Available online at www.sciencedirect.com

www.else vier.com/locate /gca

Geochi mica et Cosmochim ica Acta 111 (2013) 117–127

Variable dD values among major biochemicals in plants:Implications for environmental studies

Nicole DeBond a,⇑, Marilyn L. Fogel b, Penny L. Morrill a, Ronald Benner c,Roxane Bowden b, Susan Ziegler a

a Memorial Univers ity of Newfoundlan d, Departme nt of Earth Sciences, St. John’s, Newfoundlan d, Canada A1B 3X5 b Geophysical Labora tory, Carnegie Institution of Washington, 5251 Broad Branch Rd., N.W., Washington, DC 20015, USA c Departme nt of Biological Sciences and Marine Science Program , University of South Carolina, Columbia, SC 29208, USA

Available online 7 November 2012

Abstract

The stable hydrogen isotope composition (dD) of major plant biochemicals is variable. We present dD values for cellulose,hemicelluloses and lignin of six plant species. The dD value for lignin is consistently lower than that of bulk tissue (by �50&)and cellulose (by �100&). We show that these differences can be used to assess the extent of degradation of organic matter from a single source. A decrease in the dDbulk of decomposing Spartina alterniflora roots and rhizomes from �72& to �87&

was observed over 18 months, reflecting a relative enrichment of lignin content due to the preferential removal of polysaccha- rides from the detrital material. Similar changes in d13C were observed previously during the degradation of these plant tis- sues. These findings indicate that the extent of organic matter degradation should be considered when using stable isotope approaches to assess possible sources of organic matter in soils and sediments. We show that the change in dDbu lk of plant detritus is best described by an exponential equation, which is simpler than the multiple exponential decay (multi-G) model which best describes the change in d13Cbulk of plant detritus. Therefore correcting for isotopic shifts caused by decomposition may be more easily accomplished using dD.� 2012 Elsevier Ltd. All rights reserved.

1. INTRODUCTION

Over 30 years ago, plant structural compounds were identified as molecules that held potential environmental signals in their stable hydrogen isotope compositions (dD). For instance, the study of dD in cellulose from tree rings indicated the potential for discerning climatic change signals, particularly in long-lived tree species like the bris- tlecone pine (Epstein et al., 1977; Yapp and Epstein,1982; Feng and Epstein, 1994; Epstein, 1995 ). However,the early successes were muted somewhat when it was rea- lised that the dD of cellulose was controlled by numerous factors, some of which could obfuscate a dD climate record

0016-7037 /$ - see front matter � 2012 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.10 16/j.gca.2012.10 .043

⇑ Corresp onding author. Tel.: +1 709 864 7638.E-mail addresses: nicole.debon d@mun.ca , gca@nicole debond.

com (N. DeBond).

(DeNiro and Epstein, 1981; Terwilliger and DeNiro, 1995;Terwilliger et al., 2002 ). Measurements of dDbu lk were iden- tified as potentially problematic, in part because of the chemical complexity of plant organic matter and the effectsthat changes in the relative proportion of the biochemical constituents could have on dDbu lk measurements if they dif- fered in dD themselves (Epstein et al., 1976 ).

In addition to considering variations due to chemical composition, hydrogen isotope exchange must be ac- counted for before interpreting bulk organic dD values as primary biosynthetic signals (Epstein et al., 1976 ). As such,a strong focus has been on obtaining dD measurements that reflect the isotopic composition of source waters and are free from the complications of stable hydrogen isotope ex- change. This has led to the use of compound-specific anal- yses on materials that contain no exchangeable hydrogen such as nitrated cellulose (Epstein et al., 1976 ) or specific li- pid fractions (Northfelt et al., 1981 ). Despite the ubiquity of

Fig. 1. Flow chart schematic, modified from Van Soest and Robertson (1980), describing the sequentia l detergent extraction procedure employed to isolate four plant tissue fraction s (NDF,ADF, cellulose and lignin).

118 N. DeBond et al. / Geochi mica et Cosmochim ica Acta 111 (2013) 117–127

hydrogen in natural organic matter, few studies have at- tempted to define the stable hydrogen isotope compositions of plant components other than cellulose and lipids,including other key structural components such as lignins (Schmidt et al., 2003 ).

The primary objectives of this study were to investigate variations in the dD of major biochemical components of plant material, and to determine whether these variations provide useful information for the interpretation of dDbulk

measurements. Variations in dD should be 10 times larger than those observed for d13C based on relative mass differ-ences between each element’s stable isotopes. Measuring dD requires virtually no sample processing, indicating these measurements have the potential to reduce the work re- quired when tracking subtle changes in the composition of organic matter, for example during degradation experiments.

The dD values for a wide variety of operationally-de- fined compound classes of plants are reported in the liter- ature (e.g. Epstein et al., 1976; Smith and Jacobson, 1976;Estep and Hoering, 1980; DeNiro and Epstein, 1981;Northfelt et al., 1981; Sternberg et al., 1984; Chikaraishi and Naraoka, 2001; Schmidt et al., 2003 ). These studies provide valuable information about relative deuterium abundances in various plant components, and provide in- sights about the biosynthetic pathways that cause hydro- gen isotope fractionations (Schmidt et al., 2003 ).However, in some cases, only a single study has been per- formed. Only one value of the dD of lignin exists in the lit- erature, and it was calculated using measurements of alignocellulose fraction including hemicellulose, cellulose and lignin, and a cellulose nitrate fraction from two rings of a single tree (Epstein et al., 1976 ). The calculated dD va- lue was higher than that of whole wood. This finding is contrary to a prediction by Schimmelmann et al. (1993)who observed that whole wood was depleted in D relative to the holocellulose (cellulose and hemicelluloses) fraction,which suggests that the lignin component should have adD value that is even lower than that of whole wood (i.e. more depleted in D).

As with dD measurements for different plant metabo- lites, very few studies exist that investigate changes in dDbulk during the decomposition of plant matter. Varying trends in dDbulk were observed in these studies (Schiegl,1972; Macko et al., 1983; Fenton and Ritz, 1988 ). Here,we investigate the utility of hydrogen isotopes for tracking changes in the biochemical composition of plant detritus.We determined the dD values of cellulose, hemicellulose,lignin, and an uncharacterized component consisting of lip- ids, carbohydrates and amino acids relative to bulk plant matter for six different vascular plants, as well as the change in dDbulk of Spartina alterniflora root and rhizome detritus as it degraded over an 18 month period.

The dDbulk measurements were compared with d13Cbulk

measurements by Benner et al. (1987) to determine the effectiveness of dD for estimating the extent of degrada- tion of S. alterniflora detritus in salt marsh sediments.The change in dDbulk can be ascribed to the increasing rel- ative proportion of lignin as the extent of degradation increased.

