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We analyzed the molecular SOC composition of surface mineral soil samples spanning 32 sites in the NEON Megapit archive, along with 10 additional soils which were selected to encompass additional diversity in biogeochemical characteristics (Supplemental Table 1). This table and additional tables referenced below are reported in the manuscript associated with this dataset (Hall, Ye, et al., Accepted, Nature Geoscience). Briefly, in the dominant soil and vegetation type at each NEON terrestrial site, a soil profile was characterized and sampled by horizon with the help of US Department of Agriculture Natural Resource Conservation (NRCS) staff and archived by NEON 1-3. We requested subsamples of A horizon material from each site in the Megapit archive that was available in September 2019. The Gellisols had extensive organic (O) horizons, such that we requested material from the mineral horizon closest to the surface (described as Bg/Oajj, A/Cjj, and Bg at BONA, HEAL, and TOOL, respectively; Supplemental Table 1). The 10 non-NEON samples analyzed here were each collected from 0–10 cm depth with a clean shovel after removing any litter or O horizon material. All soils were air dried to constant mass and sieved to 2 mm. Visible root fragments were removed with tweezers and soils were finely ground with a mortar and pestle prior to subsequent analyses.

13C CPMAS NMR analyses and sample preparation All 42 samples were prepared for NMR analyses, allowing a comparative characterization of organic C molecular composition. In order to increase the NMR sensitivity and remove paramagnetic materials, soils were pre-treated with hydrochloric acid (HCl, 10% wt.) and hydrofluoric acid (HF, 10%, wt.) to remove any calcium carbonate and mineral phases, respectively. Briefly, 2–3 g of finely ground soil was weighed into a 50 mL sealed polyethylene centrifugation tube, saturated with 30 mL HCl, and allowed to settle for 30 min. After centrifugation and discarding HCl, the remaining slurry was then shaken with 40 ml of mixed HF (10% wt.) and HCl (10% wt.) for 8 h, and subsequently centrifuged. The supernatant was removed and discarded appropriately. After repeating the procedure four times, each sample was washed with distilled water three times and dried at 50 oC under a stream of dinitrogen gas.

Solid-state 13C CP-MAS and 13C DP-MAS NMR spectra were recorded at room temperature (23 degreeC) using a 300 MHz Bruker AVANCE III NMR spectrometer equipped with a 4 mm magic angle spinning (MAS) probe (Bruker BioSpin, Billerica, MA) at Baylor University (Waco, TX). The 60–130 mg HF-treated sample was placed in a zirconium rotor with a diameter of 4 mm and Kel-F caps to maximize the C mass and signal intensity. A MAS rate of 12 kHz was used for all NMR measurements. Cross polarization (CP) experiments used a ramped-amplitude (50% to 100%) contact pulse and rotor synchronized Hahn echo 4. The contact time and recycle delay were set to 2 ms and 1.2 s, respectively, and composite pulse proton decoupling was applied during signal acquisition. Direct polarization (DP) 13C spectra were acquired with a 90-degree excitation pulse and rotor-synchronized Hahn echo 5, with a recycle delay of 180 s. Glycine was used as an external standard for setting pulse angles, chemical shift and Hartman-Hahn matching conditions. DPMAS spectra were obtained for 11 HF-treated soil samples as a means against which to assess relative quantitation bias in CPMAS NMR data 6. These samples were selected to span a broad range of biogeochemical diversity (nine soil orders; Supplemental Table 1) and contained sufficient SOC ( greater than 2.9% C in the original samples) to enable timely analysis by DPMAS.

CPMAS spectra for HF-treated samples were acquired with more than 6000 scans. To assess potential impacts of HF treatment on SOC composition, 11 untreated samples with relatively high SOC concentration ( greater than 6%) were also selected for NMR analysis (this set differed slightly from the CPMAS/DPMAS comparison given the differing selection criteria). These samples included six soil orders and spanned a broad range of paramagnetic element content (14–58 mg Fe g-1). Spectra for these untreated samples were recorded using the same operation conditions of HF-treated samples and were acquired with more than 44000 scans. After baseline correction, quantification was performed by dividing the spectra into seven chemical shift regions: 0–45 ppm, 45–60 ppm, 60–95 ppm, 95–110 ppm, 110–145 ppm, 145–165 ppm and 165–215 ppm, assigned to alkyl C, N-alkyl + methoxyl C, O-alkyl C, Di-O-alkyl C, aromatic C, phenolic C, amide + carbonyl C, respectively. Subsequently, a molecular mixing model was applied to the seven integrated spectra regions, to estimate the relative abundances of six molecular SOC constituents (carbohydrate, protein, lignin, lipid, carbonyl and char) 7. The elemental concentrations of C and N were measured on the HF-treated samples by combustion/elemental analysis at Baylor University (Costech 4010, Valencia, CA) and were used as additional constraints on the molecular mixing model solutions 7.

