- Email: [email protected]
Correlation of carbon isotope ratios in the cellulose and wood extractives of Douglas-fir
ARTICLE IN PRESS
Dendrochronologia 26 (2008) 125–131 www.elsevier.de/dendro
TECHNICAL NOTE
Correlation of carbon isotope ratios in the cellulose and wood extractives of Douglas-fir Adam M. Taylora,, J. Rene´e Brooksb, Barbara Lachenbruchc, Jeffrey J. Morrellc, Steve Voelkerc a
Department of Forestry, Wildlife and Fisheries, University of Tennessee, 2506 Jacob Drive, Knoxville, TN 37996, USA Western Ecology Division US EPA/NHEERL, 200 SW 35th Street, Corvallis, OR 97333, USA c Department of Wood Science and Engineering, Oregon State University, 119 Richardson Hall, Corvallis, OR 97330, USA b
Received 17 October 2006; accepted 11 May 2007
Abstract Cellulose is usually isolated from the other components of plant material for analysis of carbon stable isotope ratios (d13C). However, many studies have shown a strong correlation between whole-wood and cellulose d13C values, prompting debate about the necessity of cellulose extraction for tree-ring studies. The d13C values were measured in whole wood, extractive-free wood, purified cellulose, acetone/water-soluble extractives and hot water-soluble extractives of Douglas-fir sapwood. Cellulose and acetone/water-soluble extractives from heartwood from the same trees also were compared. Although the various materials showed different absolute d13C values, the components of the same samples, including the extractives, were correlated. The correlations of carbon isotope ratios of cellulose, extractive-free wood and extractives, and the relatively low concentration of the extractives in the wood, suggests that extraction of purified cellulose from Douglas-fir wood samples may not be necessary for some tree-ring analyses. r 2008 Elsevier GmbH. All rights reserved. Keywords: Sapwood; Heartwood; Pseudotsuga menziesii; d13C; Extraction necessity
Introduction Wood cell walls are composed of cellulose, hemicelluloses and lignin, which are immobile compounds, but xylem also contains extractives. These substances are soluble in organic solvents or water and are mobile, potentially allowing for movement across ring boundaries (Sjostrom, 1993). Purified cellulose, isolated from wood and other plant materials, is a standard material used in analyses of variations in carbon stable isotope ratios (d13C). The analysis of a single stable compound such as cellulose is preferred to that of a whole tissue Corresponding author. Tel.: +865 946 1125; fax: +865 946 1109.
E-mail address: [email protected] (A.M. Taylor). 1125-7865/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.dendro.2007.05.005
because the variation in the d13C of a single compound is likely to reflect conditions of photosynthesis during the year of formation rather than be influenced by compound mixtures with varying isotopic signals or mobile elements (Leavitt and Long, 1989; Leavitt and Danzer, 1993). Fractionation of carbon isotopes occurs during photosynthesis in plants (O’Leary, 1993) and the isotopic signature is preserved in the products of photosynthesis and the materials derived from them. However, cellulose and other wood components such as hemicellulose and lignin made in the same year do not have the same d13C value because of differences in 13C discrimination along their biosynthetic pathways (Schmidt and Gleixner, 1998). Nevertheless, carbon
ARTICLE IN PRESS 126
A.M. Taylor et al. / Dendrochronologia 26 (2008) 125–131
isotope values of these substances can be correlated over time (Damesin and Lelarge, 2003; Loader et al., 2003; Helle and Schleser, 2004; Scartazza et al., 2004) if the differences in discrimination along the various biosynthetic routes are consistent. Alternatively, intra-annual variations in photosynthetic conditions may lead to differences between cellulose and other wood components if these substances are formed from photosynthate at different times of the year. Extractives in an individual wood increment may differ isotopically from the cellulose if they were translocated to that wood increment at some time after the wood was formed or if there are differences in biosynthetic pathways. Extractives in sapwood, often starch, simple sugars or lipids, are generally considered to be energy reserve materials for the tree (Hillis, 1987; Taylor et al., 2002). There is evidence that sapwood extractives are used and replenished on an annual basis in some species (e.g. Barbaroux and Breda, 2002), thus one could expect there would be little correlation between the carbon isotope ratios of cellulose and sapwood extractives. Energy reserves are absent from heartwood and heartwood extractives, such as phenols and terpenes, are thought to act as part of a passive defensive system (Hillis, 1987). Sapwood (carbohydrate/ lipid) extractives are believed to be converted to defensive compounds during heartwood conversion. Additionally, heartwood extractives can be formed in part by materials translocated from the cambium (Hillis and Hasegawa, 1963). Thus, heartwood extractives could be expected to differ isotopically from the cellulose of the wood in which they are located. Despite potential isotopic differences between wood extractives, cellulose and other cell wall components, some researchers have analyzed whole-wood samples instead of cellulose after finding a close correlation between whole wood and cellulose (Livingston and Spittlehouse, 1993, 1996; Warren et al., 2001). This correlation suggests that extractives and other compounds in whole wood have similar annual isotopic variation as the cellulose. Our objective in this study was to directly examine the isotopic composition of the extractives and to compare them with that of whole wood, extracted wood and cellulose to help researchers determine if time-consuming cellulose extraction is necessary for isotopic analysis of tree rings.
Materials and methods Plant materials Douglas-fir trees were sampled from one site located in the Coast Range in western Oregon, USA (latitude 441380 N, longitude 1231120 W, elevation 75 m) desig-
nated as Site Class III (McArdle et al., 1961). The site receives about 1500 mm precipitation per year, with about 85% falling between October 15 and April 15. There is no surface water on the site. The climate is mild, with fewer than 30 freezing days in winter, and fewer than 15 days in which temperature exceeds 32 1C. Soils are of the Ritner-Price series, with gravelly clay in the top 60 cm, rooting depth about 90 cm, and Siletz River basalt as parent material. Water stress can be severe in late summer. The primary anthropogenic influence relates to large-scale wildfire in the period prior to 1860, followed by grazing until the 1930s. The sample trees were part of a study of enhancement of mature forest characteristics in second-growth Douglas-fir. The stand had been thinned at intervals in the past, including in 1992 and 2000. Following the 2000 thinning, six basal disks (30 mm thick) were removed from the randomly selected, freshly cut stumps about 300 mm above the ground line. At the time the samples were taken, the stand was 58 years old. There had been no previous treering analysis of trees in the stand. The disks were airdried in the lab. The heartwood/sapwood boundary was located by staining with alizarin red (Kutscha and Sachs, 1962). The sapwood and heartwood were analyzed separately, as described below.
