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Aerial decay influence on the stable oxygen and carbon isotope ratios in tree ring cellulose
Accepted Manuscript Title: Aerial decay influence on the stable oxygen and carbon isotope ratios in tree ring cellulose Authors: Viorica Nagavciuc, Zolt´an Kern, Aurel Pers¸oiu, Kesj´ar D´ora, Ionel Popa PII: DOI: Reference:
S1125-7865(17)30157-1 https://doi.org/10.1016/j.dendro.2018.03.007 DENDRO 25508
To appear in: Received date: Revised date: Accepted date:
12-10-2017 17-3-2018 20-3-2018
Please cite this article as: Nagavciuc V, Kern Z, Perx219;oiu A, Doacute;ra K, Popa I, Aerial decay influence on the stable oxygen and carbon isotope ratios in tree ring cellulose, Dendrochronologia (2010), https://doi.org/10.1016/j.dendro.2018.03.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Aerial decay influence on the stable oxygen and carbon isotope ratios in tree ring cellulose
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Viorica Nagavciuc 1,2,3,4 Zoltán Kern 2 Aurel Perșoiu 3,5,6 Kesjár Dóra 2 Ionel Popa 7 Faculty of Forestry, Ștefan cel Mare University, Suceava, Romania Institute for Geological and Geochemical Research, Research Centre for Astronomy and Earth Sciences, Hungarian Academy of Sciences, Budapest, Hungary 3 Stable Isotope Laboratory, Ștefan cel Mare University, Suceava, Romania 4 Departement of Geography, Johannes Gutenberg University, Mainz, Germany 5 Emil Racoviță Institute of Speleology, Romanian Academy, Cluj Napoca, Romania 6 Institute of Biology, Romanian Academy, Bucharest, Romania 7 Marin Dracea National Research and Development Institute for Silviculture, Câmpulung Moldovenesc, Romania 1
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Corresponding author Nagavciuc Viorica [email protected] Address: Universității 13, Suceava 720229, Romania
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Abstract Sub-fossil wood is often affected by the decaying process that introduces uncertainties in the measurement of oxygen and carbon stable isotope composition in cellulose. Although the cellulose stable isotopes are widely used as climatic proxies, our understanding of processes controlling their behavior is very limited. We present here a comparative study of stable oxygen and carbon isotope ratios in tree ring cellulose in decayed and non-decayed wood samples of Swiss stone pine (Pinus cembra) trees. The intra-ring stable isotope variability (around the circumference of a single ring) was between 0.1 and 0.5 ‰ for δ18O values and between 0.5 and 1.6 ‰ for δ13C values for both decayed and non-decayed wood. Observed intra-tree δ18O variability is less than that reported in the literature (0.5-1.5 ‰), however, for δ13C it is larger than the reported values (0.7-1.2 ‰). The inter-tree variability for non-decayed wood ranges between 1.1 and 2.3 ‰ for δ18O values, and between 2 and 4.7 ‰ for δ13C values. The inter-tree differences for δ18O values are similar to those reported in the literature (1-2 ‰ for oxygen and 1-3 ‰ for carbon) but are larger for δ13C values. We have found that the differences for δ18O and δ13C values between decayed and non-decayed wood are smaller than the variation among different trees from the same site, suggesting that the decayed wood can be used for isotopic paleoclimate research. Keywords oxygen isotope ratios; carbon isotope ratios; tree rings; decayed wood
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Introduction The stable oxygen and carbon isotope composition (hereafter δ18O and δ13C values) of tree-ring cellulose are becoming one of the most powerful proxies for reconstructing past climate variability (Gagen et al., 2011). This results from the combination of the annual chronological control with the precisely understood environmental physiology controlling the link between climate and tree-ring δ18O and δ13C values (McCarroll and Loader, 2004). While in some areas living trees allow for millennial-scaleclimate reconstructions, most regions lack such conditions and fossil and subfossil trees are increasingly analyzed. However, these specimens are frequently (partly) decayed, thus possibly adding an extra layer of analytical uncertainty. Tree-ring width (TRW) measurements are not influenced by the state of wood (i.e., decay), as the mechanical solidity of the wood remains unchanged during decay (Schweingruber, 2007), while decayed wood is not suitable for density and blue reflectance measurements because cell walls are (partly) decomposed (Schweingruber, 1988). For stable isotope analyses uncertainties can be traced to technical errors of mass spectrometry, biological faults, and samples quality and preparation (Werner and Brand, 2001, Leavitt, 2010; Leavitt and Wright, 2002; Porté and Loustau, 2001; Robertson et al., 2011; Treydte et al., 2006). Although widespread, studies on the variability of oxygen and carbon isotopes in tree-ring cellulose imposed by wood decay are extremely rare, even though it is well documented that decayed and non-decayed wood were mixed quite frequently (Becker et al., 1991; Boettger et al., 2003; Boettger et al., 2003; Hunkeler et al., 2001; Mancini et al., 2003; Spiker and Hatcher, 1987). The reliability of δ18O and δ13C values of waterlogged subfossil black spruce (Picea mariana (Mill.)) stems, showing signs of bacterial decay due to long-time exposure to lake-bottom conditions was evaluated by Savard et al. (2012). These authors have found that even if some of the tree ring cellulose is lost during decay, the oxygen and carbon isotope composition of the remaining material largely preserves the original values. Deadwood decay is largely dependent on wood properties, enzyme activities, site-specific microbial and physical diversity of the ambient environment (Kahl et al., 2017; Klaassen and van Overeem, 2012), so different biochemical processes can be expected to dominate for aerobic and anaerobic degradation (Klaassen and van Overeem, 2012). Most of deadwood material analyzed in the previous studies was degraded by aqueous bacterial activity (Gelbrich et al., 2012, 2008; Greaves, 1971; Kłusek and Pawełczyk, 2014; Poole and Van Bergen, 2002; Savard et al., 2012). The biodegradation of lignin-cellulosic material belongs mostly to eubacteria and fungi for three major polymers (cellulose, hemicellulose and lignin) (Kačík et al., 2009; Mester et al., 2004; Pérez et al., 2002; Reese, 1957). Loader et al., (2003) analyzed the differences of stable carbon isotope ratios in the whole wood, cellulose and lignin, whereas Savard et al. (2012) examined the stability of carbon and oxygen stable isotopes in sub-fossil wood from anaerobic conditions. However, studies on the potential effect of the decay process on the stable isotope composition of cellulose under subaerial conditions are still lacking.
The aim of this study is to assess the suitability of decayed wood for stable isotope-based paleoclimatic reconstructions, by studying and comparing the oxygen and carbon isotope composition of cellulose from aerially decayed and non-decayed wood in Swiss stone pine (Pinus cembra) from the Eastern Carpathian Mountains, Romania.
