uid=HFR,o=lter,dc=ecoinformatics,dc=org all public read doi:10.6073/pasta/c84da3ceaa34323a8f7d99db8eedd0cc Serita Frey https://orcid.org/0000-0002-9221-5919 Anne Pringle https://orcid.org/0000-0002-1526-6739 Eric Morrison https://orcid.org/0000-0002-9737-9947 Researcher Christopher Sthultz Researcher Linda van Diepen Researcher 2023 English

The purpose of this study is to examine whether two environmental change stressors (warming and nitrogen deposition) differentially impact litter decomposition. We investigated this using a two year litterbag decomposition experiment at the chronic N amendment experiment and the Barre Woods Soil warming experiment, and measured litter decay dynamics, enzyme activities and litter chemistry. In both years mass loss of the mixed litter was suppressed under N addition, with most of the mass loss observed in the first year compared to the second year (70% and 30% of total mass loss, respectively). Both years showed either increased activity for some hydrolytic enzymes (e.g. cellobiohydrolase) or no difference (e.g. ß-N-acetylglucosaminidase) with increased N. The lignolytic enzymes (e.g. peroxidases) showed no difference in activity in the first year, but had a highly reduced activity in year 2 under elevated N conditions. Soil warming did not significantly affect litter mass loss, and only had an effect on the activity of a few enzymes. In the oak reciprocal litterbag study, decay of oak litter originating from the highest N addition plot was negatively affected by simulated N deposition in the first year of decomposition, while after two years, simulated N deposition negatively affected all litter, and litter originating from the highest N addition plot decayed more slowly than control litter even without added N (i.e. in the control plot). In addition, in the first year of decomposition lignolytic enzyme activities were suppressed in litter originating from the N addition treatments, but due to simulated N deposition in year two.

decomposition litter nitrogen soil warming LTER controlled vocabulary organic matter disturbance LTER core area Harvard Forest HFR LTER USA HFR default

This dataset is released to the public under Creative Commons CC0 1.0 (No Rights Reserved). Please keep the dataset creators informed of any plans to use the dataset. Consultation with the original investigators is strongly encouraged. Publications and data products that make use of the dataset should include proper acknowledgement.

Creative Commons Zero v1.0 Universal https://spdx.org/licenses/CC0-1.0.html CC0-1.0 https://harvardforest.fas.harvard.edu/exist/apps/datasets/showData.html?id=hf218 Prospect Hill Tract (Harvard Forest), Slab City Tract (Harvard Forest). Coordinates based on WGS84 datum. -72.18 -72.18 +42.54 +42.48 295 385 meter 2010 2012 complete Information Manager Harvard Forest

324 North Main Street Petersham MA 01366 USA

(978) 724-3302 hf-im@lists.fas.harvard.edu Harvard Forest

324 North Main Street Petersham MA 01366 USA

(978) 724-3302 (978) 724-3595 https://harvardforest.fas.harvard.edu

Litter was collected in litter traps placed in each of the three N treatment plots of the chronic N amendment experiment and in the control plot of the Barre Woods Soil warming experiment during leaf abscission in the fall of 2010. The collected litter was air-dried and sorted by species, stems were removed, and litter was cut into approximately 3 cm x 3 cm pieces. Litterbags (20 cm x 20 cm) were made of 0.3mm nylon mesh and filled with 10 g of air-dried litter. Each bag was sewn closed, the litter spread evenly within the bag. At the Barre Woods experiment we used mixed litter, representative of the litter fall composition at the BW site. At the Chronic N amendment experiment we implemented two different sets of litterbags; 1) mixed litter (representative of the litter fall composition at the N site), and 2) an ‘oak only’ litterbag experiment, which is a reciprocal litter transplant study using oak litter collected from each of the three N addition plots. In fall of 2010, two replicate bags of each litter type (mixed litter or oak litter, see data file for more info) were placed in five to ten subplots of the CNA or BW experiments. The bags were placed on top of the forest floor, beneath the recent litter fall, and pinned to the ground with two U-shaped gardener’s pins. At harvest (fall 2011 and fall 2012), litter and other debris attached to the outside of the bags was removed, and wet weight of the bag was recorded. Immediately after collection, a ~2 g subsample was collected from each bag in the field and flash frozen in liquid N or stored in RNA later for fungal community analysis. Then, the remainder of each bag was placed in ziplock bag, placed on ice and transported to the University of New Hampshire, where it was stored at 4°C until further processing. Within three days after harvest, the litter was removed from the harvested bags, and subsampled for subsequent analyses. Subsamples (~ 1 g) were oven-dried (60°C for 48 hours) to determine moisture content and ashed (5 hours at 450°C) to assess potential mineral contamination. Another 1 g subsample was stored at 4°C for enzyme assays and the remaining litter was frozen at -80°C, freeze-dried, ground, and analyzed for C, N, lignin and cellulose concentrations.

