Tree-ring δ<sup>15</sup>N of Qinghai spruce in the central Qilian Mountains of China: Is pre-treatment of wood samples necessary?

doi:10.1007/s40333-022-0065-1

Journal of Arid Land

2022, Vol. 14

Issue (6): 673-690 DOI: 10.1007/s40333-022-0065-1

Research article

Tree-ring δ¹⁵N of Qinghai spruce in the central Qilian Mountains of China: Is pre-treatment of wood samples necessary?

WANG Ziyi¹, LIU Xiaohong¹^,²^,³^,^*(

), WANG Keyi¹^,⁴, ZENG Xiaomin¹, ZHANG Yu¹, GE Wensen¹, KANG Huhu², LU Qiangqiang¹

¹School of Geography and Tourism, Shaanxi Normal University, Xi'an 710119, China
²State Key Laboratory of Cryospheric Science, Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou 730000, China
³Qilian Mountain National Park Research Center (Qinghai), Xining 810008, China
⁴School of Earth System Science, Tianjin University, Tianjin 300072, China

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Abstract

A knowledge of the tree-ring stable nitrogen isotope ratio (δ¹⁵N) can deepen our understanding of forest ecosystem dynamics by indicating the long-term availability, cycling and sources of nitrogen (N). However, the radial mobility of N blurs the interannual variations in the long-term N records. Previous studies of the chemical extraction of tree rings before analysis had produced inconsistent results and it is still unclear whether it is necessary to pre-treat wood samples from specific tree species to remove soluble N compounds before determining the δ¹⁵N values. We compared the effects of pre-treatment with organic solvents and hot ultrapure water on the N concentration and δ¹⁵N of tree rings from endemic Qinghai spruce (Picea crassifolia) growing in the interior of the central Qilian Mountains, China, during the last 60 a. We assessed the effects of different preparation protocols on the removal of the labile N compounds and investigated the need to pre-treat wood samples before determining the δ¹⁵N values of tree rings. Increasing trends of the tree-ring N concentration were consistently observed in both the extracted and unextracted wood samples. The total N removed by extraction with organic solvents was about 17.60%, with a significantly higher amount in the sapwood section (P<0.01). The δ¹⁵N values of tree rings decreased consistently from 1960 to 2019 in both the extracted and unextracted wood samples. Extraction with organic solvents increased the δ¹⁵N values markedly by about 5.2‰ and reduced the variations in the δ¹⁵N series. However, extraction with hot ultrapure water had little effect, with only a slight decrease in the δ¹⁵N values of about 0.5‰. Our results showed that the radial pattern in the inter-ring movement of N in Qinghai spruce was not minimized by extraction with either organic solvents or hot ultrapure water. It is unnecessary to conduct hot ultrapure water extraction for the wood samples from Qinghai spruce because of its negligible effect on the removal of the labile N. The δ¹⁵N variation trend of tree rings in the unextracted wood samples was not influenced by the heartwood-sapwood transition zone. We suggest that the δ¹⁵N values of the unextracted wood samples of the climate-sensitive Qinghai spruce could be used to explore the ecophysiological dynamics while focusing on the long-term variations.

Key words： tree rings stable nitrogen isotope ratio (δ¹⁵N) nitrogen concentration solvent-extracted wood water-extracted wood wood pre-treatment Qinghai spruce Qilian Mountains

Received: 12 November 2021 Published: 30 June 2022

Corresponding Authors: LIU Xiaohong E-mail: liuxh@lzb.ac.cn

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	WANG Ziyi
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Cite this article:

WANG Ziyi, LIU Xiaohong, WANG Keyi, ZENG Xiaomin, ZHANG Yu, GE Wensen, KANG Huhu, LU Qiangqiang. Tree-ring δ¹⁵N of Qinghai spruce in the central Qilian Mountains of China: Is pre-treatment of wood samples necessary?. Journal of Arid Land, 2022, 14(6): 673-690.

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http://jal.xjegi.com/10.1007/s40333-022-0065-1 OR http://jal.xjegi.com/Y2022/V14/I6/673

Fig. 1 Flow chart of the wood pre-treatment methods for the determination of the stable nitrogen isotope ratio (δ¹⁵N) in tree rings from the endemic Qinghai spruce. Three pre-treatment methods were used to prepare the wood samples for the measurements of the nitrogen (N) concentration and δ¹⁵N in tree rings: (1) wood extracted with organic solvents (solvent-extracted wood); (2) wood extracted with hot ultrapure water (water-extracted wood); and (3) unextracted wood (bulk wood).

