Acta Silvae et Ligni 117 (2018), 47-54 Izvirni znanstveni članek / Original scientific paper EXAMINATION OF STABLE CARBON AND OXYGEN ISOTOPIC VARIABILITY IN THE CELLULOSE AND WHOLEWOOD OF Castanea sativa Mill., Fagus sylvatica L., Pinus sylvestris L. AND Quercus robur L. ANALIZA VARIABILNOSTI STABILNIH IZOTOPOV OGLJIKA IN KISIKA V CELULOZI IN LESU BRANIK PRAVEGA KOSTANJA (Castanea sativa Mill.), BUKVE (Fagus sylvatica L.), RDEČEGA BORA (Pinus sylvestris L.) IN DOBA (Quercus robur L.) B. SLADDEN1, N.J. LOADER2, G.H.F. YOUNG3, D. McCARROLL4 (1) Department of Geography, Swansea University, Swansea SA2 8PP, United Kingdom. (2) Department of Geography, Swansea University, Swansea SA2 8PP, United Kingdom. Corresponding author, n.j.loader@swansea.ac.uk (3) Department of Geography, Swansea University, Swansea SA2 8PP, United Kingdom. (4) Department of Geography, Swansea University, Swansea SA2 8PP, United Kingdom. ABSTRACT This paper presents results from a comparison of wholewood and cellulose carbon and oxygen isotope ratios for four UK tree species. These species occur within the historic buildings archive as both primary and supplementary construction materials and have been used to reconstruct the climate of the past. New advances in the application of stable isotopes have widened the scope of the isotope approach, but require the time-consuming purification of cellulose. Comparison of the oxygen and carbon isotope signals preserved in the wood and cellulose components confirms and builds upon previous research in this field and provides additional insight into the covariance of these two sample types between species, and the potential to employ wood isotope analysis for both pre-screening trees for palaeoclimatology and chronology research. Key words: stable isotopes, tree ring, dendrochronology, dendroclimatology IZVLEČEK V prispevku so predstavljeni rezultati primerjave analize stabilnih izotopov ogljika in kisika v lesu in celulozi drevesnih branik štirih najpogostejših drevesnih vrst v Veliki Britaniji: pravega kostanja, bukve, doba in rdečega bora. Les teh drevesnih vrst pogosto najdemo tudi v različnih lesenih konstrukcijah, kar nam, v kombinaciji z lesom iz živih dreves, omogoča rekonstrukcijo klime v čas pred instrumentalnimi meritvami. Razvoj detekcijskih tehnik za stabilne izotope je omogočil uporabo stabilnih izotopov tudi tam, kjer to tradicionalno ni bilo možno (npr. za datiranje starih stavb), še vedno pa je glavna ovira za širšo uporabo teh metod zahtevna in zamudna ekstrakcija celuloze iz posameznih lesnih branik. Primerjava stabilnih izotopov kisika in ogljika v lesu in (alfa)-celulozi je potrdila dosedanje vedenje in dodala nova spoznanja o primerljivosti razmerij stabilnih izotopov, analiziranih v lesu in celulozi, kar nam bi lahko omogočilo izločitev zamudne tehnike ekstrakcije celuloze in neposredno uporabo homogeniziranega lesa branik za analizo stabilnih izotopov ogljika in kisika v branikah za potrebe paleoklimatologije in historične dendrokronologije. Ključne besede: stabilni izotopi, drevesne branike, dendrokronologija, dendroklimatologija, Velika Britanija GDK 811.4(045)=111 DOI 10.20315/ASetL.117.4 1 INTRODUCTION 1 UVOD Choosing the most appropriate wood component for isotopic analysis in tree-ring research is not a trivial matter. Factors such as research objective, cost, preparation time, sample size, intra- and inter-tree variability and the stability of the signal through time (i.e. the propensity for diagenesis to affect inter-annual variability) all play a role in the sampling protocols adopted and influence experimental design (Borella et al., 1999; McCarroll & Loader 2004; Loader et al., 2003). Prispelo / Received: 12. 11. 2018 Sprejeto / Accepted: 28. 12. 