Supplemental Material
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Influence of Forest Disturbance on Stable Nitrogen Isotope Ratio
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Profiles
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Introduction
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Many studies have suggested that ecosystem δ15N profiles—from vegetation foliage, roots,
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and wood, through the forest floor and soil—are indicative of ecosystem N status and net N
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transformations (Garten, 1993; Pardo et al., 2002). Vegetation δ15N values are affected by the
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source of N (Högberg et al., 1999; Pardo et al., 2006) as well as by fractionation that occurs
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during transfer of N from mycorrhizal fungi to plants (Evans, 2001; Hobbie et al., 2000), and
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during incorporation into biomass (Pardo et al. 2013). In woody structures (e.g., tree-rings),
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studies conclude that δ15N values are both reliable (Beghin et al. 2011; Bukata and Kyser 2005),
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and unreliable indicators of N availability (Hart and Classen 2003; Sheppard et al. 2001;
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Sheppard and Thompson 2000). Variable results may be a product of how tree-ring samples are
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processed for analysis. Sheppard and Thompson (2000) suggest that if mobile N compounds are
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not removed, variability in wood N concentrations make inferring long-term patterns in soil N
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availability difficult. We tested the effects of several wood pre-treatment methods on wood δ15N
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values and total N concentration.
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Materials and Methods
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We tested three extraction methods to remove potentially mobile or soluble N from wood,
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leaving only structurally-bound N prior to δ15N determination. Each method was tested on wood
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from three tree species: Robinia pseudoacacia L. (Black Locust), Quercus prinus L. (Chestnut
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oak), and Liriodendron tulipifera L. (Tulip poplar). Wood samples were collected using an
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increment borer to a depth of 6 inches. Air dried cores were pulverized to a powder with a ball
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mill (SPEX 8000-D Mixer/Mill, SPEX SamplePrep, LLC, Metuchen, NJ USA), making a single
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homogeneous sample of each species (n = 3). Each species by extraction method combination
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was run in triplicate, using 2 g of the homogenized wood sample for each. Extraction methods
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included: 1) boiling water, soak pulverized wood in 5 ml of hot boiled deionized H2O (H2Odi)
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for 5 min, then vacuum filter and rinse three times with 5 ml of H2Odi (Hart and Classen, 2003);
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2) toluene:ethanol, soak pulverized wood in a 50:50 (v/v) toluene:ethanol solution for 5 min,
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vacuum filter, rinse one time with 5 ml of ethanol, and rinse three times with 5 ml of H2Odi
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(Sheppard and Thompson, 2000); and 3) peroxide adjusted to pH 12.0, soak pulverized wood for
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5 min in commercial 3% hydrogen peroxide, adjusted to pH 12.0 with NaOH, vacuum filter, and
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rinse three times with H2Odi (Sheppard personal communication, 2003). Ground, extracted,
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rinsed, wood samples were left on filters and oven-dried at 50° C to a constant weight (about 1
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hour). Three replicates of untreated, dried, ground reference sample of each wood species were
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also analyzed as controls. Subsamples (30 mg) of pre-treated and control wood samples were
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weighed into tin capsules for δ15N natural abundance and total N content analysis. Analyses were
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conducted at the University of California, Davis, Stable Isotope Facility on an isotope ratio mass
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spectrometer (PDZ Europa 20-20, Sercon Ltd., Cheshire, UK) interfaced to an elemental
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analyzer (PDZ Europa ANCA-GSL). Instrument precision was 0.07 ‰ and 0.07 µg N per
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sample. N isotope ratios are presented in delta notation:
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𝛿 = (𝑅𝑠𝑎𝑚𝑝𝑙𝑒−𝑅𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑)/𝑅𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑) ∙ 1000,
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where Rsample is the 15N/14N ratio of the sample and Rstandard is the 15N/14N ratio of the atmospheric
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N2 standard (0.00367‰).