Sapwood analysis A radial strip, 50 mm wide (tangential) by 30 mm thick (longitudinal), was cut from each disk using a band saw. Working from the cambium inward, groups of three consecutive annual rings were separated from the strip using a chisel. Rings were grouped into sets of three to provide sufficient material for each of the analyses. Four sets of rings were taken, in the same manner, from each disk-comprising 12 sapwood annual rings in total, the most recent of which was formed in 2001. Wood from each three-ring set was analyzed for the carbon isotope composition of the whole-wood, acetone/water-soluble extractives (dihydroquercetin glucoside, procyanidins and pinoresinol), the hot watersoluble extractives (carbohydrates such as the simple sugars glucose and xylose), the extracted wood and the a-cellulose. The 3-year ring samples were chiseled into matchstick-sized pieces and reduced to powder by grinding them in a ball-type tissue pulverizer mill (Kleco 4100, Garcia Manufacturing, Visalia, CA) for 2 min. Samples of the wood powder (2 g oven-dried) were weighed and enclosed in heat-sealable polyester filter bags (mesh size 25 mm, ANKOM Technology, Macedon, NY). The bags were extracted with 25 ml per bag of a 70:30 mixture of acetone and water (Dellus et al., 1997) for 72 h on a rotary shaker table (100 rpm). The extractive solutions were not cloudy and there was no visual evidence that
ARTICLE IN PRESS A.M. Taylor et al. / Dendrochronologia 26 (2008) 125–131
any wood powder escaped from the bags. After removal of the sample bag, the acetone was allowed to evaporate, and the aqueous extract solution was frozen and dried under vacuum. The resulting dried extract was weighed into tin cups for isotope analysis. 13C NMR analysis of sapwood acetone/water-soluble extracts obtained in this study confirmed the presence of dihydroquercetin glucoside, procyanidins, and pinoresinol, which may be precursors for the later formation of heartwood extractives (Dellus et al., 1997). After the acetone/water extraction, the sample bags were re-dried and re-weighed and then extracted in a beaker with 75 ml of distilled water in a water bath at 95 1C for 8 h. Water was added as needed to compensate for evaporation. At the end of the extraction, the aqueous extract solution was decanted, frozen, and dried under vacuum. The resulting dried extract was weighed into tin cups for isotope analysis. After extraction, the sample bags were re-dried and re-weighed. 13C NMR analysis of the hot water-soluble sapwood extracts indicated that they were mostly carbohydrates such as the simple sugars glucose and xylose. Following extraction, cellulose was isolated from the wood powder samples using the method described by Leavitt and Danzer (1993). Any extractives that may have remained in the wood following the acetone/water and hot-water extractions were removed from the wood by successive extraction steps with toluene/ethanol (2:1), 95% ethanol, and hot water. The samples were delignified in a 70 1C sodium chlorite solution acidified with acetic acid to pH4.0. Fresh sodium chlorite and acetic acid were added over time to maintain pH4.0 and a bright yellow color solution. Delignification was continued until the pH of the solution was stable (about 4 days). The bags were rinsed with distilled water and dried. Any residual hemicelluloses were removed by submerging the bags three times in a 4.25 M sodium hydroxide aqueous solution for 45 min. The rinsed and dried a-cellulose residue was weighed into tin cups for isotope analysis. Extractives, whole wood, extracted wood, and cellulose samples were analyzed for d13C on an isotope ratio mass spectrometer (Delta Plus, Finnigan, Bremen, Germany) interfaced with an elemental analyzer (ESC 4010, Costech, Valencia, CA) located at the Integrated Stable Isotope Research Facility at the Western Ecology Division of the US Environmental Protection Agency, Corvallis, Oregon. All d13C values were expressed relative to the Pee Dee belemnite carbonate (VPDB) standard in % as d13 C ¼ ðRsample =Rstandard 1Þ 1000 where R is the ratio of 13C–12C atoms of the sample and the standard PDB. Measurement precision was 0.05% for d13C, determined as the average standard deviation of replicate analyses.
127
Heartwood analysis Heartwood samples from the same trees were also analyzed, as part of a separate analysis involving the same six wood disks. Samples of three consecutive annual increments were separated with a chisel, starting from the pith and continuing to the heartwood/sapwood boundary of the radial strips. Six to 14 samples, comprising 18–42 years of growth, were collected from each disk, depending on the size and proportion of heartwood in each disk. Purified holocellulose (NaOH extraction step inadvertently omitted, but this step is only necessary for d18O of cellulose; holo- and a-cellulose have similar d13C values (Leavitt and Danzer, 1993; Borella et al., 1998) and acetone/watersoluble extractives were prepared from each sample and analyzed as described above. Because the extractive component of Douglas-fir heartwood is almost entirely dihydroquercetin (Dellus et al., 1997), which is effectively removed using the acetone/water extraction method (Foo et al., 1992), a separate hot-water extraction step was not included for the heartwood samples. Extracted wood and whole wood also were not collected in the heartwood analysis because the mass balance and isotope comparison of the various components was not the primary objective of that study (Taylor et al., 2007).
Extractive content analysis The extractions described for the sapwood and heartwood studies were intended to remove the bulk of the extractives soluble in the various solvents. Those procedures involved only a single extraction, so it is probable that soluble extractives remained in the wood after the extraction. Furthermore, there may have been extractives present that were not soluble in those solvents. To estimate the total amount of extractable materials present in the wood, the quantitative total extractive content of the sapwood and heartwood from each tree was determined on adjacent radial strips of wood. Samples (10 mm 30 mm) comprising the entire sapwood or heartwood were ground in a Wiley mill (Arthur Thomas Co., Philadelphia, PA) to pass a 20 mesh screen. Samples of the wood powder (1.5 g) were placed in weighed filter bags, oven-dried at 103 1C for 14 h, and re-weighed. The bags were then extracted according to the ASTM standard method for the preparation of extractive-free wood D1105-84 (ASTM, 1996), which involves successive extraction steps with toluene/ethanol (2:1), 95% ethanol, and hot water. The extracted samples were oven-dried at 103 1C for 24 h and re-weighed. Total extractive content of the heartwood and sapwood of each tree was determined as the mass lost from the sample and expressed as a percentage of the oven-dry mass.
ARTICLE IN PRESS 128
A.M. Taylor et al. / Dendrochronologia 26 (2008) 125–131
Data analyses Linear regression models with an autoregressive covariance structure of the relationship between cellulose, wood, and extractives d13C values were created using the mixed procedure in SAS (Version 9.1.3, SAS Institute Inc. Cary, NC, USA) for all trees.
Results Extractives in the sapwood were a relatively small component of the wood (Table 1), 6% on average from the extractive content analysis. The two extractions used for isotope analysis recovered approximately 70% of the total sapwood extractives (sapwood and heartwood analyses). The concentration of extractives in the heartwood was about twice as high as in the sapwood. The d13C values of the cellulose, and the other wood components within the individual trees varied over a range of about 2% (Table 2). The carbon isotope ratios, and the patterns of isotopic variation over time, were different among the trees sampled. Given that we did not monitor the trees in the past, we have no explanation for this variation and this variation was not the focus of this study. However, the cellulose isotope ratios (average ¼ 23.39%) were consistently less negative than the other components. The carbohydrate (hot water-soluble) extractives were about 0.8% Table 1.
more negative than the cellulose. The whole and extracted wood values were similar, at about 1.5% more negative than the cellulose. The acetone–watersoluble extractives had d13C values that were substantially more negative than the other wood components (average ¼ 27.69%). Cellulose d13C values in sapwood were correlated significantly with those of both acetone/water-soluble extractives and the carbohydrate extractives (Fig. 1), explaining 41% and 55% of the variation, respectively (Table 3). In addition, the slope was not significantly different from one for both relationships, indicating that a 1% shift in extractives was matched by a 1% shift in the cellulose. The d13C values of the whole wood and extracted wood were both very strongly correlated with the d13C values of cellulose in the same samples, with R2 over 0.9 and slopes not significantly different from one. Cellulose and acetone/water-soluble extractives also were correlated in the heartwood samples, but the relationship was weaker and the slope was significantly o1, where a 1% shift in the cellulose was associated with only a 0.3% shift in the heartwood extractives.
