Chemical and morphological changes in archaeological seeds and fruits during preservation by desiccation

Geochimicaet CosmochimicaActa, Vol. 61, No. 9, 1919-1930, 1997 Copyright© 1997Elsevier ScienceLtd Printed in the USA. All fights reserved 0016-7037/97 $17.00 + .00

Pergamon

P I I S0016-7037(97) 00051-3

Chemical and morphological changes in archaeological seeds and fruits during preservation by desiccation P. F. VAN BERGEN, 1 H. A. BLAND, 1 M. C. nORTON, 2 and R. P. EVERSHED I ~Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, UK 2Department of Archaeology, University of Bristol, 11 Woodland Road, Bristol BS8 1TB, UK (Received June 21, 1996; accepted in revised form January 27, 1997)

Abstract--Soluble and insoluble constituents of modem and ancient (ca. 600 AD) desiccated barley kernels and radish seeds have been studied using high-temperature gas chromatography-mass spectrometry, scanning electron microscopy, and Curie-point pyrolysis-gas chromatography-mass spectrometry. Although some changes in colour are seen, the morphology and anatomy of the desiccated specimens are largely unchanged compared with their extant counterparts. The main chemical alterations arising through long-term desiccation are chemically rather than microbially mediated. Comparison between the lipid data from the modem and ancient barley and radish reveals that extensive ester hydrolysis has occurred over time, while oxidation has been retarded. The insoluble material of the modem barley kernel walls, which is composed of a lignin-cellulose complex characteristic of monocotyledons, undergoes upon desiccation chemical alterations resulting in a significant decrease in the abundance of polysaccharides and cinnamic acids moieties. In marked contrast, the insoluble constituents of the modem radish seed coat yields primarily amino acid moieties upon pyrolysis, most likely deriving from proteins. The seed coat also contains a polyphenolic macromolecule and a small contribution from a dicotyledon lignincellulose complex. This is the first time such a distinct chemical composition has been reported for modem sclerotic plant tissues. The chemical composition of the tissues of the ancient radish specimens appears little altered compared with their modern counterparts; the only obvious difference is the decrease in abundance of 2,6-dimethoxyphenol moieties in the lignin-ceUulose pyrolysis products. Comparison of the microscopic and chemical data with that of walls of propagules, i.e., fruits and seeds, deposited in aquatic environments reveals no differences between the material deposited under desiccating and aquatic conditions. Copyright © 1997 Elsevier Science Ltd 1. INTRODUCTION

changes resulting from chemical degradation to be monitored microscopically, comparisons of the ancient/fossil material with extant counterparts avoids the possible complications arising in studies of bulk organic matter due to multiple inputs. In order to obtain insights into the chemical and morphological changes occurring in organic matter upon desiccation, studies of morphologically well-defined plant structures are necessary. From a geological point of view such sites are rare, however, one very important archaeological site providing a wealth of desiccated morphologically well-preserved plant remains is the Qasr Ibrim settlement in Upper Egypt (approx. early 6 th century AD; Plumley et al., 1977; Rowley-Conwy, 1991 ). At this site large numbers of ancient seeds, fruits, and other morphologically well-defined plant organs have been recovered, all of which have extant counterparts. Initial results from DNA constituents of desiccated material have already revealed high-quality chemical preservation (O'Donoghue et al., 1994, 1996a,b). However, to date, no research on the structural and storage components, constituting the bulk of the organic matter, has been undertaken. Studying the high molecular weight constituents of desiccated seed and fruit material is a major advantage since substantial information on this fraction, in both modem and fossil propagules, i.e., seeds and fruits, is now available (Boon et al., 1989; van Bergen, 1994; van Bergen et al., 1994a,b,c, 1995, 1996; Stout and Boon, 1994; Stankiewicz et al., 1997). However, it should be noted that in all these studies the fossil material was preserved in aquatic environments.

The major factors that influence the preservation of organic matter are the molecular composition of the original biomass and the physico-chemical and biochemical conditions existing in the depositional environment (Tegelaar et al., 1989; Eglinton and Logan, 1991 ). While organic matter is deposited in a wide variety of different environments, preservation is favoured when deposition occurs under aquatic conditions. In aquatic environments the bulk of the organic matter undergoes extremely rapid partial destruction within days or years largely due to microbial activity (Eglinton and Logan, 1991 ). Apart from the microbially mediated transformations, purely chemical changes also affect the composition of the deposited organic matter. Changes in organic matter composition which are primarily autolytic or chemically mediated can be assessed through the study of materials from settings with severely reduced activities of micro-organisms, such as desiccating environments. It is becoming increasingly clear that studies of morphologically well-defined organic plant remains, i.e., leaves, wood, seeds, fruits, algae, rather than those on bulk organic matter, may provide crucial information concerning the degradative processes occurring during fossilization (de I.e.euw et al., 1991; de Leeuw and Largean, 1993). Significantly, investigations combining microscopic and chemical analyses of the resistant constituents of extant and fossil plant remains have provided a wealth of new insights (for a review see van Bergen et al., 1995 ). Apart from allowing morphological 1919

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P.F. van Bergen et al.

