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Blackwell Publishing Ltd

Stable hydrogen isotope ratios of lignin methoxyl groups as a paleoclimate proxy and constraint of the

geographical origin of wood

Frank Keppler

1

, David B. Harper

2

, Robert M. Kalin

3

, Wolfram Meier-Augenstein

3

, Nicola Farmer

3

, Simon Davis

4

, Hanns-Ludwig Schmidt

5

, David M. Brown

6

and John T. G. Hamilton

2

1

Max-Planck-Institute for Chemistry, Joh.-Joachim-Becher-Weg 2, 55128 Mainz, Germany;

2

School of Agriculture and Food Science, Newforge Lane,

Belfast BT9 5PX, UK;

3

School of Planning Architecture and Civil Engineering, Queen’s University Belfast, Belfast BT9 5AG, UK;

4

Mass Spec Solutions,

Manchester M23 9YJ, UK;

5

Technische Universität München, Lehrstuhl für Biologische Chemie, An der Saatzucht 5, D-85350 Freising-Weihenstephan,

Germany;

6

Department of Archaeology & Palaeoecology, Queen’s University Belfast, Belfast BT9 5AG, UK

Summary

• Stable isotope ratios of organic compounds are valuable tools for determining thegeographical origin, identity, authenticity or history of samples from a vast range ofsources such as sediments, plants and animals, including humans.• Hydrogen isotope ratios (

δ

2

H values) of methoxyl groups in lignin from wood oftrees grown in different geographical areas were measured using compound-specificpyrolysis isotope ratio mass spectrometry analysis.• Lignin methoxyl groups were depleted in

2

H relative to both meteoric water andwhole wood. A high correlation (

r

2

=

0.91) was observed between the

δ

2

H valuesof the methoxyl groups and meteoric water, with a relatively uniform fractionationof –216

±

19‰ recorded with respect to meteoric water over a range of

δ

2

H valuesfrom –110 in northern Norway to

+

20‰ in Yemen. Thus, woods from northernlatitudes can be clearly distinguished from those from tropical regions. By contrast,the

δ

2

H values of bulk wood were only relatively poorly correlated (

r

2

=

0.47) withthose of meteoric water.• Measurement of the

δ

2

H values of lignin methoxyl groups is potentially a powerfultool that could be of use not only in the constraint of the geographical origin oflignified material but also in paleoclimate, food authenticity and forensic investigations.

Key words:

authenticity, deuterium, geographical origin, lignified material,nonexchangeable hydrogen, paleoclimate proxy, stable hydrogen isotopes, wood.

New Phytologist

(2007)

doi

: 10.1111/j.1469-8137.2007.02213.x

© The Authors (2007). Journal compilation ©

New Phytologist

(2007)

Author for correspondence:

Frank KepplerTel:

+

49 6131305 316Fax:

+

49 6131305 388Email: keppler@mpch-mainz.mpg.de

Received:

11 May 2007

Accepted:

15 July 2007

Introduction

Stable hydrogen isotope analysis is recognized as a powerfultool in global climate research. Variations in the

δ

2

H and

δ

18

O values of water in ice cores are commonly used toreconstruct past climatic temperature fluctuations, and detailedpaleoclimate records were published as early as the 1960s and1970s (Dansgaard

et al

., 1969; Epstein

et al

., 1970)

Furthermore,

δ

2

H values of wood in the annual growth ringsof trees can provide the information necessary to reconstructpast climates and to assist in ecophysiological research(Schiegl & Vogel, 1970; Schiegl, 1974; Epstein

et al

., 1976;Switsur

et al

., 1996; McCarroll & Loader, 2004; Filot

et al

.,2006). Site- and compound-specific

δ

2

H values of biomarkersaccumulated in sediments are increasingly employed aspaleoclimatic and paleohydrological proxies (Sternberg, 1988;

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(2007)

Research2

Andersen

et al

., 2001; Sauer

et al

., 2001; Huang

et al

., 2002;Sachse

et al

., 2004).The general principles governing hydrogen isotopic

fractionation are now well delineated. The primary hydrogensource of all organic compounds in the biosphere is water and,in the case of plant biomass, plant leaf water. Hydrogenincorporated into photosynthetic products during primaryreduction steps is highly depleted in

2

H. However, a significantproportion of these hydrogen atoms are exchanged withhydrogen atoms of water during subsequent metabolism,leading to a secondary enrichment. Hayes (2001) has reviewedfractionation of carbon and, to a lesser extent, hydrogenisotopes in organic compounds produced by a single organism,principally in relation to their enrichment or depletionrelative to the total biomass of carbon or hydrogen. This workdid not attempt to consider in any depth the isotopic patternwithin individual compounds. However, recently an extensiveoverview and discussion of both intermolecular andintramolecular nonrandom

2

H distributions in naturalcompounds was provided by Schmidt

et al

. (2003), who alsoconsidered their importance in the elucidation of biosyntheticpathways and their potential to assist in assigning an origin toorganic compounds in plants.