Discussion Although cellulose remains the standard material in the analysis of tree-ring isotopes, the data in this study suggest that isolation of cellulose from whole-wood Douglas-fir samples may not be necessary for all d13C
Extractives content of Douglas-fir wood samples Fraction of whole wood (%) Average
Sapwood analysis (n ¼ 24) Hot water-soluble extractives Acetone/water-soluble extractives Total extractives
Std. deviation
Max
Min
1.41 3.13 4.53
0.42 0.79 0.87
2.39 4.84 6.32
0.64 1.90 2.83
Total extractives content using ASTM method (n ¼ 6) Sapwood 6.31 Heartwood 13.56
0.67 1.27
7.37 15.16
5.71 11.65
Table 2.
Carbon isotope composition of extractives, cellulose and wood from Douglas-fir sapwood samples, n ¼ 24 d13C (%)
Cellulose Hot water-soluble extractives Extracted wood Whole wood Acetone/water-soluble extractives
Average
Std. deviation
Max
Min
23.39 24.16 24.70 24.87 27.69
0.54 0.77 0.53 0.58 0.83
22.45 23.24 23.81 24.00 25.76
24.80 26.45 25.80 26.13 29.39
ARTICLE IN PRESS A.M. Taylor et al. / Dendrochronologia 26 (2008) 125–131
-25
Sapwood acetone/water-soluble extractives
-27
Extractives δ13C (‰)
-29
-25
Heartwood acetone/water-soluble extractives
-27
-29
-25
-27
-29
Tree A Tree B Tree C Tree D Tree E Tree F
Sapwood hot water-soluble extractives
-25
-24
-23
-22
-21
Cellulose δ13C (‰)
Fig. 1. The correlation of cellulose with extractives in the sapwood and heartwood of wood samples from six Douglas-fir trees.
129
analysis, particularly for sapwood. There were strong correlations between d13C values of cellulose and wholewood samples taken from the same wood increment, and the slope was similar to one, meaning that an isotopic shift in cellulose was matched by an isotopic shift in whole wood of a similar magnitude. This similarity in isotopic variation is likely because of the relatively low concentrations of extractives compared with the cell-wall polymers, and further because the extractives d13C values also vary directly with cellulose d13C values in the wood. This correlation between extractives, including the soluble sugars, and cellulose indicates that some of these reserves were likely deposited at the time of wood formation rather than having been deposited later (Taylor et al., 2007). Thus, extractives present in carbon isotope analyses may not significantly alter patterns of annual change that are apparent in purified cellulose samples. The close correlations observed here in the d13C values of extracted wood and cellulose samples further suggests that the other cell wall components – hemicelluloses and lignin – are isotopically closely correlated with cellulose. Given these correlations, the use of whole-wood Douglas-fir sapwood samples may be acceptable in place of the purified cellulose samples that require significant sample preparation. However, our results may not be applicable for fossil samples where the chemistry may have been altered over time. Testing the correlation between whole wood and cellulose on a subset of samples is recommended. The correlation of extractives and cellulose was weaker in the heartwood than in the sapwood. The heartwood also had relatively high concentrations of extractives. Furthermore, there is evidence that the heartwood extractive formation process can be influenced by environmental factors both at the time the (sap)wood rings were originally formed and at the time the sapwood is converted to heartwood (Taylor et al., 2003, 2007). These observations suggest that the potential influence of the isotopic signal of the
Table 3. Generalized least squares estimates from linear regression models with an autoregressive covariance structure of extractives and wood d13C values on cellulose d13C values for all trees together Wood component regressed on cellulose
R2
Slope (std. error)
Mean square error
p-Value Slope ¼ 0
Slope ¼ 1
Sapwood samples n ¼ 24 Acetone/water soluble extractives Hot water-soluble extractives Extracted wood Whole wood
0.41 0.55 0.91 0.91
0.98 0.81 0.84 0.89
(0.27) (0.28) (0.08) (0.08)
0.43 0.31 0.03 0.03
o0.01 0.01 o0.01 o0.01
0.94 0.50 0.06 0.18
Heartwood samples n ¼ 42 Acetone/water-soluble extractives
0.34
0.29 (0.10)
0.33
0.01
o0.01
ARTICLE IN PRESS 130
A.M. Taylor et al. / Dendrochronologia 26 (2008) 125–131
extractives should be considered when analyzing unextracted or whole-wood heartwood samples, especially if comparisons are to be made with sapwood samples. For tree-ring studies where samples span both heartwood and sapwood, researchers should consider extraction to avoid this potentially confounding influence. At the very least, researchers may wish to test if correlations between cellulose and whole wood differ between sapwood and heartwood for their study individuals. This study is the first to our knowledge that has measured the isotopic ratios of extractives directly and compared them with other wood components. However, the finding that cellulose and whole wood are isotopically correlated is consistent with previous observations in the wood of beech (Fagus sylvatica) (Elhani et al., 2005), mangrove (Rhizophora mucronata) sapwood (Verheyden et al., 2005), Eucalyptus globulus, Pinus radiata and P. pinaster (MacFarlane et al., 1999), oak (Quercus spp.) (Borella et al., 1998), six South African tree species (West et al., 2001), and in 44 angiosperm and gymnosperm species (Harlow et al., 2005). Still, the close relationship of d13C values between cellulose and whole-wood samples may not exist in all species or wood tissues. Total extractives content in Douglas-fir (around 6–12%) is moderate compared with some species, which can range as high as 30% and is generally greater in hardwoods than in softwoods (Hillis, 1987). Species with very high-extractives levels may not have the very close correlation between whole wood and cellulose that was observed in this study. Other species may also have more mobile and/or dynamic extractives, which may be less well correlated with cellulose, especially in the sapwood of hardwoods where annualscale depletion of starch reserves has been observed (Barbaroux and Breda, 2002).
Conclusion Stable carbon isotope signatures of wood, extractivefree wood, and cellulose in Douglas-fir sapwood are highly correlated. Cellulose and extractives in Douglasfir heartwood also are correlated. Given the relatively low concentration of extractives in the wood and the correlation of d13C values of extractives and cellulose of the rings within the same sample, in some cases it may not be necessary to isolate cellulose from increments of Douglas-fir wood for carbon isotope tree-ring analysis.