2018 Recent developments in the use of stable isotopes for dating historic building timbers in Europe (Loader et al. in review) offer the potential for dating samples previously considered undateable by dendrochronology alone (Caitling 2018; Loader et al. in review). At present this work is conducted using cellulose extracted from the wholewood of each tree ring. However, cellulose extraction can be time consuming and requires specialist equipment (McCarroll et al. 2004). If the isotopic signal contained within wholewood can be demonstrated to be similar enough to that of cellulose to 47 Sladden B., Loader N.J., Young G.H.F., McCarroll D: Examination of stable carbon and oxygen isotopic variability in . preserve a sufficiently useful signal, then this may provide a means for rapid testing or pre-screening of cores for isotope dating or dendroclimatology prior to more labour intensive cellulose-based analyses (Loader et al. 2008). Several early studies utilized unprocessed whole-wood (e.g. Farmer et al., 1974); however, Wilson and Grinsted (1977) demonstrated that different wood components differed significantly in their isotopic value within a single ring. This observation, coupled with the clearer mechanistic understanding of the timing and controls of cellulose formation in tree rings, resulted in a move away from wholewood analysis for palaeoclimate and plant physiological analyses. However, over the past few decades the benefits of using cellulose over wholewood have been periodically revisited, as the task of cellulose extraction is somewhat time consuming and labour intensive (Alessandro et al., 2004; Borella et al., 1999; McCarroll & Loader 2004; Loader et al., 2003). Even advances in batch processing, and more recently, the cellulose extraction of intact cores/laths is far from straightforward and may only be appropriate for specific tree types (Loader et al., 1997; 2002; Kagawa et al., 2015; Helle et al., 2004). Studies addressing the issues of wholewood and cellulose have generally found a strong correlation between wholewood and cellulose in both angiosperm and gymnosperm trees (Borella et al., 1998, 1999; Barbour et al., 2001; Loader et al., 2003, 2017; Alessandro et al., 2004; McCarroll et al., 2004; Cullen et al., 2006; Sidorova et al., 2008; Szymczak et al., 2011; Schleser et al., 2015). However, some studies have highlighted isotopic variability on both short and long time scales, which may represent cellulose recording some aspects of climate which wholewood analysis obscures. This could have implications for the veracity of any resulting climate reconstructions, based upon the isotopic analysis of wholewood rather than cellulose (Barbo-ur et al. 2001, Borella et al., 1999; Cullen et al., 2005; Szymczaka et al., 2011; Schleser et al., 2015). These results have been questioned by papers suggesting that the extraction of cellulose is an unnecessary step, and also by those suggesting that wholewood may hold additional, or different, environmental information than that attainable from cellulose alone (Barbour et al. 2001; Borella et al., 1998; Loader et al., 2003; Alessandro et al., 2004; McCarroll and Loader 2004). There is little doubt that, for successful dendro-climatology, it is essential that the sample signal is stable through time and unambiguously linked to the tree ring under analysis (McCarroll and Loader 2004). This is most unambiguously achieved, by analysing a single component (typically a-cellulose) from dated tree rings. However, more recent developments, which capitalise upon the inter-annual coherence in stable oxygen isotopes to date tree ring sequences, provide new scope for re-evaluation of wholewood/cellu-lose variability with this specific application in mind (Loader et al. In review). This pilot study, which builds upon an existing body of similar studies, examines four European tree species found in the UK dendrochrono-logical (historic building) arena; English oak (Quercus robur L.), European beech (Fagus sylvatica L.), Sweet Chestnut (Castanea sativa Mill.), and Scots pine (Pinus sylvestris L.). The study will investigate the extent to which observed isotopic differences between cellulose and wholewood are present and consistent in the tree species analysed. If both the cellulose and wholewood oxygen holds the same, or a very similar signal, as have been reported elsewhere for carbon isotopes (Borella et al. 1998, 1999; Loader et al. 2008), then this may widen the scope of the isotopic dating technique and provide a means for pre-screening samples, prior to more resource-intensive cellulose preparation. 