Natural archives such as ice cores, peatlands and sedimentsare already widely utilized in climate research, but trees appearto offer an additional very promising method for reconstructingprecisely detailed annual climatic histories, not only fromliving but also from subfossil trees (Schiegl, 1974; Mayr

et al

.,2003). Indeed, by careful sampling of wood within annualgrowth rings it may be possible to extract climatic informationat a much higher temporal resolution (Barbour

et al

., 2002;Loader

et al

., 1995). Early studies on tree rings analysed wholewood but, when Epstein

et al

. (1976) and Wilson & Grinsted(1977) demonstrated that the wood components lignin,cellulose and hemicellulose differed significantly in isotopiccomposition, investigations focused on cellulose. One of themain advantages of using cellulose is that it is measuredas cellulose nitrate and entirely reflects nonexchangeablehydrogen in this plant component (Epstein

et al

., 1976).From that time forward, many researchers settled on non-exchangeable hydrogen in cellulose as the best proxy forsource water. However, a general problem associated with thedetermination of the

δ

2

H values of marker compounds forthe study of climate and environmental conditions, as well asfor investigation of food authenticity investigations, is theisolation of the pure compound for analysis by isotope ratiomass spectrometry (IRMS). Exploitation of components ofwood as markers, in particular, has been restricted by the verylabour-intensive and time-consuming preparation of samples(e.g. cellulose nitrate). Any improvements to the efficiency ofsample preparation would be of immense value as these wouldnot only allow an increase in the number of sampling pointswithin an individual series but also permit replication of timeseries. Ideally, for accurate determination of the hydrogen

isotope signature, the following criteria should apply to thecompound or the chemical moiety within a compound:• hydrogen atoms that are nonexchangeable throughout thesample history and during sample preparation and analysis,so that the isotope signature measured reflects the pristineisotopic fractionation of the compound;• high natural abundance in samples;• simple extraction method;• rapid and straightforward sample preparation;• rapid and reliable analysis of the compound;• no isotopic fractionation during any stage of sampleprocessing or analysis.

On the basis of these criteria, we suggest that for

2

H analysisof wood the methoxyl groups of lignin offer great potential astarget chemical moieties. Lignin, a major component of wood(up to 31%), is produced by secondary metabolic processesand laid down in cellulose cell walls, imparting strength andrigidity to the structure. It can be considered a polymer ofthree different precursors, termed monolignols, that differin the degree of methylation of the aromatic ring. Themonolignols are

ρ

-coumaryl alcohol, which has no methoxylgroups, coniferyl alcohol, which has a methoxyl groupattached to C-3 of the aromatic ring, and synapyl alcohol,which has methoxyl groups attached to both C-3 and C-5 ofthe aromatic ring. For more information on lignin structuresand biosynthesis, we refer readers to the review by Boerjan

et al

. (2003). Overall, methoxyl groups can constitute up to20% of lignin and whole wood can possess up to 6% methoxylcontent. Methylation of hydroxyl groups attached to thearomatic ring is catalysed by

O

-methyltransferases using S-adenosylmethionine (SAM) as the methyl donor. Thisbiochemical origin from SAM has interesting consequences asregards the isotopic composition of the methoxyl groups oflignin. Recent work has shown that the methoxyl groups oflignin and pectin, which together constitute the bulk of theC

1

plant pool, have a carbon isotope signature significantlydepleted in

13

C (Keppler

et al

., 2004). The depletion betweenbulk plant biomass and plant methoxyl pools ranges from–11 to –46‰, with the pectin C

1

pool generally more depletedthan the lignin C

1

pool.Lignin methoxyl groups are considered to be stable; that is,

the hydrogen atoms of the methoxyl moiety do not exchangewith those of plant water during ongoing metabolic reactions inthe plant. Thus, the initial

δ

2

H value of the methoxyl groupsof lignin in woody tissue at formation is retained throughoutthe lifetime of the tree and in preserved tissue. The methoxylcontent of lignin in wood is usually determined by the Zeiselmethod (Zeisel, 1885) using the reaction between methyl ethersand hydroiodic acid (HI) to form methyl iodide (CH

3

I). Exploit-ing this reaction (Fig. 1) for the measurement of

δ

2

H values oflignin methoxyl groups ensures that the isotope signal is preservedthroughout the analytical procedure, as no isotopic exchangeoccurs between the methyl groups and HI, and no isotopicfractionation in the course of CH

3

I formation is observed. In

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(2007)

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Research 3

this paper, we report measurements of

δ

2

H values of both wholewood and lignin methoxyl groups from wood samples sourcedfrom various geographical locations which establish a rela-tionship between their isotopic signatures and that of thelocal precipitation. We demonstrate that the methoxyl groupsof lignin meet all the criteria listed above for ideal target chemicalmoieties for stable hydrogen isotope measurements in wood.

Materials and Methods

Plant material and field sites

Study of annual variations of early and late woods wasconducted on a section (Fig. 2) of an oak (

Quercus robur

) treefrom Killarney, Ireland (close to one of the InternationalAtomic Energy Agency (IAEA) measurement stations) whichhad been felled in 1994. A small chisel was used for separationof annual early and late woods.