Acknowledgments Our thanks to Dr. Joe Karchesy (OSU College of Forestry) for useful discussions of extractive analysis, to Dr. Mike Newton (OSU College of Forestry) for advice
and wood samples, to Warren Evans (EPA Integrated Stable Isotope Research Facility) for cellulose extraction, to Bill Griffis (EPA Integrated Stable Isotope Research Facility) for processing the isotope samples, and to Dr. E. Henry Lee (EPA Integrated Stable Isotope Research Facility) for help with statistical analysis. We also thank Dr. Nate McDowell for comments on an earlier version of this manuscript. This work was supported by a USDA Special Grant to Oregon State University for Wood Utilization Research, by a Fellowship from the Weyerhaeuser Foundation, and by the United States Environmental Protection Agency. This manuscript has been subjected to EPA peer and administrative review and has been approved for publication as an EPA document. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
References ASTM, 1996. Standard method for the preparation of extractive-free wood. Designation D1105-84. Annual Book of ASTM Standards, vol. 04-01 Wood. American Society for Testing and Materials. West Conshohocken, PA. Barbaroux, C., Breda, N., 2002. Contrasting distribution and seasonal dynamics of carbohydrate reserves in stem wood of adult ring-porous sessile oak and diffuse-porous beech trees. Tree Physiology 22, 1201–1210. Borella, S., Leuenberger, M., Saurer, M., Siegwolf, R., 1998. Reducing uncertainties in d13C analysis of tree rings: pooling milling, and cellulose extraction. Journal of Geophysics Research 103 (D16), 19519–19526. Damesin, C., Lelarge, C., 2003. Carbon isotope composition of current-year shoots from Fagus sylvatica in relation to growth, respiration and use of reserves. Plant Cell Environment 26, 207–219. Dellus, V., Scalbert, A., Janin, G., 1997. Polyphenols and colour of Douglas fir heartwood. Holzforschung 51, 291–295. Elhani, S., Guehl, J-M., Nys, C., Picard, J.-F., Dupouey, J.-L., 2005. Impact of fertilization on tree ring d15N and d13C in beech stands: a retrospective analysis. Tree Physiology 25, 1437–1446. Foo, L.Y., Helm, R., Karchesy, J., 1992. [50 ,50 ]-bisdihydroquercetin: a B-ring linked biflavonoid from Pseudotsuga menziesii. Phytochemistry 31 (4), 1444–1445. Harlow, B.A., Marshall, J.D., Robinson, A.P., 2005. A multispecies comparison of d13C from whole wood, extractivefree wood and holocellulose. Tree Physiology 26, 767–774. Helle, G., Schleser, G.H., 2004. Beyond CO2-fixation by Rubisco – an interpretation of 13C/12C variations in tree rings from novel intra-seasonal studies on broad-leaf trees. Plant Cell Environment 27, 367–380. Hillis, W.E., 1987. Heartwood and Tree Exudates. Springer, Berlin, p. 268. Hillis, W.E., Hasegawa, M., 1963. The formation of polyphenols in trees. 1. Administration of 14C glucose and subsequent distribution of radioactivity. Phytochemistry 2, 195–199.
ARTICLE IN PRESS A.M. Taylor et al. / Dendrochronologia 26 (2008) 125–131
Kutscha, N.P., Sachs, I.B., 1962. Color tests for differentiating heartwood and sapwood in certain softwood tree species. Report 2246, Forest Products Laboratory, Madison, WI, p. 16. Leavitt, S.W., Danzer, S.R., 1993. Methods for batch processing small wood samples to holocellulose for stablecarbon isotope analysis. Analysis Chemistry 65, 87–89. Leavitt, S.W., Long, A., 1989. Intertree variability of D13C in tree rings. In: Rundel, P.W., Ehleringer, J.R., Nagy, K.A. (Eds.), Stable Isotopes in Ecological Research. Springer, New York, pp. 95–104. Livingston, N.J., Spittlehouse, D.L., 1993. Carbon isotope fractionation in tree rings in relation to the growing season water balance. In: Ehleringer, J.R., Hall, A.E., Farquhar, G.D. (Eds.), Stable Isotopes and Plant Carbon–Water Relations. Academic Press, New York, pp. 141–153. Livingston, N.L., Spittlehouse, D.L., 1996. Carbon isotope fractionation in tree rings early and late wood in relation to intra-growing season water balance. Plant Cell Environment 19, 768–774. Loader, N.J., Robertson, I., McCarroll, D., 2003. Comparison of stable carbon isotope ratios in the whole wood, cellulose and lignin of oak tree-rings. Palaeogeography Palaeoclimatology 196, 395–407. MacFarlane, C., Warren, C.R., White, D.A., Adams, M.A., 1999. A rapid and simple method for processing wood to crude cellulose for analysis of stable carbon isotopes in tree rings. Tree Physiology 19, 831–835. McArdle, R.E., Meyer, W.H., Bruce, D., 1961. The yield of Douglas-fir in the Pacific Northwest. US Department of Agriculture Technical Bulletin 201. O’Leary, M.H., 1993. Biochemical basis of carbon isotope fractionation. In: Ehleringer, J.R., Hall, A.E., Farquhar, G.D. (Eds.), Stable Isotopes and Plant Carbon–Water Relations. Academic Press, San Diego, pp. 19–26.
131
Scartazza, A., Mata, C., Matteucci, G., Yakir, D., Moscatello, S., Brugnoli, E., 2004. Comparisons of d13C of photosynthetic products and ecosystem respiratory CO2 and their responses to seasonal climate variability. Oecologia 140, 340–351. Schmidt, H.-L., Gleixner, G., 1998. Carbon isotope effects on key reactions in plant metabolism and 13C patterns in natural compounds. In: Griffiths (Ed.), Stable Isotopes: Integration of Biological, Ecological and Geochemical Processes. BIOS Scientific Publishers Ltd., Oxford, pp. 13–26. Sjostrom, E., 1993. Wood Chemistry: Fundamentals and Applications. Academic Press, San Diego, p. 293. Taylor, A.M., Gartner, B.L., Morrell, J.J., 2002. Heartwood formation and natural durability—a review. Wood Fiber Science 34 (4), 587–611. Taylor, A.M., Gartner, B.L., Morrell, J.J., 2003. Co-incident variations in growth rate and extractive concentration in Douglas-fir. Forest Ecology and Management 186, 257–260. Taylor, A.M., Gartner, B.L., Morrell, J.J., Brooks, J.R., 2007. Radial patterns of carbon isotopes in the xylem extractives and cellulose of Douglas-fir. Tree Physiology 27, 921–927. Verheyden, A., Roggeman, M., Bouillon, S., Elskens, M., Beeckman, H., Koedam, N., 2005. Comparison between d13C of a-cellulose and bulk wood in the mangrove tree Rhizophora mucronata: implications for dendrochemistry. Chemistry and Geology 219, 275–282. Warren, C.R., McGrath, J.F., Adams, M.A., 2001. Water availability and carbon isotope discrimination in conifers. Oecologia 127, 476–486. West, A.G., Midgley, J.J., Bond, W.J., 2001. The evaluation of d13C isotopes of trees to determine past regeneration environments. Forest Ecology and Management 147, 139–149.
