Degradation of intracrystalline proteins and amino acids in fossil

brachiopods

DEREK WALTON

Division of Earth Sciences, University of Derby, Kedleston Road, Derby, DE22 1GB, U.K.

(Received 6 October 1996; returned to author for revision 1 April 1997; accepted 4 December 1997)

AbstractÐFour genera of Recent to Plio-Pleistocene articulated brachiopods were collected from up to16 horizons spanning the last 3.3 Ma of sediment deposition in the South Wanganui Basin, New Zeal-and, and assayed for the preservation of intracrystalline proteins and/or amino acids. The proteins pre-sent in the shells of living and Recent brachiopods undergo rapid degradation through thedecomposition of the peptide bond. Up to 95% of the constituent amino acids from the proteins arepresent in the free state by 0.12 Ma. This rate of degradation is far higher than was originally expectedfor intracrystalline proteins. Quantitative analysis of the concentrations of amino acids present withinthe shells of fossil brachiopods indicates a range of reaction rates for the subsequent degradation of in-dividual amino acids. The degradation of these amino acids may lead to the total loss of compounds,to the generation of non-standard amino acids, or to diagenetically produced proteinaceous aminoacids. These reactions do not necessarily mirror those which occur during the pyrolysis of an aqueoussolution of the pure amino acids, either in their rate or products. # 1998 Elsevier Science Ltd. Allrights reserved

Key wordsÐintracrystalline proteins, amino acids, protein degradation, decomposition pathways,brachiopods

INTRODUCTION

The fossilised hard parts of invertebrates are rich

sources of the degraded remains of organic mol-

ecules originally trapped during biomineralisation

(see, for example, Curry, 1988). Although the po-

tential signi®cance for these biomolecules in estab-

lishing molecular phylogenies for fossil material was

recognised in the early stages of their study

(Abelson, 1955), it was not clear to what extent

compositional di�erences were due to phylogenetic

di�erentiation or were re¯ecting variable degra-

dation of the original molecules (Abelson, 1955).

For phylogenetic interpretation to be meaningful, it

is important to determine precisely the degradation

characteristics of these biomolecules from a site

protected from extraneous contamination.

The discovery that molluscs (Watabe, 1963), echi-

noderms (Pilkington, 1969) and brachiopods

(Collins et al., 1988) contain organic molecules

within (intracrystalline), as well as between (inter-

crystalline), the shell crystallites provided a source

for fossil biomolecules entombed within the biomin-

eral which cannot be degraded or contaminated by

bacteria (Sykes et al., 1995). As the term ``intracrys-

talline'' remains subjective, this study follows the

operational de®nition given by Sykes et al. (1995).

Degradation (and therefore change in relative abun-

dance) of the molecules is due to the prevailing phy-

sico±chemical conditions, rather than microbial

reworking or contamination. Degradation products

remain trapped within the inorganic phase until

demineralisation or recrystallisation, allowing

sampling of both preserved indigenous biomolecules

and the degradation products of less stable com-

ponents.

Much of our understanding of protein decompo-

sition in the fossil record stems from arti®cial pyrol-

ysis experiments (for example Vallentyne, 1964,

1968). The aim of the present study was to extract

preserved amino acids, peptides and proteins from

intracrystalline sites within the shells of brachiopods

and to compare these with the previously published

simulation of amino acid degradation based on py-

rolysis experiments, in an attempt to quantify pro-

tein and amino acid degradation in the geological

record. Throughout this study, it has been assumed

that there has been no signi®cant evolution (i.e.

change in composition) of the protein and that

recorded change re¯ects diagenetic alteration.

This study has utilised the rich and diverse articu-

lated brachiopod fauna of New Zealand which

o�ers a rare opportunity to study changes in pro-

tein and amino acid composition as their shell crys-

tallites contain intracrystalline proteins (Collins et

al., 1988; Walton et al., 1993) and their shells are

composed of diagenetically stable low-Mg calcite

(Clarke and Wheeler, 1922).

Org. Geochem. Vol. 28, No. 6, pp. 389±410, 1998# 1998 Elsevier Science Ltd. All rights reserved

Printed in Great Britain0146-6380/98 $19.00+0.00PII: S0146-6380(97)00126-5

389

MATERIALS AND METHODS

Brachiopod samples

Recent brachiopods were collected from locationsgiven in Fig. 1. Fossil samples, of the same generaas those present in the Recent fauna, were collectedfrom the Plio-Pleistocene South Wanganui Basin of

North Island, New Zealand (Fig. 1). The tectonicsetting of the basin has allowed rapid subsidenceand the accumulation of up to 4 km of sediments,

mostly deposited in shallow marine conditions, withthe maximum accumulation occurring to the south

of the area considered here (Anderton, 1981). Bycomparison with Anderton (1981) and Fleming(1953), the maximum depth of burial of the part of

the basin considered here is approximately 1.5 km.Interspersed throughout the sequence are a numberof richly-fossiliferous shellbeds containing abundant

macrofossils (Fleming, 1953), ranging in age fromca. 120 ka to ca. 3.3 Ma (Table 1). From such shell-beds up to four genera of brachiopods were col-

lected, together with representatives of othergroups, for use in demonstrating the heterogeneityof the amino acid compositions.

Shell preparation

Samples were prepared according to the methodsof Walton and Curry (1994). In summary, shellswhich were excessively bored or fractured were

excluded from further study. Sediment wasscrubbed from the sample and encrusting epifaunaremoved by scraping. Articulated shells were disarti-

culated and any remaining body tissue removedbefore being soaked in an aqueous solution ofbleach (10% v/v) for 2 h at room temperature,

washed extensively with Milli ROTM water and airdried. Samples were ground using a ceramic pestleand mortar, and the powder incubated in an aqu-eous solution of bleach (10% v/v) under constant

motion for 24 h at room temperature, then washedby repeated agitation with MilliQTM water and cen-trifugation (typically ten washes) and lyophilised.

Fig. 1. Approximate locations in New Zealand from wheresamples were collected. For precise details of the sample

locations see Fleming (1953).