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Samples description and analysis The study area is located in the Călimani Mountains, Eastern Carpathians, Romania (Fig. 1) which is part of Călimani National Park, established in 1975. The samples were collected during a field trip in summer 2008 from altitudes varying between 1450 and 1850 m a.s.l. where a representative Swiss stone pine population exist (Popa and Kern, 2009). All dead wood samples were collected from fallen trees with different degrees of decay from high altitude natural mixed stands of spruce and stone pine. The substrate is composed of crystalline schist with skeletal lithic acid brown soil, with medium humidity. No sign of fires or buried wood was observed in the sampling area. From each dead tree one transversal disk was sampled using a chainsaw. All individual samples were measured and dated using millennium long reference chronology of stone pine from Călimani Mts (Popa and Kern, 2009) at annual resolution. Annual ring widths were measured using LINTAB equipment and TSAP 0.53 software, and cross-dated and checked for missing rings (Grissino-Mayer, 2001, 1997; Holmes, 1983). The detailed cross-dating process is described in Popa and Kern (2009). Three sub-fossil Swiss stone pine trees (labeled A, B, and C), with sections of both decayed and non-decayed wood around the circumference of the same ring (Fig. 2), and four non-decayed samples of the same tree (D, E, F and I) were analyzed for this study (Table 1). The A tree grew between AD 1622-1759, with the decayed and non-decayed section of wood dated to AD 1740-1759; the B tree grew between AD 1795-1894, with the decayed and nondecayed section dated to AD 1886-1889, and tree C grew between AD 1007-1705, with the decayed and non-decayed sections located in rings dated to AD 1135-1141 and AD 1143-1145. In tree A, the rings dated between AD 1747 and 1759 were in an advanced stage of degradation, while rings dated between AD 1740 and 1746 had sectors of both decayed and non-decayed wood (Fig. 2). In tree B, the rings dated between AD 1886 and AD 1889 had sectors of both decayed and non-decayed wood. The non-decayed Swiss stone pine samples used for reference grew between AD 1876-2012 (D, living tree), AD 1770-2015 (E, living tree), AD 1645-1869 (F, sub-fossil tree) and AD 1522-1863 (I, sub-fossil tree). Wood-destroying fungi that cause cell wall degradation can be classified depending on the type of decay process in three main groups: white-, brown-, and soft-rot (Blanchette et al., 1990; Karami et al., 2013). Our decayed samples were affected by brown-rot fungi, which are caused exclusively by basidiomycetes (Schwarze et al., 1997). During the destruction of wood by brown-rot fungi, the cellulose and hemicellulose are broken down with little or no overt breakdown of lignin (Blanchette et al., 1990; Eriksson et al., 1990; Mester et al., 2004; Schwarze et al., 1997). The breakdown of cellulose due to brown-rot fungi involves the enzymic and nonenzymic systems that cause oxidative reactions in the cellulose molecule, thereby enhancing the
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activity of enzymes, which split the molecule at random points along its length (Schwarze et al., 1997). As a result, the cellulose microfibrils are cut into short lengths causing the dramatic loss of tensile strength in a very short period of exposure to the brown-rot process (Schwarze et al., 1997). In advanced stages of decay, even if the wood cells appear to maintain their usual form, cellulose is depleted much more than lignin, which causes shatter and collapses of cells (Blanchette et al., 1990). Decayed wood in our samples was identified by visual inspection (Fig. 2), having a slightly altered texture (Savard et al., 2012), and black-brown coloring that is characterized for the brown-rot attack (Björdal et al., 1999). For stable isotope analyses, after separating each ring with a scalpel, we prepared the α-cellulose using the modified Jayme-Wise method (Boettger et al., 2007; Loader et al., 1997), homogenized it using the standard ultrasonic protocol (Laumer et al., 2009), and dried it at 70 °C for 24 hours. Further, 0.2 mg (±10 %) of α-cellulose was weighted into silver capsules for pyrolysis over glassy carbon at 1450 °C and simultaneous measurements of oxygen and carbon isotope ratios (δ18O and δ13C) using a ThermoQuest TCEA elemental analyzer interfaced with a Thermo Delta V Advantage IRMS (Loader et al., 2016, 2015; Saurer et al., 1998) at the Institute for Geological and Geochemical Research, Research Centre for Astronomy and Earth Sciences, Hungarian Academy of Sciences, Budapest. The samples were measured in triplicates. The results are reported in per mil (‰) relative to the Vienna Standard Mean Ocean Water (VSMOW) for oxygen, and Vienna Pee Dee Belemnite (VPDB) for carbon (Coplen, 1994), respectively, using the traditional δ notation:
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δ18O (δ13C) = (Rsample / Rstandard -1) × 1000,
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where Rsample and Rstandard are either 18O/16O (or 13C/12C) ratios of the sample and the standard, respectively. The analytical precision of the measurements was better than 0.2 ‰ for both oxygen and carbon. We have measured δ18O and δ13C values in samples from rings with both decayed and nondecayed wood, corresponding to three-time intervals (Table 1): AD 1740-1759 (samples A, F, I), AD 1886-1889 (samples B, D, E), 1135-1145 (sample C). To further test the alteration of the cellulose during the decay process, the rings with the largest difference of δ13C between the decayed and non-decayed samples along the circumference of the same ring (years AD 1744 and 1745 in sample A) were analyzed using Fourier Transform Infrared Spectroscopy (FTIR). Attenuated total reflectance (ATR) is an useful initial step to characterize organic matter with minimal sample preparation (Stuart, 2004). ATR is based on the phenomenon of total internal reflection (Bruno, 1999) and measures the changes occurring in an internally reflected infrared beam, which come in contact with the sample through a diamond crystal. When the sample is placed in contact with the ATR crystal, the resulting evanescent wave is attenuated in the regions of the IR spectrum where the sample absorbs energy (Stuart, 2004). For each sample, 16 scans were recorded in the 4000 to 400 cm-1 spectral range with a resolution of 4 cm-1. The measurements were carried out using a Bruker Vertex 70 spectrometer
(Bruker Corporation, Billerica, MA, USA) controlled by OPUS 7.2 software (Bruker Corporation), at the Institute for Geological and Geochemical Research of the Hungarian Academy of Sciences.