In this paper the insoluble constituents of ancient sclerotic kernel walls and seed coats obtained from an arid environment together with their extant counterparts are studied using scanning electron microscopy and Curie-point pyrolysis-gas c h r o m a t o g r a p h y / m a s s spectrometry. In addition, changes in lipid composition from whole propagules are studied using high-temperature-gas c h r o m a t o g r a p h y / m a s s spectrometry. The results obtained from the insoluble material are compared with the changes observed in macromolecular remains of seeds and fruits deposited under aqueous conditions. 2. SAMPLE DESCRIPTION The propagules studied were modern and ancient barley kernels (modern, Hordeum sativum, Poaceae, Monocotyledon; ancient, Hordeum spp.) and modern and ancient radish seeds (Raphanus sativus, Brassicaceae, Dicotyledon). The m o d e r n radish seed variety was black radish which is considered the closest related counterpart to the ancient material ( B r e w e r et al., 1994). The m o d e r n barley kernels (variety Palty) were from a field site at B i r m i n g h a m collected in 1989. The ancient samples were from the Qasr I b r i m settlement in Upper Egypt and were dated approximately early 6 m century A D (Plumley et al., 1977). The site is exceptionally arid resulting in a high degree of morphological preservation of desiccated material (Rowley-Conwy, 1991 ). The samples studied by pyrolysis were kernel walls o f modern and ancient barley and seed coats of m o d e r n and ancient radish whereas whole propagules were used to obtain total lipid extracts.

3. EXPERIMENTAL Whole propagules were powdered and extracted using chloroform/ methanol (2:1 v/v; 3x) to obtain a total lipid extract (TLE). The outer wails of the extant and ancient propagules were removed from the internal tissue (e.g., storage tissue, embryo, cotyledons) by dissection. Propagule walls were extracted ultrasonically using methanol (3x) and dichloromethane (3x). Residues were dried in a vacuum desiccator.

3.1. Scanning Electron Microscopy (SEM) Whole propagules, dissected propagule walls, and fractured specimens were mounted directly on stubs using carbon dag. Stubs were coated with gold and examined under a Hitachi S-2300 at 25 kV. 3.2. Derivatization Free hydroxyl and carboxylic acid groups were derivatized to their corresponding trimethylsilyl (TMS) ethers and esters, respectively, by adding 30#L of N,O-bis(trimethylsilyl)Wifluroacetamide, containing 1% trimethylchlorosilane, to sample aliquots and heating for 1 h at 70°C.

3.3. High Temperature Gas Chromatography (ItT-GC) and High Temperature Gas Chromatography/Mass Spectrometry (HT-GC/MS) HT-GC was performed using a Hewlett-Packard 5890 series II gas chromatograph equipped with a fused-silica capillary column (15 m x 0.32 mm) coated with DB-1 (film thickness 0.1 #m). Derivatized total lipid extracts ( 1.0 #1) in hexane were injected oncolumn. The temperature was programmed from 50°C (2 min) to 400"C (5 min) at a rate of 10°C rain -1. The detector was temperature 405°C. Hydrogen was used as carrier gas (head pressure 10 psi). The HT-GC/MS analyses were performed using a Carlo Erba

Table 1. Relative abundances and ratios of the lignin derived pyrolysis products calculated from Py-GC/MS. G = 2-methoxyphenols; S = 2,6-dimethoxyphenols, Ci = cinnamic acid derived pyrolysis products (4-ethenylphenol and 4-ethenyl-2-methoxyphenol), -C3 = intact aliphatic propenyl side chain, nc = not calculated. Barley modem StotJG,otat+

Ci/Stotal + G~o~tt G-C3/Gto,a~* S-CdStot~ Total S and G with different functional groups::~ ketones aldehydes non-oxygenated

0.53 1.42 0.20 0.24 13 21 65

Radish

ancient modem 1.10 0.90 0.08 0.12 20 4 76

0.40 nc 0.18 0.08 3 7 90

ancient 0.09 nc 0.07 0.06 3 0 97

+Total integrated peak areas of 2-methoxyphenols excluding 4ethenyl-2-methoxyphenol. * Percentage of total integrated peak areas of methoxyphenols excluding 4-ethenyl-2-methoxyphenol.

5160 Mega Series gas chromatograph connected to a Finnigan 4500 mass spectrometer operating at 70 eV scanning the range m/z 50850 in a cycle time of 1.5 s. The temperature was programmed from 50°C (2 min) to 350°C (10 min) at a rate of 10°C min -~ . The interface temperature was 350°C. Helium was used as cartier gas. The capillary column was as described for the HT-GC analyses. Compound identification was based on mass spectral data and retention time comparisons with reference samples.

3.4. Curie-Point Pyrolysis-Gas Chromatography (Py-GC) and Py-GC/Mass Spectrometry (MS) Py-GC analyses were performed with a Carlo Erba 4160 gas chromatograph using a Horizon Instruments Curie-point device for pyrolysis. The interface temperature of the pyrolysis unit was set at 250°C. The samples were pressed onto a ferromagnetic wire (Curie temperature 610°C) and pyrolysed for 5 s. The GC was programmed from 30°C (5 min) to 315°C (10 min) at a rate of 4°C min -~. Separation was achieved using a fused-silica capillary column (25 m x 0.32 mm) coated with CPSil-5 CB (film thickness 0.4 #m). Helium was used as the carrier gas. Py-GC/MS analyses were performed using a Carlo Erba 5160 mega series GC connected to a Finnigan 4500 mass spectrometer operated at 70 eV scanning the range m/z 50-650 in a cycle time of 1 s. The pyrolysis unit, the capillary column, and temperature programme were as described for the Py-GC. Compounds were identified based on retention time comparisons with reference samples and literature mass spectra (e.g., Pouwels et al., 1987, 1989; van Smeerdijk and Boon, 1987; Faix et al., 1990a,b, 1991a,b; Ralph and Hatfield, 1991; Chiavari and Galletti, 1992; van Bergen, 1994; van Bergen et al., 1996; Stankiewicz et al., 1996). Peak areas of the most significant lignin derived pyrolysis products were calculated from the Py-GC/MS data using the two most prominent mass ions for each of the compounds (cf. Stankiewicz et ai., 1997). Changes in lignin composition were expressed as the ratio of 2-methoxyphenols or 2,6-dimethoxyphenols with intact aliphatic C3 side chain over the total (G-C3/Gtot~, S-C3/S,ot~; Table 1 ), and the percentage of different functional groups (ketones and aldehydes) substituted at the C3 side chain (cf. van der Heijden and Boon, 1994). 4. RESULTS M o d e r n and ancient propagules of barley and radish were studied using scanning electron microscopy, high-temperature-gas c h r o m a t o g r a p h y / m a s s spectrometry, and Curie-