In 2004, samples of wood were collected from a variety ofspecies growing in their natural environments at a number ofgeographical locations around the world (Table 1). Samples inScandinavia were collected from living trees using a 8-mm-diameter borer and usually obtained across a 10–40-yr period.Samples from other regions were collected as wood slices. Siteswere selected on the basis of sample availability and also tocover a broad range of

δ

2

H values for meteoric water. Allsamples were ground to pass through a 2-mm sieve.

Treatment of wood with HI: conversion of lignin methoxyl groups to CH

3

I

Hydrogen isotope signatures of lignin methoxyl groups wereassessed by measuring

δ

2

H values of CH

3

I released upontreatment of wood samples with HI. A volume of 1 ml of 57%HI was added to the sample (10–20 mg of milled material) in

a reaction vial (5 ml). The vials were sealed with caps containingpolytetrafluoroethylene (PTFE)-lined silicone septa andincubated for 30 min at 105

°

C. After the heating cycle, the vialswere placed at room temperature for 15 min before a sample ofheadspace was removed and directly injected into the analyticalsystem. Standard solutions of CH

3

I were treated similarly to thewood samples in order to check for isotope fractionation. Nomeasurable chemical fractionation of hydrogen isotopes in CH

3

Iwas observed during the analytical procedure and no significantchanges in

δ

2

H values of CH

3

I were observed when a seriesof wood samples were incubated for varying periods from 5 to180 min (Fig. 3). It is worthy of mention that during treatmentof wood with HI there is some potential for CH

3

I productionfrom wood components other than lignin, which may interferewith the lignin methoxyl signal. For example, Goto

et al

. (2005)demonstrated that the methoxyl-free compound cellulose canform CH

3

I when treated with HI. However as the amountsproduced were some three orders of magnitude lower whencompared with wood samples, we would not expect CH

3

Iformation from cellulose to have any measurable effect on the

δ

2

H values of CH

3

I reported in this study. However, whenplant material with a very much lower lignin content is used,CH

3

I formation from carbohydrates should be considered.

Isotope ratio monitoring mass spectrometry for determination of

δ

2

H values of CH

3

I

System configuration

Hydrogen isotopic ratios of CH

3

I weremeasured using gas chromatography pyrolysis isotope ratiomass spectrometry analysis (GC-P-IRMS). A Hewlett PackardHP6890 GC (Agilent Technologies, Palo Alto, CA, USA)was interfaced with a Micromass Isoprime isotope ratio massspectrometer via a 1050

°

C pyrolysis reactor (GV Instruments,Manchester, UK). A tank of ultra-high-purity hydrogen witha known

δ

2

H value of –718.7‰ (Vienna Standard MeanOcean Water; VSMOW) was used as an internal referencegas standard. The H

3

+

factor determined daily during the2-month measurement period was in the range 9.1–10.4.The internal precision of

δ

2

H was

±

4‰ (1

σ

)

.

Throughout this paper, the conventional ‘delta’ notation,which expresses the isotopic composition of a material relativeto that of a standard on a per mil (‰) deviation basis, is used.Values of

δ

2

H (‰) relative to that for VSMOW are definedby the equation:

Fig. 1 Mechanism of the reaction between lignin methoxyl and hydroiodic acid (modified after Goto et al., 2006).

Fig. 2 Sections of an oak (Quercus robur) tree collected from Killarney, Ireland in 1995. (a) Annual growth rings from 1968 to 1993. (b) Enlarged section showing 3 yr of growth from 1985 to 1987; early and late wood formation clearly visible.

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Table 1 Wood collection sites and related isotopic composition of meteoric water

Common name (species)Country and site code

Geographical location

Altitude(m)

δ2H meteoricwater (OIPC)

95%CI δ2H meteoric water (GNIP)

Paper birch (Betula papyrifera) Canada CA1 46°01′N; 66°40′W 105 –72 3 –Sweet osmanthus (Osmanthus fragans) China CH1 31°N; 116°Ea 450 –45 8 –54.9 Nanjing (32°18′N; 118°18′E);

–46.8 Wuhan (30°62′N; 114°13′E)European birch (Betula pendula) Finland FI1 69°11′N; 20°75′E 330 –108 12 –118 Naimakka (68°41′N; 22°21′E)European birch (Betula pendula) Finland FI2 68°49′N; 20°24′E 450 –109 11 –118 Naimakka (68°41′N; 22°21′E)European birch (Betula pendula) Finland FI3, FI4 67°92′N; 24°15′E 610 –108 10 –118 Naimakka (68°41′N; 22°21′E)Norway spruce (Picea abies)Cherry (Prunus sp.) Germany GE1 51°N; 7.5°E 330 –61 3 –52.1 Koblenz (51°93′N; 10°25′W)

