DOI: 10.6071/m3310b
title: Regional differences in soil stable isotopes and vibrational features at depth in three California Grasslands

description: There is an urgent need to investigate soil carbon dynamics in grasslands
 in differing precipitation and temperature regimes in light of current and
 future climate change. In this study, we assessed differences in soil
 stability, turnover, and organic matter composition along a climatic
 gradient of California grasslands. We focused on organic matter
 composition across three California grassland sites, from a dry (~300 mm
 precipitation/year) and hot regime (MAT: 14.6 ) to a wet (~2160 mm
 precipitation/year) and cool (MAT: 11.7 ) regime. We determined changes in
 total elemental and stable isotopic concentrations of soil carbon,
 nitrogen, δ13C, δ15N, and vibrational features as measured through Diffuse
 Reflectance Infrared Fourier Transformed Spectroscopy (DRIFTS) to 1m soil
 depth. We measured two isotopic indicators of soil turnover and stability,
 a natural carbon isotope approach defined as beta ( ), and the
 relationship between δ15N and C:N values. We did observe significantly
 more carbon concentration at our driest site and observed the lowest C:N
 values (~4) at depth at our wettest site. Carbon isotope indicators
 suggested the lowest stability at the dry site. Soils at depth
 (&gt;30cm) at the wettest site had the greatest stability and had the
 greatest signal of carboxylic acid and polysaccharides. These results
 suggest differing stabilization mechanisms of organic matter at depth
 across our study sites. Results from this study suggest that taking into
 account both depth and regional differences in soil stability, turnover,
 and organic matter composition would aid with carbon sequestration efforts
 and aid biodiversity conservation efforts.  
description: <strong>Study Area </strong> Soil
 samples were collected at three annual grasslands: Sedgwick National
 Reserve (Suttle et al., 2007), University of California Hopland Research
 and Extension Center and the Angelo Coast Range Reserve in California
 (fig.1). All three sites are dominated by exotic annual grasses , while
 forbs are of both native and exotic origin. The dominant vegetation at
 Angelo is a mix of <em>Aira</em> spp.,
 <em>Bromus</em> spp, and <em>Briza</em> spp.
 (Foley et al., 2023). At Hopland, it is a mix of
 <em>Avena</em> spp., <em>Bromus </em>spp.,
 <em>Erodium </em>spp., and <em>Festuca
 </em>spp<em>. </em>(Foley et al., 2023). At Sedgwick,
 the dominant vegetation is <em>Avena</em> spp. and
 <em>Bromus</em> spp. (Foley et al., 2023). At Angelo, soils
 are part of the Holohan-Hollowtree-Casabonne Complex, are classified as
 Ultic Haploxeralfs. The parent material is largely gray wacke and
 mudstone. At Hopland, soils are part of the Yorkville series, and are
 classified as Typic Argixerolls. Parent material is largely sandstone and
 shale. Soils at Sedgwick are part of the Salinas series and are classified
 as Pachic Haploxerolls. Parent material is sandstone and shale. 
 Each of these sites is an established reserve site within the
 University of California system, and as such, has detailed information on
 vegetation and climatic factors. To assess differences in plant
 communities, we aggregated all citations associated with plant surveys at
 each of our study sites (Supplemental table 1). We then aggregated
 information about whether each species was C3/C4, native/introduced, or is
 a N fixing or non-N fixing plant. This helped us determine potential plant
 community impacts on isotopic values. Plant communities in this region are
 typically dominated by C3 annual grasses.
 <strong>Experimental Design and
 Sampling </strong> Soils were collected to 1m
 across the three study sites, Angelo, Hopland, and Sedgwick. Samples at
 Hopland and Sedgwick were collected by hand auger to 1m with 6-7
 replicates per site (with each site encompassing approximately 3 ha),
 wherein we attempted to capture spatial heterogeneity but avoid
 confounding factors at each site by evenly sampling across similar slope
 positions (only Hopland had significant relief). At Angelo, samples were
 collected by Geoprobe due to being part of a different sampling campaign.