(1)
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Statistical Analysis
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We compared wood extraction methods on δ15N values and total N concentration for each
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treatment in each tree species in a single factor analysis of variance (PROC GLM, v9.3 SAS
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(SAS, 2013)). Treatment was a fixed effect with four levels (H2O, toluene:ethanol, H2O2, or
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untreated). Each tree species was analyzed separately (black locust, white oak and tulip poplar).
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We used a post-hoc means separation test (Tukey-Kramer adjusted means) to determine
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significant differences among treatment. Significant differences of P < 0.05 are reported. We
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expected that mean wood N concentration would be higher in the untreated and lower in the
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extracted wood due to removal of mobile N compounds; we selected the extraction method that
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resulted in the lowest N concentration i.e., the greatest N removal, relative to untreated wood to
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use in processing field samples.
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Results
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Analysis of wood tissue showed that extraction method affected the total N concentration, but
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not δ15N (Table S1). Although the extractions removed a significant amount of N, methods were
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not consistent in the amount of N removed from each species. The greater the extractant polarity,
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the greater the amount of N removed. The amount of N removed with boiling H2O and the
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toluene:ethanol solution was minimal, (1–10%), followed by extraction with H2O2 (16–21%).
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Because our goal was to remove mobile N from wood samples, we selected the 3% H2O2 (pH =
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12) extractant as a wood pre-treatment to test the response of wood δ15N to forest management
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and N2-fixation.
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Table S1. Analysis of variance of wood δ15N and total N concentration (g kg-1) responses to pre-
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treatment extraction with hot boiled deionized H2O, 50:50 (v/v) toluene:ethanol solution, 3%
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hydrogen peroxide (adjusted to pH 12), and untreated wood. Tree species tested included
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Robinia pseudoacacia L. (Black Locust), Quercus prinus L. (Chestnut oak), and Liriodendron
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tulipifera L. (Tulip poplar). Shown is the effect of extraction pre-treatment, F-value and the
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probability of a value greater than F (Prob > F).
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a) Wood δ15N
Black Locust
Chestnut Oak
Tulip Poplar
F-value
0.87
1.60
1.83
Prob > F
0.50
0.26
0.22
b) Wood N
Black Locust
Chestnut Oak
Tulip Poplar
F-value
102.46
Prob > F
< 0.001
181.16
< 0.001
80.71
<0.001
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Table S2. Effect of three extraction procedures on mean (n = 3, SE) N concentration (mg kg-1)
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and δ15N (‰) of wood tissue collected at DBH (1.37 m) from trees approximately 25 years old
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(about 20 rings). Sample was ground to a powder with a SPEX 8000-D Mixer/Mill (SPEX
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SamplePrep, LLC, Metuchen, NJ USA) prior to extraction treatment. Within species, extraction
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methods followed by different lowercase letters are significantly different ( ≤ 0.05). Tree
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species tested included Robinia pseudoacacia L. (Black Locust), Quercus prinus L. (Chestnut
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oak), and Liriodendron tulipifera L. (Tulip poplar).
N concentration
δ15N
(mg kg-1)
(‰)
None †
1902 (5.3) a
-0.6 (0.11)
Boiling H2O ‡
1880 (25.4) a
-0.5 (0.13)
Toluene/EtOH (50:50) §
1853 (11.7) b
-0.5 (0.03)
H2O2 pH 12 ¶
1591 (3.1) b c
-0.6 (0.06)
None
1015 (6.1) a
-1.3 (0.06)
Boiling H2O
908 (8.6) b
-1.0 (0.16)
Toluene/EtOH
913 (2.7) b
-1.1 (0.14)
H2O2 pH 12
833 (2.5) c
-0.9 (0.18)
None
735 (3.3) a
-2.3 (0.23)
Boiling H2O
708 (14.6) b
-2.8 (0.31)
Toluene/EtOH
686 (1.9) b
-2.9 (0.05)
H2O2 pH 12
577 (2.6) c
-2.8 (0.03)
Treatment
Black Locust
Chestnut Oak
Tulip Poplar
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† No pretreatment.
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