–60 Weil (47°3′N; 7°4′E)Dark red meranti (Shorea sp.) Indonesia IN1 1°S; 114°Ea 500 –40 10English oak (Quercus robur) Ireland IR1 52°N; 9°33′W 50 –51 11 –36.2 Valentia (50°35′N; 7°58′E)Croton (Croton socotrana) Yemen YE1 12°30′N; 53°50′Ea 50 8 16Tamarisk (Tamarisk aphylla) Yemen YE2 14°25′N; 48°59′E 100 16 17Geronggang (Cratoxylum sp.) Malaysia MA1 6°N; 100°Ea 150 –36 5 –27.8 Ko Samui (13°17′N; 100°03′E)

–46.9 Singapore (1°35′S; 103°9′E)English oak (Quercus robur) Northern Ireland 54°2′N; 6°W 40 –59 7European ash (Fraxinus excelsior) NI1, NI2European birch (Betula pendula) Norway NO1 59°2′N; 10°28′E 100 –77 12Scots pine (Pinus sylvestris) Norway NO2 60°48′N; 11°04′E 120 –82 12European birch (Betula pendula) Norway NO3 61°58′N; 9°33′E 400 –88 13European birch (Betula pendula) Norway NO4 62°22′N; 9°40′E 980 –99 13 –Norway spruce (Picea abies) Norway NO5 62°42′N; 9°55′E 650 –95 13 –Scots pine (Pinus sylvestris) Norway NO6 63°60′N; 11°30′E 50 –90 12 –Norway spruce (Picea abies) Norway NO7 64°13′N; 11°55′E 280 –95 12 –Norway spruce (Picea abies) Norway NO8 64°57′N; 13°09′E 400 –98 12 –Norway spruce (Picea abies) Norway NO9, 65°04′N; 13°25′E 310 –98 12 –European birch (Betula pendula) NO10European birch (Betula pendula) Norway NO11 65°58′N; 13°27′E 60 –95 12 –Norway spruce (Picea abies) Norway NO12 66°24′N; 14°39′E 120 –98 12 –European birch (Betula pendula) Norway NO13 66°33′N; 15°18′E 620 –105 11 –Norway spruce (Picea abies) Norway NO14 67°53′N; 15°58′E 75 –100 12 –European ash (Fraxinus excelsior) Norway NO15 68°27′N; 17°29′E 30 –101 12 –European birch (Betula pendula) Norway NO16 68°40′N; 17°45′E 150 –103 12 –Norway spruce (Picea abies) Norway NO17 69°00′N; 18°33′E 80 –104 12 –European birch (Betula pendula) Norway NO18 69°21′N; 20°06′E 370 –109 12 –

Longitude, latitude and altitude were determined using a topographical map except for those marked with superscript ‘a’, which are approximations, as the geographical origin of the samples was not precisely known. Hydrogen isotope ratios (δ2H values) for meteoric water and 95% confidence intervals (CIs) were calculated using the Online Isotope Precipitation Calculator (OIPC), version 2.1. In the instances where wood sampling locations were in the broader vicinity of an International Atomic Energy Agency (IAEA) measurement station, the weighted annual values from that station using the International Atomic Energy Agency–Global Network of Isotopes in Precipitation (IAEA-GNIP) data set (International Atomic Energy Agency, 2001) are shown. All δ values are given in ‰ vs VSMOW.

© The Authors (2007). Journal compilation © New Phytologist (2007) www.newphytologist.org

Research 5

δ2H (‰) = [(2H/1H)sample/(2H/1H)standard – 1] × 1000‰

Eqn 1

Sample analysis Headspace gas (500 µl) was injected intothe gas chromatograph, which was equipped with a DB5-MScolumn (30 m, internal diameter (ID) 0.32 mm, filmthickness 0.5 µm). The injection port temperature was set at250°C in the split mode (10 : 1). The oven was maintainedfor 4 min at 30°C and then heated at 15°C min–1 to 180°C.Column flow was held constant at 0.8 ml min–1 throughoutthe analysis. Following GC separation, CH3I was transferredto a 1050°C pyrolysis reactor which contained a 0.65-mm IDquartz tube packed with chromium pellets sieved to a size of≤ 0.25 mm (Elemental Microanalysis, Devon, UK). As thesample gas passed through the furnace, a pyrolytic andcatalytic reaction resulted in its quantitative conversion to H2(Morrison et al., 2001). All δ2H values were normalizedrelative to VSMOW using a CH3I standard. The δ2H valuesof CH3I were calibrated against international referencesubstances (IA-R002, IAEA-CH-7 and NBS-22) using theoffline temperature conversion–elemental analyser (TC-EA)technique (Iso-Analytical Ltd, Cheshire, UK). The calibratedδ2H value in ‰ vs VSMOW for CH3I was –174 ± 1‰(n = 4, 1σ). The standard was measured after every thirdsample injection. If necessary, a drift correction was applied.This methodology achieved a precision, expressed as theaverage standard deviation for all samples, of 6‰ (1σ).

