Soil sampling, fractionation and carbon and nitrogen content analysis

Soil samples (0–10 cm) were collected from a long-term crop rotation experiment established in 1980 at the Elora research station, Ontario, Canada (Congreves et al. 2015). The soil at Elora was silty loam soil (Congreves et al. 2015). Samples were collected from six different crop rotations of 4 years’ duration each, including continuous alfalfa ((Medicago sativa L.), AAAA), continuous corn ((Zea mays L.), CCCC), corn–corn–alfalfa–alfalfa (CCAA), corn–corn–soybean–soybean ((Glycine max (L.) Merr.), CCSS), corn–corn–soybean–winter wheat ((Triticum aestivum L.), CCSW), and corn–corn–soybean–winter wheat with red clover ((Trifolium pretense L.), CCSW_RC), under conventional tillage. Soil samples were collected before the harvest in October 2017. It was the second year of corn or alfalfa planting in the 4-year duration. The average tissue quality of carbon inputs and estimated SOC stabilization efficiency (by the ratio of SOC stock to total carbon inputs) of each crop rotation has been published in King et al. (2020). The collected soil samples were freeze-dried and passed through a 2 mm sieve. Then, soil samples from the same crop rotation treatment were combined from four field replicates as described in Man et al. (2021) before fractionation. Four different soil fractions were separated based on the density and the aggregate sizes. Briefly, approximately 250 g of sample was mixed with 500 mL 1.7 g/mL sodium iodide solution and mixed for 24 h. The mixture was centrifuged at 1500 rpm for 50 min (Clemente et al. 2011), the supernatant was collected and filtered using a GF/F glass microfiber filter (0.7 µm cut off; Whatman, Kent, UK), and then rinsed with deionized (18 MΩ cm at 25 °C) water. This step isolated the light fraction which was then transferred into tubes and freeze-dried for further analyses. The soil was not physically disrupted before further fractionation to preserve aggregate association, which are more representative of the biogeochemical function of SOM (Sollins et al. 2009). The residual heavy fraction was passed through a 53 µm sieve with deionized water. The soil aggregate fraction which was retained on the sieve was collected (F_{53–2000 µm}). The fraction that passed through the sieve was then separated to two additional aggregate sizes (F_2–53 µm and F_{< 2 µm}) based on Stokes’ law and sedimentation rates. The suspended portion (F_<2 µm) was collected by aspiration and mixed with a 0.5 M calcium chloride solution to promote flocculation and avoid the loss of ultrafine particles. Deionized water was then used to rinse excess calcium chloride solution. All fractions were freeze-dried, ground and stored at room temperature for further analyses. Soil carbon and nitrogen concentrations of each fractionated sample were analyzed using a Thermo Flash 2000 Elemental Analyzer (Thermo Scientific, Hudson, NH, USA). Samples were combusted at 950 °C under a stream of oxygen gas. The concentration of SOC and nitrogen was reported as an average of duplicate analyses.

Solid-state ¹³C nuclear magnetic resonance (NMR) spectroscopy sample preparation and analysis

Three soil aggregate size fractions (F_{53–2000 µm}, F_{2–53 µm} and F_<2 µm) were repeatedly extracted with hydrofluoric acid (HF acid; 10% by volume in deionized water) to concentrate organic matter and decrease the concentration of paramagnetic materials which improves the signal to noise ratio in solid-state ¹³C NMR analysis (Schmidt et al. 1997; Mitchell et al. 2018). Samples were rinsed with deionized water after HF treatment to remove excess salts and freeze-dried. Light fraction samples were not HF treated due to the low yield and high SOC concentration, which did not require pretreatment before NMR analyses. Samples were packed into a 4 mm zirconium rotor with a Kel-F cap and then characterized by solid-state ¹³C cross-polarization magic angle spinning (CP-MAS) NMR spectroscopy. The NMR spectra were acquired using a 500 MHz Bruker BioSpin Avance III spectrometer (Bruker BioSpin, Rheinstetten, Germany) equipped with a 4 mm H–X MAS probe, using an 11 kHz spinning speed, a ramp-CP contact time of 1 ms, a recycle delay of 2 s with 40960 scans for light fractions and F_<2 µm and a recycle delay of 1 s with 61440 scans for F_{53–2000 µm} and F_{2–53 µm}. Spectra were processed using a line broadening of 150 Hz. Four main chemical shift regions were used for the integration of ¹³C NMR spectra and normalized to the total signal using Analysis of Mixtures (AMIX; v. 3.9.15) software (Bruker BioSpin, Rheinstetten, Germany): (i) alkyl carbon (0–50 ppm; from cutin, suberin, aliphatic side chains and lipids); (ii) O-alkyl carbon (50-110 ppm; from carbohydrates, peptides and methoxyl carbon in lignin); (iii) aromatic and phenolic carbon (110–165 ppm; from lignin and amino acids observed in peptides and black carbon) and (iv) carboxyl and carbonyl carbon (165–220 ppm; from fatty acids and amino acids in peptides and other degradation products) (Baldock et al. 1992; Preston et al. 1997; Simpson et al. 2008). The ratio of alkyl/O-alkyl carbon was calculated by dividing the integrated regions of alkyl carbon and O-alkyl carbon to establish the relative degradation stage of SOM in each fraction (Baldock et al. 1992; Baldock and Preston 1995; Preston et al. 1997; Kögel-Knabner 1997; Simpson and Simpson 2012).