Table 1. Absolute dates used in this study together with the rationale for that date. The dating of this succession is relatively poorly con-strained with a number of con¯icting ages given for the same beds

Horizon Age (Ma) Reference

Rapanui Marine Sand 0.12 Pillans (1983)

Landguard Sand 0.33 Stratigraphically between Fordell Ash (0.31 Ma, Bussell andPillans, 1992) and Rangitawa Pumice (0.3420.03 MaAlloway et al., 1993)

Upper Castlecli� Shellbed 0.36 All dates based on the accumualtion rates of Beu andEdwards (1984)

Tainui Shellbed 0.38Pinnacle Sand 0.39Lower Castlecli� Shellbed 0.40

Kupe Formation 0.50 Based on an approximate mean of the dates of Seward,1974 (0.4520.09 Ma) and Abbott, 1992 (0.55±0.6 Ma)

Kaimatira Pumice Sand 1.05 Alloway et al., 1993

Okehu Shell Grit 1.10 Based on stratigraphic position

Tewkesbury Formation 1.67 Based on the age estimates of 1.6320.15 Ma (Alloway etal., 1993) and 1.2620.16 Ma (Boellstor� and Te Punga,1977) and stratigraphic position

Waipuru Shellbed 1.75 All dates based on stratigraphic positionUpper Nukumaru (4243) 1.85Nukumaru Brown Sand 1.85Upper Okiwa Group 2.15Hautawa Shellbed 2.20Lower Okiwa Group (Te Rama) 2.25Upper Waipipi Shellbed 3.20Middle Waipipi Shellbed 3.30

D. Walton390

Table 2. A summary of the data used in this study

Horizon Age (Ma) OrganismTotal amino acid concentration

(ng mgÿ1)Free amino acid concentration

(ng mgÿ1) % Free

Mean Std Dev. Mean Std Dev.

Recent 0 Neothyris 108.88 4.03 4.55 * 4.18Terebratella 190.27 19.17 7.74* 4.07Calloria 167.08 7.93 2.04 * 1.22Notosaria 706.88 146.74 69.08 * 9.77

Rapanui Marine Sand 0.12 Calloria 267.02 13.80 202.06 24.57 75.67(Waipipi) Notosaria 469.95 47.88 449.60 18.96 95.67

Pectenid 278.33 23.84 109.35 18.31 39.29Turritellid 58.63 7.87 42.17 6.08 71.93

Rapanui Marine Sand 0.12 Terebratella 196.92 34.74 122.72 19.67 62.32(Waitotara) Calloria 203.23 46.06 160.01 * 78.73

Pectenid 138.52 28.09 125.11 1.76 90.31

Landguard Sand 0.33 Neothyris 125.85 18.50 90.41 18.39 71.84

Upper Castlecli� Shellbed 0.36 Neothyris 108.26 5.05 87.82 12.52 81.12Calloria 325.85 21.6 198.84 26.80 61.02Pectenid 326.33 28.43 180.75 * 55.39

Tainui Shellbed 0.38 Neothyris 109.24 12.94 77.45 7.50 70.90Terebratella 176.67 6.71 141.71 10.38 80.21Calloria 231.27 18.77 139.91 9.50 60.49Notosaria 318.54 47.49 322.12 54.41 100.00$Pectenid 181.64 18.02 130.41 17.81 71.79Turritellid 55.65 6.50 47.58 5.04 85.49

Pinnacle Sand 0.39 Neothyris 101.37 5.92 95.48 6.97 94.18Terebratella 236.51 7.28 184.34 18.79 77.94Calloria 165.53 41.38 150.55 13.09 90.95Notosaria 310.96 49.31 292.01 17.73 93.91Pectenid 263.33 14.55 140.62 23.64 53.40Turritellid 116.78 23.93 101.98 21.52 87.33

Lower Castlecli� Shellbed 0.4 Neothyris 81.75 17.89 72.20 23.40 88.31(Coast) Terebratella 193.39 3.74 117.09 44.94 60.54

Calloria 208.67 29.78 163.84 33.65 78.52Pectenid 264.99 20.65 164.46 29.59 62.06Turritellid 164.62 42.52 130.13 53.18 79.05

Lower Castlecli� Shellbed 0.4 Neothyris 77.60 8.72 62.36 4.53 80.35(Waipuka Road) Terebratella 221.47 31.58 198.17 10.67 89.48

Calloria 180.09 14.11 195.90 22.18 100.00$

Kupe Formation 0.5 Neothyris 75.00 16.36 74.03 4.70 98.70Terebratella 227.18 20.02 215.71 * 94.95Calloria 259.97 59.46 220.68 26.29 84.89Turritellid 73.77 4.45 42.78 9.17 57.98

Kaimatira Pumice Sand 0.95 Terebratella 146.57 20.24 ND ND NDCalloria 138.43 17.57 152.28 * 100.00$

Okehu Shell Grit 1.1 Terebratella 173.88 72.51 178.68 26.04 100.00$Pectenid 125.67 1.18 ND ND NDTurritellid 55.94 0.18 37.08 * 66.29

Tewkesbury Formation 1.67 Calloria 101.19 11.77 92.64 6.12 91.55Pectenid 57.91 12.55 44.81 2.29 77.38Neothyris 47.71 4.26 49.72 16.19 100.00$

Waipuru Shellbed 1.75 Calloria 101.49 13.24 95.46 15.19 94.06

Undi�erentiated Shellbed 1.85 Neothyris 53.63 6.05 58.09 5.64 100.00$(4243 of Fleming, 1953) Calloria 123.09 28.76 102.30 20.17 83.11

Nukumaru Brown Sand 1.85 Calloria 155.28 30.96 132.04 35.06 85.03

Undi�erentiated Shellbed 2.15 Neothyris 21.93 5.45 16.82 6.68 76.69(4207 of Fleming, 1953) Calloria 54.57 4.81 41.39 2.84 75.84

Notosaria 89.26 7.98 75.14 11.65 84.18

Hautawa Shellbed 2.2 Neothyris 31.73 4.47 27.83 1.15 87.69Calloria 69.70 11.69 48.83 3.01 70.05Notosaria 100.23 18.58 83.91 16.17 83.71Turritellid 21.32 2.25 10.95 1.17 51.37

Te Rama Shellbed 2.25 Neothyris 53.50 11.41 46.09 7.23 86.16

Upper Waipipi Shellbed 3.2 Neothyris 40.75 6.05 28.66 7.54 70.34Terebratella 96.22 21.44 77.88 9.96 80.94

Middle Waipipi Shellbed 3.3 Neothyris 79.31 9.59 59.77 11.88 75.36

*One analysis only.$Corrected to 100% (see Section 2).ND, no data recorded.Full details for individual amino acids are available from the author on request.