Bulk δ2H isotope analysis by TC-EA/IRMS

A DeltaPlus-XP High Temperature Conversion/ElementalAnalyser (Thermo Electron Corporation, Bremen, Germany)was used for 2H:1H isotope ratio measurement of groundwood samples. Typically, 0.2 mg of sample was weighed intoa silver capsule and placed in a desiccator for a week before

the samples were introduced into the TC-EA by means of asolid Costech Zero-Blank autosampler (Pelican Scientific Ltd,Alford, UK). The reactor tube was self-packed and comprisedan AlsintTM ceramic tube containing a glassy carbon tube, filledwith glassy carbon granulate, silver and quartz wool (SerCon,Crewe, UK). Reactor temperature was set to 1425°C whilethe postreactor GC column was maintained at 85°C. Helium(99.99% purity; Air Products Plc, Walton-on-Thames, UK)pressure was set to 1.45 bar. Data were processed using ISODAT

NT software, version 2.0 (Thermo Electron Corporation,Bremen, Germany). The run time per analysis was 350 s.Measured 2H:1H isotope ratios are expressed as δ values in[‰] relative to VSMOW.

Isotopic calibration and quality control of TC/EA-IRMSmeasurement The working reference gas, H2 (BOC Gases,Guildford, UK), was calibrated against VSMOW using aninternational reference material, IAEA-CH-7 (IAEA, Vienna,Austria) polyethylene (δ2HVSMOW = –100.3‰), and checkedagainst the international reference materials VSMOW andStandard Light Antarctic Precipitation (SLAP). Cross-checkingof the working δ2HVSMOW value of H2 against the internationalreference material Greenland Ice Sheet Precipitation (GISP)yielded a δ2HVSMOW value for GISP of –194.6‰ (expected:–189.5‰). The H3+ factor was determined on reference H2 gaspulses of different signal size and was found to be 4.43. A batchanalysis, comprising 10 samples analysed in triplicate, waspreceded and followed by a set of standards, as reportedpreviously (Farmer et al., 2005). Reproducibility of 2H isotopeanalysis as monitored using the IRMS and laboratory standardswas 1.15‰ or better. Each batch was preceded and followedby a blank capsule. Measured δ2H values were normalizedaccording to the method described by Coplen (1988), with Zfactors (or ‘stretch’ factors) typically being of the order of 1.08.

δ2H of water samples and the Online Isotope Precipitation Calculator

The Online Isotope Precipitation Calculator (OIPC; accessibleat http://www.waterisotopes.org/), employing the IAEAdatabase and interpolation algorithms developed by Bowen& Wilkinson (2002) and refined by Bowen & Revenaugh(2003) and Bowen et al. (2005), was used to calculate annualδ2H values of meteoric water from wood sampling sites.Long-term precipitation isotope data were taken from theInternational Atomic Energy Agency–Global Network ofIsotopes in Precipitation (IAEA-GNIP) database (InternationalAtomic Energy Agency, 2001), accessible on the Internet athttp://isohis.iaea.org.

Calculation of isotopic fractionation ε

The isotopic difference between the δ2H value of the methoxylgroups and the δ2H value of precipitation was calculated using

Fig. 3 Hydrogen isotope ratios (δ2H values) of CH3I released from oak (Quercus robur) wood (NI1) over time following treatment with hydroiodic acid (HI). Data show the mean of two measurements, except for those with error bars, which are the means of at least three independent replicate measurements (n = 3–15, 1σ).

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Research6

Eqn 2

Throughout the paper we use the short form εm/w forεmethoxyl/water.

Results and Discussion

2H values of lignin methoxyl groups in early- and late-season wood from annual growth rings of oak from Killarney in Ireland

Annual δ2H values of lignin methoxyl groups of early-seasonoak wood for the 25-yr period 1968–1993 (Fig. 2) were in therange of –209 to –242‰, with a mean value of –228‰,while annual values of late-season wood were slightly moredepleted, ranging from –211 to –255‰, with a mean valueof –237‰ (Fig. 4). Variation (expressed as SD) in the annualδ2H values for early- and late-season wood for the 25-yrperiod was 10 and 12‰, respectively. The δ2H value measuredfor lignin methoxyl groups of a composite wood samplerepresenting the entire 25-yr period was –233 ± 4‰ (n = 5,1σ). The oak tree sampled grew in close vicinity to an IAEA-GNIP sampling station (Valentia, Ireland) where δ2H valuesof precipitation have been recorded on a nearly monthly basissince the early 1960s. The weighted annual δ2H value forprecipitation at this site (International Atomic EnergyAgency, 2001) is –36.2‰, and over the last 40 yr weightedmonthly values have ranged from ∼–10 to –65‰. The annualvariation (expressed as SD) of δ2H values is approximately11‰, a similar range to that observed for the δ2H values oflignin methoxyl groups in both early- and late-season wood.If we assume –36.2 and –233‰ as mean values of precipitationand lignin methoxyl groups, respectively, we calculate theisotopic fractionation, εm/w, to be –205‰. It should be

borne in mind that, as δ2H values for precipitation during thegrowing season are enriched in 2H relative to the weightedannual δ2H value, the actual εm/w may be slightly larger.The apparent discrepancy between the observation that theδ2H value of early-season wood is more positive than that oflate-season wood and the fact that the primary hydrogensource, leaf water, is relatively enriched in 2H in the summercan be resolved by bearing in mind that the first wood inspring is mainly synthesized from reserve starch transportedfrom the tree roots (Hill et al., 1995; Helle & Schleser, 2004).This starch is also the source of NADPH via the oxidativepentose phosphate cycle before photosynthesis begins.Secondary hydrogen exchange with xylem water results inreserve starch possessing a more positive δ2H value than theimmediate products of photosynthesis (McCarroll & Loader,2004).