Dendrochronologia 26 (2008) 125–131 www.elsevier.de/dendro
TECHNICAL NOTE
Correlation of carbon isotope ratios in the cellulose and wood extractives of Douglas-fir Adam M. Taylora,, J. Rene´e Brooksb, Barbara Lachenbruchc, Jeffrey J. Morrellc, Steve Voelkerc a
Department of Forestry, Wildlife and Fisheries, University of Tennessee, 2506 Jacob Drive, Knoxville, TN 37996, USA Western Ecology Division US EPA/NHEERL, 200 SW 35th Street, Corvallis, OR 97333, USA c Department of Wood Science and Engineering, Oregon State University, 119 Richardson Hall, Corvallis, OR 97330, USA b
Received 17 October 2006; accepted 11 May 2007
Abstract Cellulose is usually isolated from the other components of plant material for analysis of carbon stable isotope ratios (d13C). However, many studies have shown a strong correlation between whole-wood and cellulose d13C values, prompting debate about the necessity of cellulose extraction for tree-ring studies. The d13C values were measured in whole wood, extractive-free wood, purified cellulose, acetone/water-soluble extractives and hot water-soluble extractives of Douglas-fir sapwood. Cellulose and acetone/water-soluble extractives from heartwood from the same trees also were compared. Although the various materials showed different absolute d13C values, the components of the same samples, including the extractives, were correlated. The correlations of carbon isotope ratios of cellulose, extractive-free wood and extractives, and the relatively low concentration of the extractives in the wood, suggests that extraction of purified cellulose from Douglas-fir wood samples may not be necessary for some tree-ring analyses. r 2008 Elsevier GmbH. All rights reserved. Keywords: Sapwood; Heartwood; Pseudotsuga menziesii; d13C; Extraction necessity
Introduction Wood cell walls are composed of cellulose, hemicelluloses and lignin, which are immobile compounds, but xylem also contains extractives. These substances are soluble in organic solvents or water and are mobile, potentially allowing for movement across ring boundaries (Sjostrom, 1993). Purified cellulose, isolated from wood and other plant materials, is a standard material used in analyses of variations in carbon stable isotope ratios (d13C). The analysis of a single stable compound such as cellulose is preferred to that of a whole tissue Corresponding author. Tel.: +865 946 1125; fax: +865 946 1109.
E-mail address: [email protected] (A.M. Taylor). 1125-7865/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.dendro.2007.05.005
because the variation in the d13C of a single compound is likely to reflect conditions of photosynthesis during the year of formation rather than be influenced by compound mixtures with varying isotopic signals or mobile elements (Leavitt and Long, 1989; Leavitt and Danzer, 1993). Fractionation of carbon isotopes occurs during photosynthesis in plants (O’Leary, 1993) and the isotopic signature is preserved in the products of photosynthesis and the materials derived from them. However, cellulose and other wood components such as hemicellulose and lignin made in the same year do not have the same d13C value because of differences in 13C discrimination along their biosynthetic pathways (Schmidt and Gleixner, 1998). Nevertheless, carbon
ARTICLE IN PRESS 126
A.M. Taylor et al. / Dendrochronologia 26 (2008) 125–131
isotope values of these substances can be correlated over time (Damesin and Lelarge, 2003; Loader et al., 2003; Helle and Schleser, 2004; Scartazza et al., 2004) if the differences in discrimination along the various biosynthetic routes are consistent. Alternatively, intra-annual variations in photosynthetic conditions may lead to differences between cellulose and other wood components if these substances are formed from photosynthate at different times of the year. Extractives in an individual wood increment may differ isotopically from the cellulose if they were translocated to that wood increment at some time after the wood was formed or if there are differences in biosynthetic pathways. Extractives in sapwood, often starch, simple sugars or lipids, are generally considered to be energy reserve materials for the tree (Hillis, 1987; Taylor et al., 2002). There is evidence that sapwood extractives are used and replenished on an annual basis in some species (e.g. Barbaroux and Breda, 2002), thus one could expect there would be little correlation between the carbon isotope ratios of cellulose and sapwood extractives. Energy reserves are absent from heartwood and heartwood extractives, such as phenols and terpenes, are thought to act as part of a passive defensive system (Hillis, 1987). Sapwood (carbohydrate/ lipid) extractives are believed to be converted to defensive compounds during heartwood conversion. Additionally, heartwood extractives can be formed in part by materials translocated from the cambium (Hillis and Hasegawa, 1963). Thus, heartwood extractives could be expected to differ isotopically from the cellulose of the wood in which they are located. Despite potential isotopic differences between wood extractives, cellulose and other cell wall components, some researchers have analyzed whole-wood samples instead of cellulose after finding a close correlation between whole wood and cellulose (Livingston and Spittlehouse, 1993, 1996; Warren et al., 2001). This correlation suggests that extractives and other compounds in whole wood have similar annual isotopic variation as the cellulose. Our objective in this study was to directly examine the isotopic composition of the extractives and to compare them with that of whole wood, extracted wood and cellulose to help researchers determine if time-consuming cellulose extraction is necessary for isotopic analysis of tree rings.
Materials and methods Plant materials Douglas-fir trees were sampled from one site located in the Coast Range in western Oregon, USA (latitude 441380 N, longitude 1231120 W, elevation 75 m) desig-
nated as Site Class III (McArdle et al., 1961). The site receives about 1500 mm precipitation per year, with about 85% falling between October 15 and April 15. There is no surface water on the site. The climate is mild, with fewer than 30 freezing days in winter, and fewer than 15 days in which temperature exceeds 32 1C. Soils are of the Ritner-Price series, with gravelly clay in the top 60 cm, rooting depth about 90 cm, and Siletz River basalt as parent material. Water stress can be severe in late summer. The primary anthropogenic influence relates to large-scale wildfire in the period prior to 1860, followed by grazing until the 1930s. The sample trees were part of a study of enhancement of mature forest characteristics in second-growth Douglas-fir. The stand had been thinned at intervals in the past, including in 1992 and 2000. Following the 2000 thinning, six basal disks (30 mm thick) were removed from the randomly selected, freshly cut stumps about 300 mm above the ground line. At the time the samples were taken, the stand was 58 years old. There had been no previous treering analysis of trees in the stand. The disks were airdried in the lab. The heartwood/sapwood boundary was located by staining with alizarin red (Kutscha and Sachs, 1962). The sapwood and heartwood were analyzed separately, as described below.