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Results and discussions The IR ATR spectra show perfect agreement between the cellulose extracted from decayed and non-decayed sections of the rings (Fig. 3). All the identified peaks are characteristic to cellulose (Richard et al., 2014) excluding any significant difference between extraction residuals from decayed and non-decayed wood. The wood from samples C was weighed before extracting the cellulose and also the mass of the resulting cellulose in order to compare the differences of cellulose content in decayed and non-decayed wood. The structure of the xylem cell walls is usually composed of roughly 45 % cellulose (Chen, 2014; Novaes et al., 2010), the percentage that was also measured in the non-decayed samples from tree C, whereas for the decayed samples the wood is composed of ~37% cellulose. The obtained percentage of cellulose in the decayed tree rings match those in the slightly altered sub-fossil wood class described by (Savard et al., 2012), and in the range reported from two Stone Pine stand (31.6-39.7 %) from the Alps by Ziehmer et al., (2017). The differences of cellulose content between decayed and non-decayed wood vary between 0 % (AD 1135) and 12 % (AD 1143), the decayed samples being depleted by 7 %, on average. This is in agreement with the reported increase in the lignin: carbohydrate peak area ratio during brown-rot decay (Pandey and Pitman, 2003). Cellulose percentage in the slightly altered wood definitively indicates the progressive decomposition degree from the wellpreserved wood to highly altered wood (Savard et al., 2012). The results of stable isotope analyses are presented in Table 2 (AD 1740-1759), Table 3 (AD 1886-1889) and Table 4 (AD 1135-1141, 1143-1145). The intra-ring differences in δ18O and δ13C values (Table 5), measured on the decayed and non-decayed wood in samples from all three trees are between 0.1 and 0.4 ‰ for δ18O values and among 1.0 and 1.6 for δ13C values in tree A; between 0.3 and 0.5 ‰ for δ18O values and 0.2 and 0.5 ‰ for δ13C values in tree B; and between 0.5 and 0.3 ‰ for δ18O values and 0.0 and 0.5 ‰ for δ13C values in tree C. The intra-tree differences are only marginally larger than the analytical precision for δ18O values, but somewhat higher for δ13C values. The δ18O values on intra-tree differences are lower than those reported in the literature (e.g., 0.5 – 1.5 ‰), measured among the three different radii (Leavitt, 2010), as well as than two measurements for the same year from the two radii of eight trees (Li et al., 2015). However, the δ13C values are larger than those reported in the literature (0.5 – 1.5 ‰, Leavitt, 2010) for various for measurement on four different radii at three different heights (Leavitt and Long, 1986), or between eight radii of the same tree (Leavitt and Long, 1984) and the values reported in the review article (Leavitt, 2010). The differences in δ13C values (up to 1.5 ‰, Table 5) imply the possible alteration of the original isotopic signal due to wood decay. Schleser et al. (1999) have found a 1 ‰ difference in the carbon isotopic values in affected wood while English et al. (2011) found no effect of blue fungus on δ13C and δ18O values. Previous studies have shown that for samples collected from different sections along the circumference of
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a given ring, δ18O and δ13C values ranged in a narrow 0.5-1.5 ‰ interval (Leavitt, 2010). However, our δ13C differences are not consistent for the two analyzed trees: in tree A and C, the decayed samples are enriched in the heavy isotopes compared to non-decayed samples, while in tree B, they are depleted. Interestingly, the δ18O differences show a similar sign of enrichment in heavy isotopes during wood decomposition, with only five samples (tree A, year AD 1742; tree C, years AD 1143-1145 and tree C, year AD 1141) being slightly depleted (Table 2, 4). Thus, while the differences in δ13C values are large, the opposing sign in different trees suggest that the decay process is not responsible for the shift in the stable isotope composition. Considering the strong coherence of δ18O values in tree rings and the common variability of the δ18O values in xylem water, needle water and phloem organic matter in all the measured trees, Treydte et al. (2014) conclude that individual tree characteristics do not influence oxygen isotope fractionation until assimilate production. The inter-tree differences in stable isotope values for non-decayed wood range from 1.1 to 2.3 ‰ for δ18O values, and from 2 to 4.7 ‰ for δ13C values, for the combined studied intervals (AD 1740-1746 and AD 1886-1889) and for all analyzed samples (D, E, I, F). The differences for δ18O values are between those reported (1 - 2 ‰) by Leavitt and Wright (2002), Wright and Leavitt (2006) and (Treydte et al., 2006), but are larger for δ13C values; Leavitt (2010) and Porté and Loustau (2001) reporting differences in the 1 - 3 ‰ range. The inter-tree differences are larger than the intra-tree ones affected by decay (Fig. 4), thus suggesting that the stable isotope values change during wood-decay processes and are affecting the original isotopic signal to a degree that should not impede the preservation of the climatic signal by the stable isotope composition in tree ring cellulose. The coherence in the inter-tree differences of decayed and non-decayed wood and the similar trends reflect a strong common signal of trees in the area, a finding confirmed also by the validity of the pooling method for isotopic measurement (Borella et al., 1998; Leavitt, 2008; Treydte et al., 2001). Excellent linear relation was found between the δ18O values measured on α-cellulose derived from decayed and non-decayed wood, while the correlation for δ13C values is much poorer. The observed relationship (Fig. 5) between the δ18O (δ13C) values of decayed and non-decayed samples was above (below) the mean inter-tree correlation value reported for pine (Pinus ponderosa) and Coast redwood (Sequoia sempervirens) species studied at five sites (Roden, 2008). In addition, the best-fit slopes are close to unity for δ18O values, whereas lower slopes were obtained for δ13C values. Our results are in agreement with Savard et al. (2012), who found that slightly altered wood preserved similar isotopic ratios with non-decayed wood, while the highly altered wood (containing less than 21 % cellulose), preserved reliable δ13C values but depleted δ18O values, thus suggesting that site-specific analysis are necessary when decayed wood is to be used in paleoclimatic research. The differences noted in the transition zone between different stages of decomposition can be observed visually (Savard et al., 2012), thus eliminating the risk of contamination of samples with highly decayed wood.
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Conclusions The brown-rot decaying process of wood analyzed in this study has a limited influence on the stable oxygen isotope composition in α-cellulose, and a larger one on the stable carbon isotope composition. We therefore suggest that the slightly decayed Pinus cembra wood from our site can be used for isotopic analysis in paleoclimatic research. The differences in δ13C values between decayed and non-decayed wood are lower than those between different trees from the same site, thus suggesting a limited effect of wood decay on the original stable isotope composition of cellulose, that would not impact tentative paleoclimatic reconstructions that are using the cellulose stable isotope composition as a proxy for past climates. However, in the absence of comparative studies between decayed and non-decayed wood, the safest approach would still be to avoid the decayed wood when analyzing the stable isotope composition of tree rings. The intra-tree differences between decayed and non-decayed wood are lower than the inter-site (between different trees) variability, but our limited data-set for circumferential variability renders our conclusion to be preliminary. A general methodological recommendation for future stable isotope studies on subfossil tree-rings is to provide a detailed documentation and presentation of the type of decay observed in any sampled section as it can help to better understand the potential distinct isotope effects accompanying the different decay processes.
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Acknowledgements The research leading to these results has received funding from EEA Financial Mechanism 2009 - 2014 under the project contract no CLIMFOR18SEE. Thanks for the support of “Lendület” program of the Hungarian Academy of Sciences (LP2012-27/2012). This is contribution No.54. of ‘2ka Palæoclimatology’ Research Group. The research was supported partial by CTR 663/2013 and 1993/2014 and PN-III-P4-ID-PCE-2016-0253 projects.