 At Angelo, samples were collected to depth of resistance (approximately
 3m) with 4 replicates. Depths greater than 1m are not reported for Angelo
 in this study. At all sites, samples were collected at consistent 10cm
 intervals (0-10, 10-20, and so on). After soils were
 collected, they were stored in coolers with ice packs in transport and
 then they were stored in a 4  cold room for approximately 4 months until
 they could be subsampled and analyzed. Storage took place during the
 pandemic due to a lack of access to laboratory facilities. When samples
 could be processed, a subsample was removed from each sample, and air
 dried for 7 days at room temperature. No carbonates were observed in these
 samples. Following air drying, the sample was then sieved through a 2mm
 sieve. A further subsample was taken for ball milling (using a Sample Prep
 8000M Ball Mill) to a homogenous particle size. We
 collected bulk density at Angelo and Hopland through Geoprobe cores, and
 calculated carbon stocks with these bulk density estimates. At Angelo, we
 subsampled each depth increment to estimate water content, and then
 calculated the dry mass of soil in a 10cm increment. Bulk density was
 calculated as the mass of the dry &gt;2mm fraction to correct for the
 impact of rock and root volume on soil carbon and nitrogen stocks (Throop
 et al., 2012). However, we did not observe a high coarse fraction at
 Hopland. <strong>Elemental and Isotopic
 Analyses </strong> The
 d<sup>13</sup>C and d<sup>15</sup>N values and
 elemental carbon and nitrogen contents of all samples were measured in the
 Stable Isotope Ecosystem Laboratory at the University of California,
 Merced (SIELO). Briefly, samples were weighed into tin capsules and
 combusted in a Costech 4010 Elemental Analyzer coupled with a Delta V Plus
 Continuous Flow Isotope Ratio Mass Spectrometer. Carbon and nitrogen
 isotope compositions were corrected for instrumental drift, mass
 linearity, and standardized to the international VPDB
 (d<sup>13</sup>C) and AIR (d<sup>15</sup>N) scales
 using the USGS 41A and USGS 40 standard reference materials. Mean
 d<sup>13</sup>C values for reference materials were USGS 40 =
 -26.4 ± 0.1‰ (n = 173) and USGS 41a = 36.5 ±0.2‰ (n = 87) and
 corresponding mean d<sup>15</sup>N values were USGS 40 -4.5 ±
 0.1‰ (n = 173) and USGS 41a 47.5 ± 0.1‰ (n = 87). Elemental carbon and
 nitrogen content were determined via linear regression of
 CO<sub>2</sub> and N<sub>2</sub> sample gas peak
 areas against the known carbon and nitrogen contents of USGS 40, USGS 41a,
 and Costech acetanilide. All isotope compositions are expressed in
 standard delta notations. <strong>Diffuse
 Reflectance Infrared Fourier transform Spectroscopy
 (DRIFTS)</strong> To characterize the chemical
 composition of soil C across our study systems, we used diffuse
 reflectance mid-infrared Fourier Transform spectroscopy (DRIFTS) analyses
 on bulk soil samples. DRIFTS measures the vibrational frequencies of
 functional groups in a sample, and allows us to measure the presence of
 functional groups that are important for organic matter and mineral
 surfaces. DRIFTS is informative on the abundance of organic and inorganic
 substances by measuring the excitation of molecular bonds when exposed to
 infrared radiation (Parikh et al., 2014). We used a Bruker IFS 66v/S
 Spectrophotometer (Ettlingen, Germany) with a praying Mantis apparatus
 (Harrick Scientific, Ossining, NY).  Potassium bromide (KBr) was used as a
 background reference, btu samples were not diluted with KBr. Samples were
 first dried in a desiccator following homogenization to remove
 interference from water. Absorption was measured between 4000 and 400
 cm<sup>-1</sup> averaged over 300 scans with an aperture of
 4mm. Functional group assignments are based on Parikh et al.