Sapwood analysis A radial strip, 50 mm wide (tangential) by 30 mm thick (longitudinal), was cut from each disk using a band saw. Working from the cambium inward, groups of three consecutive annual rings were separated from the strip using a chisel. Rings were grouped into sets of three to provide sufficient material for each of the analyses. Four sets of rings were taken, in the same manner, from each disk-comprising 12 sapwood annual rings in total, the most recent of which was formed in 2001. Wood from each three-ring set was analyzed for the carbon isotope composition of the whole-wood, acetone/water-soluble extractives (dihydroquercetin glucoside, procyanidins and pinoresinol), the hot watersoluble extractives (carbohydrates such as the simple sugars glucose and xylose), the extracted wood and the a-cellulose. The 3-year ring samples were chiseled into matchstick-sized pieces and reduced to powder by grinding them in a ball-type tissue pulverizer mill (Kleco 4100, Garcia Manufacturing, Visalia, CA) for 2 min. Samples of the wood powder (2 g oven-dried) were weighed and enclosed in heat-sealable polyester filter bags (mesh size 25 mm, ANKOM Technology, Macedon, NY). The bags were extracted with 25 ml per bag of a 70:30 mixture of acetone and water (Dellus et al., 1997) for 72 h on a rotary shaker table (100 rpm). The extractive solutions were not cloudy and there was no visual evidence that
ARTICLE IN PRESS A.M. Taylor et al. / Dendrochronologia 26 (2008) 125–131
any wood powder escaped from the bags. After removal of the sample bag, the acetone was allowed to evaporate, and the aqueous extract solution was frozen and dried under vacuum. The resulting dried extract was weighed into tin cups for isotope analysis. 13C NMR analysis of sapwood acetone/water-soluble extracts obtained in this study confirmed the presence of dihydroquercetin glucoside, procyanidins, and pinoresinol, which may be precursors for the later formation of heartwood extractives (Dellus et al., 1997). After the acetone/water extraction, the sample bags were re-dried and re-weighed and then extracted in a beaker with 75 ml of distilled water in a water bath at 95 1C for 8 h. Water was added as needed to compensate for evaporation. At the end of the extraction, the aqueous extract solution was decanted, frozen, and dried under vacuum. The resulting dried extract was weighed into tin cups for isotope analysis. After extraction, the sample bags were re-dried and re-weighed. 13C NMR analysis of the hot water-soluble sapwood extracts indicated that they were mostly carbohydrates such as the simple sugars glucose and xylose. Following extraction, cellulose was isolated from the wood powder samples using the method described by Leavitt and Danzer (1993). Any extractives that may have remained in the wood following the acetone/water and hot-water extractions were removed from the wood by successive extraction steps with toluene/ethanol (2:1), 95% ethanol, and hot water. The samples were delignified in a 70 1C sodium chlorite solution acidified with acetic acid to pH4.0. Fresh sodium chlorite and acetic acid were added over time to maintain pH4.0 and a bright yellow color solution. Delignification was continued until the pH of the solution was stable (about 4 days). The bags were rinsed with distilled water and dried. Any residual hemicelluloses were removed by submerging the bags three times in a 4.25 M sodium hydroxide aqueous solution for 45 min. The rinsed and dried a-cellulose residue was weighed into tin cups for isotope analysis. Extractives, whole wood, extracted wood, and cellulose samples were analyzed for d13C on an isotope ratio mass spectrometer (Delta Plus, Finnigan, Bremen, Germany) interfaced with an elemental analyzer (ESC 4010, Costech, Valencia, CA) located at the Integrated Stable Isotope Research Facility at the Western Ecology Division of the US Environmental Protection Agency, Corvallis, Oregon. All d13C values were expressed relative to the Pee Dee belemnite carbonate (VPDB) standard in % as d13 C ¼ ðRsample =Rstandard 1Þ 1000 where R is the ratio of 13C–12C atoms of the sample and the standard PDB. Measurement precision was 0.05% for d13C, determined as the average standard deviation of replicate analyses.
127
Heartwood analysis Heartwood samples from the same trees were also analyzed, as part of a separate analysis involving the same six wood disks. Samples of three consecutive annual increments were separated with a chisel, starting from the pith and continuing to the heartwood/sapwood boundary of the radial strips. Six to 14 samples, comprising 18–42 years of growth, were collected from each disk, depending on the size and proportion of heartwood in each disk. Purified holocellulose (NaOH extraction step inadvertently omitted, but this step is only necessary for d18O of cellulose; holo- and a-cellulose have similar d13C values (Leavitt and Danzer, 1993; Borella et al., 1998) and acetone/watersoluble extractives were prepared from each sample and analyzed as described above. Because the extractive component of Douglas-fir heartwood is almost entirely dihydroquercetin (Dellus et al., 1997), which is effectively removed using the acetone/water extraction method (Foo et al., 1992), a separate hot-water extraction step was not included for the heartwood samples. Extracted wood and whole wood also were not collected in the heartwood analysis because the mass balance and isotope comparison of the various components was not the primary objective of that study (Taylor et al., 2007).
Extractive content analysis The extractions described for the sapwood and heartwood studies were intended to remove the bulk of the extractives soluble in the various solvents. Those procedures involved only a single extraction, so it is probable that soluble extractives remained in the wood after the extraction. Furthermore, there may have been extractives present that were not soluble in those solvents. To estimate the total amount of extractable materials present in the wood, the quantitative total extractive content of the sapwood and heartwood from each tree was determined on adjacent radial strips of wood. Samples (10 mm 30 mm) comprising the entire sapwood or heartwood were ground in a Wiley mill (Arthur Thomas Co., Philadelphia, PA) to pass a 20 mesh screen. Samples of the wood powder (1.5 g) were placed in weighed filter bags, oven-dried at 103 1C for 14 h, and re-weighed. The bags were then extracted according to the ASTM standard method for the preparation of extractive-free wood D1105-84 (ASTM, 1996), which involves successive extraction steps with toluene/ethanol (2:1), 95% ethanol, and hot water. The extracted samples were oven-dried at 103 1C for 24 h and re-weighed. Total extractive content of the heartwood and sapwood of each tree was determined as the mass lost from the sample and expressed as a percentage of the oven-dry mass.
ARTICLE IN PRESS 128
A.M. Taylor et al. / Dendrochronologia 26 (2008) 125–131
Data analyses Linear regression models with an autoregressive covariance structure of the relationship between cellulose, wood, and extractives d13C values were created using the mixed procedure in SAS (Version 9.1.3, SAS Institute Inc. Cary, NC, USA) for all trees.
Results Extractives in the sapwood were a relatively small component of the wood (Table 1), 6% on average from the extractive content analysis. The two extractions used for isotope analysis recovered approximately 70% of the total sapwood extractives (sapwood and heartwood analyses). The concentration of extractives in the heartwood was about twice as high as in the sapwood. The d13C values of the cellulose, and the other wood components within the individual trees varied over a range of about 2% (Table 2). The carbon isotope ratios, and the patterns of isotopic variation over time, were different among the trees sampled. Given that we did not monitor the trees in the past, we have no explanation for this variation and this variation was not the focus of this study. However, the cellulose isotope ratios (average ¼ 23.39%) were consistently less negative than the other components. The carbohydrate (hot water-soluble) extractives were about 0.8% Table 1.
more negative than the cellulose. The whole and extracted wood values were similar, at about 1.5% more negative than the cellulose. The acetone–watersoluble extractives had d13C values that were substantially more negative than the other wood components (average ¼ 27.69%). Cellulose d13C values in sapwood were correlated significantly with those of both acetone/water-soluble extractives and the carbohydrate extractives (Fig. 1), explaining 41% and 55% of the variation, respectively (Table 3). In addition, the slope was not significantly different from one for both relationships, indicating that a 1% shift in extractives was matched by a 1% shift in the cellulose. The d13C values of the whole wood and extracted wood were both very strongly correlated with the d13C values of cellulose in the same samples, with R2 over 0.9 and slopes not significantly different from one. Cellulose and acetone/water-soluble extractives also were correlated in the heartwood samples, but the relationship was weaker and the slope was significantly o1, where a 1% shift in the cellulose was associated with only a 0.3% shift in the heartwood extractives.