2. METHODS

2.1. Plant fractions

Four plant tissue fractions were isolated using the sequential detergent extraction approach (Fig. 1) com- monly employed for forage (i.e. edible plant) analysis (Van Soest and Robertson, 1980 ). Samples were dried,ground in a Wiley mill, and boiled in a neutral detergent,after which the insoluble residue (neutral detergent fibre,NDF), composed primarily of hemicelluloses, cellulose and lignin, was collected. The NDF was then boiled in an acid detergent and again the insoluble residue was collected.This fraction, termed the acid detergent fibre (ADF) is com- posed primarily of lignin and cellulose. The cellulose frac- tion was obtained by extracting lignin from an aliquot of ADF using a potassium permanganate (KMnO4) solution.A second aliquot of ADF was mixed with 12 M sulphuric acid (H2SO4) at room temperature, and the insoluble com- ponent recovered from this treatment was lignin. The esti- mate of lignin content from this extraction process was verified using CuO oxidation and gas chromatography (GC) measurements of lignin phenols from ground plant tissues that did not undergo the sequential extraction (Ben-ner et al., 1987 ).

After the sequential detergent extraction, d13C values as well as the ash free dry weight (AFDW) percentages for each fraction were determined by Benner et al. (1987).

N. DeBond et al. / Geochi mica et Cosmochim ica Acta 111 (2013) 117–127 119

The dried plant fractions were subsequently stored in the dark in sealed glass containers until 2011, when stable hydrogen isotope analyses were performed as described below.

Although the fractionation technique employed works well for many samples, under some circumstances the tech- nique is less effective. For example, lignin content can be overestimated in samples with high concentrations of tan- nins and cutin, because these compounds are not com- pletely removed from the sample (Benner et al., 1990 ).We include three samples that were prepared but were ulti- mately excluded from the investigation of variations in d13Cby Benner et al. (1987). These samples were excluded by Benner et al. (1987) due to a lack of carbon mass and stable isotope balance, however they are included here in order to investigate the robustness of dD values in different plants.Those samples are: Pinus eliotti and Juniperus virgniana

needles, and mangrove leaves.

2.2. Calculations

Sequential extraction procedures can lead to material loss and a lack of mass balance, which has the potential for causing isotopic fractionation. Using a pair of mass and isotope balance approaches, we were able to assess and verify the reliability of the isotopic measurements and calculations for the four major chemical fractions investi- gated. The dD value of the hemicellulose fraction was cal- culated using the differences between the neutral detergent fibre and acid detergent fibre fractions, and the dD value of the uncharacterized fraction was calculated in a similar manner using the differences between whole plant and the neutral detergent fibre fractions.

In order to account for differences in hydrogen content among the plant fractions, the amount of hydrogen (H_amt) present in each fraction was calculated by multi- plying the ash free dry weight (AFDW) of a given fraction with its percentage hydrogen content as determined from direct measurement of the sample. The H_amt value for hemicelluloses was then determined two different ways. It was first calculated as the difference between the H_amt val- ues for NDF and ADF:

H amthemi ¼ H amtNDF �H amtADF ð1Þ

where H_amt is the amount of hydrogen in each fraction,and the subscript denotes the chemical fraction (hemi = hemicellulose, NDF = neutral detergent fibre,ADF = acid detergent fibre). The H_amt value for hemicel- lulose was then recalculated using the sum of the H_amt values for cellulose and lignin in place of the ADF value:

H amthemi ¼ H amtNDF � ðH amtcell þH amtligÞ ð2Þ

where the subscript cell denotes the cellulose and lig denotes the lignin fractions. A similar pair of calculations was per- formed to calculate the H_amt values for the uncharacter- ized fraction, using first the NDF value, and then the sum of the cellulose, lignin and hemicellulose H_amt values in place of the NDF value:

H amtunchar ¼ H amtwhole �H amtNDF ð3Þ

H amtunchar ¼ H amtwhole � ðH amtcell þH amtlig

þH amthemiÞ ð4Þ

where unchar denotes the uncharacterized fraction, and whole denotes whole plant material. These H_amt values are then used to determine the isotopic composition of the hemicellulose and uncharacterized fractions. For exam- ple, the result from Eq. (1) was used to solve for the dD of hemicellulose:

dDhemi ¼ðdDNDF �H amtNDFÞ � ðdDADF �H amtADFÞ

H amthemi

ð5Þ

where dD denotes the stable hydrogen isotopic composition of a given fraction, and the remainder of the variables are described above. The H_amt values obtained from Eqs.(2)–(4) were used to calculate dD for hemicellulose and the uncharacterized fraction in a similar manner to that ex- pressed in Eq. (5). Similar calculations were also used to determine the dD value of lignin for S. alterniflora, as sam- ples for direct measurement were not available. In this case,the H_amt of lignin was calculated as the difference be- tween the ADF and cellulose fractions.

2.3. Degradation experiment

Roots and rhizomes from the short form of S. alternifl-

ora were placed in litter bags and buried at depths of 5–10 cm in salt-marsh sediments on Sapelo Island, Georgia in January, 1985. The bags were retrieved at 1–3 month intervals over an 18-month period, and the lignin compo- nent of the recovered material was measured as described previously (Benner et al., 1991 ). Recovered material was dried and then stored in the dark in sealed glass containers as with the plant fractions described above, until stable hydrogen analysis in 2011.

2.4. Stable hydrogen isotope measurements

All samples were equilibrated with laboratory condi- tions for at least 24 h prior to analysis. Stable hydrogen iso- tope and percent hydrogen measurements were performed concurrently using a ThermoQuest Finnigan thermochemi- cal reactor elemental analyzer (TC/EA) coupled to a Ther- moQuest Finnigan Delta Plus XL isotope ratio mass spectrometer via a Conflo III open split interface. Prior to analysis, �300 lg of sample was weighed into silver cap- sules in duplicate, which were folded shut and formed into balls. Samples were further dried in a heated (50 �C) nitro- gen atmosphere oven for a minimum of 24 h. Samples were introduced into the TC/EA using a Costech Zero Blank Autosampler, which was purged with helium gas for 5 min prior to each run. Hydrogen isotope ratios were mea- sured relative to a reference gas and calibrated against the following standards: stearic acid (�233&, 12.76%H), Iso- analytical IA-R002 mineral oil (�111&), and pump oil (�112&), reported relative to V-SMOW. Accuracy and precision were determined by calculating the absolute value of the difference between the measured value of each stan- dard and the known value for multiple measurements.