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in: Kück, U. (Ed.), Genetics and Biotechnology. Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 355–368. doi:10.1007/978-3-662-07426-8_17. Novaes, E., Kirst, M., Chiang, V., Winter-Sederoff, H., Sederoff, R., 2010. Lignin and Biomass: A Negative Correlation for Wood Formation and Lignin Content in Trees. Plant Physiol. 154, 555–561. doi:10.1104/pp.110.161281. Pandey, K.K., Pitman, A.J., 2003. FTIR studies of the changes in wood chemistry following decay by brown-rot and white-rot fungi. Int. Biodeterior. Biodegrad. doi:10.1016/S09648305(03)00052-0. Pérez, J., Muñoz-Dorado, J., De La Rubia, T., Martínez, J., 2002. Biodegradation and biological treatments of cellulose, hemicellulose and lignin: An overview. Int. Microbiol. 5, 53–63. doi:10.1007/s10123-002-0062-3. Poole, I., Van Bergen, P.F., 2002. Carbon isotope ratio analysis of organic moieties from fossil mummified wood: Establishing optimum conditions for off-line pyrolysis extraction using gas chromatography/mass spectrometry. Rapid Commun. Mass Spectrom. 16, 1976–1981. doi:10.1002/rcm.818. Popa, I., 2004. Fundamente metodologice şi aplicaţii de dendrocronologie (in romanian). Editura Tehnică Silvică, Bucureşti. Popa, I., Kern, Z., 2009. Long-term summer temperature reconstruction inferred from tree ring records from the Easter Carpathians. Clim. Dyn. 32, 1107–1117. Porté, A., Loustau, D., 2001. Seasonal and interannual variations in carbon isotope discrimination in a maritime pine (Pinus pinaster) stand assessed from the isotopic composition of cellulose in annual rings. Tree Physiol. 21, 861–868. Reese, E.T., 1957. Biological Degradation of Cellulose Derivatives. Ind. Eng. Chem. 49, 89–93. doi:10.1021/ie50565a033. Richard, B., Quilès, F., Carteret, C., Brendel, O., 2014. Infrared spectroscopy and multivariate analysis to appraise cellulose extracted from wood for stable carbon isotope measurements. Chem. Geol. doi:10.1016/j.chemgeo.2014.05.010 Robertson, I., Leavitt, S., Loader, N., Buhay, W., 2011. Progress in isotope dendroclimatology. Chem. Geol. 252, 2001–2004. doi:10.1016/S0009-2541(08)00177-0. Roden, J., 2008. Cross-dating of tree ring d18O and d13C time series. Chem. Geol. 252, 72–79. doi:10.1016/j.chemgeo.2008.01.007. Saurer, M., Robertson, I., Siegwolf, R., Leuenberger, M., 1998. Oxygen Isotope Analysis of Cellulose: An Interlaboratory Comparison. Anal. Chem. 70, 2074–2080. doi:10.1021/ac971022f. Savard, M.M., Bégin, C., Marion, J., Arseneault, D., Bégin, Y., 2012. Evaluating the integrity of C and O isotopes in sub-fossil wood from boreal lakes. Palaeogeogr. Palaeoclimatol. Palaeoecol. 348–349, 21–31. doi:10.1016/j.palaeo.2012.06.003. Schleser, G.H., Helle, G., Lu, A., Vos, H., 1999. Isotope signals as climate proxies : the role of transfer functions in the study of terrestrial archives. Quat. Sci. Rev. 18, 927–943. Schwarze, F.W.M.R., Fink, S., Lonsdale, D., 1997. An overview of wood degradation patterns and their implications for tree hazard assessment. Arboric. J. 21, 1–32. doi:10.1080/03071375.1997.9747146. Schweingruber, F.H., 2007. Wood structure and environment. Springer Ser. wood Sci. 279. doi:10.1007/978-3-540-48548-3. Schweingruber, F.H., 1988. Tree Rings Basics and Applications of Dendrochronology, Kluwer Academic Publishers. doi:10.1007/978-94-009-1273-1.
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Spiker, E.C., Hatcher, P.G., 1987. The effects of early diagenesis on the chemical and stable carbon isotopic composition of wood. Geochim. Cosmochim. Acta 51, 1385–1391. doi:10.1016/0016-7037(87)90323-1. Stuart, B.H., 2004. Infrared Spectroscopy: Fundamentals and Applications, Methods. Wiley, Chichester. doi:10.1002/0470011149 Treydte, K., Schleser, G.H., Schweingruber, F.H., Winiger, M., 2001. The climatic significance of delta 13C in subalpine spruces (Loetschental, Swiss Alps); a case study with respect to altitude, exposure and soil moisture. Tellus, Ser. B Chem. Phys. Meteorol. 53, 593–611. doi:10.1034/j.1600-0889.2001.530505.x. Treydte, K.S., Schleser, G.H., Helle, G., Frank, D.C., Winiger, M., Haug, G.H., Esper, J., 2006. The twentieth century was the wettest period in northern Pakistan over the past millennium. Nature 440, 1179–1182. Werner, R.A., Brand, W.A., 2001. Referencing strategies and techniques in stable isotope ratio analysis. Rapid Commun. Mass Spectrom. 15, 501–519. doi:10.1002/rcm.258 Wright, W.E., Leavitt, S.W., 2006. Boundary layer humidity reconstruction for a semiarid location from tree ring cellulose δ18O. J. Geophys. Res. Atmos. 111, 1–9. doi:10.1029/2005JD006806. Ziehmer, M.M., Nicolussi, K., Schlüchter, C., Leuenberger, M., 2017. The potential of tree-ring cellulose content as a novel supplementary proxy in dendroclimatology. Biogeosciences Discuss. 1–30. doi:10.5194/bg-2017-117.
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Fig.1. Location of the sampling site in N Romania. The inset photograph shows the appearance of the investigated forest.
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Fig. 2. Example of decayed and non-decayed wood form the same sample (B).
Fig. 3 Spectra of infra red absorbance over the 1800-700 cm-1 spectral region of cellulose extracted from decayed and non-decayed sections of the same annual increment from A. Top:
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AD 1744, Bottom: AD 1745. Decayed (black line) and non-decayed (pink line) sub-sample plotted. Characteristic spectral peaks are annotated.
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Fig. 4 Individual isotope trends and the resulting maximum intra-tree and inter-tree differences for oxygen and carbon stable isotopes.
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Fig. 5 Linear relationship between the measured δ18O and δ13C values in α-cellulose derived from decayed and non-decayed wood. Brown – sample A, blue – sample B, green – sample C.
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Non-decayed wood 1740-1746 1886-1889 1135-1145 1886-1889 1886-1889 1740-1759 1740-1759
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Decayed wood 1740-1759 1886-1889 1135-1145 -
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Tree code A B C D E F I
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Table 1. Range of calendar years of the investigated samples. In Tree “C” the ring corresponding to the year 1142 was not analyzed.
Table 2 Stable isotope composition of decayed and non-decayed wood in sample A, and nondecayed wood in samples F and I.
F
I
A
Status
Non-decayed δ18O δ13C
Non-decayed δ18O δ13C
Non-decayed δ18O δ13C
Decayed δ O δ13C
1740
27.8
-20.5
26.9
-22.1
27.3
-24.7
27.5
-23.6
1741
28.4
-20.7
27.4
-22.4
27.7
-25.1
27.8
-23.5
1742
29.1
-20.9
27.9
-22.9
29.0
-25.2
28.9
-23.7
1743
28.2
-21.1
27.1
-23.2
28.1
-25.4
28.2
-24.0
1744
27.8
-20.5
27.0
-22.9
27.3
-25.2
27.5
-23.6
1745
28.4
-20.3
27.6
-22.7
27.9
-24.7
28.3
-23.5
1746
29.1
-20.5
27.7
-22.6
28.7
-24.8
28.7
-23.8
1747
29.3
-20.9
28.7
-22.0
28.1
-25.1
1748
29.6
-20.8
27.4
-22.1
29.8
-24.6
1749 1750 1751 1752 1753 1754 1755 1756 1757 1758 1759
29.1 28.8 28.4 28.7 28.8 30.2 29.1 29.1 29.5 29.0 29.5
-20.8 -20.9 -20.4 -20.3 -20.4 -20.3 -20.3 -20.6 -20.9 -21.3 -21.6
27.8 27.8 27.7 28.2 29.1 28.0 28.0 28.5 27.5 27.8 27.6
-22.4 -22.2 -22.4 -22.6 -22.2 -22.3 -22.1 -22.4 -23.1 -22.6 -22.5
28.4 28.1 27.4 27.9 27.8 29.4 28.9 27.9 28.8 28.3 29.2
-24.9 -24.6 -24.6 -23.7 -24.1 -23.8 -23.6 -23.8 -23.8 -23.9 -24.4
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Tree name
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Table 2
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Table 3 Stable isotope composition of decayed and non-decayed wood in sample B and nondecayed wood in samples E and D.
Table 3
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Tree name Status
E
D
B
Non-decayed δ O δ13C
Non-decayed δ18O δ13C
Non-decayed δ18O δ13C
Decayed δ O δ13C
1886
27.9
-23.1
28.3
-24.4
28.0
-22.8
28.2
-23.0
1887
28.9
-23.0
27.6
-24.1
28.4
-22.4
28.8
-22.9
1888
28.6
-23.0
29.2
-23.9
27.9
-21.9
28.4
-22.2
1889
30.2
-22.7
30.4
-23.8
28.7
-22.6
29.2
-22.4
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Table 4 Stable isotope composition of decayed and non-decayed wood in sample C.