Activities of the hydrolytic enzymes cellobiohydrolase (CBH), acid phosphatase (PHOS), N-acetyl-ß-glucosaminidase (NAG), and ß-glucosidase (BG) were assayed using the methylumbelliferyl-(MUB) linked substrates (200µM) ß-D-cellobioside, phosphate, N-acetyl-ß-D-glucosaminide, and β-D-glucopyranoside, respectively. The hydrolytic enzyme leucine aminopeptidase (LAP) was assayed using a 7-amido-4-methylcoumarin (AMC)-linked substrate L-Leucine (200µM). Activities of oxidative enzymes phenol oxidases (OX1 and OX2), and peroxidases (PER1 and PER2), were assayed using the substrates L-3,4-dihydroxyphenylalanine (L-DOPA, 25mM), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, 3mM), L-DOPA+H2O2 (0.3% hydrogen peroxide), and 3,3',5,5'-Tetramethylbenzidine (TMB + H2O2), respectively. Briefly, ~0.5 g of field-moist litter was homogenized in 125 ml sodium acetate buffer (50mM, pH = 4.7, average pH of the litter) for 30 seconds using a Magic Bullet (Homeland Housewares LLC). Sample homogenate (200 µl) was transferred to 96-well microplate, followed by addition of 50 µl substrate. Microplates were incubated for 15 min to 18 hours, depending on the enzyme substrate. After incubation, fluorescence was measured at excitation wavelength of 360 nm and an emission wavelength of 450 nm (hydrolytic enzymes) and absorbance was measured at 450nm (OX1, PER1, PER2) or 420nm (OX2) on a Biotek HT plate reader (Biotek, Winooski, USA). All enzyme assays were done with eight or sixteen replicate wells per sample, and corrected for background fluorescence or absorbance of substrate (negative control). For hydrolytic enzymes, the conversion of fluorescence was based on a standard line of the reference standard MUB (BG, CBH, NAG, PHOS) or AMC (LAP). Conversion of OX1 and PER1 was determined based on an empirically determined extinction coefficient of 7.9 μmole-1 used in other studies. Conversion of PER2 (TMB substrate), and OX2 (ABTS substrate) was determined based on an empirically determined extinction coefficient of ε450= 59,000 M-1 cm-1, and ε420=36,000 M-1 cm-1 respectively. Final enzyme activity was expressed as µmol of substrate converted per hour per g litter dry mass (µmol h-1 g-1).

Litter C and N concentrations were measured by dry combustion using a CN Analyzer (Perkin Elmer 2400 Series II). Lignin and cellulose concentrations were analyzed at Cumberland Valley Analytical Services (Hagerstown, Maryland, USA) using the acid detergent fiber (ADF) procedure.