Degradation of intracrystalline proteins and amino acids 391

An aqueous solution of 2 M HCl (at 19218C) ata ratio of 11 ml mgÿ1 was used to dissolve the shell

powder and release the entrapped biomolecules.Once demineralisation was complete, insoluble par-ticles were removed by centrifugation (20 g hÿ1).

Amino acid analysis

All samples were subjected to vapour-phase 6NHCl automated hydrolysis (Applied Biosystems420A). Amino acid analysis was completed follow-ing the standard protocols for the 420A which are

given in West and Crabb (1989) and Dupont et al.(1989). Standard proteins and peptides were usedduring every analysis to ensure that hydrolysis pro-

ceeded to completion and blank analyses wereincluded to check for background levels of contami-

nation. An aliquot of the same sample was analysed

without hydrolysis to ascertain the quantities of free

amino acids present. Individual amino acids were

derivatised using phenylisothiocyanate (PITC;

Heinrikson and Meredith, 1984), and transferred to

a dedicated narrow bore HPLC system for separ-

ation and quanti®cation. Amino acids were ident-

i®ed by comparison with Pierce amino acid

standard H (proteinogenic) or single commercial

standards (non-standard). Analyses with hydrolysis

were repeated at least three times, and those with-

out generally twice to ensure reproducibility

(Table 2).

As acid hydrolysis of samples causes variable loss

of amino acids (Hill, 1965) the concentrations listed

in Table 2 are likely to be minimum estimates. This

Fig. 2. Absolute concentration of amino acids (free + combined, given as nanograms of amino acid permilligrams of shell material, (A) and proportion of the total amino acid which are uncombined (free,given as a percentage; (B) plotted against sample age. In (A) note the overall decline in the concen-tration of the amino acids and the di�erence in the rate of decline between the genera. (B) demonstratesthat the original proteins have undergone natural hydrolysis (c.f. Hare, 1974; Goodfriend et al., 1992).Free amino acids are not leached out (c.f. Hare, 1974) as the degradation products remain within the

shell (Sykes et al., 1995; Walton et al., 1993).

D. Walton392

complicates determination of the proportion of theamino acids that are present in the free state, as

samples without hydrolysis will not su�er the sameloss of amino acids. In samples where a high pro-portion of amino acids are present in the free state,

the loss caused by hydrolysis may therefore yield avalue for free amino acids greater than that fortotal amino acids after hydrolysis (i.e. greater than

100%; these are corrected to 100%).

RESULTS AND DISCUSSION

Bulk amino acid composition

The absolute concentration and proportion ofamino acids in the free state are given in Table 2.Means and standard deviations are given for mul-

tiple analyses. Figure 2(A) shows the total aminoacids compared with the age of the sample. Allsamples contain appreciable amounts of amino acid

and show an overall general decreasing trend inconcentration with time, although with some scat-ter. In three of the species investigated (Calloria,

Neothyris and Terebratella) there is an initial rise inthe concentration between Recent and the youngestfossils, which di�ers in magnitude and duration andwhich has also been observed following arti®cial

diagenesis of gastropod shells (Qian et al., 1995). Inthis study, the increase is attributed to the solubil-isation of originally acid-insoluble proteins from the

intracrystalline fraction, similar to that noted byWeiner and Lowenstam (1980) where fossil sampleswere dominated by the soluble fraction and Recent

samples by the insoluble fraction.The proportion of amino acids which are present

in the free state increases from negligible amounts

in the Recent (indicating that the amino acids areall bound into proteins), to ca. 80% in less than0.5 Ma [Fig. 2(B)], indicating rapid hydrolysis, es-pecially during the ®rst 120 ka. The proportion of

free amino acids ¯uctuates with time, althoughremaining greater than 60% in all fossil samples.This is higher than that reported by Abelson (1955)

who determined that 1±5% of peptide bonds wouldbe broken in 0.1 Ma, but who acknowledged a lossof soluble peptides from the shell due to leaching.

This resulted in a false impression of the state ofpreservation, with the residual protein containing arelatively high proportion of peptide bonds to freeamino acids. In brachiopods, intercrystalline pro-

teins largely decay in under a year (Collins, 1986),

perhaps due to microbial activity between the shell

crystallites (Gaspard, 1989), which con®rms the po-

tential for loss hypothesised by Abelson (1955).

Some amino acids remain peptide-bound even in

the oldest of the samples analysed [Fig. 2(B)],

although there is no information regarding their

size nor their primary sequence.

The rate of hydrolysis depends on available

water, temperature and the chemical characteristics

of the amino acids on either side of the bond.

Inclusions of water have been observed within the

shell of living and fossil molluscs (Hudson, 1967),

and articulated brachiopods (B. Stern, University of

Newcastle upon Tyne, U.K., pers. comm.); brachio-

pod shells contain up to 3% water by mass (Ga�ey,

1988). As the fossil proteins are in a highly

degraded state, water has been present in relatively

large quantities within the shell, and hydrolysis has

proceeded largely unhindered. The proportion of

free amino acids in the shells of molluscs from the

same horizon as the brachiopods are broadly simi-

lar (Table 2), suggesting similar hydrolysis mechan-

isms. There is no evidence as to the relationship

between the protein and water and it is possible

that the proteins are contained in aqueous solution,

which provides a ready source of water for natural

hydrolysis. If, however, the protein is discrete from

any large included source of water, it may be better

preserved (Towe and Thompson, 1972).

Decomposition of some amino acids (Ser, Thr and

Glu; see Table 3) yields water, which would be

available to continue the degradative process.

However, it is important to note that hydrolysis

does not proceed to completion, suggesting a lim-

ited availability of water and a closed or partially

closed decompositional environment.

A second factor which can in¯uence hydrolysis is

temperature. The South Wanganui Basin has under-

gone successive periods of subsidence and uplift,

although seismic evidence demonstrates that this

burial is unlikely to exceed 1.5 km (Anderton,

1981). Samples are likely to have been heated to

20±308C, increasing the rate of hydrolysis by an

order of magnitude compared to average sediment

surface conditions.