C Non-decayed δ O δ13C
Non-decayed δ O δ13C
29.8 28.6 27.6 28.5 28.7 29.1 27.9 27.9 28.4 27.8
29.7 28.1 27.5 28.4 28.8 29.1 28.0 28.0 28.5 28.1
-21.7 -22.2 -22.3 -22.4 -22.7 -22.1 -22.1 -22.1 -22.6 -22.8
D TE EP CC A
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-22.3 -22.3 -22.2 -22.5 -22.8 -22.2 -22.2 -22.4 -22.9 -23.0
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1145 1144 1143 1141 1140 1139 1138 1137 1136 1135
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Tree name Status
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Table 4
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Table 5. Intra- and inter-tree differences in stable isotope values of oxygen and carbon, calculated as the differences between the measured isotopic values. Intra-tree differences were measured between decayed (D) and non-decayed (ND) wood on tress A and B. The inter-tree variability was calculated on non-decayed wood, only. The color shading varies from maximum (red) to minimum (yellow) differences.
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Table 5 δ18O differences
1740
-0.3
0.8
0.5
1741
-0.1
1.0
1742
0.1
1.2
1743
-0.1
1.0
1744
-0.1
0.9
1745
-0.4
1746
0.0
F-I
inter-tree F-AND
I-AND
δ13C
intra-tree CD-ND CD-ND
-1.1
1.6
4.2
2.6
1135
0.3
0.3
-0.3
-1.5
1.7
4.3
2.7
1136
0.1
0.4
0.1
-1.1
-1.5
2.0
4.3
2.3
1137
0.1
0.3
0.1
-1.0
-1.4
2.1
4.3
2.2
1138
0.0
0.2
0.5
-0.4
-1.6
2.4
4.7
2.2
1139
0.0
0.0
0.8
0.6
-0.2
-1.2
2.4
4.5
2.0
1140
0.1
0.1
1.4
0.4
-1.0
-1.0
2.1
4.3
2.2
1141
-0.1
0.2
E-D
E-BND
D-BND
BND-D
E-D
E-BND
D-BND
1142
PT
-0.3
0.7
CC E
BND-D
I-AND
intra-tree AND-D
M
F-I
inter-tree F-AND
δ18O
ED
intra-tree AND-D
δ13C differences
-0.3
-0.4
-0.1
0.4
0.2
1.3
-0.3
-1.6
1143
-0.1
0.0
1887 1888 1889
-0.4 -0.5 -0.5
1.3 -0.5 -0.3
0.6 0.7 1.4
-0.8 1.3 1.7
0.5 0.3 -0.2
1.2 1.0 1.1
-0.6 -1.0 -0.1
-1.8 -2.0 -1.2
1144 1145
-0.5 -0.1
0.1 0.5
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1886
S1125-7865(17)30157-1 https://doi.org/10.1016/j.dendro.2018.03.007 DENDRO 25508
To appear in: Received date: Revised date: Accepted date:
12-10-2017 17-3-2018 20-3-2018
Please cite this article as: Nagavciuc V, Kern Z, Perx219;oiu A, Doacute;ra K, Popa I, Aerial decay influence on the stable oxygen and carbon isotope ratios in tree ring cellulose, Dendrochronologia (2010), https://doi.org/10.1016/j.dendro.2018.03.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Aerial decay influence on the stable oxygen and carbon isotope ratios in tree ring cellulose
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Viorica Nagavciuc 1,2,3,4 Zoltán Kern 2 Aurel Perșoiu 3,5,6 Kesjár Dóra 2 Ionel Popa 7 Faculty of Forestry, Ștefan cel Mare University, Suceava, Romania Institute for Geological and Geochemical Research, Research Centre for Astronomy and Earth Sciences, Hungarian Academy of Sciences, Budapest, Hungary 3 Stable Isotope Laboratory, Ștefan cel Mare University, Suceava, Romania 4 Departement of Geography, Johannes Gutenberg University, Mainz, Germany 5 Emil Racoviță Institute of Speleology, Romanian Academy, Cluj Napoca, Romania 6 Institute of Biology, Romanian Academy, Bucharest, Romania 7 Marin Dracea National Research and Development Institute for Silviculture, Câmpulung Moldovenesc, Romania 1
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Corresponding author Nagavciuc Viorica [email protected] Address: Universității 13, Suceava 720229, Romania
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Abstract Sub-fossil wood is often affected by the decaying process that introduces uncertainties in the measurement of oxygen and carbon stable isotope composition in cellulose. Although the cellulose stable isotopes are widely used as climatic proxies, our understanding of processes controlling their behavior is very limited. We present here a comparative study of stable oxygen and carbon isotope ratios in tree ring cellulose in decayed and non-decayed wood samples of Swiss stone pine (Pinus cembra) trees. The intra-ring stable isotope variability (around the circumference of a single ring) was between 0.1 and 0.5 ‰ for δ18O values and between 0.5 and 1.6 ‰ for δ13C values for both decayed and non-decayed wood. Observed intra-tree δ18O variability is less than that reported in the literature (0.5-1.5 ‰), however, for δ13C it is larger than the reported values (0.7-1.2 ‰). The inter-tree variability for non-decayed wood ranges between 1.1 and 2.3 ‰ for δ18O values, and between 2 and 4.7 ‰ for δ13C values. The inter-tree differences for δ18O values are similar to those reported in the literature (1-2 ‰ for oxygen and 1-3 ‰ for carbon) but are larger for δ13C values. We have found that the differences for δ18O and δ13C values between decayed and non-decayed wood are smaller than the variation among different trees from the same site, suggesting that the decayed wood can be used for isotopic paleoclimate research. Keywords oxygen isotope ratios; carbon isotope ratios; tree rings; decayed wood
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Introduction The stable oxygen and carbon isotope composition (hereafter δ18O and δ13C values) of tree-ring cellulose are becoming one of the most powerful proxies for reconstructing past climate variability (Gagen et al., 2011). This results from the combination of the annual chronological control with the precisely understood environmental physiology controlling the link between climate and tree-ring δ18O and δ13C values (McCarroll and Loader, 2004). While in some areas living trees allow for millennial-scaleclimate reconstructions, most regions lack such conditions and fossil and subfossil trees are increasingly analyzed. However, these specimens are frequently (partly) decayed, thus possibly adding an extra layer of analytical uncertainty. Tree-ring width (TRW) measurements are not influenced by the state of wood (i.e., decay), as the mechanical solidity of the wood remains unchanged during decay (Schweingruber, 2007), while decayed wood is not suitable for density and blue reflectance measurements because cell walls are (partly) decomposed (Schweingruber, 1988). For stable isotope analyses uncertainties can be traced to technical errors of mass spectrometry, biological faults, and samples quality and preparation (Werner and Brand, 2001, Leavitt, 2010; Leavitt and Wright, 2002; Porté and Loustau, 2001; Robertson et al., 2011; Treydte et al., 2006). Although widespread, studies on the variability of oxygen and carbon isotopes in tree-ring cellulose imposed by wood decay are extremely rare, even though it is well documented that decayed and non-decayed wood were mixed quite frequently (Becker et al., 1991; Boettger et al., 2003; Boettger et al., 2003; Hunkeler et al., 2001; Mancini et al., 2003; Spiker and Hatcher, 1987). The reliability of δ18O and δ13C values of waterlogged subfossil black spruce (Picea mariana (Mill.)) stems, showing signs of bacterial decay due to long-time exposure to lake-bottom conditions was evaluated by Savard et al. (2012). These authors have found that even if some of the tree ring cellulose is lost during decay, the oxygen and carbon isotope composition of the remaining material largely preserves the original values. Deadwood decay is largely dependent on wood properties, enzyme activities, site-specific microbial and physical diversity of the ambient environment (Kahl et al., 2017; Klaassen and van Overeem, 2012), so different biochemical processes can be expected to dominate for aerobic and anaerobic degradation (Klaassen and van Overeem, 2012). Most of deadwood material analyzed in the previous studies was degraded by aqueous bacterial activity (Gelbrich et al., 2012, 2008; Greaves, 1971; Kłusek and Pawełczyk, 2014; Poole and Van Bergen, 2002; Savard et al., 2012). The biodegradation of lignin-cellulosic material belongs mostly to eubacteria and fungi for three major polymers (cellulose, hemicellulose and lignin) (Kačík et al., 2009; Mester et al., 2004; Pérez et al., 2002; Reese, 1957). Loader et al., (2003) analyzed the differences of stable carbon isotope ratios in the whole wood, cellulose and lignin, whereas Savard et al. (2012) examined the stability of carbon and oxygen stable isotopes in sub-fossil wood from anaerobic conditions. However, studies on the potential effect of the decay process on the stable isotope composition of cellulose under subaerial conditions are still lacking.