δ2H values of lignin methoxyl groups in wood samples from various geographical locations and their relation to δ2H values of local precipitation

To further test our hypothesis we collected wood samplesfrom several locations around the world (Table 1, Fig. 5) inorder to compare the δ2H values of lignin methoxyl groupswith those of local precipitation. Sampling sites were selectedin order that a broad range of δ2H values for meteoric waterwas covered. δ2H values of precipitation were assessed by twomethods, and are given in Table 1 together with detailedinformation on the locations at which samples were collected.In instances where sampling points were in the vicinity of anIAEA measurement station, we show weighted annual valuesfor that station. However, there is only one station (Valentia,Ireland) situated in close proximity to one of our samplepoints and only a few in the broader vicinity of our othersampling points. Therefore, we decided to use the OIPC(http://www.waterisotopes.org/) to calculate δ2H values oflocal precipitation at collection sites. Sachse et al. (2004)found a good correlation between measured δ2H values forwater in lakes on a north–south European transect and thosecalculated for local precipitation using the OIPC. We makethe simplifying assumption that the predicted mean annualδ2H values of the OIPC are a valid proxy for tree source water.However, as trees mainly incorporate hydrogen during thegrowing season and have highest productivity in spring andlate summer, particularly in temperate regions, the δ2H valuesobtained from the OIPC for these regions may be slightlylower than the actual average values over the growing season.In this work we do not attempt to account for this effect. Ascan be seen in Table 1, the annual weighted δ2H values ofprecipitation in the areas where wood was collected rangedfrom ∼–110 (close to the North Cape, Norway) to ∼+20‰(Yemen).

δ2H values for lignin methoxyl groups and those for bulkwood of samples used in our investigations are presented in

εδ

δmethoxyl/watermethoxyl

water

H

H

=

++

−

×

2

2

1000

10001 1000

Fig. 4 Yearly record of hydrogen isotope ratios (δ2H values) of lignin methoxyl groups from early (diamonds) and late (squares) wood of an Irish oak (Quercus robur) tree covering a 25-yr period. Data are single measurements except for those with error bars, which are the means of three independent replicate measurements (< ± 5‰, 1σ). Solid lines, linear regression of early and late wood; dashed line, composite wood.

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Research 7

Table 2 together with those for local precipitation. Alsoshown for each location is the isotopic fractionation betweenwater and lignin methoxyl groups. The data are presentedgraphically in Fig. 6. It is clear that, while lignin methoxylgroups are relatively depleted in 2H with respect to bulkwood, the δ2H values of the methoxyl groups are highlycorrelated with the OIPC δ2H values of meteoric water(r2 = 0.91). By contrast, the δ2H values for whole wood areonly relatively poorly correlated with those of meteoric water(r2 = 0.47).

Although early studies used whole wood (Schiegl & Vogel,1970; Schiegl, 1974), it must be pointed out that nowadaysδ2H measurement of bulk wood is not the method of choicein paleoclimate research using tree rings because bulk woodcontains a significant portion of exchangeable hydrogenatoms. When Epstein et al. (1976) and Wilson & Grinsted(1977) demonstrated that the main components of wood –lignin, cellulose and hemicellulose – differed in isotopiccomposition, investigations began to focus on cellulose. Thisshift has opened up the debate on whether, if whole woodcontains the same isotopic record as cellulose, the analysis ofindividual plant components such as cellulose can be justifiedgiven the onerous nature of the extraction process (Barbouret al., 2001; Loader et al., 2003). However, while this argumentmay be persuasive for 13C and 18O isotope analysis, therelatively poor correlation observed in this study between theδ2H values of whole wood and meteoric water suggests thatthis approach is not suitable for analysis of hydrogen isotopes.Nevertheless, a complex extraction procedure to isolate a single

component, and, in the case of cellulose, nitration to replaceexchangeable hydrogen atoms might introduce unquantifiableuncertainties, even if improved correlation coefficients wereachieved. The use of lignin methoxyl groups for determina-tion of the environmental δ2H signal in wood as discussedhere circumvents such problems because of the simplicity ofthe sample preparation procedure. In addition, the use oflignin methoxyl groups is a highly specific wood component,presumably because of a more restricted metabolic source ofhydrogen.