2 MATERIALS AND METHODS 2 MATERIALI IN METODE Wood samples were collected from living trees growing in Broadmoor woods, southern England, UK (51.38 °N, -0.74 °E) (Figure 1). This woodland contains a mosaic of broadleaved stands and conifer plantations, allowing for the collection of the Castanea sativa (sweet chestnut), Fagus sylvatica (European beech), Pinus sylvestris (Scots pine), and Quercus robur (English oak) used in this study. All trees were located on sandy, moderately well-draining soils overlaying free-draining Eocene gravels of the Camberley Formation (Bracklesham group). Sampled trees were located towards the base of shallow slopes. 12 mm diameter cores were obtained using an increment borer at a height of c. 1.1 m. One core was taken per species, samples were air-dried and sanded to enable clear ring identification. Each sample was measured to an accuracy of 0.01 mm using a horizontally-travelling Velmex™ measuring stage, under binocular magnification. The tree ring width data were statistically cross-dated (synchronised) using the Time Series Analysis and Presentation (TSAP™) software. Ring separation for isotopic analysis was conducted using scalpel and microscope. Earlywood and latewood were separated in oak and chestnut, due to their ring-porous nature, and therefore the potential for signal carry-over from the previous year in the earlywood (Switsur et al., 1995). The separation of earlywood and latewood was 48 Acta Silvae et Ligni 117 (2018), 47-54 Fig. 1: Map of the southern UK showing the location of the Broadmoor woods (red circle) from where samples were collected for this study. deemed unnecessary in pine due to the coherence of their isotopic ratios (Kress et al., 2009). Due to the diffuse porous wood anatomy of beech, separation was also considered unnecessary (Schleser et al., 2015). Sample material was limited and so it was not possible to cut and mill the wholewood and to have sufficient material remaining for cellulose analysis. To overcome this limitation a small number of wholewood slivers were removed from the cut sample for wholewood isotope analysis. Wood samples 0.30-0.35mg were then sub-sampled from these wood slivers and weighed into silver capsules. Significant intra-ring variability has been previously reported and we recognise that this sub-sampling strategy, forced by material availability is sub-optimal. A conscious effort was therefore made, to include parts of different shavings for each year to obtain a value more broadly representing the isotopic ratio of the whole ring. In addition, ten sub-samples were taken from a single year from both the wholewood and cellulose samples to demonstrate the intra-annual variability of the wholewood and allow for the error of this sub-sampling strategy to be assessed. Cellulose samples were prepared using standard batch methods (after Loader et al., 1997), followed by homogenisation using an ultrasonic probe and freeze drying. Cellulose samples were weighed using an identical protocol to the wood sub-samples, including replication to demonstrate isotopic variability. Both Slika 1: Karta južnega dela Velike Britanije z vrisano lokacijo mesta vzorčenja - Broadmoor woods (rdeči krog) cellulose and wholewood samples were converted to carbon monoxide (CO) by high temperature pyrolysis over glassy carbon at 1400 °C using a Flash HT elemental analyser and the stable carbon and oxygen isotopic ratios measured simultaneously using a Thermo Delta V isotope ratio mass spectrometer. Results are expressed using the delta notation as per mille deviations from the VPDB and VSMOW standards (Coplen, 1995). Analytical