120 N. DeBond et al. / Geochi mica et Cosmochim ica Acta 111 (2013) 117–127

The mean ± root-mean-square deviations (RMSD) of these differences were 3 ± 6&, 4 ± 5& and 2 ± 3& for stearic acid, mineral oil, and pump oil, respectively (n = 49 for stearic acid, n = 12 for both mineral and pump oils).

2.5. Constraining the effects of hydrogen isotope exchange

Plant fractions were analysed on 3 days during a 6 week period in the spring of 2011, with all fractions from a single plant analysed on the same day, allowing comparisons be- tween the fractions and bulk material for a given plant.All samples from the degradation experiment were analysed on a single day 5 months later. In order to assess the effectsof changes in the hydrogen isotope composition of the atmosphere over this period, keratin laboratory standards (CCHIX and TURK-1) underwent the same atmospheric equilibration and analysis as the samples.

The amount of exchangeable hydrogen in a subset of the samples analysed was also determined using the steam equilibration technique and calculations described by Saueret al. (2009). The ADF, NDF and cellulose fractions of S.

alterniflora roots and rhizomes, three samples from the deg- radation experiment (months 1, 8 and 18), commercially available cellulose, vanillic acid (as a model for lignin)and the keratin laboratory standards (CCHIX and TURK-1) were exposed to waters with hydrogen isotope compositions of �59 ± 1& and 1162 ± 5& (n = 5).

3. RESULTS AND DISCUSSION

3.1. Assessment of the effects of stable hydrogen isotope

exchange

The mean ± RMSD of the difference between the known nonexchangeable hydrogen isotope composition and the measured dD value of the atmospherically equilibrated ker- atin standards analysed with the plant fractions was 6 ± 10 & for CCHIX and 9 ± 10 & for TURK-1 (n = 6for both). These variations are similar in magnitude to the accuracy and precision obtained for the stearic acid and oil standards, described in Section 2 above, which con- tain no exchangeable hydrogen. This indicates that changes in the hydrogen isotopic composition of the water vapour the samples were equilibrated with was minimal, and results from analysis on the different days may be compared.

It was also important to consider any hydrogen isotope exchange that may have occurred during the sequential extraction procedure used to isolate the plant fractions studied here. Although it has been shown that the tertiary structure of organic macromolecules involves rigid hydro- gen bonds, once these structures are destroyed, exchange with water does occur (Estep and Hoering, 1980 ). The sequential extraction procedure used to isolate the various plant fractions involves boiling plant matter with a series of detergent solutions. It is possible that these conditions could result in isotopic mass balance not being preserved throughout the extraction procedure (e.g. due to increased hydrogen isotope exchange during the acid detergent and lignin extractions). It should be noted, however, that the insoluble residues are the products subsequently analysed,

not the dissolved fractions where exchange would be ex- pected to occur to a greater extent. Hydrogen isotope ex- change during the extractions could both (1) alter the measured dD value of a fraction, and (2) result in errors in the calculations used to determine the dD values of the hemicellulose and uncharacterized fractions.

It is difficult to quantify such exchange, however, calcu- lating the dD values of the hemicellulose and uncharacter- ized fractions using two independent approaches should indicate whether major changes in isotopic mass balance occurred over the course of the extractions. After propagat- ing experimental error through these calculations, the two approaches described above (Eqs. (1)–(5)) yielded the same isotopic values for both the hemicellulose and uncharacter- ized fractions of all the plants discussed in the original d13Cstudy (see Electronic Annex ). This indicates that any mass loss that occurred during the extractions had no significanteffects on the calculated isotopic compositions, and suggests the effects of any hydrogen isotope exchange during the sequential extraction procedure are minimal.

Of the three plant materials not included in the Benneret al. (1987) study, the mangrove leaves yielded significantlydifferent isotopic values for the hemicelluose fraction when calculated using both approaches, and the H_amt values for uncharacterized fraction using a form of Eq. (4) modi- fied to include cutin was zero. Both of these findings are indications that the separation techniques did not work well for this plant’s tissues, and that other compounds are pres- ent in the fractions, such as cutin and tannins in the lignin fraction (Van Soest and Robertson, 1980 ). Results for the mangrove leaves have, therefore, been excluded from fur- ther discussion. The other two materials (P. eliotti and J.

virgniana needles) were not ideal candidates for the extrac- tion procedure due to their tannin and cutin contents, and because no cellulose fraction was available, the calculations could not be performed with both approaches. However,the fractionations relative to bulk material are similar to those of the other plants, suggesting the presence of tannins and cutin may not have a large effect on the dD measure- ments for the P. eliotti and J. virginiana needles, and these dD values may indeed be reliable.

Finally, steam equilibration experiments following the methods of Sauer et al. (2009) found that the fraction of exchangeable hydrogen (fe) for the samples discussed in this study varied from 10% to 20% (Table 1). The effect of the exchangeable hydrogen depends in part on the similarity between the nonexchangeable hydrogen isotope composi- tion (dDn), and the exchangeable hydrogen isotope compo- sition – the more similar these values, the greater the fe mustbe in order to affect the total measured dD value. Assuming equilibration with atmospheric water vapour that has acomposition of �59& (equal to the dD value of laboratory deionized water), and an equilibrium isotopic enrichment factor (e) of 80, varying the fe from 10% to 20% causes a2& change in the measured dD value for a compound with a dDn of 0&. The same change in fe, however, for a com- pound with dDn of �150& is 17 &. Based on these observa- tions, we assume that any differences >20 & betweendifferent plant fractions reflect differences in the dDn ofthe fractions, rather than simply being the result of different

Table 1The fraction of hydrogen that is exchang eable (fe) for a subset of the materials investiga ted in this study, as determin ed from steam equilibration with waters with hydrogen isotope compositions of –59& and 1162 &, calculated assumi ng ee-w = 80 &, and using Eqs. (1) and (2) from Sauer et al. (2009). The letters ‘na’ denotes samples for which a predicted fe is not availabl e (i.e. comple x natural organic matter samples for which exact structur al informat ion is not known or well understood), and ‘–’ denotes samples for which no other investigat ions have occurred.

Sample fe fe predicted from structure fe from literature

Cellulose 0.192 0.300 0.205 ± 0.002 a

Vanillic Acid 0.182 0.250 –CCHIX 0.130 0.421 �0.13b, �0.14c, d

TURK-1 0.136 0.421 �0.13b, �0.14c,d

S. alterniflora ADF 0.106 na –S. alterniflora cellulose 0.144 0.300 0.205 ± 0.002 a

S. alterniflora NDF 0.105 na –Degradatio n experiment month 1 0.102 na –Degradatio n experiment month 8 0.107 na –Degradatio n experiment month 18 0.093 na –

a Cellulose (IAEA-C3) measurement from Sauer et al. (2009).b Amino acid compo sition of keratin from O’Donnell and Inglis (1974) used to predict fe for TURK-1 and CCHIX .c Determin ed for feather keratin by Chamberl ain et al. (1997).d Qi and Coplen (2011) use 14% exchangeable hydrogen as a threshold indicating a fully dried keratin sample.