Targeted soil organic matter (SOM) compounds extraction and analysis with gas chromatography – mass spectrometry (GC–MS)

Targeted SOM compounds with various microbial- and plant-derived sources were isolated and quantified using gas chromatography – mass spectrometry (GC–MS). Soil fractions were sequentially extracted in duplicate using solvent (dichloromethane/methanol), base hydrolysis and copper (II) oxide (CuO) oxidation extractions, which are described in detail in the Appendix A (Otto and Simpson 2005, 2006a, 2006b). The isolated compounds (including aliphatic lipids, steroids, as well as the cutin-, suberin-, and lignin-derived compounds) from each extraction step were analyzed by GC–MS using established methods (Otto and Simpson 2005, 2006a, 2006b). Briefly, soil fractions (light fraction 0.2 g; F_{53–2000 µm} and F_{2–53 µm} 15 g and F_<2 µm 3 g) were solvent-extracted sequentially with dichloromethane, dichloromethane and methanol mixture (1:1 by volume), and methanol to isolate the solvent-extractable SOM components in each fraction (Otto and Simpson 2005). Air-dried solvent extracted samples were then base hydrolyzed with methanolic potassium hydroxide (KOH, 1 M in methanol) solution in Teflon lined bombs at 100 °C for 3 h to obtain ester-bound lipids (Goñi and Hedges 1990b; Otto and Simpson 2006a). The base hydrolyzed samples were then oxidized with 1 g CuO, 100 mg ammonium iron (II) sulphate hexahydrate [Fe(NH₄)₂(SO₄)₂·6H₂O] and 15 mL of sodium hydroxide (NaOH, 2 M in deionized water) in Teflon lined bombs at 170 °C for 2.5 h (Hedges and Ertel 1982; Hedges et al. 1988; Otto and Simpson 2006b). Then the supernatants were solid-phase extracted using HLB SPE cartridges (Waters Limited, Mississauga, ON, Canada) to obtain the lignin-derived compounds (Pinto et al. 2010).

All the soil extracts were derivatized before GC–MS analysis so that the targeted compounds are more volatile and amenable to GC separation and analysis (Otto and Simpson 2005, 2006a, 2006b). Compounds isolated via solvent and CuO oxidation extraction were derivatized using N, O-bis(trimethylsilyl)trifluoroacetamide and pyridine. Base hydrolysis extraction products were first methylated using N, N-dimethylformamide dimethyl acetal and then derivatized with N, O-bis(trimethylsilyl)trifluoroacetamide and pyridine. The derivatized samples were analyzed by GC–MS using an Agilent 7890B gas chromatograph equipped with a 5977B mass spectrometer operated with electron impact (70 eV) ionization. Samples were diluted with hexane and 1 µL of each sample was injected using an Agilent 7603 A automatic sampler at an inlet temperature of 280 °C onto an HP-5MS fused silica capillary column (30 m × 0.25 mm i.d. × 0.25 µm film thickness). The oven temperature gradient started at 65 °C for 2 min, followed by an increase of 6 °C min⁻¹ to 300 °C, and then a 20 min isothermal hold. The flow rate of carrier gas (helium) was 1 mL min⁻¹. Data acquisition was performed using Agilent Mass Hunter GC–MS Acquisition (version B.07.03.2129) and data were processed using Agilent Enhanced ChemStation software (version E.02.02.1431). The identification of the targeted compounds was obtained by comparing the mass spectra with Wiley Registry (9th edition) plus the National Institute of Standards and Technology (NIST) mass spectral databases, the mass spectral library of soil compounds and published mass spectra. External standards were used to quantify the concentration of targeted compounds from extractions (Otto and Simpson 2005, 2006a, 2006b). Tetracosane, 1-docosanol, methyl tricosanoate and cholesterol were used as external standards for solvent extractable compounds. Methyl tricosanoate was used as an external standard for cutin- and suberin-derived compounds, and vanillic acid and vanillin were used as external standards for lignin-derived phenols.