 (2014)
description: The only programs are Excel and R (though R is only used to
 process/clean/plot the data, all of this can be done in Excel). 
description: # Organic Matter Dynamics at Depth Across a Precipitation Gradient of
 California Grasslands There are 4 xlsx files associated with this dataset:
 -Sample_Metadata_Precipitation_Grad gives associated metadata with each
 sample ID. This metadata includes the location where it was sampled, the
 replicate, the depth, and so on. \- Precipitation_gradient_iso_data gives
 all the isotopic and elemental data assocaited with each sample ID. NAs
 refer to missing or lacking data. This data sheet represents data that
 comes out of the Stable Isotope Ecosystem Laboratory at UC Merced, with
 metadata attached for each sample. The measurements included in this
 datasheet make (C% and N%) make it possible to calculate carbon stocks, an
 important bulk density corrected factor, for each site. Measurements of
 stable isotopes (d13C and d15N) were also important markers of
 decomposition and turnover for the sites we were analyzing.
 -Precip_grad_C_inventories gives all the necessary data to calculate
 carbon stocks for the sites in this study, including bulk density and
 coarse fraction. NAs refer to missing or lacking data. \-
 Precip_Grad_C_stock_Mean is just the c stock data but averaged per 1ocm
 depth interval per site. \## Description of the data and file structure
 The information included in Sample_Metadata_Precipitation_Grad is largely
 descriptive. This information is largely redundant, and is also included
 in the files with elemental/isotopic data. The sample ID column in this
 file refers to the unique 3 digit code I assigned every sample I
 collected. The field site column refers to the site at which the sample
 was collected (see Wahab et al. 2024). The replicate column refers to the
 numbered replicate it was at that particular field site. The date
 collected column refers to the calendar date the sample was collected on.
 The depth column refers to the depth at which the sample was collected in
 cm. The raw data for the elemental and isotopic analyses is included in
 Precipitation_Gradient_Iso_Data, and can be fed into the two included R
 scripts to produce the plots/statistical tests with the associated
 manuscript in prep. The identifier column in this dataset refers to the
 unique identifier assigned by the Stable Isotope lab at UC Merced. The
 Sample ID refers to the unique 3 digit code assigned to each sample. The
 amount column refers to the mass of sample in mg that was weighed and
 combusted for the elemental analysis. The Area 28 column is the area under
 the curve for the N2 peak, and is used to compute the d15N_corrected
 within the isotope lab software. The Area 44 column is the curve for the
 CO2 peak, and is used to compute the d13C_corrected within the isotope lab
 software. The d13C_corrected is the stable isotope value in per mil. The
 d15N corrected is the stable isotope value in per mil. The N_wt_percent
 value is the N content of the sample reported as percent (%). The
 C_wt_percent value is the C content of the sample reported as percent (%).
 The C/N is the C:N ratio computed based on the N and C weight percents.
 The rest of the columns in this data are from
 Sample_Metadata_Precipitation_Grad, and are described in the above
 paragraph. The Precip_Grad_C_Inventories file includes data to compute C
 stocks. The first columns in this dataset, Sample Code, Field site,
 Location, Replicate, Date collected, Depth, are all from
 Sample_Metadata_Precipitation_Grad and are described there. The treatment
 column in this file refers to field site location. Bulk density refers to
 the bulk density (reported as g/cm^3) either collected or calculated
 depending on the site (see Wahab et al. 2024). The coarse fraction refers
 to the lab measured coarse fraction (reported as a fraction, unitless).
 The C_stock is the computed C stock value with a coarse fraction
 correction, and reported as g/cm^2. The C_stock_CF column is the computed
 C stock value without a coarse fraction correction, reported as g/cm^2.
 The Precip_Grad_C_stock_mean file is data from Precip_grad_C_Inventories
 but just averaged within each 10cm interval at each site for ease of
 visualization. \## Sharing/Access information Data available only on Dryad
 as of 6/20/23 \## Code/Software There are two R code scripts that were
 used for the production of the elemental and isotopic figures for this
 study, and also an R script for all the statistical tests peformed in this
 study -Precip_Grad_Analsysis_Final_Plots to process all the data and plot
 it -Precip_Grad_Stats_Test is to performed one-way ANOVAS per depth
 interval -Precip_Grad_Sedgwick_PTF_analysis is to calculate carbon stocks
 at Sedgwick using a pedo-transfer function as described in Wahab et al.
 2024 R scripts Precip_grad_Analysis_Final_Plots and Precip_Grad_stats_test
 intake the raw data from Precipitation_Gradient_Iso_Data.csv