The third factor important in hydrolysis is the

nature of the residues on either side of the bond

(for example Hill, 1965; Powell, 1994; Qian et al.,

Table 3. The three letter abbreviations used for amino acids

Amino acid Three letter code Amino acid Three letter code

Alanine Ala Lysine LysArginine Arg Phenylalanine PheAspartic acid Asp Proline ProGlutamic acid Glu Serine SerGlycine Gly Threonine ThrIsoleucine Ile Tyrosine TyrLeucine Leu Valine Val

Degradation of intracrystalline proteins and amino acids 393

1995). Protein sequencing (Cusack et al., 1992), im-munology (Endo et al., 1994) and amino acid analy-sis (Walton et al., 1993) have shown that the aminoacid composition and the size of intracrystalline

proteins are di�erent in the various brachiopodsstudied here. Variations in sequence and structuremay be expected to cause species level variation in

the rate of hydrolysis, as they appear to do in thecase of amino acid racemization (Wehmiller, 1980).

Individual amino acids

Acidic side chains. During preparative hydrolysis,asparagine (Asn) and glutamine (Gln) are deami-dated to aspartic acid (Asp) and glutamic acid

(Glu) respectively (Hill, 1965), hence no distinctionis made between the amino acids with the acidicside chains and their non-charged derivatives and

they are referred to as Asx and Glx respectively.Rapid and irreversible deamidation also occurswithin peptides with a half life of days to years

(Robinson and Rudd, 1974; Brinton and Bada,

1995), and it is unlikely that Asn and Gln would

persist in the fossil record.

In brachiopods, Asx and Glx both show an expo-

nential decrease in concentration, following an in-

itial rise in Calloria, with >80% lost by 3.3 Ma

indicating some parity in the degradation of the

acidic amino acids, although Glx is more stable

than Asx (Fig. 3). The proportion of the Asx pre-

sent in the free state varies between samples,

although in each case approximately 95% is free by

0.5 Ma and complete destruction in the free state in

Calloria takes place by 2.15 Ma. However, for Glx

the proportion in the free state in all samples is

low, generally below 40% of the total present, and

it maintains this level throughout the rest of the

period under study (Fig. 4).

Asx may decompose by two main pathways:

®rstly by reversible deamination to produce fumaric

acid and ammonia (Bada, 1971; Sohn and Ho,

1995). If the ammonia remains trapped, an equili-

brium will be established, leading to the persistence

Fig. 3. The concentration (free and combined) of Asx (A) and Glx (B) plotted against sample age. Notethe initial rise in the concentration of Asx in Calloria, due to the solubilisation of an originally acidinsoluble component in the Recent sample (see text). Notosaria is omitted from (A) as its concentration

is far higher than the other samples, and obscures the detail.

D. Walton394

of Asx in older samples, although this reaction can-

not take place when Asx is peptide-bound (Bada

and Man, 1980). Secondly, decomposition occursby decarboxylation of the a- or b-carbons to form

b-Alanine (b-Ala) or Ala respectively.

Evidence for deamination is di�cult to obtain, asthere may be many sources and sinks for the

ammonia produced by deamination, and fumaric

acid cannot be identi®ed on the analysis systemused. Ammonia reacts with PITC to give a systems

peak (phenylthiourea (PTU), Bidlingmeyer et al.,

1984; Cohen and Strydom, 1988), and the size ofthis peak rises in the fossil record, indicating that

the concentration of ammonia increases with time,

although this was not quanti®ed.

Decarboxylation, however, results in the for-mation of b-Ala which can be recognised on the

system used and has an elution time identical to

that of Thr. Hence a-decarboxylation of Asx wouldcause a rise in the concentration of ``Thr''. Of the

samples analysed here, Notosaria contains the high-

est concentration of Asx and fossil samples might

be expected to contain b-Ala in relatively high con-

centrations. In Notosaria, the concentration of Thr

shows a very large increase over the period 0.12±0.5 Ma (Fig. 5; Table 4), compared to a loss in

Calloria of >80%. This indicates that some diage-

netic reaction product in Notosaria co-elutes withThr, probably b-Ala, and that the a-decarboxyla-tion reaction may take place in geological samples.

A similar rise also occurs in samples of the pecte-nids analysed. However, as there is no direct corre-

lation between the concentrations of Asx and

``Thr'' not all Asx degrades by decarboxylation.Samples such as Calloria which have a lower initial

concentration of Asx do not have a rise in ``Thr'',

but it is likely some conversion to b-Ala is occur-ring, but only after 120 ka (Fig. 5).

Pyrolysis has indicated that decarboxylation reac-

tions are rare (Bada, 1971) and less than 0.2% dec-

arboxylation occurs during pyrolysis (Bada andMiller, 1969, 1970). However, these estimates were

based on reactions at elevated temperatures of the

pure compound alone, and in this case may not be

Fig. 4. The proportion of Asx (A) and Glx (B) present in the free state. Note the di�erences in thescale of the y-axis. Glx is present in much lower concentrations than Asx due to the formation of pyro-glutamic acid. This lactam reverts back to glutamic acid on hydrolysis, resulting in a depressed concen-

tration of Glx in the free state.

Degradation of intracrystalline proteins and amino acids 395

directly applicable to fossil biomolecules (c.f. Cowie

and Hedges, 1994, although this study does not dis-

tinguish between chemical and biologically

mediated decomposition). If a-decarboxylationoccurs it is possible that b-decarboxylation will also

occur, resulting in Ala, which may explain some of

the increase in Ala concentration found in fossils

[see Fig. 6(A)].

Glx may undergo degradation by two reaction

pathways. Firstly, by g-decarboxylation to produce

g-aminobutyric acid (Hare and Mitterer, 1967),

and secondly by lactam formation to produce

pyroglutamic acid (Wilson and Cannan, 1937).

g-decarboxylation in brachiopods is indicated by g-aminobutyric acid, although there is no direct corre-

lation between the age of the sample, the decrease

in concentration of Glx and the increase in size of

the peak at the position of g-aminobutyric acid.

The proportion of Glx in the free state is low (ca.

40%), which may be due to either the selective pres-

ervation of poly-Glx, or the formation of pyrogluta-

mic acid by lactamisation. The low proportion of

Glx in the free state is here attributed to lactam for-

mation, rather than the preservation of poly-Glx.

This reaction, however, does not explain the

decrease in the concentration of Glx, which either

occurs via decarboxylation to form g-aminobutyric

acid, or via the decomposition of pyroglutamic

acid. Lactam formation may only take place when

Glx is in the free state, hence the protein must have

undergone hydrolysis prior to lactamisation.

Pyroglutamic acid may be converted back to Glx

through protein hydrolysis.

Aliphatic hydroxyl side chains. Serine (Ser) and

threonine (Thr) initially decay rapidly, with ca.