Solvent extractable compounds included: short-chain aliphatic lipids (<C₂₀, n-alkanes, n-alkanols, and n-alkanoic acids), long-chain aliphatic lipids (≥C₂₀, n-alkanes, n-alkanols, and n-alkanoic acids), and cyclic lipids (cholesterol, ergosterol, campesterol, stigmasterol, sitosterol and stigmastanol) (Otto and Simpson 2005). Cutin-derived compound concentrations (ΣC) were calculated as the sum of mid-chain hydroxy C₁₄, C₁₅, C₁₇ acids, C₁₆ mono- and dihydroxy acids and diacids (Otto and Simpson 2006a). The ω-hydroxy C₁₆ acids (ω-C₁₆) are relatively more persistent than other fatty acids, thus the increased abundance of ω-C₁₆ in C₁₆ hydroxy acids (ω-C₁₆/ΣC₁₆) is indicative of cutin decomposition (Otto and Simpson 2006a). The concentration of suberin-derived compounds (ΣS) was calculated as the sum of long-chain ω-hydroxyalkanoic acids (C₂₀–C₃₂), α,ω-diacids (C₂₀–C₃₂) and 9,10-epoxy-18-hydroxy C₁₈ dioic acid (Otto and Simpson 2006a). Suberin- or cutin-derived compounds concentration (ΣSvC) was calculated as the sum of ω-hydroxyalkanoic acids C₁₆, C₁₈, C₁₈ di- and trihydroxy acids, 9,10-epoxy-ω-hydroxy C₁₈ acid, and α, ω-diacids C₁₆, C₁₈ (Otto and Simpson 2006a). The ratio of suberin- to cutin-derived compounds (suberin/cutin ratio, which was calculated as (ΣS+ΣSvC)/(ΣC+ΣSvC)) informs root-derived (ratio > 1) or leave-derived (ratio < 1) sources (Otto and Simpson 2006a). The concentrations of lignin-derived phenols were calculated as the sum of cinnamyl (p-coumaric acid and ferulic acid), syringyl (syringic acid, syringaldehyde and acetosyringone), and vanillyl (vanillic acid, vanillin and acetovanillone) phenols (Goñi and Hedges 1990a; Otto and Simpson 2006b). Ratios of syringyl to vanillyl (syringyl/vanillyl) phenols versus the ratios of cinnamyl to vanillyl (cinnamyl/vanillyl) phenols can discriminate different lignin sources (Hedges and Ertel 1982; Hedges et al. 1988; Goñi and Hedges 1990a; Otto and Simpson 2006b). The oxidation state of lignin was determined by the acid to aldehyde ratios of two lignin monomer classes, vanillyl ((Ad/Al)_v) and syringyl ((Ad/Al)_s), which increase with progressive degradation (Hedges et al. 1988).

Statistical analyses

Statistical analyses were performed using IBM SPSS Statistics version 22. Independent samples t-test was used to examine the differences in soil chemical properties (i.e., soil carbon and nitrogen contents as well as concentration and degradation of groups of SOM components) between crop rotations in each soil fraction with a significant level of P < 0.05 and n = 2. The independent samples t-test analysis was also used to examine the significant differences (P < 0.05) of SOM characteristics (e.g., groups of SOM components concentration and degradation) between relatively low (AAAA, CCCC, CCAA, and CCSS, in total n = 8) and high (CCSW and CCSW_RC, in total n = 4) crop rotational diversity in each soil fraction. Linear regression was used to evaluate the relationships of the SOM characteristics between whole soil and fractionated samples (including SOC concentration, carbon to nitrogen (C: N) ratio, the relative contribution of different functional groups and alkyl/O-alkyl carbon ratio from ¹³C NMR spectroscopy, targeted compounds concentration and the degradation proxies for specific groups).