N. DeBond et al. / Geochi mica et Cosmochim ica Acta 111 (2013) 117–127 121

fractions of exchangeable hydrogen. However, these calcu- lations likely overestimate the amount of exchange that oc- curs between the samples and the laboratory atmosphere.The dD of the laboratory atmosphere is likely much lower than �59&, due to large liquid–vapour fractionation for water. This would result in a smaller change in dD for com- pounds that have dDn of �150& than estimated by the cal- culations above. In any case, given that the fraction of exchangeable hydrogen only varies by 1.5% for the degra- dation experiment, the effect of the exchangeable fraction is assumed to be negligible for those measurements.

3.2. Plant fractions

The biochemical constituents of plants varied signifi-cantly in their stable hydrogen isotope composition within a species and between plant species. The dD values of the various plant fractions are reported in Table 2 along with the variations in d13C for the same fractions from Benneret al. (1987). Whole tissue, cellulose and lignin values are from direct measurement unless otherwise indicated.

The analysed plants came from a variety of locations in the United States, except the mangrove samples which came from the Bahamas. In order to account for differences in source water, hydrogen isotope compositions of the four fractions relative to bulk material (DDfraction-bul k) were cal- culated (Fig. 2). A similar calculation was performed to ob- tain D13Cfraction-bul k for each fraction so that differences in both isotope compositions are expressed relative to bulk material. Cellulose and lignin plotted as well-defined fields,while the other components were more scattered. All cellu- lose values fell in the upper right quadrant indicating they were more positive in both dD and d13C relative to bulk,while all lignin values fell in the lower left quadrant, and were more negative in both dD and d13C relative to the bulk. Most hemicellulose values were not offset from bulk,while the uncharacterized ranged from no offset from bulk to being depleted in D relative to bulk.

Cellulose was enriched in D by �50&, and lignin was depleted in D by �50& relative to whole plant material,such that the difference in dD between cellulose and lignin was 106 ± 15 &. The hydrogen isotopic values of the cellu- lose and lignin fractions were not significantly correlated with their ash free dry weights (P = 0.150 and 0.236 respec- tively), suggesting variations in the proportion of material each fraction represents did not have major influence on their isotopic compositions. The dD and d13C values for cel- lulose were negatively correlated (Pearson correlation,r2 = 0.66, P = 0.025), and a weak positive correlation was observed between dD and d13C values for lignin (r2 = 0.56, P = 0.053, Fig. 2). Other correlations have also been identified for other plant fractions, such as a negative correlation between the lipid and pigment fraction dD and d13C (Chikaraishi and Naraoka, 2001 ). In combination such results, though not conclusive, suggest different factors such as different reactions regulate the changes in dD and d13C within specific chemical fractions of plant biomass during biosynthesis (Schmidt et al., 2003 ).

The dD values of hemicellulose were typically similar to the dDbulk values observed for each plant, and were typi- cally �50& less than those for cellulose. The difference be- tween the cellulose and hemicellulose fractions likely reflects differences in the hydrogen isotopic composition of the water present in the locations where these com- pounds are synthesized (cell-membrane bound complexes for cellulose (Brown, 1996 ), and the Golgi apparatus for hemicelluloses (Scheller and Ulvskov, 2010 )), as carbon- bound hydrogen can be partially exchanged with cell water during biosynthesis. A similar process results in cellulose having dD values �80& higher than those of plastidic starches within the same plants (Luo and Sternberg,1991). Additionally, the dD and d13C values for hemicellu- lose are not correlated, indicating they are due to differentprocesses (i.e. fractionation of carbon isotopes during bio- synthesis, and exchange of hydrogen isotopes with cell water during biosynthesis).

Table 2Stable hydroge n (dD) and carbo n (d13C) isotope comp osition s, and ash-fre e dry weight (AFDW) pe rcentag es of the maj or chemica l co mpone nts of vascula r plant tissues. d13C and AFDWs values are taken from Be nner et al. (1987). The table sec tion be low the be low the gap depict s results for sam ples whe re the extra ction proced ure did not work as effectively, ho wever the dD valu es for these sam ples appe ar unaffected (see Section 3 in text). Valu es in par enthe ses represen t mean de viations (for dire ctly measu red sam ples, n = 2). ‘nm’ denotes sam ples that were no t avail able for dDanal ysis. Bla nk cell s indicate no co rrespo nding d13C valu e in Benner et al. (1987). ‘–’ indica tes insta nces where the he micellulo se or unch aracteriz ed fractio n co uld not be calculat ed due to missing measu rement s. Valu es for dD are repor ted rela tive to V-SMO W. Valu es for d13C (±0.1 &) are rep orted rela tive to PDB.

Plant spe cies, photo synthe tic mode and tissue anal yzed

Whole tissue Hemicel lulose Cellu lose Lignin Unchara cteri zed

dD d13C dD d13C AFDW dD d13C AF DW dD d13C AF DW dD d13C AF DW

Spartina alt erniflora C4 Le af �85 (1) T �12.6 �110 (6) �11.2 43.6 �39 (1) �11.1 30.8 �135 (8)* �17.0 4.3 – �16.8 21.3 Rhi zome �74 (2) �13.2 �77 (4) �11.4 37.0 �36 (5) �12.3 30.6 �136 (12)* �17.9 9.3 �95 (17) �15.4 23.1

Cynod on dacty lon C4 Le af nm �18.7 – �14.5 32.8 nm �18.2 25.5 nm �25.4 7.9 �95 (13) �24.2 33.8

Juncus roem eria nus C3 Whole plant �77 (1) �80 (3) 37.1 �18 (2)* 30.3 �129 (1) 7.7 �142 (8) 24.9 Le af nm �23.6 – �22.5 46.3 nm �22.5 28.3 nm �26.8 6.0 – �26.8 19.4 Rhi zome nm �23.7 – �22.9 36.7 nm �23.0 21.0 nm �27.3 5.1 – �24.4 37.2

Pin us elliotti C3 Wood �74 (1) �25.3 �69 (7) �24.3 25.4 �28 (3) �23.8 36.3 �120 (1) �27.9 31.0 �125 (32) �25.2 7.3

Junip erus virg inian a C3 Wood nm �24.0 – �22.5 23.0 �9 (3) �22.1 34.4 �120 (1) �26.5 31.4 – �25.9 11.2

Que rcus nige r C3 Wood �82 (1) �27.5 �90 (28) �28.4 26.5 �15 (1) �27.2 41.6 �144 (2) �30.8 20.8 �133 (57) �20.3 11.1

Pin us elliotti C3 Ne edles �96 (4) 3 (3) 13.4 nm 19.1 �124 (1) 16.2 �115 (8) 51.3 Junip erus virg inian a C3 Ne edles �70 (1) �35 (6) 14.3 nm 14.6 �101 (1) 5.1 �81 (2) 66.0

Subs cript T indicat es tho se Spartina altern iflora me asurem ents from the tall form. All others were from the short form.All fractio ns were me asured directly , with the except ion of hemic ellulos e and the unchara cterize d fraction and a few other excep tions indica ted by ‘*’ whic h were calcul ated by mas s balanc e.