The aim of this study is to assess the suitability of decayed wood for stable isotope-based paleoclimatic reconstructions, by studying and comparing the oxygen and carbon isotope composition of cellulose from aerially decayed and non-decayed wood in Swiss stone pine (Pinus cembra) from the Eastern Carpathian Mountains, Romania.
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Samples description and analysis The study area is located in the Călimani Mountains, Eastern Carpathians, Romania (Fig. 1) which is part of Călimani National Park, established in 1975. The samples were collected during a field trip in summer 2008 from altitudes varying between 1450 and 1850 m a.s.l. where a representative Swiss stone pine population exist (Popa and Kern, 2009). All dead wood samples were collected from fallen trees with different degrees of decay from high altitude natural mixed stands of spruce and stone pine. The substrate is composed of crystalline schist with skeletal lithic acid brown soil, with medium humidity. No sign of fires or buried wood was observed in the sampling area. From each dead tree one transversal disk was sampled using a chainsaw. All individual samples were measured and dated using millennium long reference chronology of stone pine from Călimani Mts (Popa and Kern, 2009) at annual resolution. Annual ring widths were measured using LINTAB equipment and TSAP 0.53 software, and cross-dated and checked for missing rings (Grissino-Mayer, 2001, 1997; Holmes, 1983). The detailed cross-dating process is described in Popa and Kern (2009). Three sub-fossil Swiss stone pine trees (labeled A, B, and C), with sections of both decayed and non-decayed wood around the circumference of the same ring (Fig. 2), and four non-decayed samples of the same tree (D, E, F and I) were analyzed for this study (Table 1). The A tree grew between AD 1622-1759, with the decayed and non-decayed section of wood dated to AD 1740-1759; the B tree grew between AD 1795-1894, with the decayed and nondecayed section dated to AD 1886-1889, and tree C grew between AD 1007-1705, with the decayed and non-decayed sections located in rings dated to AD 1135-1141 and AD 1143-1145. In tree A, the rings dated between AD 1747 and 1759 were in an advanced stage of degradation, while rings dated between AD 1740 and 1746 had sectors of both decayed and non-decayed wood (Fig. 2). In tree B, the rings dated between AD 1886 and AD 1889 had sectors of both decayed and non-decayed wood. The non-decayed Swiss stone pine samples used for reference grew between AD 1876-2012 (D, living tree), AD 1770-2015 (E, living tree), AD 1645-1869 (F, sub-fossil tree) and AD 1522-1863 (I, sub-fossil tree). Wood-destroying fungi that cause cell wall degradation can be classified depending on the type of decay process in three main groups: white-, brown-, and soft-rot (Blanchette et al., 1990; Karami et al., 2013). Our decayed samples were affected by brown-rot fungi, which are caused exclusively by basidiomycetes (Schwarze et al., 1997). During the destruction of wood by brown-rot fungi, the cellulose and hemicellulose are broken down with little or no overt breakdown of lignin (Blanchette et al., 1990; Eriksson et al., 1990; Mester et al., 2004; Schwarze et al., 1997). The breakdown of cellulose due to brown-rot fungi involves the enzymic and nonenzymic systems that cause oxidative reactions in the cellulose molecule, thereby enhancing the
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activity of enzymes, which split the molecule at random points along its length (Schwarze et al., 1997). As a result, the cellulose microfibrils are cut into short lengths causing the dramatic loss of tensile strength in a very short period of exposure to the brown-rot process (Schwarze et al., 1997). In advanced stages of decay, even if the wood cells appear to maintain their usual form, cellulose is depleted much more than lignin, which causes shatter and collapses of cells (Blanchette et al., 1990). Decayed wood in our samples was identified by visual inspection (Fig. 2), having a slightly altered texture (Savard et al., 2012), and black-brown coloring that is characterized for the brown-rot attack (Björdal et al., 1999). For stable isotope analyses, after separating each ring with a scalpel, we prepared the α-cellulose using the modified Jayme-Wise method (Boettger et al., 2007; Loader et al., 1997), homogenized it using the standard ultrasonic protocol (Laumer et al., 2009), and dried it at 70 °C for 24 hours. Further, 0.2 mg (±10 %) of α-cellulose was weighted into silver capsules for pyrolysis over glassy carbon at 1450 °C and simultaneous measurements of oxygen and carbon isotope ratios (δ18O and δ13C) using a ThermoQuest TCEA elemental analyzer interfaced with a Thermo Delta V Advantage IRMS (Loader et al., 2016, 2015; Saurer et al., 1998) at the Institute for Geological and Geochemical Research, Research Centre for Astronomy and Earth Sciences, Hungarian Academy of Sciences, Budapest. The samples were measured in triplicates. The results are reported in per mil (‰) relative to the Vienna Standard Mean Ocean Water (VSMOW) for oxygen, and Vienna Pee Dee Belemnite (VPDB) for carbon (Coplen, 1994), respectively, using the traditional δ notation:
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δ18O (δ13C) = (Rsample / Rstandard -1) × 1000,
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where Rsample and Rstandard are either 18O/16O (or 13C/12C) ratios of the sample and the standard, respectively. The analytical precision of the measurements was better than 0.2 ‰ for both oxygen and carbon. We have measured δ18O and δ13C values in samples from rings with both decayed and nondecayed wood, corresponding to three-time intervals (Table 1): AD 1740-1759 (samples A, F, I), AD 1886-1889 (samples B, D, E), 1135-1145 (sample C). To further test the alteration of the cellulose during the decay process, the rings with the largest difference of δ13C between the decayed and non-decayed samples along the circumference of the same ring (years AD 1744 and 1745 in sample A) were analyzed using Fourier Transform Infrared Spectroscopy (FTIR). Attenuated total reflectance (ATR) is an useful initial step to characterize organic matter with minimal sample preparation (Stuart, 2004). ATR is based on the phenomenon of total internal reflection (Bruno, 1999) and measures the changes occurring in an internally reflected infrared beam, which come in contact with the sample through a diamond crystal. When the sample is placed in contact with the ATR crystal, the resulting evanescent wave is attenuated in the regions of the IR spectrum where the sample absorbs energy (Stuart, 2004). For each sample, 16 scans were recorded in the 4000 to 400 cm-1 spectral range with a resolution of 4 cm-1. The measurements were carried out using a Bruker Vertex 70 spectrometer
(Bruker Corporation, Billerica, MA, USA) controlled by OPUS 7.2 software (Bruker Corporation), at the Institute for Geological and Geochemical Research of the Hungarian Academy of Sciences.