Soil carbon and nitrogen analysis

The mass recovery of samples from different crop rotations was greater than 97% (Table A1). The carbon recovery of the samples ranged from 86% to 95% (Table A1). The SOC concentration was highest in the light fractions followed by the F_<2 µm, F_{53–2000 µm} to F_2–53 µm fractions (Table 1). Higher SOC concentrations were observed in light and F_<2 µm fractions compared to whole soil (Table 1). Within the same soil fraction, SOC concentrations from different crop rotations exhibited significant differences (P < 0.05, Table 1). For example, the SOC concentrations in light fractions were significantly different between crop rotations (Table 1). Within the same mineral fraction (F_{53–2000 µm}, F_2–53 µm or F_<2 µm), there were fewer significant differences observed between rotations compared to the light fractions. The SOC concentrations in mineral fractions (F_{53–2000 µm}, F_2–53 µm and F_<2 µm) with the AAAA treatment were the highest compared to other crop rotations (Table 1). Significant differences were observed in SOC concentrations in light fractions and F_2–53 µm between relatively high and low crop rotational diversities (Table 2). Higher SOC concentrations were observed in light fractions from relatively high diversified crop rotations (CCSW and CCSW_RC) and in F_2–53 µm from low diversified crop rotations (AAAA, CCCC, CCAA and CCSS; Table 2). The C: N ratios decreased from light fractions, F_{53–2000 µm}, F_2–53 µm to F_<2 µm among all crop rotations whereas the C: N ratio of the unfractionated soil samples was only higher than F_<2 µm ratios.

Table 1.

Soil organic carbon (SOC) concentration (with the unit g OC/kg soil for whole soil and g OC/kg fraction for soil fractions) and carbon to nitrogen (C: N) ratios (standard error from duplicate samples) of F_{53–2000 µm}, F_2–53 µm, F_{<2 µm}, and light fraction was compared to whole soil (Man et al. 2021).

Table 2.

Soil organic carbon (SOC) concentration, carbon to nitrogen ratio, the relative contribution of alkyl, O-alkyl, aromatic and phenolic and carboxyl and carbonyl carbon, alkyl/O-alkyl carbon ratio, and specific groups of compounds (solvent extractable compounds, cutin- and/or suberin-derived compounds, and lignin-derived phenols) concentrations (which were normalized to SOC concentration of fractions, µg/g OC) and degradation between relatively low (AAAA, CCCC, CCAA and CCSS; mean ± standard error) and high diversified (CCSW and CCSW_RC; mean ± standard error) crop rotations.

Solid-state ¹³C nuclear magnetic resonance (NMR) analysis

The NMR spectra from all crop rotations exhibited similar chemical signatures (Fig. A1) but different abundances of SOM components (Table 3). Alkyl carbon was relatively higher in the F_<2 µm (29%–31%) than in other fractions analyzed (Table 3). The relative abundance of O-alkyl carbon were observed to be higher in F_{53–2000 µm} (45%–46%) and F_2–53 µm (42%–43%) than in F_<2 µm (27%–32%). The highest relative contribution of aromatic and phenolic carbon was observed in light fractions (20%–30%) and the lowest relative contribution was found in F_<2 µm (16%–18%). The highest relative abundance of carboxylic and carbonyl carbon was in F_<2 µm (22%–26%) and lowest was in F_{53–2000 µm} (8%–9%) across crop rotations. The relative degradation state of SOM (alkyl/O-alkyl carbon), which increases with progressive organic matter degradation (Baldock et al. 1992; Preston et al. 1997; Kögel-Knabner 1997), was higher from F_{53–2000 µm}, F_2–53 µm to F_<2 µm. The light fraction had the lowest alkyl/O-alkyl carbon ratio compared to mineral-bound fractions. The alkyl/O-alkyl carbon ratio of F_{<2 µm} was closer to the ratio of the whole soil than other fractions (Table 3).

Table 3.

Relative contribution (%) of alkyl (0–50 ppm), O-alkyl (50–110 ppm), aromatic and phenolic (110–165 ppm) and carboxyl and carbonyl (165–220 ppm) carbon (Baldock et al. 1992; Preston et al. 1997; Simpson et al. 2008) integrated from solid-state ¹³C NMR spectra with the alkyl/O-alkyl carbon ratios (Baldock and Preston 1995; Preston et al. 1997; Kögel-Knabner 1997; Simpson and Simpson 2012) from whole soil (Man et al. 2021) and different soil fractions.