Fig. 2 N concentration (a) and δ¹⁵N values (b) in tree rings from Qinghai spruce as a function of time determined by the three different pre-treatment methods: wood extracted with organic solvents (solvent-extracted wood), wood extracted with hot ultrapure water (water-extracted wood) and unextracted wood (bulk wood). The vertical gray-shaded area indicates the heartwood-sapwood transition zone around the time period of 1995-1998.

Fig. 3 Comparisons of the time series of the tree-ring N concentration and δ¹⁵N values for the extracted wood (solvent-extracted wood and water-extracted wood) and unextracted wood (bulk wood) during the time period of 1960-2019, as well as relationships of the Z-scores of the N concentration and δ¹⁵N values between the extracted wood (solvent-extracted and water-extracted wood) and unextracted wood (bulk wood). (a and b), the N concentration; (c and d), the δ¹⁵N values. The curves in the left panel were standardized by the Z-scores. The solid lines in the right panel represent the linear regression of each dataset. The 1:1 line is shown by the dashed line. The coefficient of determination (R²) and P-value are also given.

Table 1 Summary statistical analyses among the pre-treatment methods of wood samples

Fig. 4 Box-whisker plots of the tree-ring N concentration (a) and δ¹⁵N values (b), as well as relationships between the N concentration and δ¹⁵N values (c) of the extracted wood (solvent-extracted wood and water-extracted wood) and unextracted wood (bulk wood). The boundaries of the boxes indicate the 25^th and 75^th percentiles. The solid lines are median values, the black dots are mean values and the black squares are outliers. Bars represent the change range of the N concentration or δ¹⁵N values. Different lowercase letters indicate significant differences (P<0.05) between the pre-treatment methods by one-way ANOVA. The solid curves represent the correlations simulated by exponential fitting.

Fig. 5 Box-whisker plots of the changes in the tree-ring N concentration (a) and δ¹⁵N values (b) for the heartwood and sapwood sections in the extracted wood (solvent-extracted wood and water-extracted wood) compared with the unextracted wood (bulk wood), as well as relationships between differences in the N concentration and δ¹⁵N values between the extracted wood and unextracted wood (c). The boundaries of the boxes indicate the 25^th and 75^th percentiles. The solid lines are median values, the black dots are mean values and the black squares are outliers. The bars represent the change range of differences in the N concentration or δ¹⁵N values. Offsets were calculated between the extracted wood and unextracted wood. Different lowercase letters indicate significant differences (P<0.05) between the sapwood and heartwood by the independent sample t-test.

Fig. 6 Offset in the tree-ring δ¹⁵N values (a) and percentage of offset in the N concentration (b) for different tree species after the extraction of bulk wood. Offset in the δ¹⁵N was calculated as the difference between the extracted wood and unextracted wood, so offset in the N concentration did. The percentage of offset in the N concentration was calculated as the quotients of the offset in the N concentration divided by the actual value of the unextracted wood. Tree rings in white oak were extracted with deionized water. Tree rings of other species were extracted with organic solvents. Data were obtained from previous studies as follows (see Table S3 for details): [1], Sheppard and Thompson (2000); [2], Elhani et al. (2003); [3], Elhani et al. (2005); [4], Kwak et al. (2009); [5] Hietz et al. (2010); [6], Larry et al. (2011); [7], Bukata and Kyser (2005); [8], Hart and Classen (2003). S-e, solvent-extracted wood; W-e, water-extracted wood.

Table S1 Summary statistical analyses, including mean sensitivity, 1^st autocorrelation and coefficient of variance of the N concentration and stable nitrogen isotope ratio (δ¹⁵N) in tree rings of the extracted wood (solvent-extracted wood and water-extracted wood) and unextracted wood (bulk wood)

Fig. S1 Trends simulated by the linear regression of the N concentration (a) and δ¹⁵N (c) in tree rings of the extracted wood (solvent-extracted wood and water-extracted wood) and unextracted wood (bulk wood), and trends of the N concentration (b) and δ¹⁵N (d) of the heartwood and sapwood sections in tree rings of the extracted wood (solvent-extracted wood and water-extracted wood) and unextracted wood (bulk wood) from 1960 to 2019. The vertical grey-shaded area indicates the heartwood-sapwood transition zone around the period of 1995-1998.