80% of the original concentration lost by 1 Ma,

and their concentration remains at a similar level in

older samples (Fig. 7). The proportion of Ser pre-

sent in the free state rapidly increases to a maxi-

mum (approximately 90% free) and then decreases

until none remains in the uncombined state (Fig. 8).

Any Ser which remains in the sample after approxi-

mately 0.7 Ma is bound via an HCl sensitive bond.

Most of the decomposition therefore takes place in

Fig. 5. Histogram showing the change in concentration (free and combined) of Asx and ``Thr'' inNotosaria. Note the increase in the concentration of ``Thr'' and the decrease in the concentration ofAsx over the time period, probably due to the production of b-alanine from the decarboxylation ofAsx. This production does not take place until after 120 ka. There is no dramatic increase in the con-centration of ``Thr'' in the other samples which have a much lower initial concentration of Asx, poss-ibly indicating that there is a relatively low proportion of b-alanine production through this

mechanism.

Table 4. The changing concentrations (free and combined) of Asx and Thr over time. Note the decrease in the concentration of Asx anda corresponding increase in Thr which is attributed to the co-elution of b-Ala formed from decarboxylation of Asp

Concentration (ng mgÿ1)

Sample Age (Ma) Aspartic acid/asparagine Threonine

Recent 0 223.64 7.86Rapanui Marine Sand 0.12 109.19 4.49Tainui Shellbed 0.38 42.65 53.78Pinnacle Sand 0.39 41.72 34.21Upper Okiwa Group 2.15 1.61 23.27Hautawa Shellbed 2.20 1.98 22.29

D. Walton396

the free state, although some may decay whilst

remaining peptide-bound (Akiyama, 1980).

Decomposition of Ser and Thr may occur via

three pathways (Vallentyne, 1964; Bada et al.,

1978): ®rstly, by dehydration of the hydroxyl

group, forming Ala (from Ser) or a-aminobutyric

acid (a-ABA, from Thr). Secondly, by aldol clea-

vage resulting in the formation of Gly and formal-

dehyde (from Ser) or Gly and acetaldehyde (from

Thr). Thirdly, by decarboxylation, resulting in the

formation of ethanolamine (Ser) or propanolamine

(Thr). Deamination is not a major pathway (Sohn

and Ho, 1995). Dehydration is the prevalent reac-

tion when Ser is in the free state and aldol cleavage

is dominant when the molecule remains bound

(Bada and Man, 1980). Aldol cleavage of Thr

occurs more rapidly when the reactions are cata-

lysed by metal ions, or when it is in peptides

(Vallentyne, 1964). Although the 0.37-life gained by

pyrolysis for Thr at 108C is in the region of 30 Ma

(Vallentyne, 1964), greater than is shown here

(where the 0.37-life would be ca. 0.5 Ma), the pyrol-

ysis experiments were completed on solutions of

pure amino acids and may not be directly compar-

able.

The increase in concentration of Ala in the bra-

chiopods [Fig. 6(A)] is suggestive of dehydration

reactions, although there is no direct correlation

between the concentration of Ser and Ala, and the

increase in concentration of Ala cannot be

explained solely by this decomposition.

Decomposition by aldol cleavage is negligible, as

there is only a small rise in the corresponding con-

centration of Gly: most of the Ser has decomposed

to Ala.

Complete destruction of Thr occurs within 1 Ma

(Bada et al., 1978), consistent with the results pre-

sented here. a-ABA has been identi®ed in the fossil

species examined in this study, but not quanti®ed,

indicating some decomposition by dehydration,

although Bada et al. (1978) demonstrate that only

Fig. 6. The concentration (free and combined) (A) and the proportion in the free state (B) of Alaplotted against sample age. In (A) the very large increase in the concentration of this molecule is due todiagenetic production and the concentration in the oldest samples is approximately the same as in theRecent. Note that the maxima of molecules in the free state correlates to the maxima in the diagenetic

increase.

Degradation of intracrystalline proteins and amino acids 397

approximately 10% of the Thr decomposes by this

mechanism.

Basic side chains. Arginine (Arg) proceeds to

complete destruction in less than 1 Ma (with the

exception of two results of older samples of Calloria

and one from Terebratella). In contrast, the concen-

tration of lysine (Lys) is variable (Fig. 9) showing

an initial increase and then a decrease with time.

The third amino acid with a basic side chain, histi-

dine, was not quanti®ed, although it is only found

in small concentrations in brachiopods (Walton et

al., 1993).

The proportion of free Arg shows a rapid

increase, until almost 100% is free by 0.38 Ma. This

proportion decreases rapidly to 0% free by 1.5 Ma

(Fig. 10), indicating that any Arg which remains in

older samples must be bound via an HCl sensitive

bond. The proportion of Lys present in the free

state is highly variable between species.

Pyrolysis of Arg (Vallentyne, 1968) indicates that

the 0.37-life is ca. 100 yr at 208C, and decompo-

sition yields urea, ornithine, ammonia, proline and

an unidenti®ed compound which is associated both

with ornithine and with Arg (Murray et al., 1965;

Vallentyne, 1964, 1968; Sohn and Ho, 1995).

Ornithine occurs in Mercenaria (Hare and Mitterer,

1967) and brachiopods (Fig. 11). Although the

height of the peak is variable, it does show a re-

lationship between the decrease in concentration of

Arg and the increase in ornithine. Low concen-

trations of ornithine are identi®ed from Recent

samples which may be caused by the hydrolysis of

peptides and proteins containing Arg, although at

the temperatures and time of hydrolysis used there

would be a negligible degradation (Murray et al.,

1965). The concentration of ornithine rises rapidly

in the ®rst fossil shells, re¯ecting the decrease in

Arg, but then begins to decline, probably re¯ecting

decay via decarboxylation to putrescine (Murray et

al., 1965).

Lys is one of the least stable of the amino acids

(Vallentyne, 1964). This is not unexpected, as it

contains a secondary amino group susceptible to

deamidation and is also prone to condensation with

Fig. 7. The concentration (free and combined) of Ser (A) and Thr (B) plotted against sample age.Notosaria is omitted from (B) as the rise in the concentration of b-alanine, co-eluting with Thr,obscures the detail. Some of the Thr for the remaining samples may also be due to b-alanine (see also

D. Walton398

reducing sugars via the Maillard reaction.