122 N

.D

eBo

nd

et al./G

eoch

i mica et C

osm

och

im ica A

cta 111(2013)

117–127

Fig. 2. Hydrogen isotope compositions of the four fraction srelative to bulk material (DDfraction-bulk) versus the carbon isotope composition s of the four fractions relative to bulk material (D13Cfraction-bulk) for S. alterniflora , P. elliotti , J. roemerianus , and Q. niger . The four fraction s depicted are lignin, celluose, hemicel- lulose, and an uncharacter ized fraction . See details in Section 2 for how these factions were obtained and defined.

N. DeBond et al. / Geochi mica et Cosmochim ica Acta 111 (2013) 117–127 123

The root and rhizome biomass of short-form S. alternifl-

ora differed in hydrogen isotope composition from its stems and leaves. The dD of stem and leaf cellulose was 10 & low-er than the cellulose in roots and rhizomes. Stem and leaf hemicellulose and lignin were also lower (�30& and�50&, respectively) than their root and rhizome counter- parts. Unfortunately, stem and leaf bulk (i.e., whole) mate- rial was only available from a tall-form plant, and cannot necessarily be directly compared to the short-form roots and rhizomes bulk. Similar to the cellulose fraction from the short-form, however, the tall-form stem and leaf bulk dD value was 10 & lower than the roots and rhizomes of the short-form plant.

The differences in hydrogen isotope composition indi- cate a plant water dD gradient, the use of precursor mole- cules with differing dD values, and/or different reaction pathways causing different fractionations during biosynthe- sis in the roots and rhizomes versus the stem and leaves of the plant. The production of a dD gradient via evaporation of leaf water does not account for the differences observed,as it results in stem and leaf water that is enriched in D rel- ative to water in the roots and rhizomes (Wershaw et al.,1966; Bricout, 1978 ). The use of primary photosynthate during the biosynthesis of more complex compounds versus biosynthesis from carbohydrates stored in roots has been shown to cause variations in the dD values of leaf lipids,where compounds produced from primary photosynthate have lower dD values than those produced from stored car- bohydrates (Sessions, 2006 ). The S. alterniflora from this

study was collected during the month of September, while photosynthesis was still very active. It may be the case that the stem and leaf material reflects immediate access to pri- mary photosynthate, while the roots and rhizomes may uti- lise a greater proportion of stored carbohydrates during biosynthesis.

The stable hydrogen isotopic composition of cellulose is �50& higher than bulk plant material, which is in agree- ment with the findings of Epstein et al. (1976). However,a review by White (1989) uses mass balance and the data from Epstein et al. (1976) to calculate a dD value for lignin that is even more enriched (�90& increase in dD) in D rel- ative to bulk material. This is in stark contrast to our obser- vation of a consistent �50& decrease in the dD value of lignin relative to bulk tissue, which is in agreement with the prediction by Schimmelmann et al. (1993).

Although Epstein et al. (1976) and Benner et al. (1987)both isolated a biochemical fraction containing cellulose,hemicelluloses and lignin, they were achieved using differentmethods. Epstein et al. (1976) used a benzene-methanol extraction to separate lipids and resins from an insoluble cellulose, lignin and hemicellulose fraction. The cellulose,lignin and hemicellulose fraction was found to be �50&

more enriched in D than the starting material, which is �40& more enriched than the equivalent fraction extracted by Benner et al. (1987). The disagreement between these re- sults is surprising. Cellulose, hemicelluloses, and lignin comprise the majority of wood, with cellulose and hemicel- luloses representing 65–75% of the biomass, while lignin ac- counts for 18–35% (Pettersen, 1984 ). Mass balance suggests that any chemical fraction representing nearly the total bulk mass should have an isotopic composition that is quite similar to that of the bulk material, such as was observed for d13C by Benner et al. (1987). Some lignin is soluble in organic solvents (Katzen et al., 1945 ), therefore removal of lipids and resins via benzene-methanol extraction is also likely to remove portions of the lignin fraction. This poten- tial extraction may have resulted in hydrogen isotopic frac- tionation of the lignin pool, causing the dD value for lignin reported by Epstein et al. (1976) to be higher.

Our observation, that lignin is more depleted in D than cellulose and bulk tissue, suggests that earlier lines of evi- dence for this relationship are in fact robust. The depletions in both D and 13C observed in lignin are likely in large part a result of fractionations during biosynthesis. Lignin is acomplex metabolite that is derived primarily from three hydroxycinnamyl alcohol monomers with methoxy hydro- gen contents ranging from 0% to 43%, which are themselves derived from the amino acid phenylalanine (see review by Boerjan et al. (2003) for additional information regarding lignin structure and biosynthesis). Hydrogen isotope fracti- onations during biosynthesis result in amino acids having lower dD values than bulk organic matter, which suggests that lignin should be depleted in D relative to bulk tissue as well (Estep and Hoering, 1981 ). Methoxylation that oc- curs during lignin biosynthesis results in further depletion in D. Lignin methoxyl groups, which may constitute up to 20% of total lignin mass, and likely a larger percentage of lignin hydrogen content, have been demonstrated to be depleted in D both relative to bulk wood (DD ffi �170&)

124 N. DeBond et al. / Geochi mica et Cosmochim ica Acta 111 (2013) 117–127

and environmental water (DD ffi �200&, e = �216 ± 19 &;Keppler et al. (2007)). Finally, our lignin values (�101 ± 1to �144 ± 2&) fall within the range reported by a previous review of global dD values for phenylpropanoids (�70 to �150&), a compound class including lignin (Schmidtet al., 1998 ).