Preservation of archeological seeds and fruits

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Fig. 1. Cross-sections of modern (a) and ancient (b) barley kernels, and modern (c) and ancient (d) radish seeds as observed by scanning electron microscopy. For sources of the material see text. (a) SEM of extant Hordeum sativum (barley) kernel. From top to bottom, husk (H) with on top small thick walled cells (T), compressed pericarp (P) and seed coat (S), and the large cells of the underlying aleuron layer (A); 560x. (b) SEM of ancient Hordeum spp. kernel. The composition of the kernel wall is identical to that of the modern Hordeum; 840x. (c) SEM of modern Raphanus sativus seed. From top to bottom, remnants of seed coat epidermis (E), thick-walled columnar cells (L; palisade layer), compressed pigment layer (G) and thick walled aleurone layer (A); 880x. (d) SEM of ancient Raphanus sativus. The seed coat composition is very similar to that of the modem counterpart; 880x.

point pyrolysis-gas chromatography/mass spectrometry in order to assess morphological and chemical changes in the organic matter composition occurring during preservation under desiccating conditions.

4.1. Microscopic Examination Comparisons of the modern and ancient specimens by electron microscopy showed that the overall morphology and wall anatomy of both the barley kernels and radish seeds had remained unaltered (cf. Fig. la,c vs. lb,d). Moreover, no visible evidence of microbial activity, i.e., fungal remains and/or bacterial sheets, was observed in the desiccated specimens. Although morphologically identical to the extant fruits, the overall colour, as determined visually, of the barley kernels and storage tissue was darker in the ancient samples. The outside of the ancient kernel was dark reddish-brown whereas the modern kernels were light brown. The storage tissue was dark brown to black, while that of the modern fruits was white. The grain walls of the modern (Fig. la)

and ancient (Fig. lb) barley kernels were found to comprise a thin compressed seed coat (S) and pericarp (P; fruit wall) attached to a sclerotic outer layer, husk (H; cf. Freeman and Palmer, 1984). This husk consisted of a layer of thick-walled cells with large lumina, which was two to three cells deep, under a layer of smaller thick-walled cells, which was one cell deep (T; Fig. la,b). The husk constituted the bulk of the kernel wall. Comparison of the modern and ancient radish seeds also revealed some differences in colour of the whole seed and the storage tissue. All ancient seeds were dark brown, whereas the modern radish seeds varied in colour from light grey-brown to reddish brown. The storage tissue of the ancient sample was dark brown to black, whereas that of the modern seeds was yellow. The ancient seeds were relatively brittle and soft compared with the modern specimens. The modern (Fig. lc) and ancient (Fig. ld) radish seed coats were composed of two distinct zones. The outer zone was composed of columnar thick-walled sclerotic cells with relatively large lumina (L; cf. palisade layer sensu Lyshede, 1982) underlying a thin sheet-like layer (E). This latter

1922

P.F. van Bergen et al. TAG f

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Fig. 2. Partial HT-GC traces of the total lipid extracts of whole kernels of (a) modern and (b) ancient barley. IS = interna/standard, DAG = Diacylgtycerols, TAG = Triacylglycerols. Numbers annotated for the DAG and TAG indicate total acyl carbons. For additional information about the source of the material see text.

layer is most likely derived from remnants of the seed coat epidermis and subepidermal cell layers (Lyshede, 1982). The inner zone was composed of a layer of compressed cells (G; cf. pigment layer sensu Lyshede, 1982) and a layer of thick-walled cells with very small lumina which was one cell deep (cf. aleurone layer sensu Lyshede, 1982). The outer layer was approximately one and a half to two times the thickness of the inner layer.

4.2. Chemical Composition 4.2.1. Total lipid extracts Barley. The total lipid extract (TLE) of the modem barley sample consisted primarily of triacylglycerols (TAGs) bearing 50, 52, 54, and 56 acyl carbon atoms (Fig. 2a). The main fatty acyl moieties were C16:0and C~s:2, although analysis by mass spectrometry also revealed the presence of monounsaturated and triunsaturated C~8 components. Aside from TAGs, diacylglycerols (DAGs), bearing 34 or 36 acyl carbon atoms, and free fatty acids, Ci6:0 and C~8:2, were present. Sterols, mainly sitosterol and campesterol, were also apparent, as were steryl esters which elute just prior to the triacylglycerols. In sharp contrast, the TLE of ancient specimens was dominated by free fatty acids, C)6:0, CIS:~, and C~8:2, with little

evidence of TAGs (Fig. 2b). However, diacylglycerois were still present, as were the sterois and steryl esters observed in the modem barley. The results from the modem specimens are in close agreement with data reported in the literature (de Man and Cauberghe, 1988; de Man and Vervenne, 1988). Radish. Like the TLE of the modem barley kernels, that of the radish seeds was dominated by a mixture of triacylglycerols (Fig. 3a). These, however, consisted of a much wider range of homologues (C50 to C64) representing the greater variety of component fatty acid moieties present (C~6:o, C~8:l, C18:~, C18:3, C2o:~, and Cn:)). Sterols, mainly brassicasterol, sitosterol, and campesterol, were also identified from the extracts of the modem seeds. In contrast to the modem seeds, the TLE of the ancient specimens was dominated by free fatty acids and diacylglycerols with only small quantities of triacylglycerols still present (Fig. 3b). The free fatty acids present were in the carbon number range C16:0 to C24:j and comprised predominantly saturated or monounsaturated components with a small contribution from the diunsaturated C~8 acid. 4.2.2. Pyrolysates