precision for repeat analysis of standard (Sigma) cellulose for S13C and S18O is typically ±0.1 %%, and ±0.3 % (anJ (Sigma Aldrich, UK. No. C-8002 Lot. 92F-0243) (Woodley et al., 2012, Loader et al. 2015). This "standard" analytical uncertainty should be considered when comparing standard deviations in carbon and oxygen isotopes, in the context of the results and may play a role in the lower than expected r values for the S18O series of the less-homogenous wood chips (see Tables 1 & 2). Although not a climate study, carbon isotopes were corrected (after McCarroll and Loader 2004) for changes in the carbon isotopic value of the CO2 used by the plant for photosynthesis, as this has been progressively modified through time since the onset of global industrialisation (the atmospheric S13C "Suess" effect). Employing this adjustment reduces the risk of artificial inflation of correlation arising from the industrial S13C decline in atmospheric CO2 post-industrialisation which dominates the stable carbon isotope signal. 49 Sladden B., Loader N.J., Young G.H.F., McCarroll D: Examination of stable carbon and oxygen isotopic variability in . 3 RESULTS AND DISCUSSION 3 REZULTATI IN RAZPRAVA 3.1 Intra-annual isotopic variability 3.1 Intraanualna (znotrajletna) variabilnost stabilnih izotopov A possible consequence of the wholewood sampling strategy which measured only a small part of the tree-ring rather than an homogenous mix from the entire ring, is that the resulting isotopic value would likely exhibit greater variability, relative to the cellulose samples. This in turn could influence the degree of coherence between the two sampled media. Table 1 displays intra-annual variability for a single year of each species. Values differ between species and without additional trees; it is not possible to determine whether intra-annual isotopic variability differs systematically between tree species, or whether these results are indicative of specific tree or ring properties. Results do however demonstrate that, as anticipated, the unhomogenized (wood chip) samples show significantly higher variability in all cases. This is most clearly demonstrated in chestnut, with S18O having a standard deviation of 1.14 %o in wholewood compared to 0.35 % in S18O cellulose (Table 1)(Borella et al. 1998). Furthermore, all samples displayed at least double the variability in wholewood compared to cellulose in S18O, indicating that the lack of homogenization has a clear Table 1: Results obtained from the repeat isotopic of manually-sampled wood chips and alpha-cellulose prepared from a single tree-ring and run to explore intra-annual isotopic variability in S18O & S13C wholewood and cellulose analysis (Standard deviation (an-1) , n=10 per species and sample type are presented). effect on accuracy. This relationship is similar for S13C, however, to a significantly lesser extent, with the difference between wholewood and cellulose variability in chestnut only 0.12 %o compared to 0.78 %o in S18O. The intra-annual variability found in the S18O of this study are comparable to those found in other works such as; 0.8-3 % (Roden et al., 2009) and 0.5-1.8 % (Helle et al., 2004). However, S13C intra-annual variability of 0.23-0.36 % is markedly lower than the results reported by Roden et al. (2009), which showed an increased variability of 2 % compared to the S18O of the same tree. 3.2 Wholewood and cellulose analyses 3.2 Analiza lesa in celuloze For all four species studied, a significant Pearson's r correlation of p < 0.01 was demonstrated between cellulose and wholewood for both S13C and S18O, with average offsets varying considerably between isotopes and species. Table 2 displays average offset between cellulose and wholewood, as well as the Pearson's rvalues and the significance of correlation between cellulose and wholewood. We observed that for S18O wholewood is isotopically depleted (less of the heavier isotope) by 5.46-4.33 % in comparison to cellulose, these results are similar