3.3. Degradation experiment

Many experiments have demonstrated that the chemical components of plant tissues degrade at different rates. The lignin component of vascular plants degrades 2–5 times slower than the polysaccharide components which include cellulose (Benner et al., 1984, 1986; Maccubbin and Hod- son, 1980; Opsahl and Benner, 1995; Tenney and Waks- man, 1929; Wilson, 1985 ). As a result, the proportion of lignin in a S. alterniflora detritus sample reflects the amount of degradation that has occurred. In this study the dDbu lk and d13Cbulk of the root and rhizome detritus were positively correlated (r2 = 0.67, P = 0.001), and de- creased as the proportion of lignin increased (Fig. 3).The difference in dD values from the initial to final mea- surement was 15 &, representing the total variation in dD observed, which slightly exceeded the expected approx- imate ten-fold increase over the d13C variation due to the mass differences between the stable isotopes of carbon and hydrogen.

The dD of bulk S. alterniflora detritus samples was neg- atively correlated with lignin content (Pearson correlation,r2 = 0.80, P < 0.001, Fig. 3a). A similar relationship existed between the dD and time of decomposition (r2 = 0.78,P < 0.001), which verified that lignin concentration is agood proxy for the extent of degradation that has occurred.The dDbulk value of degraded organic matter reflects the

Fig. 3. Variations in (a) the stable hydroge n (dDbulk, this study) and (b) cbulk S. alterniflora root and rhizome detritus versus lignin concentraticomposition s became more depleted (in D and 13C) as the proportio n odecomposit ion. Linear fits are plotted for each relationsh ip (see Section 3noted next to each point.

isotopic signatures of its biochemical components, and de- creases when the lignin component is relatively resistant to biodegradation.

It is possible that the correlation between dDbulk and lig- nin content is reinforced by an increase in the proportion of lipids which are also relatively resistant to degradation, and have dD values which are lower than that of bulk tissue.Lipids were not isolated in this study, and instead are in- cluded as a component of the uncharacterized fraction.As a result, the relative increase in the lipid component of S. alterniflora during the 18 month degradation is not known. However, many of the lipids present in S. alternifl-

ora biomass have been shown to have dD values that are similar to those found for lignin in this study (Sessions,2006). An additional study quantifying the relative increase in lipid content in the detrital matter is required in order to quantify its contribution to the trend toward lower dDbulk

values over time as S. alterniflora biomass degrades. How- ever, this study suggests materials with high lignin content (i.e. woody materials which have �30% lignin) will have dD values that decrease with increased degradation, even when their lipid content is low.

Although it appears that a linear relationship exists be- tween lignin concentration and the isotopic composition (d13C and dD) of the bulk detritus over 18 months (Fig. 3), extrapolating the linear fit to 100% lignin content did not predict the isotopic composition of the lignin frac- tion (see Table 3). Changes in d13C during the degradation of S. alterniflora were best described by Benner et al. (1987)using a multiple component exponential decay model (mul-ti-G model), whereas a simple exponential fit best predicted both the dDbulk value for S. alterniflora detritus after degra- dation for 18 months and the hydrogen isotopic composi- tion of the lignin fraction.

arbon isotope composit ions (d13Cbulk, from Benner et al. (1987)) of o n expressed as a fraction of ash free dry weight. Both isotopic f lignin increased, indicating the isotopic integrity of lignin during for statistics ), and the number of months each sample degraded is

Table 3Measurem ents and model predictions for the total percenta ge of material remaining, lignin concentrat ion, and stable carbon and hydrogen isotope composition for decaying Spartina alterniflora rhizome material buried in salt-marsh sediments, modified from Benner et al. (1987).Linear fits were obtained by linear regression of the data in Fig. 3. The multi-G model is discussed in Benner et al. (1987), and was recalculated using the hydroge n stable isotope information presented in Table 2. Stable carbon isotope values are reported relative to PDB. Stable hydrogen isotope values are reported relative to V-SMOW .

Decomposit ion period (yr) 0.0 1.5 5.0 10.0 100% lignin

Observed Predicted Observed Predicted Predicted Predicted Observed Predicted

Material remainin g (%AFDW) 100 100 44.9 45.8 9.2 1.9 n/a n/a Lignin concentratio n (% AFDW) 9.8 9.8 15.4 16.3 42.6 82.7 100 100

d13C (&)Predicte d, linear fit �13.0 �14.3 to �14.6 �20.9 �30.5 �34.7 Predicte d, exponential fit �13.1 �13.0 �14.1 �14.3 to �14.5 �17.2 �19.2 �17.9 ± 0.2 �19.7 Predicte d, multi-G model �13.1 �14.0 �15.4 �17.2 n/a

dD (&)Predicte d, linear fit �74 �87 to �89 �154 �253 �295 Predicte d, exponential fit �72 �73 �87 �86 to �88 �116 �136 �145 ± 12 �141 Predicte d, multi-G model �76 �80 �99 �125 n/a

N. DeBond et al. / Geochi mica et Cosmochim ica Acta 111 (2013) 117–127 125

The simple exponential relationship that describes the change in dDbu lk value for S. alterniflora detritus over time indicates that dDbulk measurements can potentially provide useful information for determining the extent of degrada- tion of a simple detritus sample where the type of plant lit- ter is identified (e.g. in salt marsh sediments dominated by S. alterniflora). Applying such a relationship to more com- plex mixtures of organic matter from various sources, how- ever, may be difficult. Each organic matter source material may degrade at different rates and exhibit different changes in dDbulk over the same period of time. The dDbulk of acomplex mixture of detritus will reflect the sum of these ef- fects, which would likely mask overall source identificationor degradation state. Understanding how dDbulk changesfor each potential source material with degradation, and what the ecosystem level inputs of each source to the depo- sitional environment would be, is required in order to assess the extent of degradation of such a detritus sample using adDbulk approach.

It is important to note that the same challenges that sur- round interpreting dDbu lk values for a system with multiple inputs are also encountered when using other isotopic and molecular approaches to understand organic matter degra- dation. However, measuring dDbulk values has two poten- tial advantages which may reduce the amount of work required to investigate the degradation state of organic matter. Bulk dD measurements require minimal processing (i.e. no acid fumigation necessary) and no additional char- acterization of the organic matter (i.e. degradation rates and the development of a multi-G model). The potential application of dDbulk measurements in assessing the extent of organic matter degradation in depositional environments should be investigated in the future.

Time scale and starting material composition are key factors in the interpretation of changes in bulk isotope val- ues during organic matter degradation. Over a period of 18 months, this study observed a negative correlation be- tween the dD of bulk S. alterniflora detritus samples and the degradation time. In contrast, studies of the degrada- tion of algal matter performed over much shorter period

of times (�2 months) have found no consistent relationship between dD and time (Macko et al., 1983; Fenton and Ritz,1988). The chemical composition of the starting material is important to the changes in dD expected. Algae are not vas- cular plants, and do not contain lignin, so the same degra- dation pattern as observed for S. alterniflora is not necessarily expected, nor was it observed in these studies.