All pyrolysates were dominated by phenols, e.g., alkylated phenols and methoxyphenols, and polysaccharide pyrolysis

Preservation of archeological seeds and fruits

1923

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Fig. 3. Partial HT-GC traces of the total lipid extracts of whole seeds of (a) modern and (b) ancient radish. IS = internal standard, DAG = Diacylglycerols, TAG = Triacylglycerols. Numbers annotated for the DAG and TAG indicate total acyl carbons. For additional information about the source of the material see text.

products (Figs. 4, 5 ). However, the distributions of the individual pyrolysis products and the relative concentrations of the various compounds classes differed substantially. Barley. The pyrolysate of the modern barley kernel wall was dominated by 4-ethenyl-2-methoxyphenol (Fig. 4a; Note that 4-ethenyl-2-methoxypbenol is off-scale in order to allow annotation of the other pyrolysis products). In addition, other 2-methoxyphenols (G; guaiacyl moieties), 2,6dimethoxyphenols (S; syringyl moieties), 4-ethenyl-phenol, and polysaccharide pyrolysis products (ps), in particular 4hydroxy-5,6-dihydro-(2H)-pyran-2-one (HE1) and levoglucosan (LG), were abundant. Ferulic acid and its methyl ester were also detected as were C~6 and Ct8 fatty acids, which are commonly encountered in pyrolysates of modern plant material. The pyrolysis data are consistent with those reported for monocotyledonous sclerotic materials (e.g., Boon, 1989; Galletti and Reeves, 1991; Ralph and Hatfield, 1991; van der Hage et al., 1993; Terron et al., 1993; van Bergen, 1994). 4-Ethenyl-2-methoxyphenol also dominated the pyrolysate of the ancient barley specimens (Fig. 4b), however, the relative concentration of this compound was significantly lower compared with that of the modem counterpart (Table

1 ). The same methoxyphenols were observed in the pyrolysates of the modern and ancient specimens. However, it should be noted that 2-methoxyphenol, 2,6-dimethoxyphenol, and methoxyphenol derivatives with ketone side-chains (e.g., 4-acetyl-2-methoxyphenol, 4-acetyl-2,6-dimethoxyphenol) showed an increase in relative abundance in the pyrolysate of the ancient barley (Fig. 4b; Table 1). In addition, the relative decrease of 4-hydroxy-5,6-dihydro-(2H)pyran-2-one and levoglucosan should be noted. The same fatty acids as those in the pyrolysate of the modern barley were detected whereas ferulic acid and its methyl ester were absent from the pyrolysate of the ancient specimen. Radish samples. In sharp contrast to the barley, the pyrolysate (Fig. 5a) of the seed coat of the modern radish was dominated by pyrrole, (alkylated)phenols, and fatty acids (Cl6, Cls, C20, and C22). In addition, several polysaccharide pyrolysis products (ps), various other nitrogen-containing products, such as pyridine, phenylalkyl nitriles, indole, methylindole, diketodipyrrole (DK), diketopiperazines (DKPs), and 2-methoxy-and 2,6-dimethoxyphenols were detected. The distribution patterns of the methoxyphenols were largely similar to those reported for the pyrolysates of dicotyledonous sclerotic plant remains (e.g., wood: Saiz-Jimenez and

1924

P.F. van Bergen et al.

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Fig. 4. GC traces of the Curie-pointpyrolysates (610°C) of the insoluble kernel wall remains of (a) modern and (b) ancient barley. Note that 4-ethenyl-2-methoxyphenolis off-scale in Fig. 4a. Key: P = Phenol; 2P = 2-methoxyphenol;3 + 4P = coeluting 3-and 4-methylphenol; G = 2-methoxyphenol; S = 2,6-dimethoxyphenol;ps = polysaccharide pyrolysis products; psi = polysaccharide pyrolysis product (m/z 55,86,114, cf. #51 in Faix et al., 1991b); HEI = hemicellulose marker, 4-hydroxy-5,6-dihydro-(2H)-pyran-2-one; LG = Levoglucosan; * = contaminants. Side chains of the G and S components are attached at carbon position 4 of the ring. CI6FA refers to hexadecanoic acid. For additional information about the source of the material see text.

de Leeuw, 1984, 1986; Boon et al., 1987; van der Heijden and Boon, 1994; propagules: Boon et al., 1989; van Bergen et al., 1994b,c 1995, 1996; Stout and Boon, 1994). The abundant polysaccharide products present in the pyrolysate of the barley kernels (Fig. 4a), levoglucosan, and 4-hydroxy5,6-dihydro-(2H)-pyran-2-one, were present only in low concentrations in the radish sample whereas several other polysaccharide derived pyrolysis products, 1,4-anhydrofuranose (HE2) and products with a molecular weight of 128 (RH), indicating rhamnose (van Smeerdijk and Boon, 1987), were characteristic in the radish pyrolysate. Although the pyrolysate of the ancient specimen was dominated by the same compounds as that of the modem sample, the polysaccharide pyrolysis products were slightly less abundant when compared with the modern samples. The same nitrogen-containing compounds, phenols, and methoxyphenols were present, however, it should be noted that the 2,6-dimethoxyphenols were relatively decreased in abundance compared with those observed in the modem specimens (Stotal/Gtotal; Table 1 ). 5. DISCUSSION The results obtained from the modem and ancient propagules of barley and radish are discussed below in terms of