The mean isotopic fractionation εmethoxyl/water (εm/w)between methoxyl groups and the modelled water δ2H valuesof the OIPC was –216 ± 19‰. Assuming constant isotopefractionation between methoxyl groups and meteoric water,we obtain an average fractionation factor (αm/w) of 0.784(αm/w ≡ (δ2Hm + 1000)/(δ2Hw + 1000), where δ2Hm is thehydrogen isotope ratio of wood methoxyl groups and δ2Hw isthe hydrogen isotope ratio of meteoric water). The constantfractionation line obtained using this equation is shown inFig. 6(b), and it is clear that almost all samples lie within theanalytical uncertainty of this line except for a sample frommainland Yemen (YE2), for which the measured δ2H valuewas slightly higher than that predicted by the equation. Theregion has a hot, arid tropical climate, with temperatures insummer reaching up to 50°C. From the GNIP map, δ2Hvalues in precipitation show a broad range from c. –20 to+30‰ throughout the year and the weighted annual value(16 ± 17‰) employed in our study from the OIPC fits quitewell within that range. However, a plausible explanation for

Fig. 5 Sites of collection of wood samples (n = 30) and the mean annual hydrogen isotope ratio (δ2H value) of precipitation. Because of limited space, not all locations in Scandinavia can be shown. The map was provided by G. J. Bowen and made using the Bowen & Wilkinson (2002) method. VPDB; Vienna Peedee Belemnite.

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fractionation being lower than calculated is that, in the lowrelative humidity of this extreme climate, evaporative enrich-ment of leaf water is much greater than normal. It is knownthat the leaf-to-air vapour pressure deficit is the dominantcontrol on the degree of leaf enrichment for δ2H (McCarroll& Loader, 2004). Significantly, the sample from the Yemeniisland of Socatra, several hundred miles offshore in the IndianOcean, showed the predicted fractionation, possibly reflectingthe higher relative humidity associated with a maritimeenvironment. Interestingly, recent paleoclimate studies usingbiomarkers from aquatic sediments have found isotopefractionations in a similar range to those we report for

lignin methoxyl groups in wood. For example, the isotopefractionation between sedimentary lipids (Sauer et al., 2001),palmitic acid (Huang et al., 2002) and n-alkanes (Sachseet al., 2004) and environmental water was found to be–201 ± 10, –171 ± 15 and –157 ± 13‰, respectively.

Origin and δ2H values of H atoms in O-CH3 groups

The methoxyl groups of the phenylpropanoids originate fromposition C-3 of serine, and hence from carbohydrates,perhaps even directly from 3-phosphoglycerate. The CH2OHgroup of serine is transferred by serine hydroxymethyltransferaseto tetrahydrofolic acid (FH4) to form N5,N10-methylene-FH4(‘active formaldehyde’), which is then reduced by N5,N10-

Table 2 Hydrogen isotope ratios (δ2H values) of source water (Online Isotope Precipitation Calculator (OIPC) data) and lignin methoxyl groups, bulk biomass and isotopic fractionation, εmethoxyl/water (εm/w), of wood samples from around the world

Country andsite code

δ2H sourcewatera

δ2H bulkwood

δ2H methoxyl εm/w

Norway NO18 –109 ± 12 –143 –292 –205Finland FI2 –109 ± 11 –145 –309 –225Finland FI1 –108 ± 12 – –313 –230Finland FI3 –108 ± 10 –110 –324 ± 4 (3) –243Finland FI4 –108 ± 10 –148 –320 –230Norway NO13 –105 ± 12 –139 –287 –203Norway NO17 –104 ± 12 –118 –316 –237Norway NO16 –103 ± 12 –126 –313 –234Norway NO15 –101 ± 12 –108 –288 –208Norway NO14 –100 ± 12 –96 –298 –220Norway NO4 –99 ± 13 –142 –294 –216Norway NO8 –98 ± 12 –99 –325 ± 8 (3) –251Norway NO9 –98 ± 12 –91 –309 –234Norway NO10 –98 ± 12 –135 –287 –210Norway NO12 –98 ± 11 –94 –289 –211Norway NO5 –95 ± 13 –96 –309 –236Norway NO7 –95 ± 12 –80 –311 –239Norway NO11 –95 ± 12 –131 –284 –209Norway NO6 –90 ± 12 –77 –283 –212Norway NO3 –88 ± 13 –153 –301 –234Norway NO2 –82 ± 12 –79 –262 –196Norway NO1 –77 ± 12 –122 –270 –209Canada CA1 –72 ± 3 –132 –285 –230Germany GE1 –61 ± 3 –122 –269 –222Ireland NI1 –59 ± 7 –89 –264 ± 2 (12) –218Ireland NI2 –59 ± 7 –97 –244 ± 2 (15) –197Ireland IR1 –51 ± 11 –44 –233 ± 4 (5) –205China CH1 –45 ± 8 –76 –240 –204Indonesia IN1 –40 ± 10 –94 –244 ± 14 (3) –213Malaysia MA1 –36 ± 5 –97 –220 –191Yemen YE1 8 ± 16 –66 –177 ± 5 (3) –183Yemen YE2 16 ± 17 –29 –141 ± 8 (3) –155

All δ2H values presented are in ‰. δ2H values for methoxyl groups and bulk wood are means of two samples unless otherwise stated.aδ2H values of source water and 95% confidence intervals were calculated using the OIPC, except for IR1, which was taken from the International Atomic Energy Agency–Global Network of Isotopes in Precipitation (IAEA-GNIP) data set.