Harvard Forest

324 North Main Street Petersham MA 01366 USA

(978) 724-3302 (978) 724-3595 https://harvardforest.fas.harvard.edu https://ror.org/059cpzx98 pointOfContact The Harvard Forest Long-Term Ecological Research (LTER) program examines ecological dynamics in the New England region resulting from natural disturbances, environmental change, and human impacts. National Science Foundation LTER grants: DEB-8811764, DEB-9411975, DEB-0080592, DEB-0620443, DEB-1237491, DEB-1832210. hf218-01-litter.csv litter decomposition hf218-01-litter.csv 28873 ab34b4618f3b85f32bb6d5c57b4dd99c 1 \r\n column , https://harvardforest.fas.harvard.edu/data/p21/hf218/hf218-01-litter.csv id litterbag identification number. Odd numbers were harvested in year one, even numbers were harvested in year two. litter bag identification number. Odd numbers were harvested in year one, even numbers were harvested in year two NA missing value start.date date that litterbag was harvested from the field YYYY-MM-DD NA missing value harvest.date date that litterbag was harvested from the field YYYY-MM-DD NA missing value days number of days that litterbag was out in the field decomposing number whole NA missing value site experiment code CNA chronic nitrogen amendment experiment BW barre woods soil warming experiment NA missing value treatment treatment code CN control nitrogen (0 kg N per ha per year) LN low nitrogen (50 kg N per ha per year) HN high nitrogen (150 kg N per ha per year) CW control warming (no heating, ambient temperature) HW heated warming (5C above ambient temperature) NA missing value plot plot ID where bag was placed within the SITE plot ID where bag was placed within the SITE NA missing value litter code for treatment where litter in litterbag originated from and litter type mixedCN litter originating from the CN treatment (TRT): 8.5 g oak (Quercus velutina and rubra), 1.5 g other species; red and striped maple (Acer rubrum and pennsylvanicum), black birch (Betula lenta), and American beech (Fagus grandifolia) mixedCW litter originating from the CW treatment (TRT): 7 g oak (Quercus velutina and rubra), 2g maple (Acer rubrum and pennsylvanicum),1 g other species; black birch (Betula lenta), and American beech (Fagus grandifolia) oakCN litter originating from the CW treatment (TRT): 7 g oak (Quercus velutina and rubra), 2g maple (Acer rubrum and pennsylvanicum),1 g other species; black birch (Betula lenta), and American beech (Fagus grandifolia) oakLN oak (Quercus velutina & rubra) litter originating from the LN treatment oakHN oak (Quercus velutina & rubra) litter originating from the HN treatment NA missing value moist percentage moisture in litter at harvest date dimensionless 0.01 real NA missing value mass.loss percentage mass loss over the period (DAYS) that bag was out in the field number 0.01 real NA missing value decay decay rate (k) calculated based on first order exponential decay function (y = e-kt), where y is percentage mass remaining at a time point, and t is time in years since decomposition start. dimensionless 0.01 real NA missing value n.per percentage nitrogen in litter at harvest dimensionless 0.01 real NA missing value c.per percentage carbon in litter at harvest dimensionless 0.01 real NA missing value cn carbon to nitrogen ratio in litter at harvest dimensionless 0.01 real NA missing value lap leucine aminopeptidase activity measured in litter at harvest nanomolePerGramPerHour 0.01 real NA missing value cbh cellobiohydrolase activity measured in litter at harvest nanomolePerGramPerHour 0.01 real NA missing value bg ß-glucosidase activity measured in litter at harvest nanomolePerGramPerHour 0.01 real NA missing value phos acid phosphatase activity measured in litter at harvest nanomolePerGramPerHour 0.01 real NA missing value nag N-acetyl-ß-glucosaminidase activity measured in litter at harvest nanomolePerGramPerHour 0.01 real NA missing value ox1 phenol oxidase activity measured in litter at harvest using L-DOPA as substrate micromolePerGramPerHour 0.01 real NA missing value ox2 phenol oxidase activity measured in litter at harvest using ABTS as substrate micromolePerGramPerHour 0.01 real NA missing value per1 gross peroxidases activity measured in litter at harvest using (L-DOPA+ H2O2 as substrate micromolePerGramPerHour 0.01 real NA missing value per2 peroxidase activity measured in litter at harvest using TMB + H2O2 as substrate micromolePerGramPerHour 0.01 real NA missing value adf acid Detergent Fiber concentration in litter at harvest dimensionless 0.1 real NA missing value lignin lignin concentration in litter at harvest dimensionless 0.01 real NA missing value cellulose cellulose concentration in litter at harvest dimensionless 0.01 real NA missing value 190 plot soil short-term measurement https://harvardforest.fas.harvard.edu/exist/apps/datasets/showData.html?id=hf018 https://harvardforest.fas.harvard.edu/exist/apps/datasets/showData.html?id=hf008