Vallentyne (1968) used column chromatography to

detect the presence of ammonia in the pyrolysed

solutions, indicating that either primary or second-

ary deamination was occurring. Under di�erent

conditions, Sohn and Ho (1995) found little ammo-

nia produced by thermal degradation. There is no

evidence in this study to indicate which decay path-

way Lys follows, although the production of ammo-

nia may be recorded in the increase in the size of

the systems peak PTU in the current study.

Aromatic side chains. The concentration of both

tyrosine (Tyr) and phenylalanine (Phe), after an in-

itial increase in Calloria, show a rapid decrease in

concentration over time to greater than 80% loss

(Fig. 12). Tryptophan, a third amino acid which

contains an aromatic group in its side chain, is

completely destroyed during the acid hydrolysis of

peptides (Hill, 1965) and is therefore not quanti®ed

here. The proportion of Tyr and Phe present in the

uncombined state rises up to 100% by 0.5 Ma in all

species studied (Fig. 13), but is very variable.

Pyrolysis of Tyr has not been studied in detail,

although Gly is formed in small quantities

(Vallentyne, 1964). Tyr is almost totally destroyed

by 3.3 Ma, but it is not apparent by which reaction

pathway. The main pathway of decomposition for

Phe during pyrolysis is decarboxylation to form

phenethylamine, which is then further decomposed

to benzylamine (Vallentyne, 1964), following ®rst

order kinetics, with a 0.37-life of 100 Ma at 108C,much slower than that found in the present study.

The decomposition of Phe in the pyrolysis of mix-

tures of amino acids is more rapid than the single

compound (Vallentyne, 1964), and it is this

phenomenon which may cause such rapid decompo-

sition in fossils.

As the initial increase in the concentration of Ala

[Fig. 6(A)] cannot be accounted for by the dehy-

dration of Ser alone, other decomposition reactions

must also have an in¯uence on the increase in con-

centration of this amino acid. Tyr, with the poten-

tial for cleavage of the ring from the remainder of

the molecule must be considered for the origin of

Fig. 8. The proportion of Ser (A) and Thr (B) present in the free state. Notosaria is omitted for thereasons given in the caption to Fig. 7. Note that some Ser remains in all samples (Fig. 7; except 2 ofNeothyris) throughout the period under study, but that it is not present in the free state, only when

Degradation of intracrystalline proteins and amino acids 399

some of the diagenetic Ala. Other reactions, such as

decarboxylation and deamidation could proceed in

Tyr, although further work is required to determine

potential products and to con®rm reaction path-

ways.

Aliphatic side chains. The six amino acids with

aliphatic side chains show variable decomposition

pathways and will be grouped accordingly. Concen-

trations of valine (Val) are variable through time,

although there is a trend representing 50±60%

destruction [Fig. 14(A)]. All samples show a large

(up to 100%) initial increase in Val concentration

between Recent and young fossil samples, with a

maxima at 0.5 Ma, after which there is a decrease.

The proportion of Val present in the free state rises

to greater than 80% by 0.12 Ma [Fig. 14(B)], but

then shows a slight decrease indicating that the rate

of degradation is higher in the free than in the

bound state, and that once the susceptible peptide

bonds are broken, the rate of release slows dramati-

cally. Pyrolysis experiments have not conclusively

identi®ed the decomposition products of Val,

although Gly has been tentatively identi®ed, with a

very high 0.37-life (Vallentyne, 1964). Decarboxyla-

tion of Val produces 2-methylpropylamine (Meister,

1965), which was not identi®ed.

Leucine (Leu) and Isoleucine (Ile) decompose

rapidly in the fossil record until approx. 80% of

that found in the Recent samples of the same

species has decomposed by 3.3 Ma (Fig. 15). Most

samples show an increase between the Recent and

the youngest fossil samples. The proportion of free

amino acids rises rapidly to around 80% by 0.5 Ma

for both Leu and Ile, and this level is maintained

for most of the period under study, although in

older samples the proportion of free amino acids

decreases. Such levels of free amino acid indicate

that some Ile remains bound by an HCl sensitive

bond in the fossils (Fig. 16).

Pyrolysis of Ile did not yield identi®able products

(Vallentyne, 1964), although Leu released ammonia

on decomposition. The formation of ammonia only

represented 25±40% conversion when 85±98% of

the Leu was lost, indicating that deamination was

not the sole pathway of decomposition (Vallentyne,

1968). The data presented in this study identi®es a

Fig. 9. The concentration (free and combined) of Arg (A) and Lys (B) plotted against sample age. Note

D. Walton400

much more rapid rate of decay of Leu than is indi-

cated by the pyrolysis reactions. Such rapid de-

composition of this amino acid (when compared to

pyrolysis experiments) has broad implications for

previous studies, some of which (for example Bada

and Man, 1980) have represented the data as being

Leu equivalents, or have used the concentration of

Leu as a constant on the basis of the stability in

pure aqueous solution (Bada et al., 1978). Leu is

very stable when pure solutions of the amino acid

are pyrolysed, but this does not appear to be the

case when the amino acids are peptide-bound, in

free mixtures or when associated with an inorganic

phase. Comparisons with the results of pyrolysis of

pure compounds need to be drawn with caution in

these samples, and this is con®rmed by the pyrolysis

of oyster shell powders (Totten et al., 1972), where

Leu is also rapidly decomposed.

Glycine (Gly) is one of the most thermally stable

amino acids (Abelson, 1954). However, although

Calloria, Neothyris and Terebratella demonstrate an

initial increase, there is considerable degradation

over the period studied, resulting in a highly vari-

able loss [Fig. 17(A)]. All samples show a general

decreasing trend over time, with up to 80% of the

molecules being degraded by 3.3 Ma.

Fig. 10. The proportion of Arg (A) and Lys (B) present in the free state. Note that unlike other aminoacids, the proportion in the free state rapidly declines. A value of 0% free in older samples corresponds

here to complete destruction of the amino acid (see also Fig. 9).

Fig. 11. Graph to show the changing concentration (freeand combined) of Arg and its decomposition productornithine in Calloria. A similar pattern is observed in theother samples, but Calloria is shown as this is the genus

with the highest number of samples.

Degradation of intracrystalline proteins and amino acids 401

The proportion of Gly present in the free state is

similar in all species examined, with a rapid rise to

greater than 75% free, followed by a maintenance

of this level [Fig. 17(B)]. The oldest samples show a

slight decrease in the proportion of free amino

acids, indicating that Gly decays whilst in the free

state. The major pathway for the degradation of

Gly under pyrolysis is via decarboxylation to pro-

duce methylamine (Vallentyne, 1964), although this

product was not positively identi®ed in this study.