Time scales are also important since biomolecules will not remain intact across all timescales. Kerogen degrades at much longer timescales, where the degradation rates of individual biomolecules are less relevant, and consequently kerogens have been shown to generally become more en- riched in D as their maturity increases (Schimmelmannet al., 2006 ). In contrast, change in dD during the transfor- mation of plant material into peat is similar to the degrada- tion of material S. alterniflora in this study: a decrease of 2.8& in measured dD for every 1% increase in carbon con- tent from fresh material with 50% carbon to mature peat with 63% carbon (Schiegl, 1972 ). The differences in these trends for kerogen and peat illustrate the importance of selecting timescales relevant to the specific biomolecules affecting dD values when assessing changes in dD and iden- tifying potential sources for the analysed detritus.

4. CONCLUSIONS

This study provides direct evidence that lignin has a sta- ble hydrogen isotope composition that is more depleted in D relative to bulk plant matter and cellulose. During the biodegradation of S. alterniflora, polysaccharides are pref- erentially removed resulting in a relative enrichment of lig- nin and a depletion of D in the remaining material. This isotopic shift in dD with increasing biodegradation is con- sistent with shifts in d13C observed previously (Benneret al., 1987 ).

The stable hydrogen and carbon isotope compositions of organic matter which has degraded over a time scale of years change due to varying degradation rates of differentplant constituents. Shifts in isotopic compositions during selective biodegradation should be considered when inter-

126 N. DeBond et al. / Geochi mica et Cosmochim ica Acta 111 (2013) 117–127

preting dD and d13C in the geologic record, as the bulk iso- topic compositions of organic matter reflect diagenetic pro- cesses as well as climate change. The trend observed in dDis effectively modelled with a simple exponential equation and combined with the fact that measuring dDbulk requiresvirtually no sample processing suggests that for simple sys- tems (i.e. where the source of organic matter is identified),constructing a model to estimate the extent of degradation would be more easily accomplished using dD than d13C.Further investigation is needed, however, to assess the application of dDbulk to the study of other natural organic matter reservoirs which are often complex mixtures of com- pounds derived from multiple sources exhibiting variable degradation rates.

ACKNO WLEDGEM ENTS

This work was funded by the Carnegie Institution of Washing- ton and also supporte d by Grants from the Carnegie Instituti on of Canada to M.L.F, P.L.M., and S.Z., an NSERC Alexander Gra- ham Bell Canada Graduate Scholarship to N.D., and NSERC Dis- covery Grants to S.Z. and P.L.M. We would like to thank N.Szponar for providing helpful comments on an early version of the manuscript. Alex L. Sessions and an anonymous reviewe r pro- vided comme nts that greatly improved the content and clarity of this manuscript.

APPENDIX A. SUPPLEMENTARY DATA

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.gca.2012.10.043.

REFERE NCES

Benner R., Maccubbin A. E. and Hodson R. E. (1984) Anaerob ic biodegrada tion of the lignin and polysacchar ide compone nts of lignocellulo se and syntheti c lignin by sediment microflora. Appl.

Environ. Microbiol. 47, 998–1004.

Benner R., Maccubb in A. E. and Hodson R. E. (1986) Temporal relationsh ip between the depositio n and microbial degradatio nof lignocellulo sic detritus in a Georgia salt marsh and the Okefeno kee Swamp. Microbial Ecol. 12, 291–298.

Benner R., Fogel M. L., Sprague E. K. and Hodson R. E. (1987)Depletion of 13C in lignin and its implicati ons for stable carbon isotope studies. Nature 329, 708–710.

Benner R., Hatcher P. G. and Hedges J. I. (1990) Early diagenesis of mangrove leaves in a tropical estuary: bulk chemica lcharacteri zation using solid-state 13C NMR and elemental analyses. Geochim . Cosmochim. Acta 54, 2003–2013.

Benner R., Fogel M. L. and Sprague E. K. (1991) Diagenesis of belowgroun d biomass of Spartina alterniflora in salt-marsh sediment s. Limnol. Oceanogr. 36, 1358–1374.

Boerjan W., Ralph J. and Baucher M. (2003) Lignin biosynthesis.Annu. Rev. Plant. Biol. 54, 519–546.

Bricout J. (1978) Recherches sur le fractionneme nt des isotopes stables de l’hydroge `ne et de l’oxyge `ne dans quelqu es ve ´ge ´taux.Rev. Cytol. Biol. Ve ´ge ´t. Bot. 1, 133–209.

Brown, Jr., R. M. (1996) The biosynthe sis of cellulose. J.

Macromo l. Sci. 33, 1345–1373.

Chamberl ain C. P., Blum J. D., Holmes R. T., Feng X., Sherry T.W. and Graves G. R. (1997) The use of isotope tracers for

identifying populatio ns of migratory birds. Oecologia 109, 132–

141.

Chikaraishi Y. and Naraoka H. (2001) Organic hydrogen–carbon isotope signatur es of terrestrial higher plants during biosynthe -sis for distinctiv e photosyn thetic pathways. Geochem. J. 35,

451–458.

DeNiro M. J. and Epstein S. (1981) Isotopic compo sition of cellulose from aquatic organisms . Geochim . Cosmochim. Acta

45, 1885–1894.

Epstein S. (1995) The isotopi c climatic records in the Allero ¨d–Bølling–Younger Dryas and post-Youn ger Dryas events.Global Biogeochem. Cycles 9, 557–563.

Epstein S., Yapp C. J. and Hall J. H. (1976) The determination of the D/H ratio of non-exc hangeable hydrogen in cellulose extracted from aquatic and land plants. Earth Planet. Sci. Lett.

30, 241–251.

Epstein S., Thompson P. and Yapp C. J. (1977) Oxygen and hydrogen isotopic ratios in plant cellulose. Science 198, 1209–

1215.

Estep M. F. and Hoering T. C. (1980) Biogeochem istry of the stable hydroge n isotopes. Geochim. Cosmochi m. Acta 44, 1197–

1206.

Estep M. F. and Hoering T. C. (1981) Stable hydrogen isotope fractionation s during autotroph ic and mixotrophic growth of microalgae. Plant Physiol. 67, 474–477.

Feng X. and Epstein S. (1994) Climat ic implications of an 8000- year hydrogen isotope time series from Bristlecone pine trees.Science 265, 1079–1081.

Fenton G. E. and Ritz D. A. (1988) Changes in carbon and hydrogen stable isotope ratios of macroalgae and seagrass during decomposition . Estuar. Coast. Shelf Sci. 26, 429–436.