Fig. 6 Hydrogen isotope ratios (δ2H values) for wood lignin methoxyl groups, bulk wood and meteoric water derived from the Online Isotope Precipitation Calculator (OIPC). (a) Measured δ2H values of wood lignin methoxyl groups and bulk wood relative to δ2H values of meteoric water. Error bars for δ2H values of water from the OIPC reflect the calculated 95% confidence interval. Error bars for methoxyl groups are the average standard deviation of all measurements (±6‰, 1σ). Error bars for δ2H values of bulk wood are within the symbols. Solid lines show linear regression. For reference, the 1 : 1 δ2H values (dashed line) of meteoric water are displayed. (b) Measured δ2H values of lignin methoxyl groups relative to δ2H values of meteoric water. The solid line represents constant fractionation, εmethoxyl/water (εm/w), between the OIPC δ2H values of meteoric water (dashed line) and methoxyl groups.

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methylene-FH4 reductase to N5-methyl-FH4. From here,the methyl group is transferred to homocysteine to formmethionine and, after activation of this amino acid to SAM,to the phenolic OH groups of the phenylpropanoids (Boerjanet al., 2003)

The δ2H values of ∼–107‰ (Rinne et al., 2005), and–52‰ (Betson et al., 2006) that have been reported for celluloseextracted from whole wood grown in northern Finland (Pinussylvestris) and Northern Ireland (Quercus robur), respectively,directly reflect the δ2H values of the carbohydrate pool ofthese species at these locations. As the C-3 position of serineis generally slightly more depleted (20–50‰) than that ofcarbohydrates of C3 plants (Zhang et al., 2002; Augusti et al.,2006), we assume δ2H values of around –127 and –72‰for this position at these locations. N5,N10-methylene-FH4reductase is a flavoprotein, and Schmidt et al. (2003) havedemonstrated that hydrogen transfer by certain flavoproteinsis accompanied by large isotope discriminations, explainingthe alternating 2H abundance in the even and odd positions,respectively, of fatty acids and the extreme 2H depletion atdefined positions of isoprenoids from the deoxyxylulosephosphate pathway. Depletions down to –580‰ in the firstcase (Billault et al., 2001) and down to –780‰ in the secondcase (Martin et al., 2004) have been reported. If a depletionbetween these values (–680‰) is assumed for the third Hatom of the O-CH3 groups of the phenylpropanoids, δ2Hvalues of –311 and –275‰ are thus expected for methoxylgroups of wood from northern Finland and Northern Ireland,respectively. These values are in good agreement with meandata reported here for CH3I derived from lignin of wholewood from northern Finland (–317‰) and Northern Ireland(–254‰).

Conclusions

In summary, the methoxyl group of lignin exhibits all thefeatures required of the ideal target moiety for stable hydrogenisotope studies listed in the Introduction. The methodemployed in measurement of the δ2H values of lignin methoxylgroups, which requires only a small quantity of sample (10–20 mg of wood) and minimal and straightforward samplepreparation, clearly has major benefits. However, the greatestadvantage for isotope studies is derived from the fact thatlignin methoxyl groups contain nonexchangeable hydrogenatoms which can be quantitatively converted to CH3I,avoiding isotopic fractionation. Thus, lignin methoxyl groupsof wood may be considered a plant component that trulyrecords δ2H values of meteoric water, with a mean isotopicfractionation that is uniform (–216 ± 19‰) over a range ofδ2H values for meteoric water from +20 to –110‰. However,our work should be regarded as a pilot study providing thefirst δ2H values for lignin methoxyl groups for a limitednumber of wood samples from around the world. Many moremeasurements at a higher geographical resolution, both

regionally and globally, are essential to provide us with aclearer understanding of the exact relationship betweenthe isotopic compositions of meteoric water and ligninmethoxyl groups. Direct measurements of the δ2H values ofenvironmental water available to the plants and comparisonwith the δ2H values of their lignin methoxyl groups shouldfurther help to improve our knowledge. We envisage the δ2Hvalues of lignin methoxyl groups becoming a powerful tool foruse not only in the constraint of the geographical origin oflignified material but also in paleoclimatic, origin authenticityand forensic studies.

Acknowledgements

We are grateful to Simone Keppler for her support in collectingwood samples from Scandinavia. We thank Peter and SusanChristie, Peter Silk and H. F. Schöler for providing woodsamples from Yemen; Fredericton, Canada and Hau, Germany,respectively. We also thank Gabriel Bowen for providing theglobal map used in Fig. 4. The European Commission isacknowledged for a Marie Curie Research Training Grant(MCFI-2002-00022) awarded to FK.

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