In most samples, there is a rise in the concen-

tration of Gly, indicating formation as a decompo-

sition product from Val, Ser, Thr or Tyr. These

reactions are considered in the relevant sections.

Where Gly is the decomposition product, the

``new'' molecule will decompose in the same way as

the original Gly, and hence the overall concen-

tration of this amino acid will decrease once pro-

duction from other sources has ceased or declined.

The diagenetic production of this amino acid will

distort the pattern of its occurrence in fossil shells.

Alanine (Ala) is one of the group of amino acids

which are thermally very stable (Abelson, 1954;

Vallentyne, 1964). The concentration within the

shells is an essentially random spread with respect

to age, with a large increase between the Recent

and the fossil, with the maximum at approx.

0.5 Ma [Fig. 6(A)]. This is followed by a decreasing

trend, although in all cases the concentration of Ala

in the oldest samples is similar to, or greater than,

the initial concentration found in Recent samples.

The maxima of Ala concentration correlates with

the highest proportion in the free state. There is a

rapid increase in the proportion of free amino acids

with greater than 90% being free in most cases by

0.5 Ma [Fig. 6(B)]. A high proportion of free Ala

remains throughout the samples studied, indicating

that the increase in concentration occurs in the free

state, rather than by degradation of peptide-bound

amino acids. Decay of Ala is via decarboxylation to

form ethylamine (Abelson, 1954), although pyrol-

ysis is very slow under nitrogen, indicating that

thermal decomposition may not be important

Fig. 12. The concentration (free and combined) of Tyr (A) and Phe (B) plotted against sample age.Both show very rapid decomposition, in some cases after an initial increase (Calloria and Neothyris; see

D. Walton402

(Abelson, 1954). In the presence of oxygen, how-

ever, the reaction rates would increase dramatically

(Conway and Libby, 1958). The rate of decay of

Ala appears, from both this and previous studies

(Hare and Mitterer, 1967, 1969), to be much more

rapid than that recorded for pyrolysis of a solution

of the pure amino acid. This phenomenon could

have a number of causes, notably the presence of

oxygen, surviving peptide bonds within the shell

and the e�ect of mixtures of amino acids

(Vallentyne, 1964).

The concentration of Ala present in the Recent

samples is almost identical to that recovered from

samples dated at 3.3 Ma. The intervening period,

however, shows major variations in concentration,

with the increase being due, at least in part, to its

diagenetic formation. There is no di�erence between

the original and diagenetic Ala in terms of de-

composition pathways, hence some formed diagene-

tically will also decompose. As there is no direct

correlation between the decomposition of Ser and

the production of Ala, it is likely that other diage-

netic reactions may produce Ala, for example the b-decarboxylation of Asx.

Proline (Pro) has an aliphatic side chain which is

bonded to both the nitrogen and the a-carbonatoms. Pro is the most stable of the amino acids

tested by pyrolysis at temperatures below 2128C(Vallentyne, 1968), although it shows rapid decay in

the fossil record to a level representing greater than

80% loss over the 3.3 Ma of the study (Fig. 18).

The proportion of Pro present in the free state

rapidly increases to greater than 80% in 0.2 Ma,

and maintains that proportion throughout the rest

of the period under study.

In pyrolysis experiments, Pro decomposed to

form ammonia, indicative of decay by deamidation

(Vallentyne, 1968), although calculated 0.37-lives in-

dicate that no decomposition would be expected at

the temperatures the fossils have been subjected to.

Again, the data presented here contradicts this and

shows that there is an increased rate of decompo-

sition in fossil samples, not mimicked by pyrolysis

of the pure compound. Other factors, such as the

Fig. 13. The proportion of Tyr (A) and Phe (B) present in the free state. The irregular pattern is caused

Degradation of intracrystalline proteins and amino acids 403

inorganic component of the shell or the e�ect ofmixtures of amino acids, clearly have a major e�ecton the rate of decomposition.

Sulphur-containing side chains. Cysteine andmethionine contain sulphur within their side chains.During the hydrolysis of the samples in preparationfor analysis, these amino acids undergo variable

degradation, and these molecules were therefore notquanti®ed.

The e�ect of carbohydrates on the destruction ofamino acids

The reaction between carbohydrates and aminocompounds is well documented (for exampleHoering, 1973; Collins et al., 1992). The reactions

begin with interactions between the reducing groupsof sugars and amino groups of other compounds toform glycosylamines. The end product of the reac-

tion is a dark heteropolymer referred to as melanoi-din (Hoering, 1973). Carbohydrates are presentwithin the shells of brachiopods (Collins et al.,

1991), but it is not certain whether they are

attached to the protein in these samples (M.Cusack, University of Glasgow, U.K., pers.comm.). However, at least some of the proteins in

the brachiopod Terebratulina retusa are glycosylated(J. Laing, University of Derby, U.K., pers. comm.).Although not examined in this study, carbo-

hydrates have an e�ect on amino acid decompo-

sition. Pyrolysis reactions involving amino acidsand glucose (Vallentyne, 1964) indicate that thehigher the concentration of glucose in a standard

solution of Ala, the faster the decomposition rate ofthe amino acid. The 0.37-life of the Ala at 1678Cwithout glucose is just over 10 yr, but is reduced to

approx. 2 h when 0.05 M glucose is present. This in-teraction of carbohydrates with amino acids couldexplain some of the accelerated degradation reac-

tions detected in this study.

The use of pyrolysis as a predictive tool

Pyrolysis experiments have been the only methodby which amino acid degradation over an equival-

ent time scale has been examined prior to this

Fig. 14. The concentration (free and combined) (A) and proportion in the free state (B) of Val plottedagainst sample age. Note in (A) the initial increase in concentration and the relatively slow rate of de-composition, demonstrating that Val is one of the most stable of the amino acids and the slowing in

D. Walton404

study. Several authors have considered the pyrolysis

of shell powders, in addition to the pure solutions

of amino acids (for example Jones and Vallentyne,

1960; Vallentyne, 1964; Hare and Mitterer, 1969;

Totten et al., 1972). Some of the amino acids most

stable to pyrolysis as solutions of the pure amino

acids, such as Phe, Lys and Asx were some of the

least stable of the amino acids when fossilised

(Jones and Vallentyne, 1960). The decomposition of

Ala was studied by the pyrolysis of shell powders,

where it occurred more rapidly than in an aqueous

solution of the pure compound, a ®nding con®rmed

by this study. This is likely to be due to one of

three possibilities (Jones and Vallentyne, 1960;

Vallentyne, 1964). Firstly, the act of heating does

not mimic the e�ect of time accurately. Implicit in

this statement is the e�ect of pressure on the de-

composition of solutions of pure amino acids. The

reaction vessels were sealed before heating, and the

evaporation of the water from the aqueous solution

would increase the pressure. No measure of this

pressure was made, although Vallentyne (1964)

noted that the vessels frequently shattered during

heating, indicating the great increase in pressure.