Biogeochemical analyses Megapit soil samples and vegetation in proximity to the soil pit were subjected to numerous physical and chemical analyses 8. Here, we utilized measurements of total elemental content and particle size from the Megapit samples. We also used measurements of the copy number of functional genes from bacteria/archaea (16S) and fungi (ITS) calculated by quantitative polymerase chain reaction (qPCR), which were conducted on separate fresh soil samples collected in the vicinity of each sampled Megapit profile 8. Briefly, these soils were flash frozen in the field on dry ice and shipped to an analytical facility for DNA extraction and amplification. Soil samples for qPCR analysis were collected periodically (approximately three times per year) from each site from 0–30 cm depth, and cores were visually separated according to organic and mineral horizons; only samples from mineral soil were used here. We selected samples from plots in proximity to each Megapit (i.e., within several hundred m; denoted as Tower plots in NEON terminology) and averaged the mean 16S and ITS abundance for each site based on the 2016–2018 data. Fine root biomass was measured by depth in three pit profiles within the Megapit and sorted into live/dead classes for fine ( less than 2 mm or less than 4 mm, depending on the site) and coarse diameter classes. Here, we denoted the combined less than 2 mm and less than 4 mm fractions as fine roots for subsequent analyses. Roots were dried, weighed, and combusted for analysis of carbon (C) and nitrogen (N) content. We averaged root data from 0–30 cm depth for use in subsequent analyses. No NEON root data were available from TOOL, so we used previous published data from the same site 9. Samples for foliar and/or litter chemistry were available from a subset of the NEON sites (15 and 16 sites, respectively), as these are collected from each site on a five-year rolling schedule. Foliar samples represented clips of bulk herbaceous samples from the plant community. Litter samples included debris from trees and shrubs. We used measurements of foliar and litter C:N and a proxy for lignin content (acid-unhydrolyzable residue) 8. No root or microbial or litter chemistry data were available from the non-NEON samples from which we collected 13C NMR spectra.

We conducted several additional soil extractions of all samples to quantify reactive metals. Subsamples were extracted in parallel with sodium dithionite (1:150 ratio of soil:solution) to quantify pedogenic iron (denoted Fed) and ammonium oxalate (1:60 ratio of soil:solution) to quantify Fe and aluminum in short-range-ordered phases and organo-metal complexes (termed Feo and Alo). The concentration of crystalline Fe minerals was then calculated as the difference between Fed and Feo (Fed-o). Subsamples were also sequentially extracted with deionized water and sodium sulfate (1:150 ratio of soil:solution). The calcium and magnesium concentration of the sodium sulfate extraction (termed Cas + Mgs), which followed the water extraction, was interpreted as a proxy for Ca and Mg that may have participated in divalent cation bridging between clays and organic matter 10. All metals were analyzed by inductively coupled plasma optical emission spectroscopy at Iowa State University (ICP-OES; Perkin Elmer Optima 5300 DV, Waltham Massachusetts). Mean annual precipitation and temperature data were estimated for each NEON site using previously synthesized data 11. Potential evapotranspiration (PET) data were extracted from a global 1-km resolution mean annual evapotranspiration dataset from 2000-2014 12.

References

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3. Ayres, E. NEON procedure and protocol: producing TIS soil archive subsamples for users. NEON.DOC.001306. https://data.neonscience.org/data-products/DP1.00097.001 (2017).

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10. Ye, C. et al. Reconciling multiple impacts of nitrogen enrichment on soil carbon: plant, microbial and geochemical controls. Ecol. Lett. 21, 1162–1173 (2018).

11. SanClements, M. et al. Collaborating with NEON. BioScience 70, 107 - 107 (2020).

12. Mu, Q., Zhao, M. & Running, S. W. Improvements to a MODIS global terrestrial evapotranspiration algorithm (MOD16 method). Remote Sens. Environ. 115, 1781–1800 (2011).