Report

Identification of Carboniferous (320 Million Years Old) Class Ic Amber

See allHide authors and affiliations

Science  02 Oct 2009:
Vol. 326, Issue 5949, pp. 132-134
DOI: 10.1126/science.1177539

Extra Ancient Amber

Amber is fossilized tree resin, typically produced by trees in response to an injury. Most amber is Mesozoic or Cenozoic in age (dating back as far as 250 million years ago), and the most common class, produced primarily by angiosperms, is formed from distinct complex polyterpenoids. Bray and Anderson (p. 132; see the Perspective by Grimaldi) now find that amber from the Carboniferous, dating to more than 300 million years ago, long before the evolution of angiosperms, has a similar chemistry. Thus, the biosynthetic mechanism for producing complex ambers evolved long before the appearance of flowering plants.

Abstract

The presence of amber, the fossil form of the resins produced by many types of higher plants, has been reported from many localities in Mesozoic and Cenozoic rocks. We have found Class I (polylabdanoid) amber in Carboniferous sediments dating to ~320 million years ago. This result demonstrates that preconifer gymnosperms evolved the biosynthetic mechanisms to produce complex polyterpenoid resins earlier than previously believed and that the biosynthetic pathways leading to the types of polylabdanoid resins that are now typically found in conifers and those now typically found in angiosperms had already diverged by the Carboniferous.

Terpenoid resins are produced by nearly all modern conifers and by many angiosperms. These products serve a variety of ecological functions, including sealing and protecting wounds, repelling insects, and discouraging herbivores. (1). Many resins contain terpenoid compounds that readily polymerize when exposed to light or air (1), resulting in the formation of the hardened resinous masses commonly observed on modern trees. Solidified resins are often highly resistant to many typical degradation mechanisms and are commonly preserved in sediments. The fossil form of plant resin is known as amber. Ambers are classified on the basis of the chemical nature of the polymerized terpenoids composing the macromolecular structure (2, 3). The most common class of ambers (Class I), based on polymerized labdanoid diterpenes, has been shown to exist since at least the early Cretaceous (4). Previous investigations of unequivocal pre-Cretaceous terpenoid ambers have been limited to spectroscopic techniques, such as infrared spectroscopy or nuclear magnetic resonance (57). These analyses only survey the functional groups present and do not yield definite chemical structures. Previous pyrolysis–gas chromatography–mass spectrometry (Py-GC-MS) analyses on equivocal Carboniferous resin rodlets and resinite separated from coal resulted in waxy compositions (810) totally unlike the Class I terpenoid ambers presented here. Therefore, the chemical nature of ambers before the Cretaceous and the evolutionary development of terpenoid resin are unknown. Here we present Py-GC-MS analyses of macroscopic amber from Carboniferous [~320 million years old] coal, and demonstrate that this material is a mature Class Ic amber. This result demonstrates that preconifer gymnosperms evolved the biosynthetic mechanisms to produce complex polyterpenoid resins earlier than previously believed.

Class I ambers are subclassified on the basis of the stereochemical nature of the labdanoids that make up their macromolecular structure and on the presence or absence of succinic acid in the macromolecular structure (3). Two types of labdanoid diterpenes are found in Class I ambers: the so-called regular and enantio series (Fig. 1) (11). Regular polylabdanoid ambers containing succinic acid are classified as Class Ia (the abundant and well-known ambers from the Baltic region are generally of this class). Class Ib ambers are also based on regular polylabdanoids, but these do not incorporate succinic acid. This type of amber is the most common on a global basis. Class Ic ambers are based on polymers of enantio-series labdanoids and also lack succinic acid (3). In modern species, resins based on regular polylabdanoids, equivalent to Class Ib ambers, are commonly derived from conifers, whereas resins based on enantio-series labdanoids, equivalent to Class Ic ambers, are commonly associated with angiosperms (there is no known extant species that produces resin analogous to Class Ia ambers). Nonpolylabdanoid ambers are also known, but are much less common than Class I ambers.

Fig. 1

Characteristic products of regular polylabdanoid ambers, I to IV (a to d), are illustrated. Epimeric products derived from the so-called enantio-series labdanoids (11) characteristic of Class Ic ambers, V to VIII (a to d), are also shown [after (3)]. Other identified compounds include 1,2,3,4-tetrahydro-1,5,6-trimethyl-naphthalene (IX); 1,5,6-trimethyl-napthalene (X); 1,2,3,4-tetrahydro-1,1,5,6-tetramethyl-naphthalene (XI); 1,2,3,4-tetrahydro-1,1,6-trimethyl-naphthalene (XII); dehydroabietane (XIII); methyl dehydroabietate (XIV); methyl callistrate (XV); and related compounds (for example, XVI and XVII).

Pyrolysis of Class I ambers produces characteristic bicyclic products derived from the A/B ring system of the original labdanoid monomers (3) (Fig. 1, I to IV, a to d; or V to VIII, a to d). These characteristic products, which are readily identified by GC-MS, retain the original stereochemistry of the precursors and hence are a reliable means of identifying and differentiating Class Ia/Ib and Class Ic ambers.

Polylabdanoid macromolecular resin structures are common across a wide range of taxa. However, it is also common for nonpolymerizable terpenes to occur as occluded products within the polylabdanoid structure (1, 3). The nature and distributions of these compounds, which may include abietanes, pimaranes, totaranes, and kuaranes, among many others, tend to vary more widely between families and genera. These diterpenes are frequently specific to the originating family, and therefore have substantial chemotaxonomic value in reconstructing paleoenvironments, as well as in assisting in the determination of phylogenetic relationships (12).