Katzen R., Sawyer F. G. and Othmer D. F. (1945) Extraction of lignin from hydrolyzed lignocellulo se. Ind. Eng. Chem. 37,

1218–1222.

Keppler F., Harper D. B., Kalin R. M., Meier-Au genstein W.,Farmer N., Davis S., Schmid t H.-L., Brown D. M. and Hamilton J. T. G. (2007) Stable hydrogen isotope ratios of lignin methoxyl groups as a paleoclim ate proxy and constraint of the geographica l origin of wood. New Phytol. 176, 600–609.

Luo Y.-H. and Sternberg L. (1991) Deuterium heterogeneit y in starch and cellulose nitrate of CAM and C3 plants. Phyto-

chemistry 30, 1095–1098.

Maccubbin A. E. and Hodson R. E. (1980) Mineralizatio n of detrital lignocellulo ses by salt marsh sediment microflora. Appl.

Environ. Microbiol. 40, 735–740.

Macko S. A., Estep M. L. F. and Lee W. Y. (1983) Stable hydrogen isotope analysis of foodwebs on laboratory and field popula- tions of marine amphipod s. J. Exp. Mar. Biol. Ecol. 72, 243–

249.

Northfelt D. W., DeNiro M. J. and Epstein S. (1981) Hydrogen and carbon isotopic ratios of the cellulose nitrate and sapon- ifiable lipid fraction s prepared from annual growth rings of aCalifornia redwood. Geochim. Cosmochi m. Acta 45, 1895–1898.

O’Donnell I. J. and Inglis A. S. (1974) Amino acid sequen ce of afeather keratin from silver gull (Larus novae-hollan diae ) and acomparison with one from emu (Dromaius novae-hollan diae ).Aust. J. Biol. Sci. 27, 369–382.

Opsahl S. and Benner R. (1995) Early diagenes is of vascular plant tissues – lignin and cutin decomposit ion and biogeoche mical implications. Geochim . Cosmochi m. Acta 59, 4889–4904.

Pettersen R. C. (1984) The chemica l composit ion of wood. In The

Chemistry of Solid Wood (ed. R. M. Rowell). American

Chemical Society, Washington D.C.

Qi H. and Coplen T. B. (2011) Investigation of preparation techniques for d2H analysis of keratin materials and a proposed

N. DeBond et al. / Geochi mica et Cosmochim ica Acta 111 (2013) 117–127 127

analytical protoco l. Rapid Commun. Mass Spectrom. 25, 2209–

2222.

Sauer P. E., Schimmelman n A., Sessions A. L. and Topalov K.(2009) Simplified batch equilibration for D/H determin ation of non-exchan geable hydroge n in solid organic material. Rapid

Commun. Mass Spectrom. 23, 949–956.

Scheller H. V. and Ulvskov P. (2010) Hemicellulose s. Annu. Rev.

Plant Biol. 61, 263–289.

Schiegl W. E. (1972) Deuterium content of peat as a paleoclimat ic recorder. Science 175, 512–513.

Schimmelm ann A., Miller R. F. and Leavitt S. W. (1993) Hydroge nisotopic exchange and stable isotope ratios in cellulose, wood,chitin, and amino compo unds. Geophys. Monogr. Ser. 78, 367–

374.

Schimmelm ann A., Sessions A. L. and Mastalerz M. (2006)Hydrogen isotopic (D/H) composit ion of organic matter during diagenes is and thermal maturation . Annu. Rev. Earth Planet.

Sci. 34, 501–533.

Schmidt H.-L., Roßmann A. and Werner R. A. (1998) Stable isotope ratio analysis in quality control of flavourings. In Flavouring s, production, composi tion, applicatio ns, regulations

(eds. E. Ziegler and H. Ziegler). Wiley-VCH, Weinheim, pp.

539–594.

Schmidt H.-L., Werner R. A. and Eisenreich W. (2003) Systemat ics of 2H patterns in natural compo unds and its importance for the elucidatio n of biosynthet ic pathway s. Phytochem. Rev. 2, 61–85.

Sessions A. L. (2006) Seasonal changes in D/H fractionation accompan ying lipid biosynthesis in Spartina alterniflora . Geo-

chim. Cosmochim. Acta 70, 2153–2162.

Smith B. N. and Jacobson B. S. (1976) 2H/1H and 13C/12C ratios for classes of compounds isolated from potato tuber. Plant Cell

Physiol. 17, 1089–1092.

Sternberg L., DeNiro M. J. and Ajie H. (1984) Stable hydroge nisotope ratios of saponifiable lipids and cellulose nitrate from CAM, C3 and C4 plants. Phytochem istry 23, 2475–2477.

Tenney F. G. and Waksman A. (1929) Compositio n of organic material and their decomposit ion in the soil: 5. The nature and rapidity of decompo sition of the various organi c comple xes in different plant materials, under aerobic conditions. Soil Sci. 28,

55–84.

Terwilliger V. J., Betanco urt J. L., Leavitt S. W. and Van de water P. K. (2002) Leaf cellulose dD and d18O trends with elevation differ in direction among co-occurrin g, semiarid plant species.Geochim. Cosmochi m. Acta 66, 3887–3900.

Terwilliger V. J. and DeNiro M. J. (1995) Hydrogen isotope fractionation in wood-pro ducing avocado seedlings : biological constraints to paleoclimatic interpretatio ns of dD values in tree ring cellulose nitrate. Geochim . Cosmochi m. Acta 24, 5199–

5207.

Van Soest P. J. and Robertson J. B. (1980) Systems of analysis for evaluating fibrous feeds. In Standardiz ation of Analytical

Methodolo gy for Feeds (eds. W. I. Pigden , C. C. Balch and M. Graham). International Development Reseearch Centre,

Ottawa, Canada, pp. 49–60.

Wershaw R. L., Friedman I., Heller S. J. and Frank P. A. (1966)Hydrogen isotopic fractionation of water passing through trees.In Advances in Organic Geochemistry (eds. G. D. Hobson and M. Speers). Pergamon Press, New York, N.Y., pp. 55–67.

White J. W. C. (1989) Stable hydroge n isotope ratios in plants: areview of current theory and some potential applicatio ns. In Stable Isotopes in Ecologica l Research (eds. P. Rundel, J. R.Ehleringer and K. A. Nagy). Springer-Verlag, New York, N.Y.,

pp. 142–162.

Wilson J. O. (1985) Decompositio n of [14C]lignocellulo ses of Spartina alterniflora and a comparison with field experiments .Appl. Environ. Microbiol. 49, 478–484.

Yapp C. J. and Epstein S. (1982) Climatic significance of the hydrogen isotope ratios in tree cellulose. Nature 297, 636–639.

Associate editor: Jack J. Middelburg