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Results and discussions The IR ATR spectra show perfect agreement between the cellulose extracted from decayed and non-decayed sections of the rings (Fig. 3). All the identified peaks are characteristic to cellulose (Richard et al., 2014) excluding any significant difference between extraction residuals from decayed and non-decayed wood. The wood from samples C was weighed before extracting the cellulose and also the mass of the resulting cellulose in order to compare the differences of cellulose content in decayed and non-decayed wood. The structure of the xylem cell walls is usually composed of roughly 45 % cellulose (Chen, 2014; Novaes et al., 2010), the percentage that was also measured in the non-decayed samples from tree C, whereas for the decayed samples the wood is composed of ~37% cellulose. The obtained percentage of cellulose in the decayed tree rings match those in the slightly altered sub-fossil wood class described by (Savard et al., 2012), and in the range reported from two Stone Pine stand (31.6-39.7 %) from the Alps by Ziehmer et al., (2017). The differences of cellulose content between decayed and non-decayed wood vary between 0 % (AD 1135) and 12 % (AD 1143), the decayed samples being depleted by 7 %, on average. This is in agreement with the reported increase in the lignin: carbohydrate peak area ratio during brown-rot decay (Pandey and Pitman, 2003). Cellulose percentage in the slightly altered wood definitively indicates the progressive decomposition degree from the wellpreserved wood to highly altered wood (Savard et al., 2012). The results of stable isotope analyses are presented in Table 2 (AD 1740-1759), Table 3 (AD 1886-1889) and Table 4 (AD 1135-1141, 1143-1145). The intra-ring differences in δ18O and δ13C values (Table 5), measured on the decayed and non-decayed wood in samples from all three trees are between 0.1 and 0.4 ‰ for δ18O values and among 1.0 and 1.6 for δ13C values in tree A; between 0.3 and 0.5 ‰ for δ18O values and 0.2 and 0.5 ‰ for δ13C values in tree B; and between 0.5 and 0.3 ‰ for δ18O values and 0.0 and 0.5 ‰ for δ13C values in tree C. The intra-tree differences are only marginally larger than the analytical precision for δ18O values, but somewhat higher for δ13C values. The δ18O values on intra-tree differences are lower than those reported in the literature (e.g., 0.5 – 1.5 ‰), measured among the three different radii (Leavitt, 2010), as well as than two measurements for the same year from the two radii of eight trees (Li et al., 2015). However, the δ13C values are larger than those reported in the literature (0.5 – 1.5 ‰, Leavitt, 2010) for various for measurement on four different radii at three different heights (Leavitt and Long, 1986), or between eight radii of the same tree (Leavitt and Long, 1984) and the values reported in the review article (Leavitt, 2010). The differences in δ13C values (up to 1.5 ‰, Table 5) imply the possible alteration of the original isotopic signal due to wood decay. Schleser et al. (1999) have found a 1 ‰ difference in the carbon isotopic values in affected wood while English et al. (2011) found no effect of blue fungus on δ13C and δ18O values. Previous studies have shown that for samples collected from different sections along the circumference of
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a given ring, δ18O and δ13C values ranged in a narrow 0.5-1.5 ‰ interval (Leavitt, 2010). However, our δ13C differences are not consistent for the two analyzed trees: in tree A and C, the decayed samples are enriched in the heavy isotopes compared to non-decayed samples, while in tree B, they are depleted. Interestingly, the δ18O differences show a similar sign of enrichment in heavy isotopes during wood decomposition, with only five samples (tree A, year AD 1742; tree C, years AD 1143-1145 and tree C, year AD 1141) being slightly depleted (Table 2, 4). Thus, while the differences in δ13C values are large, the opposing sign in different trees suggest that the decay process is not responsible for the shift in the stable isotope composition. Considering the strong coherence of δ18O values in tree rings and the common variability of the δ18O values in xylem water, needle water and phloem organic matter in all the measured trees, Treydte et al. (2014) conclude that individual tree characteristics do not influence oxygen isotope fractionation until assimilate production. The inter-tree differences in stable isotope values for non-decayed wood range from 1.1 to 2.3 ‰ for δ18O values, and from 2 to 4.7 ‰ for δ13C values, for the combined studied intervals (AD 1740-1746 and AD 1886-1889) and for all analyzed samples (D, E, I, F). The differences for δ18O values are between those reported (1 - 2 ‰) by Leavitt and Wright (2002), Wright and Leavitt (2006) and (Treydte et al., 2006), but are larger for δ13C values; Leavitt (2010) and Porté and Loustau (2001) reporting differences in the 1 - 3 ‰ range. The inter-tree differences are larger than the intra-tree ones affected by decay (Fig. 4), thus suggesting that the stable isotope values change during wood-decay processes and are affecting the original isotopic signal to a degree that should not impede the preservation of the climatic signal by the stable isotope composition in tree ring cellulose. The coherence in the inter-tree differences of decayed and non-decayed wood and the similar trends reflect a strong common signal of trees in the area, a finding confirmed also by the validity of the pooling method for isotopic measurement (Borella et al., 1998; Leavitt, 2008; Treydte et al., 2001). Excellent linear relation was found between the δ18O values measured on α-cellulose derived from decayed and non-decayed wood, while the correlation for δ13C values is much poorer. The observed relationship (Fig. 5) between the δ18O (δ13C) values of decayed and non-decayed samples was above (below) the mean inter-tree correlation value reported for pine (Pinus ponderosa) and Coast redwood (Sequoia sempervirens) species studied at five sites (Roden, 2008). In addition, the best-fit slopes are close to unity for δ18O values, whereas lower slopes were obtained for δ13C values. Our results are in agreement with Savard et al. (2012), who found that slightly altered wood preserved similar isotopic ratios with non-decayed wood, while the highly altered wood (containing less than 21 % cellulose), preserved reliable δ13C values but depleted δ18O values, thus suggesting that site-specific analysis are necessary when decayed wood is to be used in paleoclimatic research. The differences noted in the transition zone between different stages of decomposition can be observed visually (Savard et al., 2012), thus eliminating the risk of contamination of samples with highly decayed wood.
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Conclusions The brown-rot decaying process of wood analyzed in this study has a limited influence on the stable oxygen isotope composition in α-cellulose, and a larger one on the stable carbon isotope composition. We therefore suggest that the slightly decayed Pinus cembra wood from our site can be used for isotopic analysis in paleoclimatic research. The differences in δ13C values between decayed and non-decayed wood are lower than those between different trees from the same site, thus suggesting a limited effect of wood decay on the original stable isotope composition of cellulose, that would not impact tentative paleoclimatic reconstructions that are using the cellulose stable isotope composition as a proxy for past climates. However, in the absence of comparative studies between decayed and non-decayed wood, the safest approach would still be to avoid the decayed wood when analyzing the stable isotope composition of tree rings. The intra-tree differences between decayed and non-decayed wood are lower than the inter-site (between different trees) variability, but our limited data-set for circumferential variability renders our conclusion to be preliminary. A general methodological recommendation for future stable isotope studies on subfossil tree-rings is to provide a detailed documentation and presentation of the type of decay observed in any sampled section as it can help to better understand the potential distinct isotope effects accompanying the different decay processes.