We have recovered well-preserved macroscopic blebs of amber (Fig. 2) from an Illinois coal seam located within the Lower Desmoinesian Series, Tradewater formation, which is stratigraphically dated to ~320 million years ago, and have analyzed these by Py-GC-MS. Amber blebs were golden yellow and specimens were infrequent. The pyrolysates of these samples showed that they contain characteristic bicyclic products associated with the pyrolysis of Class I (labdanoid based) ambers (Fig. 1, V to VIII, a, d, and e). Methyl ether and alcohol products (Fig. 1, V to VIII, b and c) appear to be absent. These samples lack succinic acid and are composed of enantio-type labdanoids and are, therefore, classified as Class Ic ambers. Analyses of five discrete blebs resulted in nearly identical compound distributions, indicating that these ambers are all derived from the same botanical source. No amber specimens were found in association with plant macrofossils, and the botanical origin of these materials is as yet unknown.

Fig. 2

Bleb of Carboniferous amber. The amber is brittle with resinous luster. Average bleb size is approximately 5 mm.

The stereochemical nature of the labdanoids present in the macromolecular structure of these samples was determined by co-pyrolysis with the following well-characterized reference ambers: Raritan amber, Class Ib, regular labdanoids, coniferous origin (Cupressaceae), from New Jersey, United States (13); and Chiapas amber, Class Ic, enantio labdanoids, angiospermous origin (Hymenaea), from Chiapas, Mexico (3) (Fig. 3). Both regular and enantio labdanoid products are present in the co-pyrolysate of the Carboniferous amber and the Raritan amber (Fig. 3A), whereas only enantio products are observed in the co-pyrolysate of the Carboniferous amber with the Chiapas sample (Fig. 3B).

Fig. 3

Multiple ion traces of co-pyrolysis analyses. (A) Co-pyrolysis of Raritan amber (regular, Class Ib) with the Carboniferous amber and (B) co-pyrolysis of Chiapas amber (enantio, Class Ic) with the Carboniferous amber. Multiple ion traces were constructed using ions of mass-to-charge ratios of 161, 173, 175, and 176, corresponding to the methyl ester pyrolysis products of polylabdanoid ambers (Ia to VIIIa). All four characteristic products are present.

Several bicyclic compounds with structures similar to the characteristic products were also present in the pyrolysates, including 1,2,3,4-tetrahydro-1,5,6-trimethyl-naphthalene (Fig. 1, IX), trimethyl-naphthalenes (Fig. 1, X), 1,2,3,4-tetrahydro-1,1,5,6-tetramethyl-naphthalene (Fig. 1, XI), and 1,2,3,4-tetrahydro-1,1,6-trimethyl-naphthalene (Fig. 1, XII), among others. These compounds are interpreted to be the pyrolysis products of degraded labdanoids within the macromolecular structure and generally differ by having fewer methyl groups and/or by having increased aromaticity. The presence of these compounds is not surprising, considering the age and maturity of these samples.

In addition to the characteristic labdanoid products, several abietane-class diterpenes were also identified in the pyrolysates. These included dehydroabietane (Fig. 1, XIII), methyl dehydroabietate (Fig. 1, XIV), methyl callistrate (Fig. 1, XV), related compounds (Fig. 1, XVI and XVII α and β), and others. These compounds are common in Class I ambers (1, 3), as well as in modern resins (12). The distributions of these products in pyrolysates generated by pyrolysis at temperatures Tpy = 300°C and Tpy = 480°C are essentially identical, indicating that these diterpenes are present in these samples as occluded materials. The exact stereochemistry of dehydroabietane (Fig. 1, XIII) or methyl 16,17-dinor dehydroabietate and methyl 16,17-dinor callitrisate (Fig. 1, XVII α and β) is unknown in these samples, but is drawn in Fig. 1 as commonly found in Class I ambers.

The observation of Class Ic ambers in Carboniferous sediments suggests that preconifer gymnosperms were using complex polyterpenoid resin in a manner similar to that seen in a wide variety of modern species. Modern resins that are structurally analogous to Class Ic ambers are primarily derived from angiosperms. Our data do not imply that angiosperms existed in the Carboniferous, because the fossil record does not record unequivocal angiosperm fossils until the Cretaceous. However, our data do suggest that the divergence of the biosynthetic mechanisms needed to produce resins based on regular and enantio-series labdanoid diterpenes predates both the emergence of true conifers and the differentiation of angiosperms and gymnosperms. Furthermore, these basic biosynthetic pathways have been retained in both gymnosperms and angiosperms through several major extinction events and over 300 million years of evolution. Based on genomic evidence, previous workers have postulated initial differentiation of terpene synthase genes associated with the production of resin-related diterpenes during the Carboniferous (14). Our data support this hypothesis.

Supporting Online Material

  • * Present address: Department of Earth and Planetary Sciences, Macquarie University, Sydney, New South Wales 2109, Australia.

References and Notes

  1. Despite the common terminology used to describe labdanoids related to communic acids as regular and those related to ozic acid as enantio, these products are in fact epimeric, not enantiomers. The same is true of the bicyclic products (Fig. 1, I to IV and V to VIII) derived from the polymers of these terpenoids by Py-GC-MS. This terminology, although imprecise, is deeply entrenched in the literature related to these compounds and is retained here for consistency with numerous previously published works.
  2. This contribution constitutes part 14 of the authors’ series of publications under the general title, “The Nature and Fate of Natural Resins in the Geosphere.”
View Abstract

Stay Connected to Science

Navigate This Article