Discussion Although cellulose remains the standard material in the analysis of tree-ring isotopes, the data in this study suggest that isolation of cellulose from whole-wood Douglas-fir samples may not be necessary for all d13C
Extractives content of Douglas-fir wood samples Fraction of whole wood (%) Average
Sapwood analysis (n ¼ 24) Hot water-soluble extractives Acetone/water-soluble extractives Total extractives
Std. deviation
Max
Min
1.41 3.13 4.53
0.42 0.79 0.87
2.39 4.84 6.32
0.64 1.90 2.83
Total extractives content using ASTM method (n ¼ 6) Sapwood 6.31 Heartwood 13.56
0.67 1.27
7.37 15.16
5.71 11.65
Table 2.
Carbon isotope composition of extractives, cellulose and wood from Douglas-fir sapwood samples, n ¼ 24 d13C (%)
Cellulose Hot water-soluble extractives Extracted wood Whole wood Acetone/water-soluble extractives
Average
Std. deviation
Max
Min
23.39 24.16 24.70 24.87 27.69
0.54 0.77 0.53 0.58 0.83
22.45 23.24 23.81 24.00 25.76
24.80 26.45 25.80 26.13 29.39
ARTICLE IN PRESS A.M. Taylor et al. / Dendrochronologia 26 (2008) 125–131
-25
Sapwood acetone/water-soluble extractives
-27
Extractives δ13C (‰)
-29
-25
Heartwood acetone/water-soluble extractives
-27
-29
-25
-27
-29
Tree A Tree B Tree C Tree D Tree E Tree F
Sapwood hot water-soluble extractives
-25
-24
-23
-22
-21
Cellulose δ13C (‰)
Fig. 1. The correlation of cellulose with extractives in the sapwood and heartwood of wood samples from six Douglas-fir trees.
129
analysis, particularly for sapwood. There were strong correlations between d13C values of cellulose and wholewood samples taken from the same wood increment, and the slope was similar to one, meaning that an isotopic shift in cellulose was matched by an isotopic shift in whole wood of a similar magnitude. This similarity in isotopic variation is likely because of the relatively low concentrations of extractives compared with the cell-wall polymers, and further because the extractives d13C values also vary directly with cellulose d13C values in the wood. This correlation between extractives, including the soluble sugars, and cellulose indicates that some of these reserves were likely deposited at the time of wood formation rather than having been deposited later (Taylor et al., 2007). Thus, extractives present in carbon isotope analyses may not significantly alter patterns of annual change that are apparent in purified cellulose samples. The close correlations observed here in the d13C values of extracted wood and cellulose samples further suggests that the other cell wall components – hemicelluloses and lignin – are isotopically closely correlated with cellulose. Given these correlations, the use of whole-wood Douglas-fir sapwood samples may be acceptable in place of the purified cellulose samples that require significant sample preparation. However, our results may not be applicable for fossil samples where the chemistry may have been altered over time. Testing the correlation between whole wood and cellulose on a subset of samples is recommended. The correlation of extractives and cellulose was weaker in the heartwood than in the sapwood. The heartwood also had relatively high concentrations of extractives. Furthermore, there is evidence that the heartwood extractive formation process can be influenced by environmental factors both at the time the (sap)wood rings were originally formed and at the time the sapwood is converted to heartwood (Taylor et al., 2003, 2007). These observations suggest that the potential influence of the isotopic signal of the
Table 3. Generalized least squares estimates from linear regression models with an autoregressive covariance structure of extractives and wood d13C values on cellulose d13C values for all trees together Wood component regressed on cellulose
R2
Slope (std. error)
Mean square error
p-Value Slope ¼ 0
Slope ¼ 1
Sapwood samples n ¼ 24 Acetone/water soluble extractives Hot water-soluble extractives Extracted wood Whole wood
0.41 0.55 0.91 0.91
0.98 0.81 0.84 0.89
(0.27) (0.28) (0.08) (0.08)
0.43 0.31 0.03 0.03
o0.01 0.01 o0.01 o0.01
0.94 0.50 0.06 0.18
Heartwood samples n ¼ 42 Acetone/water-soluble extractives
0.34
0.29 (0.10)
0.33
0.01
o0.01
ARTICLE IN PRESS 130
A.M. Taylor et al. / Dendrochronologia 26 (2008) 125–131
extractives should be considered when analyzing unextracted or whole-wood heartwood samples, especially if comparisons are to be made with sapwood samples. For tree-ring studies where samples span both heartwood and sapwood, researchers should consider extraction to avoid this potentially confounding influence. At the very least, researchers may wish to test if correlations between cellulose and whole wood differ between sapwood and heartwood for their study individuals. This study is the first to our knowledge that has measured the isotopic ratios of extractives directly and compared them with other wood components. However, the finding that cellulose and whole wood are isotopically correlated is consistent with previous observations in the wood of beech (Fagus sylvatica) (Elhani et al., 2005), mangrove (Rhizophora mucronata) sapwood (Verheyden et al., 2005), Eucalyptus globulus, Pinus radiata and P. pinaster (MacFarlane et al., 1999), oak (Quercus spp.) (Borella et al., 1998), six South African tree species (West et al., 2001), and in 44 angiosperm and gymnosperm species (Harlow et al., 2005). Still, the close relationship of d13C values between cellulose and whole-wood samples may not exist in all species or wood tissues. Total extractives content in Douglas-fir (around 6–12%) is moderate compared with some species, which can range as high as 30% and is generally greater in hardwoods than in softwoods (Hillis, 1987). Species with very high-extractives levels may not have the very close correlation between whole wood and cellulose that was observed in this study. Other species may also have more mobile and/or dynamic extractives, which may be less well correlated with cellulose, especially in the sapwood of hardwoods where annualscale depletion of starch reserves has been observed (Barbaroux and Breda, 2002).
Conclusion Stable carbon isotope signatures of wood, extractivefree wood, and cellulose in Douglas-fir sapwood are highly correlated. Cellulose and extractives in Douglasfir heartwood also are correlated. Given the relatively low concentration of extractives in the wood and the correlation of d13C values of extractives and cellulose of the rings within the same sample, in some cases it may not be necessary to isolate cellulose from increments of Douglas-fir wood for carbon isotope tree-ring analysis.