the morphological and chemical changes in organic matter composition which occur upon preservation under conditions of desiccation. The observations on the insoluble remains of the kernel walls and seed coats are compared with those reported for the same plant structures, although not the same species, deposited under aquatic conditions. 5.1. Morphology and Anatomy of the Desiccated Propagules The general morphology and anatomy of the ancient propagules has remained unchanged by desiccation (Fig. 1 ). This is not surprising considering that the Qasr Ibr~m site is located in a virtually rainless environment (Rowley-Conwy, 1991 ) and the moisture content is below the minimum ( 2 0 30%) necessary for microbially mediated alterations (Hedges, 1990). Unaltered morphology and anatomy has been reported previously for other ancient and fossilized fruits and seeds (Boon et al., 1989; van Bergen et al., 1994b,c, 1995, 1996), however, in sharp contrast to the specimens studied here, these latter propagules were derived from sediments deposited in aquatic settings. Our microscopic observations, revealing no visible evidence of microbial activity, imply, therefore, that any chemical alteration

Preservation of archeological seeds and fruits

1925

?

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Fig. 5. GC traces of the Curie-point pyrolysates (610°C) of the insoluble seed coat remains of (a) modern and (b) ancient radish. Key: P = Phenol; 2P = 2-methoxyphenol; 3 + 4P = coeluting 3- and 4-methylphenol; C = 1,2benzenediol (catechol); G = 2-methoxyphenol; S = 2,6-dimethoxyphenol;ps = polysaccharide pyrolysis products; HE1 = hemicellulosemarker, 4-hydroxy-5,6-dihydro-(2H)-pyran-2-one;HE2 = hemicellulosemarker, 1,4-anhydrofuranose; RH = rhamnose marker; LG = Levoglucosan; DK = diketodipyrrole; DKP = diketopiperazines (1, rrdz 57, 86, 125, 154; 2, rrdz 55, 70, 84, 86, 154; 3, m/z 70, 194; 4, m/z 70, 86, 125, 154), * = contaminants. Side chains of the G and S componentsare attached at carbon position 4 of the ring. C~rFArefers to hexadecanoicand hexadecenoic acids. For additional information about the source of the material see text.

observed in the soluble and insoluble constituents of the desiccated material are chemically, e.g., oxidatively, limited hydrolytically or autolytically, rather than microbially mediated (cf. van Bergen et al., 1994c). This is, however, based on the assumption that chemical changes microbially induced are expressed as changes on a microscopic scale. But, to date, all studies reporting chemical alterations of plant materials due to biodegradation have shown either changes in gross morphology or visible evidence of remnants of microbial organisms (e.g. Hedges et al., 1985; Stout and Spackman, 1989; Blanchette and Simpson, 1992; van der Heijden and Boon, 1994).

5.2. Barley Kernels 5.2.1. Modem specimens The data from the total lipid extract of the whole barley kernels (Fig. 2a) is consistent with the general composition known for cereal lipids (Gunstone et al., 1986) with TAGs and sterol derived compounds predominating. Previous results obtained for the fatty acid composition (acyl esters and unesterified fatty acids) of modem barley kernels (de Man and Canberghe, 1988) compare closely with results gained

in this study (and also those of Bland and Evershed, unpubl. data) showing dominance of the Ct6:o and C~8:2 members amongst the TAG fatty acyl moieties and free acids. The pyrolysate of the modem barley kernel wall (Fig. 4a) clearly indicates the presence of monocotyledon lignin (so called grass-lignin; Saiz-Jimenez and de Leeuw, 1986), based on 2-methoxyphenols, 2,6-dimethoxyphenols, and the presence of ferulic acid and its abundant pyrolysis product 4-ethenyl-2-methoxyphenol (e.g., Boon, 1989; Galletti and Reeves, 1991; Galletti et al., 1991; Ralph and Hatfield, 1991; van Bergen, 1994). In addition, polysaccharide pyrolysis products, such as levoglucosan, indicate cellulose (Pouwels et al., 1989) whereas, 4-hydroxy-5,6-dihydro-(2H)-pyran2-one is indicative of hemicellulose (e.g., Pouwels et al., 1987, 1989). These results are consistent with pyrolysis data from other lignin-contalningmonocotyledon samples, in particular, cereals (e.g., Boon, 1989; Galletti and Reeves, 1991; Galletti et al., 1991; Ralph and Hatfield, 1991). Two of the main pyrolysis products in such samples are usually 4-ethenylphenol and 4-ethenyl-2-methoxyphenol. They are almost exclusively considered to be derived from the cinnamic acids, ferulic and p-coumaric acid, which are both esterified and etherified to lignins as well as ester-linked to

1926

P.F. van Bergen et al.

the carbohydrate constituents (e.g., Hartley and Jones, 1977; Scaibert et al., 1985, 1986; Iiyama et al., 1990; Ralph and Hatfield, 1991; Ralph et al., 1992; Lam et al., 1992). An alternative source for the 4-ethenyl-2-methoxyphenol could be ferulic acid esterified to other compounds, such as sterols, which have been isolated from monocotyledon propagules (Collins, 1986; Evershed et al., 1988). However, as no abundant sterol pyrolysis products were detected, a contribution of those steryl esters appears relatively minor. The fatty acids ( F A ) , which are commonly observed in pyrolysates of modern plant remains, are most likely present as polyester-linked moieties which are common plant constituents (Walton, 1990).