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Acknowledgements The research leading to these results has received funding from EEA Financial Mechanism 2009 - 2014 under the project contract no CLIMFOR18SEE. Thanks for the support of “Lendület” program of the Hungarian Academy of Sciences (LP2012-27/2012). This is contribution No.54. of ‘2ka Palæoclimatology’ Research Group. The research was supported partial by CTR 663/2013 and 1993/2014 and PN-III-P4-ID-PCE-2016-0253 projects.
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Fig.1. Location of the sampling site in N Romania. The inset photograph shows the appearance of the investigated forest.
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Fig. 2. Example of decayed and non-decayed wood form the same sample (B).
Fig. 3 Spectra of infra red absorbance over the 1800-700 cm-1 spectral region of cellulose extracted from decayed and non-decayed sections of the same annual increment from A. Top:
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AD 1744, Bottom: AD 1745. Decayed (black line) and non-decayed (pink line) sub-sample plotted. Characteristic spectral peaks are annotated.
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Fig. 4 Individual isotope trends and the resulting maximum intra-tree and inter-tree differences for oxygen and carbon stable isotopes.
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Fig. 5 Linear relationship between the measured δ18O and δ13C values in α-cellulose derived from decayed and non-decayed wood. Brown – sample A, blue – sample B, green – sample C.
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Non-decayed wood 1740-1746 1886-1889 1135-1145 1886-1889 1886-1889 1740-1759 1740-1759
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Decayed wood 1740-1759 1886-1889 1135-1145 -
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Tree code A B C D E F I
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Table 1. Range of calendar years of the investigated samples. In Tree “C” the ring corresponding to the year 1142 was not analyzed.
Table 2 Stable isotope composition of decayed and non-decayed wood in sample A, and nondecayed wood in samples F and I.
F
I
A
Status
Non-decayed δ18O δ13C
Non-decayed δ18O δ13C
Non-decayed δ18O δ13C
Decayed δ O δ13C
1740
27.8
-20.5
26.9
-22.1
27.3
-24.7
27.5
-23.6
1741
28.4
-20.7
27.4
-22.4
27.7
-25.1
27.8
-23.5
1742
29.1
-20.9
27.9
-22.9
29.0
-25.2
28.9
-23.7
1743
28.2
-21.1
27.1
-23.2
28.1
-25.4
28.2
-24.0
1744
27.8
-20.5
27.0
-22.9
27.3
-25.2
27.5
-23.6
1745
28.4
-20.3
27.6
-22.7
27.9
-24.7
28.3
-23.5
1746
29.1
-20.5
27.7
-22.6
28.7
-24.8
28.7
-23.8
1747
29.3
-20.9
28.7
-22.0
28.1
-25.1
1748
29.6
-20.8
27.4
-22.1
29.8
-24.6
1749 1750 1751 1752 1753 1754 1755 1756 1757 1758 1759
29.1 28.8 28.4 28.7 28.8 30.2 29.1 29.1 29.5 29.0 29.5
-20.8 -20.9 -20.4 -20.3 -20.4 -20.3 -20.3 -20.6 -20.9 -21.3 -21.6
27.8 27.8 27.7 28.2 29.1 28.0 28.0 28.5 27.5 27.8 27.6
-22.4 -22.2 -22.4 -22.6 -22.2 -22.3 -22.1 -22.4 -23.1 -22.6 -22.5
28.4 28.1 27.4 27.9 27.8 29.4 28.9 27.9 28.8 28.3 29.2
-24.9 -24.6 -24.6 -23.7 -24.1 -23.8 -23.6 -23.8 -23.8 -23.9 -24.4
SC RI PT
Tree name
U
Table 2
TE
D
M
A
N
18
CC
EP
Table 3 Stable isotope composition of decayed and non-decayed wood in sample B and nondecayed wood in samples E and D.
Table 3
A
Tree name Status
E
D
B
Non-decayed δ O δ13C
Non-decayed δ18O δ13C
Non-decayed δ18O δ13C
Decayed δ O δ13C
1886
27.9
-23.1
28.3
-24.4
28.0
-22.8
28.2
-23.0
1887
28.9
-23.0
27.6
-24.1
28.4
-22.4
28.8
-22.9
1888
28.6
-23.0
29.2
-23.9
27.9
-21.9
28.4
-22.2
1889
30.2
-22.7
30.4
-23.8
28.7
-22.6
29.2
-22.4
18
18
Table 4 Stable isotope composition of decayed and non-decayed wood in sample C.
C Non-decayed δ O δ13C
Non-decayed δ O δ13C
29.8 28.6 27.6 28.5 28.7 29.1 27.9 27.9 28.4 27.8
29.7 28.1 27.5 28.4 28.8 29.1 28.0 28.0 28.5 28.1
-21.7 -22.2 -22.3 -22.4 -22.7 -22.1 -22.1 -22.1 -22.6 -22.8
D TE EP CC A
U
-22.3 -22.3 -22.2 -22.5 -22.8 -22.2 -22.2 -22.4 -22.9 -23.0
M
1145 1144 1143 1141 1140 1139 1138 1137 1136 1135
18
N
18
A
Tree name Status
SC RI PT
Table 4
N U SC RI PT
Table 5. Intra- and inter-tree differences in stable isotope values of oxygen and carbon, calculated as the differences between the measured isotopic values. Intra-tree differences were measured between decayed (D) and non-decayed (ND) wood on tress A and B. The inter-tree variability was calculated on non-decayed wood, only. The color shading varies from maximum (red) to minimum (yellow) differences.
A
Table 5 δ18O differences
1740
-0.3
0.8
0.5
1741
-0.1
1.0
1742
0.1
1.2
1743
-0.1
1.0
1744
-0.1
0.9
1745
-0.4
1746
0.0
F-I
inter-tree F-AND
I-AND
δ13C
intra-tree CD-ND CD-ND
-1.1
1.6
4.2
2.6
1135
0.3
0.3
-0.3
-1.5
1.7
4.3
2.7
1136
0.1
0.4
0.1
-1.1
-1.5
2.0
4.3
2.3
1137
0.1
0.3
0.1
-1.0
-1.4
2.1
4.3
2.2
1138
0.0
0.2
0.5
-0.4
-1.6
2.4
4.7
2.2
1139
0.0
0.0
0.8
0.6
-0.2
-1.2
2.4
4.5
2.0
1140
0.1
0.1
1.4
0.4
-1.0
-1.0
2.1
4.3
2.2
1141
-0.1
0.2
E-D
E-BND
D-BND
BND-D
E-D
E-BND
D-BND
1142
PT
-0.3
0.7
CC E
BND-D
I-AND
intra-tree AND-D
M
F-I
inter-tree F-AND
δ18O
ED
intra-tree AND-D
δ13C differences
-0.3
-0.4
-0.1
0.4
0.2
1.3
-0.3
-1.6
1143
-0.1
0.0
1887 1888 1889
-0.4 -0.5 -0.5
1.3 -0.5 -0.3
0.6 0.7 1.4
-0.8 1.3 1.7
0.5 0.3 -0.2
1.2 1.0 1.1
-0.6 -1.0 -0.1
-1.8 -2.0 -1.2
1144 1145
-0.5 -0.1
0.1 0.5
A
1886