Acknowledgments Our thanks to Dr. Joe Karchesy (OSU College of Forestry) for useful discussions of extractive analysis, to Dr. Mike Newton (OSU College of Forestry) for advice
and wood samples, to Warren Evans (EPA Integrated Stable Isotope Research Facility) for cellulose extraction, to Bill Griffis (EPA Integrated Stable Isotope Research Facility) for processing the isotope samples, and to Dr. E. Henry Lee (EPA Integrated Stable Isotope Research Facility) for help with statistical analysis. We also thank Dr. Nate McDowell for comments on an earlier version of this manuscript. This work was supported by a USDA Special Grant to Oregon State University for Wood Utilization Research, by a Fellowship from the Weyerhaeuser Foundation, and by the United States Environmental Protection Agency. This manuscript has been subjected to EPA peer and administrative review and has been approved for publication as an EPA document. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
References ASTM, 1996. Standard method for the preparation of extractive-free wood. Designation D1105-84. Annual Book of ASTM Standards, vol. 04-01 Wood. American Society for Testing and Materials. West Conshohocken, PA. Barbaroux, C., Breda, N., 2002. Contrasting distribution and seasonal dynamics of carbohydrate reserves in stem wood of adult ring-porous sessile oak and diffuse-porous beech trees. Tree Physiology 22, 1201–1210. Borella, S., Leuenberger, M., Saurer, M., Siegwolf, R., 1998. Reducing uncertainties in d13C analysis of tree rings: pooling milling, and cellulose extraction. Journal of Geophysics Research 103 (D16), 19519–19526. Damesin, C., Lelarge, C., 2003. Carbon isotope composition of current-year shoots from Fagus sylvatica in relation to growth, respiration and use of reserves. Plant Cell Environment 26, 207–219. Dellus, V., Scalbert, A., Janin, G., 1997. Polyphenols and colour of Douglas fir heartwood. Holzforschung 51, 291–295. Elhani, S., Guehl, J-M., Nys, C., Picard, J.-F., Dupouey, J.-L., 2005. Impact of fertilization on tree ring d15N and d13C in beech stands: a retrospective analysis. Tree Physiology 25, 1437–1446. Foo, L.Y., Helm, R., Karchesy, J., 1992. [50 ,50 ]-bisdihydroquercetin: a B-ring linked biflavonoid from Pseudotsuga menziesii. Phytochemistry 31 (4), 1444–1445. Harlow, B.A., Marshall, J.D., Robinson, A.P., 2005. A multispecies comparison of d13C from whole wood, extractivefree wood and holocellulose. Tree Physiology 26, 767–774. Helle, G., Schleser, G.H., 2004. Beyond CO2-fixation by Rubisco – an interpretation of 13C/12C variations in tree rings from novel intra-seasonal studies on broad-leaf trees. Plant Cell Environment 27, 367–380. Hillis, W.E., 1987. Heartwood and Tree Exudates. Springer, Berlin, p. 268. Hillis, W.E., Hasegawa, M., 1963. The formation of polyphenols in trees. 1. Administration of 14C glucose and subsequent distribution of radioactivity. Phytochemistry 2, 195–199.
ARTICLE IN PRESS A.M. Taylor et al. / Dendrochronologia 26 (2008) 125–131
Kutscha, N.P., Sachs, I.B., 1962. Color tests for differentiating heartwood and sapwood in certain softwood tree species. Report 2246, Forest Products Laboratory, Madison, WI, p. 16. Leavitt, S.W., Danzer, S.R., 1993. Methods for batch processing small wood samples to holocellulose for stablecarbon isotope analysis. Analysis Chemistry 65, 87–89. Leavitt, S.W., Long, A., 1989. Intertree variability of D13C in tree rings. In: Rundel, P.W., Ehleringer, J.R., Nagy, K.A. (Eds.), Stable Isotopes in Ecological Research. Springer, New York, pp. 95–104. Livingston, N.J., Spittlehouse, D.L., 1993. Carbon isotope fractionation in tree rings in relation to the growing season water balance. In: Ehleringer, J.R., Hall, A.E., Farquhar, G.D. (Eds.), Stable Isotopes and Plant Carbon–Water Relations. Academic Press, New York, pp. 141–153. Livingston, N.L., Spittlehouse, D.L., 1996. Carbon isotope fractionation in tree rings early and late wood in relation to intra-growing season water balance. Plant Cell Environment 19, 768–774. Loader, N.J., Robertson, I., McCarroll, D., 2003. Comparison of stable carbon isotope ratios in the whole wood, cellulose and lignin of oak tree-rings. Palaeogeography Palaeoclimatology 196, 395–407. MacFarlane, C., Warren, C.R., White, D.A., Adams, M.A., 1999. A rapid and simple method for processing wood to crude cellulose for analysis of stable carbon isotopes in tree rings. Tree Physiology 19, 831–835. McArdle, R.E., Meyer, W.H., Bruce, D., 1961. The yield of Douglas-fir in the Pacific Northwest. US Department of Agriculture Technical Bulletin 201. O’Leary, M.H., 1993. Biochemical basis of carbon isotope fractionation. In: Ehleringer, J.R., Hall, A.E., Farquhar, G.D. (Eds.), Stable Isotopes and Plant Carbon–Water Relations. Academic Press, San Diego, pp. 19–26.
131
Scartazza, A., Mata, C., Matteucci, G., Yakir, D., Moscatello, S., Brugnoli, E., 2004. Comparisons of d13C of photosynthetic products and ecosystem respiratory CO2 and their responses to seasonal climate variability. Oecologia 140, 340–351. Schmidt, H.-L., Gleixner, G., 1998. Carbon isotope effects on key reactions in plant metabolism and 13C patterns in natural compounds. In: Griffiths (Ed.), Stable Isotopes: Integration of Biological, Ecological and Geochemical Processes. BIOS Scientific Publishers Ltd., Oxford, pp. 13–26. Sjostrom, E., 1993. Wood Chemistry: Fundamentals and Applications. Academic Press, San Diego, p. 293. Taylor, A.M., Gartner, B.L., Morrell, J.J., 2002. Heartwood formation and natural durability—a review. Wood Fiber Science 34 (4), 587–611. Taylor, A.M., Gartner, B.L., Morrell, J.J., 2003. Co-incident variations in growth rate and extractive concentration in Douglas-fir. Forest Ecology and Management 186, 257–260. Taylor, A.M., Gartner, B.L., Morrell, J.J., Brooks, J.R., 2007. Radial patterns of carbon isotopes in the xylem extractives and cellulose of Douglas-fir. Tree Physiology 27, 921–927. Verheyden, A., Roggeman, M., Bouillon, S., Elskens, M., Beeckman, H., Koedam, N., 2005. Comparison between d13C of a-cellulose and bulk wood in the mangrove tree Rhizophora mucronata: implications for dendrochemistry. Chemistry and Geology 219, 275–282. Warren, C.R., McGrath, J.F., Adams, M.A., 2001. Water availability and carbon isotope discrimination in conifers. Oecologia 127, 476–486. West, A.G., Midgley, J.J., Bond, W.J., 2001. The evaluation of d13C isotopes of trees to determine past regeneration environments. Forest Ecology and Management 147, 139–149.