5.2.2. Ancient specimens The lipid data of the whole ancient kernels (Fig. 2b) clearly reveals degradation of the triacylglycerols which are the dominant components in the modern specimens. The high abundance of free fatty acids indicates that the most likely pathway for TAG degradation is hydrolysis which can occur either spontaneously (i.e., initiated by lipid oxidation products within the propagule) or catalysed by lipases from micro-organisms (Gunstone et ai., 1986). However, the lack of evidence for the presence of micro-organisms suggests, in this case, that hydrolysis occurred autolytically. Furthermore, the overall fatty acid distribution shows an exceptional degree of preservation. This is highlighted by the survival of C~8:2which, although depleted compared with the modem specimens, is rarely seen in lipid extracts of archaeological finds (Simic et al, 1992) due to depolymerization or oxidation (Chan, 1987; Davfdek et ai., 1990). Moreover, no significant changes are seen in the sterol and steryl ester composition again emphasizing that the dominant mechanism of chemical alteration is hydrolysis rather than oxidation. Substantial consideration has been given to the chemical changes occurring in lignin-containing plant structures upon fossilization (e.g., Hedges et al., 1985; Stout et al., 1988; Hatcher, 1988, 1990; Hatcher et al., 1989a,b, 1994). However, it should be emphasized that all these studies focused on material deposited under aquatic conditions. Moreover, only one study reports on the chemical changes upon fossilization occurring in monocotyledonous lignin (van Bergen et ai., 1994c). The major changes in chemical composition that were observed include: ( 1 ) removal of carbohydrates, (2) loss of ester-linked phenolic acids attached to carbohydrates, (3) loss of 2,6-dimethoxyphenol moieties, (4) C3 side-chain degradation, and (5) a dramatic relative increase of alkylphenols when compared with the methoxyphenols. It should, however, be noted that all this earlier work was based on material much older than the material under consideration herein (several million years vs. 1,400 years). Moreover, the lignin composition of their specimens, to a certain extent, differed from the normal grass-lignin. Despite these differences, the data presented here on the ancient barley kernel (Fig. 4b) appear in agreement with the earlier observations, especially with respect to the first two processes; the most obvious differences in chemical composition between the modem and ancient specimens (Fig. 4a vs. 4b) are the marked decreases in the relative abundance of pyrolysis

products of cellulose, based on levoglucosan, hemicellulose, and ester-linked cinnamic acids (4-ethenylphenol and 4ethenyl-2-methoxyphenol; Ci/Stotal + Gtot~l, Table 1 ). The relative decrease of these latter compounds may be due to oxidation processes as the double bond present in the C3 side-chain of the cinnamic acids is prone to autoxidation and photo-oxidation (Davfdek et al., 1990). In contrast, there is no apparent removal of 2,6-dimethoxyphenols but rather a relative increase as evidenced by their abundance in the pyrolysate of the ancient specimens (Stotal/Gtotal, Table t ). Whether this is an effect of the desiccation process causing preferential degradation of other constituents or because of a slightly different chemical composition (greater abundance of 2,6-dimethoxyphenol moieties) in the original material cannot be assessed at this stage. The ancient sample shows evidence of changes in the C3 side-chain as revealed by the decrease of methoxyphenols with intact C3 side chains (GC3/Gtotal, S-C3/Stotal; Table 1) and the increase of methoxyphenols with side chains containing ketones (e.g., 4acetyl-2-methoxyphenol, 4-acetyl-2,6-dimethoxyphenol; Table 1). C3 side-chain alterations appear to be a common phenomenon in lignin modifications (Hatcher et al., 1989a,b; van Bergen et ai., 1994c; van der Heijden and Boon, 1994) and are suggested to be one of the major reasons for the formation of alkylphenols in diageneticaily altered lignin samples (Hatcher et al., 1989a,b; van Bergen et al., 1994c). In view of this, it is not surprising that the (alkyl)phenols are also slightly more abundant in the ancient specimen compared with the modern one. 5.3. Radish Seeds

5.3.1. Modem specimens The total lipid extract results of the modern radish seeds (Fig. 3a) are consistent with literature data (O'Donoghue et al, 1996a) showing that C~8:2and Ct8:3 fatty acids are present in significant relative quantities. Furthermore, the sterol composition corroborates that known for radish, especially the presence of brassicasterol, which is a characteristic component of crucifer seeds (O'Donoghue et al., 1996a). In sharp contrast to the barley kernel wails, the insoluble components of the extant radish seed coats (Fig. 5a) are only partly composed of a lignin-cellulose complex, characteristic of dicotyledons, as revealed by 2-methoxyphenols, 2,6-dimethoxyphenols, and polysaccharide pyrolysis products indicating the presence of cellulose and hemicellulose (e.g., Saiz-Jimenez and de Leeuw, 1986; Pouwels et al., 1987; Boon et al., 1989; van Bergen et al., 1994c). Moreover, several of the polysaccharide pyrolysis products, such as 1,4-anhydrofuranose (HE2), indicating hemicellulose and the rhamnose derived compounds, are characteristic of distinctly different polysaccharides from those found in lignincellulose complexes. In contrast, almost all of the most abundant pyrolysis products, other than those derived from lignin-cellulose, can be explained from amino acid moieties, most probably present as proteins in the seed coat (Lyshede, 1982). Pyrrole is produced upon pyrolysis from proline, hydroxyproline, or glutamic acid moieties (Bracewell and Robertson, 1984;