The possibility of the increased pressure increasing

the rate of reaction has not been considered in any

of the pyrolysis experiments except in the consider-

ation on the rate of hydrolysis (Qian et al., 1995).

The burial history of the horizons containing the

fossils will have a marked e�ect on the rate of de-

composition reaction. Pyrolysis data (Vallentyne,

1964, 1968) shows that the rate of reaction will

increase by an order of magnitude between 20 and

408C. Such an increase in temperature represents

burial of approximately a kilometre, a possibility

which exists for the samples under study (Fleming,

1953; Anderton, 1981). The results of pyrolysis ex-

periments cannot therefore be directly applied to

fossil amino acids unless a detailed burial history

for each bed is known. Without this, the pyrolysis

results cannot be used as a predictive technique for

assessing the age or the molecular state of preser-

vation of fossil biomolecules.

Fig. 15. The concentration (free and combined) of Leu (A) and Ile (B) plotted against sample age. Bothdecompose rapidly in contrast to the ®ndings of previous studies.

Degradation of intracrystalline proteins and amino acids 405

Secondly, the stabilities of the amino acids may

be a�ected by factors other than temperature. Forexample, metal ions catalyse oxidative deamination,by chelation of the amino acid (Ikawa and Snell,

1954). Ca2+ or Mg2+ ions present within the shellcarbonate could act as a chelation site for theamino acids. Thirdly, interactions between carbo-hydrates and amino acids which have already been

discussed.

The molecular state of preservation

The proteins of these fossil brachiopods are

almost completely decomposed, with only a limitednumber of peptide bonds surviving fossilisation.The presence of relatively unstable molecules suchas Asx in peptide-bound compounds is likely to be

due to the stabilising e�ect of the peptide bond, thestability of which is a function of the nature of theresidues on the other side of the bond (Hill, 1965).

All samples have a proportion of free amino acidswhich rapidly (within 0.5 Ma) rises to greater than80%. The rate of hydrolysis then slows and may

decline, corresponding to the destruction of the

most labile peptide bonds and the preservation of

less labile ones.

Individual amino acids also undergo degradative

reactions, the majority of which take place when

the molecule has been released from the protein

and is present in the free state. Degradative reac-

tions produce a range of reaction products, includ-

ing the diagenetic generation of other amino acids

which may be either proteinogenic or non-standard

and which may distort any taxonomic relationship

through time (Walton, 1998). For example, Jope

(1967) found that the insoluble fraction of the inter-

crystalline protein from fossil brachiopods showed

marked di�erences from the nearest living relatives.

This study also found raised Asx in the insoluble

fraction, possibly indicating either post-mortem

alteration or contamination. Both the present study,

and that of Jope (1967) contrast with the results of

Kolesnikov and Prosorovskaya (1986), who recog-

nised ``very familiar'' compositions between Recent

and fossil brachiopods. Such results need to be trea-

ted with caution as this study has shown that, at

least in the soluble fraction, the amino acids are un-

Fig. 16. The proportion of Leu (A) and Ile (B) present in the free state. Some molecules remain in pep-

D. Walton406

Fig. 17. The concentration (free and combined) (A) and the proportion in the free state (B) of Glyplotted against sample age. Although in (A) there is a general decreasing trend, the concentration ishighly variable, representing the diagenetic formation of Gly. Older samples have a lower proportion

Fig. 18. The concentration (free and combined) of Pro plotted against sample age. Although pyrolysisof the pure compound indicates that this is one of the most stable of amino acids, the data here suggest

that this is not the case.

Degradation of intracrystalline proteins and amino acids 407

stable to di�ering degrees which results in changingamino acid ratios over time. Fossil samples will

have di�erent amino acid ratios to those of extantspecies.The advantage of using intracrystalline biomole-

cules is therefore to be certain of in situ degra-dation, where the products of degradative reactionsare contained within the shell and not lost.

Although the bulk amino acid composition ofsamples represents an oversimpli®cation of thenature of the proteins, it is likely to be the sole

method of analysis in older samples, where the pro-teins have been totally degraded. If the biomole-cules are undergoing in situ decay, then the onlycause of this loss will be from chemical degradation,

rather than di�usion of original amino acids out ofthe shell or microbial degradation, as is the case forintercrystalline biomolecules.

CONCLUSIONS

These results indicate that pyrolysis experimentsof pure compounds can act as a useful guide for thestudy of the relative rates of decomposition of

amino acids in fossils, but they are not representa-tive of the natural system. Therefore pyrolysis ofshells must also be undertaken to discern the e�ects

of the carbonate, and other factors associated withthe shell. The burial history of any given samplewill have a great e�ect on the degradation of theintracrystalline biomolecules. As the samples are

successively buried and exhumed, temperature andpressure regimes will change, either increasing ordecreasing the rate of reaction. Pyrolysis of pure

compounds should not be applied directly to thefossil record, but only in combination with shell py-rolysis experiments and information on the burial

history of the fossils.Due to the level of both natural hydrolysis and

amino acid decomposition that has taken place inthe fossil samples, it is highly unlikely that proteins

from these samples will survive in a state wherebythey may be routinely separated and analysed forprimary sequence data. The identi®cation of path-

ways and products is required to reconstruct theoriginal amino acid composition.

Associate EditorÐR. L. Patience

AcknowledgementsÐThis work was conducted during thetenure of a U.K. NERC studentship (GT4/89/GS/42) inthe Department of Geology and Applied Geology,University of Glasgow, which is gratefully acknowledged.I wish to thank Drs Maggie Cusack, Matthew Collins,Heather Clegg and Phil Jackson for advice throughout thework and two anonymous referees who added enormouslyto the manuscript. I am indebted to Sandra McCormackfor technical assistance in sample preparation. This workwas written during a sabbatical funded by the Universityof Derby.

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