar to that of modern beetles (Fig. 1B), although the organic matrix surrounding the fibers has partially degraded. The preservation of chitin in the beetles and not in the flies may reflect the greater thickness and degree of cross-linking in the cuticle of the former. This study demonstrates that the primary control on the preservation of these biomolecules in ancient rocks is not time but the nature of the depositional environment and the inhibition of diagenetic alteration. In the case of Enspel, the combination of high productivity (evidenced by the abundance of diatoms in the matrix) and strongly reducing bottom conditions (23) played a key role in the enhanced preservation of the chitin-protein complex.

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