Table S2 Statistical analyses of the variations in the N concentration and δ¹⁵N in tree rings of the extracted wood (solvent-extracted wood and water-extracted wood) and unextracted wood (bulk wood)

Method	Extraction protocol	Tree species	N concentration	δ¹⁵N	Determination	Reference
Sheppard/ Thompson	S1: 4 h in a 1:1 mixture of toluene/ethanol; S2: 4 h in ethanol; S3: 1 or 4 h in distilled water	Ponderosa pine (Pinus ponderosa) and Douglas-fir	Decrease (about 45.000%)	NA	The variation in N concentration of tree rings was reduced substantially.	Sheppard and Thompson (2000)
		Beech (Fagus sylvatica)	Decrease (about 36.000%, from 0.220% to 0.150%)	Decrease (about 0.4‰)	The interannual resolution of the N and δ¹⁵N was improved, but not all mobile N was removed.	Elhani et al. (2003)
		Beech (F. sylvatica)	NA	Increase (-6.0‰- -5.0‰ to -4.5‰- -3.5‰ in the control trees)	Variation in the δ¹⁵N signal was decreased, but differences between trees before and after treatment were enhanced.	Elhani et al. (2005)
		Red pine (Pinus densiflora)	Decrease (19.400%- 31.600%)	NA	Higher extractable N in the sapwood was removed, while not affecting the overall trend of N.	Kwak et al. (2009)
		Spanish cedar (Cedrela odorata) and big-leaf mahogany (Swietenia macrophylla)	Decrease (not significantly lower)	Increase (about 1.0‰ higher in the treated wood)	The proportion of N extractable in the sapwood was not higher than that in the heartwood.	Hietz et al. (2010)
		Norway spruce (Picea abies)	Decrease	Increase (slight)	No significant effect on the tree-ring N and δ¹⁵N.	Tomlinson et al. (2014)
		Western redcedar (Thuja plicata) and Douglas-fir	NA	Elevated ¹⁵N signal found 10 a before the ¹⁵N-labeled	The mobile N in tree rings was eliminated ineffectively.	Bunn et al. (2017)
Simplified Sheppard/ Thompson	Keep overnight in a 1:1 mixture of toluene/ethanol	Japanese black pine (Pinus thunbergii)	NA	Increase of 3.0‰ in the control trees, and decrease of -7.0‰ in the sites with the high N	Significant effect on the tree-ring δ¹⁵N under high-N conditions.	Larry et al. (2011)
Altered Sheppard/ Thompson	S1: in 1:1 mixture of benzene/methanol; S2: 12 or 48 h in acetone; S3: 1 h in deionized (DI) water	Beech (Fagus grandifolia) and red spruce (Picea rubens)	Decrease (8.000% and 7.000%, respectively)	Decrease (0.4‰ and 0.3‰, respectively)	The difference is always within the analytical error, with no significant change in the N or δ¹⁵N values or temporal trends.	Doucet et al. (2011)
Methods	Extraction protocol	Tree species	N concentration	δ¹⁵N	Determination	Reference
Water-bath	Fresh DI water (shaking 4 times each for 3 d)	Red oak (Quercus rubra) and white oak (Quercus alba)	Decrease (10.500% in the sapwood)	Increase (0.6‰ in the sapwood)	No significant change in the δ¹⁵N.	Bukata and Kyser (2005)
Holocellulose extraction	S1: 16-18 h in a 2:1 mixture of toluene/ ethanol; S2: 16-18 h in ethanol; S3: 6 h in DI water	Ponderosa pine (P. ponderosa)	Decrease (about 10.000%)	Decrease (2.0‰-3.0‰ in the control trees; 4.0‰-34.0‰ in the labeled trees)	Relatively small and consistent effect on the control trees, while relatively large and variable effect on the highly ¹⁵N.	Hart and Classen (2003)

Table S3 Retrospect of extraction protocols used in the tree-ring δ¹⁵N analysis from previous studies

Fig. S2 Growth pattern of Qinghai Spruce during the period of 1840-2019. Variations in the tree growth were estimated as the basal area increment (BAI) during the period of 1840-2019. The orange line is smoothed using a 20 a low-pass Fast Fourier Transform (FFT) filter to emphasize the low-frequency variations.

Fig. S3 Temporal trends of annual mean temperature (a) and total precipitation (b) from 1958 to 2019 based on averaging data from two nearby meteorological stations (Qilian and Minle) of the study area. The dashed lines represent the trends simulated by the linear regressions. Annual variation is represented by the slope of the trend line. The coefficient of determination (R²) and significance P-value are also provided.


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