to those established by the literature, with offsets of 4.3 % (Borella et al., 1999), Preglednica 1: Rezultati ponovljenih meritev stabilnih izotopov vzorcev lesa in alfa celuloze iz ene drevesne branike z namenom preučiti intra-anualno variabilnost stabilnih izotopov ogljika 513C in kisika 518O v lesu in celulozi (standardni odklon (an-1), n=10 vzorcev na drevesno vrsto in tip vzorca (les / celuloza)). Species Drevesna vrsta 5«O an, (%„) Wholewood / Les 5«O a„, (%) Cellulose / Celuloza 5"C a„, (%) Wholewood / Les 5"C a„, (% Cellulose / Celuloza Oak / dob 0.67 0.29 0.23 0.12 Chestnut / pravi kostanj 1.14 0.35 0.36 0.14 Pine / rdeči bor 0.51 0.25 0.29 0.14 Beech / bukev 0.72 0.26 0.28 0.10 Table 2: Pearson's correlation coefficients (r) between who- Preglednica 2: Pearsonovi korelacijski koeficienti med raz-lewood and cellulose and average offset between the two merjem stabilnih izotopov ogljika in kisika v lesu in celulozi (wholewood and cellulose) constituents for each tree species. glede na drevesno vrsto Species Drevesna vrsta 518O (r) Average Offset (%) Povprečni odklon (%) 5"C (r) Average Offset (%) Povprečni odklon (%) Oak / dob 0.60** 4.50 0.81** -0.62 Chestnut / pravi kostanj 0.42** 5.46 0.89** -0.46 Pine / rdeči bor 0.51** 4.54 0.82** +0.30 Beech / bukev 0.74** 4.33 0.90** -0.34 ** Significance / Značilnost 99%, *Significance / Značilnost 95% 50 3.6 %o for oak, 3.9 %o pine (Barbour et al., 2001) and 4.8 ± 0.72 %o (Szymczak et al., 2011). Although slightly higher levels of isotopic depletion were observed in this study, they are in general agreement with the literature. Average offsets are markedly lower for S13C; -0.62 to -0.34 %o, when compared with results from the literature and our S18O results, with the offset for pine at +0.3 relative to (non-resin extracted) wholewood. Previous work has demonstrated S13C offsets with S13C wholewood depletion of 1.17 ± 0.4 % (Cullen et al., 2006), 1.20 ± 0.50 % (Szymczak et al., 2011), 1.50 ± 0.36 %o (Schleser et al., 2015) and c. 1.2 %o (Loader et al., 2003). These studies examined several species of pine and oak, as well as the tropical wood Cariniana micrantha and produced considerably higher offsets than those obtained in this study. The isotopic enrichment (more of the heavier isotope) between wood and cellulose in pine could be attributed to the lack of resin extraction for the Pinus samples or/and high intra-an-nual isotopic variability in the wholewood of the rings subsampled in this study. Pine would be especially affected due to its resinous nature, and this may be the reason for the carbon isotopic enrichment of cellulose in its result (Rinne et al., 2005). Across all species and isotopes, significant correlations (p < 0.01) were found between wholewood and Acta Silvae et Ligni 117 (2018), 47-54 cellulose, with especially high r values of between r 0.81-0.90 found for S13C. The results of this study are generally comparable with the existing literature, with S13C values: r = 0.98, p < 0.001 (Loader et al., 2003); r = 0.64, p < 0.001 (Cullen et al., 2006); r = 0.71, p < 0.01 (Szymczak et al., 2011); and r = 0.96, p < 0.01 (Schleser et al., 2015). These findings were split over several species including oak, Cariniana micrantha and pine, agreeing with our data in showing that the correlation between cellulose and wholewood is not restrained by species. Furthermore, the significance of correlations found in this studies S13C series and their comparability to the literature suggest that even using a simple wholewood preparation techniques, cogent results can be produced. Pearson's correlation (r) values for S18O calculated in this study were found to be between r = 0.42 and 0.74 (p < 0.01), with all species showing significant correlations between cellulose and whole-wood. Our results are comparable with those found in the existing literature: (Cullen et al., 2006) r = 0.68p < 0.001; (Szymczak et al., 2011) r = 0.77 p < 0.01; (Barbour et al., 2001) r = 0.79 p < 0.001. These studies