SAMPLING DESIGN
This study was performed at the Hubbard Brook Experimental Forest
(HBEF) in central New Hampshire, USA. Most of the HBEF is northern
hardwood forest, dominated by sugar maple (Acer saccharum Marsh.),
American beech (Fagus grandifolia Erhr.), and yellow birch (Betula
alleghaniensis Britt.). Together, these three species accounted for
86% of the above-ground living tree biomass in a similarly situated
stand at the HBEF when this study was initiated in 1991 (Siccama et
al. 2007). This work was conducted in the area of the south-facing
experimental watersheds 1-6 at the HBEF. In July 1990 and May 1991, 71
trees of the three dominant species were felled with a chain saw. The
trees were approximately 50-70 years old. From each felled tree, two
adjacent segments of the bole, each approximately 1.5-m long (range:
0.99 – 2.22 m), were isolated with a chain saw. After measuring the
length and the diameter at each end, one of these segments was then
placed on the forest floor under fully intact forest canopy. The
incubated bole segments were placed on sloping ground, approximately
perpendicular to contours, in two similar stands approximately 200 m
apart. The other ‘fresh’ segment was taken to the lab for sampling.
Each of the 71 samples incubated in situ was therefore paired with a
fresh sample from the same tree.
INITIAL LABORATORY WORK
After measuring the dimensions of the fresh bole-segments, disks about
8-10 cm thick were cut from one end of each segment. The bark was
separated from the wood of each disk, and both were dried at 80o C to
constant weight. Subsamples were collected from the fresh wood disks
by drilling from the side to the center with a 2.5-cm drill bit. The
dried bark samples and the wood shavings were ground in a Wiley mill.
Subsamples from the same log were composited for chemical analysis.
FIELD COLLECTION PROCEDURES
Incubated boles were collected from the field in April, 1993 (T1; 2Y);
May, 1997 (T2; 6Y); May-July, 2001 (T3; 10Y); July, 2007 (T4; 16Y) and
July of 2015 and 2016 (T5; 25Y). Three boles of each species (nine
total) were collected in 1993, 1997 and 2001. In 2007, three beech,
six maple and six birch boles were collected. In 2015/2016, all
identifiable remaining boles were collected. After removing any
surface litter the bole was gently rolled onto a sampling tarp, and
any loose material was collected in a bag. The log was placed in the
bag and returned to the laboratory for processing.
POST-INCUBATION SAMPLE PREPARATION
The incubated boles were dried at 80 oC to constant weight, which was
recorded. The dry boles were laid out on kraft paper, along with the
loose debris collected in the field. After measuring the dimensions of
each log, we then removed the bark from the bole and gathered the bark
fragments from the debris. The mass of the bark and the wood
(log+loose debris) was recorded. Subsamples of the log were collected
by drilling to the center of the log with a 2.5-cm bit. Log, loose
debris, and bark samples were ground in a Wiley mill for chemical
analysis. A wood sample for chemical analysis was created by mixing
loose debris and log samples proportionally by mass. Two beech boles
collected in 2001 (10Y) had so little bark remaining that it was not
sampled and judged to be completely decomposed. The samples collected
in 1997 (6Y) were inadvertently discarded after mass determination, so
we have no chemical data for those samples. In 2015/2016, there was so
much fine loose rotten wood and bark that a separate sample (mixed) is
included in the archive. In total, 42 incubated boles have been
collected from the field to date. A total of 106 bark samples (54
fresh, 52 decomposed), 108 wood samples (54 fresh, 54 decomposed), and
21 mixed tissue samples (all from T5) were collected and stored in
glass jars.
CHEMICAL ANALYSIS
Cross polarization with magic-angle spinning (CPMAS) 13C NMR analyses
were performed on a Bruker AVANCE 300 spectrometer, operating at 75.47
MHz for 13C. Spectra were acquired using a 1-ms contact time,
acquisition time of 17.5 ms, and a recycle delay of 3 s. The samples
were spun in 7-mm zirconia rotors with Kel-F caps. A study performed
to examine the effects of spinning speed determined that there were no
significant differences between samples run at 5kHz and samples run at
7 kHz. This data set includes results from samples were spun at both
speeds. The CPMAS experiments were set up using glycine.
Additional chemical analyses on these samples, including elemental
carbon, nitrogen, and hydrogen, are available in a companion data set
(Johnson et al. 2019).
DATA PROCESSING
The CPMAS spectra were zero-filled to 8192 data points and processed
with 50-Hz Lorentzian line broadening. Chemical shift values were
internally referenced to the anomeric C peak at 105 ppm. After phasing
and baseline correction, we integrated the spectra between 280 and –50
ppm. All processing was performed using the mNova data processing
software tool (Mestrelab Research, S.L., Santiago, Spain).
Assignments for various spectral regions are given in Table 1 and were
based on those used by Baldock and Smernik (2002). The 45–60 ppm
region includes signals from methoxyl and N-alkyl C. However, since
the N content of wood and bark is generally low, we attributed the
intensity in this region solely to methoxyl C. We accounted for the
effect of spinning sidebands as described in Table 1. The integrated
area between 0 and –50 ppm was never more than 1.8% of total spectral
area. Thus, we estimated the fraction of C associated with various
structures by dividing the integrated intensities shown in Table 1 by
the spectral area between 0 and 280 ppm.
Table 1. Chemical shift assignments for carbon structures and spinning
sidebands (SSB); assignments are based on those of Baldock and Smernik
(2002).
Chemical shift region (ppm) Assignment Integrated intensity
calculation (ppm)
5-kHz spinning speed
0–45 Alkyl C (0–45)
45–60 Methoxyl C (45–60)
60–94 O-alkyl C (60–94) – (215–230)
94–110 Di-O-alkyl C (94–110) – (230–250)
110–144 Aryl and unsaturated C (110–144)
144–165 O-aryl C (144–165) + 2(215–230)
165–190 Carbonyl and amide C (165–190) + 2(230–250)
190–215 Ketone C (190–215)
215–230 O-aryl SSB
230–250 Carbonyl SSB
250–280 No assignment
7-kHz spinning speed
0–45 Alkyl C (0–45) – (215–235)
45–60 Methoxyl C (45–60) – (235–260)
60–94 O-alkyl C (60–94) – (260–280)
94–110 Di-O-alkyl C (94–110)
110–144 Aryl and unsaturated C (110–144) + 2(215–235)
144–165 O-aryl C (144–165) + 2(235–260)
165–190 Carbonyl and amide C (165–190) + 2(260–280)
215–235 Aryl SSB
235–260 O-aryl SSB
260–280 Carbonyl SSB T
REFERENCES
Johnson, C.E., R.J. Smernik, T.G. Siccama, D.M. Keimle, Z. Xu, and D.
J. Vogt. 2005. Using 13C nuclear magnetic resonance spectroscopy for
the study of northern hardwood tissues. Canadian Journal of Forest
Research. 35:1821-1831.
Johnson, C., W. Clymans, and T. Siccama. 2019. Mass and Nutrient Loss
in Decomposing Hardwood Boles on Watershed 1 at the Hubbard Brook
Experimental Forest, 1990 - present ver 2. Environmental Data
Initiative.
https://doi.org/10.6073/pasta/db7ff4e756631bd50256d3d14c288bc1
(Accessed 2022-04-27).