Preservation of archeological seeds and fruits Chiavari and Galletti, 1992; Stankiewicz et al., 1996). Furthermore, proline moieties account for the presence of the various diketopiperazines detected (DKP, Fig. 5; cf. Chiavari and GaUetti, 1992; Stankiewicz et al., 1996), whereas diketodipyrrole (DK) is produced upon the pyrolysis of hydroxyproline (Chiavari and Galletti, 1992; Stankiewicz et al., unpubl. results). In addition, phenol, 4-methylphenol, and 4ethenylphenol are pyrolysis products of tyrosine (Bracewell and Robertson, 1984; Tsuge and Matsubara, 1985; Chiavari and Galletti, 1992), toluene, and the two phenylnitriles can derive from phenylalanine amino acid moieties (Tsuge and Matsubara, 1985; Chiavari and Galletti, 1992), whereas indole and methylindole may originate from tryptophan units (Tsuge and Matsubara, 1985; Chiavari and Galletti, 1992). Various proteins present in radish seed coats (e.g., Lyshede, 1982; Monsalve et al., 1994; Terras et al., 1995) contain, in particular, abundant proline and glutamic acid moieties which could account for the high abundance of pyrrole in the pyrolysate. However, the recognition of diketodipyrrole (DK) implies that hydroxyproline moieties are also present and these will, therefore, also have contributed to pyrrole. Furthermore, hydroxyproline, which is particularly abundant in animal structural-proteins such as collagen (Voet and Voet, 1995), is to be expected as it is known to occur in glycoproteins of cell walls, which are believed to play a structural role in the primary cell-wall of dicotyledons (Dey and Brinson, 1984). In contrast, tyrosine moieties, which would yield phenols upon pyrolysis, are not very abundant. It is, therefore, concluded that phenol and methylphenol are derived, at least in part, from a nonproteinaceous polyphenolic macromolecule. It should be mentioned that we know of no studies to date reporting abundant amino acid-derived products in pyrolysates of modem or ancient propagule wails. As in the barley kernel walls, the fatty acids observed in the pyrolysates could derive from naturally occurring polyesters, but are, in this case, believed to originate from the abundant triacylglycerols (TAGs; Fig. 3a). As the tissues were exhaustively extracted, we believe that the TAGs have to be strongly absorbed or physically occluded into the insoluble polymeric matrix of the seed coat. 5.3.2. Ancient specimens As with the ancient barley kernels, the total lipid extract of the whole ancient radish seeds shows that extensive hydrolysis has taken place during the desiccation process. Again, because of the lack of evidence for microbial activity, this process is thought to have occurred autolytically. Studies on the total fatty acid composition, analysed as methyl ester derivatives (O'Donoghue et al, 1996a; Bland and Evershed, unpubl, data), have shown the preservation of C1s:2, C20:2, and Cls:3 fatty acids implying that, as for the barley specimens, oxidation processes were retarded. The pyrolysis data indicate that the insoluble constituents of the ancient seed coats (Fig. 3c) are largely similar to those of their extant counterparts. The only difference is that there is a slight decrease in the relative abundance of lignin derived 2,6-dimethoxyphenols (StoJGtot~, Table 1 ). Chemical alterations during fossilization of dicotyledonous lignin

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are well-documented (e.g., Hatcher et al., 1989b; Stout and Boon, 1994; van Bergen et al., 1994c) and, primarily, involve analogous degradative processes as in the monocotyledonous material namely: (1) removal of carbohydrates, (2) loss of 2,6-dimethoxyphenols, (3) formation of 1,2-benzenediol moieties, (4) C3 side-chain degradation, (5) alkylation at the carbon next to the original ether linkage, and (6) a relative increase in the abundance of alkylphenols compared with the methoxyphenols (cf. van Bergen et al., 1994c). The lignin-cellulose complex in the ancient radish seed coat appears to have undergone some chemical alteration upon desiccation as revealed by the decrease of dimethoxyphenols (Sto~/G~ot~, Table 1) and methoxyphenols with intact C3 side chains (G-C3/Gtoua, S-C3/S~I; Table 1) which is in concordance with previous observations (e.g., Hatcher et al., 1989b). Despite the exhaustive extraction, the fatty acids appear to exist primarily as free moieties since they are present in high abundance upon thermal evaporation (Curie-wire 358°C; data not shown) as well as in the TLE (Fig. 3b). However, as TAGs are still present in the ancient material (Fig. 3b), the fatty acids may be derived from these compounds. A contribution from chemically bound fatty acids in pyrolysates at higher Curie-temperatures cannot be excluded. As with the modem sample, most pyrolysis products of the ancient seed coat are believed to derive from amino acid moieties most likely present as proteins. An interesting aspect of these latter compounds is the fact that antifungal proteins have been reported from radish seed coats which create a microenvironment around the seeds in which fungal growth is suppressed (Terras et al., 1995). The presence of these compounds may also have contributed to the high quality morphological and chemical preservation (i.e., preservation of nucleic acids; O'Donoghue et al., 1994, 1996a,b) of these seeds in the archaeological record. The phenolic macromolecule present within the seed coat would provide additional protection against microbial degradation as phenols are known for there fungicidal properties (e.g., Collins, 1986; Waller, 1987). Despite the presence of antifungal proteins in modem seed coats, the actual presence of proteins itself in these ancient seed coats is rather surprizing as they are known to be chemically labile (Tegelaar et al., 1989). Additional explanantions for their presence could be physical occlusion within the actual plant structure itself or protection by chemically more resistant molecules (cf. van der Heijden and Boon, 1994). Alternatively, the amino acid moieties are present as polypeptides, different from the original proteins, or could have reacted with carbohydrate monomers to form condensation products, the so-called melanin complexes (e.g., Eglinton and Logan, 1991). The distinctive composition of the insoluble constituents in sclerotic material is rather unusual. To date, the insoluble constituents of all but one of the sclerotic propagule walls studied have been shown to be dominated by lignin-cellulose or lignin-hemicelhilose (for a review see van Bergen et al., 1995). The only exception was the propagule wall of the water lily, Nelumbo, which was composed of a polysaccharide-tannin complex (van Bergen et al., 1996, 1997). However, the Nelumbo fruit wall revealed distinctly different products from those of the radish seed coat upon pyrolysis.