were conducted on oak and chestnut series, but as earlier suggested, the wholewood and cellulose relationship does not seem to be significantly impacted by differences in species. The isotopic timeseries for S18O and S13C are displayed in Figures 2 and 3, which demon- A '20.00 -20.60 -21.00 „ -2150 £ -22.00 m r, -22.50 >. -23.00 P -23.60 io -24.00 -24.50 -25.00 1950 J 0 1 0.01) were observed between wholewood and cellulose for each of the four European tree species examined. We demonstrated that even using unho-mogenised wholewood, significant correlation can be found with cellulose from the same tree rings and that this relationship was preserved across species. This relationship was stronger with S13C than S18O. Expected wholewood isotopic depletion (in 18O and 13C), relative Slika 3: Časovne vrste stabilnega izotopa kisika (518O VSMOW) za obdobje 1950 - 2015 (celuloza - črna črta, črni krogci, les -siva črta, beli krogci), pravi kostanj (A), dob (B), bukev (C) in rdeči bor (D) to cellulose, was observed in the oxygen and carbon isotopic series. Our findings therefore support those of a number of similar studies. S13C wholewood does not appear to carry similar enough signal to cellulose to be suitable for correlative dating (Loader et al., in review). For oxygen however, correlation between cellulose and wholewood, using our rudimentary approach for sampling wholewood, were lower than for carbon, suggesting that S18O in our sampled wholewood did not reflect the signal preserved in cellulose as faithfully, or that a more homogeneous wholewood sample is required. The results gathered from this study suggest that for specific applications, S13C measured on wholewood may be sufficient for screening non-resinous species, providing a time efficient and cost effective alternative to cellulose isolation. However for S18O, additional comparative studies focussing upon the preparation of homogenous wholewood samples are likely to be required. 4 ZAKLJUČKI V študiji smo primerjali meritve razmerja stabilnih izotopov ogljika in kisika, merjenih v lesu branike in celulozi, ekstrahirani iz istih branik. Analiza je bila opravljena na lesu štirih drevesnih vrst - pravemu kostanju (Castanea sativa Mill.), bukvi (Fagus sylvatica L.), rdečem boru (Pinus sylvestris L.) in dobu (Quercus 52 Acta Silvae et Ligni 117 (2018), 47-54 robur L.). Korelacijska analiza razmerja stabilnih izotopov v lesu in celulozi iz iste branike je bila statistično značilna za vse štiri drevesne vrste. Uporaba nehomog-eniziranih vzorcev lesa, ločeno po drevesnih vrstah, je pokazala, da so bili rezultati do neke mere primerljivi z rezultati meritev stabilnih izotopov v celulozi. Povezava med razmerjem stabilnih izotopov v lesu in celulozi je bila močnejša pri stabilnem izotopu ogljika (S13C) kot pri kisiku (S18O). Pričakovano osiromašenje (ang. depletion) razmerja stabilnih izotopov ogljika in kisika v lesu v primerjavi s celulozo je bilo opaženo v vseh izotopskih kronologijah ogljika in kisika. Rezultati te študije zato podpirajo rezultate drugih podobnih študij. Zdi se, da razmerje stabilnega izotopa ogljika v lesu ni dovolj podobno signalu v celulozi, da bi bil lahko primeren za primerjalno datiranje (Loader et al., v recenziji). Za stabilni izotop kisika je bila korelacija med razmerjem stabilnega izotopa kisika v celulozi in ne-homogeniziranem lesu nižja kot pri stabilnem izotopu ogljika. To pomeni, da stabilni izotop kisika v lesu nima enakega signala kot celuloza, zato (1) ni možno preskočiti faze ekstrakcije celuloze, ali pa (2) da je treba les za analizo stabilnega izotopa kisika homog-enizirati. Rezultati te pilotne študije nakazujejo, da bi lahko v raziskavah, kjer nas zanimajo meritve stabilnega izotopa ogljika, potencialno uporabili tudi nehomoge-niziran les branike, še posebej če gre za drevesne vrste, ki nimajo smole (npr. listavci). Tako bi lahko preskočili zamudno in zahtevno ekstrakcijo celuloze iz lesnih branik. 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