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Whether the distinct chemical composition of the insoluble remains of radish seed coats is diagnostic for radish or also occurs in sclerotic tissues of other plants from the same family (Brassicaceae) or order (Capparales s e n s u Cronquist, 1981 ) is as yet unknown. Systematic studies of the insoluble constituents of closely related plants within the family Brassicaceae may provide a clearer insight into this phenomenon.

changes in lignin-cellulose containing plant structures, such as propagules, occurring under desiccating or aqueous conditions. However, it should be emphasized that the material under consideration has undergone a relatively short fossilization history and, therefore, changes specific to desiccation may occur later in the preservation process.

6. SUMMARY 5.4. Comparison of the Insoluble Remains of Propagules Obtained from Desiccating and Aquatic Environments As mentioned earlier, the main difference between desiccating and aquatic environments is the virtual absence of microbial activity under desiccated conditions. Our data concur with this assertion based on the absence of visible morphological and anatomical changes of the specimens studied. However, unaltered morphologies of propagules have also been reported for fossil propagules deposited in aquatic environments (Boon et al., 1989; van Bergen, 1994; van Bergen et al., 1994b,c). Moreover, the chemical alterations of the insoluble constituents of the desiccated specimens are not significantly different from those reported for the lignincellulose complexes in other fossil seeds and fruits. The absence of distinct morphological alterations of propagules walls under both aquatic and desiccating conditions could imply that the actual physical structure of those tissues is the determining factor for the high degree of morphological preservation of the whole propagule rather than the chemical composition. This thesis would be in concordance with the fact that the propagule walls are resistant covering envelopes protecting the genetic material inside. Recent studies of propagule walls, however, have shown that the chemical composition may play an equally, if not more, important role in determining the preservation potential of these plant organs (van Bergen et al., 1996, 1997). However, it should be mentioned that the chemical composition of the specimen with the low preservation potential (van Bergen et al., 1996, 1997) was completely different from those studied here, the chemical constituents of which are more similar to the propagules studied from aquatic environments. The similarities between materials from the desiccating and aqueous environments imply, perhaps surprisingly, that the microscopic and chemical effects of desiccation on plant remains, containing lignin-cellulose complexes, are very similar to those occurring under the more usual aquatic depositional settings. Based on the findings from the aquatic material it has already been postulated (van Bergen et al., 1994b,c) that changes of the lignin-cellulose were chemically rather than microbially mediated. The main problem with this hypothesis is the difficulty in establishing the absence of microbial degradation in an aquatic setting; enzymes secreted by micro-organisms could cause transformations without leaving obvious microscopic evidence. We believe that the chances of microbial degradation of the material studied here are very unlikely and, therefore, provide the only feasible source of evidence of purely chemically (including autolytic enzymatic reactions) mediated changes in lignin-cellulose-complexes. So far, there are few indications of differences in the chemical and microscopic

Total lipid extracts (TLE) and pyrolysates of c a . 1400 y old desiccated barley kernels and radish seeds were compared with the TLEs and pyrolysates of their modern counterparts to obtain insight into the chemical and morphological changes arising through long-term desiccation. The main observations are summarized below. The morphology and anatomy of the desiccated specimens were largely unchanged compared with their extant counterparts and revealed no visible evidence of biodegradation. Comparison between the total lipid extract data from both the barley kernels and radish seeds revealed extensive ester hydrolysis of triacylglycerols while oxidation processes have been retarded. The modern barley kernel walls were composed of a lignin-cellulose complex characteristic of monocotyledons. The main chemical alterations arising through desiccation were chemically rather than microbially mediated resulting in a significant decrease in the abundance of polysaccharides and cinnamic acids moieties. Additional alterations to the lignin molecule were C3 side-chain degradation and oxidation. The insoluble constituents of the modern radish seed coat yielded primarily amino acid moieties upon pyrolysis which are most likely derived from proteins. In addition, the seed coat also contained a polyphenolic macromolecule and a small contribution from a dicotyledon lignin-cellulose complex. The chemical composition of the tissues of the ancient specimens appeared little altered compared with their modern counterparts. The only obvious difference was the decrease in abundance of 2,6-dimethoxyphenol moieties in the lignin-cellulose pyrolysis products. Comparison of the microscopic and chemical data with that of walls of propagules, i.e., fruits and seeds, preserved in aquatic settings revealed no obvious differences between the material deposited under desiccating and aquatic conditions. Acknowledgments--This project was undertaken whilst the authors

were in receipt of NERC Grant GR3/9578 to RPE and a NERC studentship (JT4/95/24/b) to HAB which are gratefully acknowledged. We thank Dr. R. Sallares for providing the modem barley sample and the Egyptian Exploration Society for providing the ancient material. The use of the NERC Mass Spectrometry Facilities (Grants GR3/2951, GR3/3758, FG6/36/01 ) is gratefully acknowledged. Mr. J. Dimery is thanked for his help with the scanning electron microscopy. Drs. J.J. Boon, G.A. Logan, and S. Peulv6 are thanked for critically reading an earlier version of the manuscript. Editorial handling: R. E. Summons

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