Within the same fraction class, similar trends of the relative contribution of groups of SOM were observed, except for light fractions and F_<2 µm with monoculture crops (AAAA and CCCC). In diversified crop rotations, the relative contribution of SOM components from light fraction samples decreased from O-alkyl carbon, aromatic and phenolic carbon, alkyl carbon to carboxyl and carbonyl carbon (Table 3). The AAAA light fraction had relatively higher alkyl carbon (23%) than aromatic and phenolic carbon (20%), and the CCCC had slightly higher carboxyl and carbonyl carbon (17%) than alkyl carbon (16%). In F_<2 µm, the relative contribution of components from high to low was alkyl carbon, O-alkyl carbon, carboxyl and carbonyl carbon and aromatic and phenolic carbon for samples from diversified crop rotations (Table 3). AAAA and CCCC had relatively higher O-alkyl carbon (32%) than alkyl carbon (29%–30%) in the F_<2 µm.

Targeted soil organic matter (SOM) compounds analysis by gas chromatography – mass spectrometry (GC–MS)

The concentration of solvent extractable compounds (n-alkanes, n-alkanols, n-alkanoic acids and steroids) differed among soil fractions (Fig. 1). Higher concentrations of plant-derived steroids and n-alkanoic acids were observed in light fractions and F_{53–2000 µm} in comparison to F_2–53 µm and F_<2 µm. The concentrations of n-alkanoic acids and plant-derived steroids in light fractions were much higher than in the unfractionated soil (Fig. 1). In contrast, n-alkane and n-alkanol concentrations were higher in F_2–53 µm and F_<2 µm than in light fraction and F_{53–2000 µm} for all crop rotations (Fig. 1). F_<2 µm contained a much higher concentration of n-alkanols than the whole soil (Fig. 1). The concentrations of long-chain n-alkanes and n-alkanols (≥C₂₀) in F_{53–2000 µm} and soil samples were inversely correlated (Fig. A2). Positive linear correlations were observed between concentrations of long-chain n-alkanes and n-alkanols (≥C₂₀) in F_{2–53 µm} and whole soils across crop rotations (Fig. A2). N-alkanes, n-alkanols and steroids from all fractionated samples were predominantly plant-derived (≥C₂₀, Fig. 1 and Table A2). For the light fractions, F_{53–2000 µm} and F_2–53 µm, the main contribution of n-alkanoic acids are microbial-derived compounds (<C₂₀,Fig. 1 and Table A2) for all crop rotations.

Fig. 1.

The concentrations of solvent extractable compounds (n-alkanes, n-alkanols, n-alkanoic acids and steroids, which the concentrations were normalized to soil organic carbon content of whole soil or fractions) among different crop rotational diversity within the whole soil (Man et al. 2021), light fractions, F_{53–2000 µm}, F_{2–53 µm} and F_{<2 µm} (error bars indicate the standard error, n = 3 for whole soil (Man et al. 2021) and n = 2 for fraction samples). The concentration ranges on the y-axis were different for each graph. AAAA, CCCC, CCAA, CCSS, CCSW, and CCSW_RC represent continuous alfalfa, continuous corn, corn–corn–alfalfa–alfalfa, corn–corn–soybean–soybean, corn–corn–soybean–winter wheat, and corn–corn–soybean–winter wheat with red clover, respectively.

Fig. 2.

The distribution of (a) cutin-derived compounds and (b) suberin-derived compounds concentrations (which were normalized to soil organic carbon content of whole soil or fraction), (c) cutin degradation state and (d) suberin/cutin ratio among varied crop rotational diversities in the whole soil (Man et al. 2021), light fraction, F_{53–2000 µm}, F_{2–53 µm}, and F _{<2 µm} (error bars indicate standard error, n = 3 for whole soil (Man et al. 2021) and n = 2 for fraction samples). AAAA, CCCC, CCAA, CCSS, CCSW, and CCSW_RC represent continuous alfalfa, continuous corn, corn–corn–alfalfa–alfalfa, corn–corn–soybean–soybean, corn–corn–soybean–winter wheat, and corn–corn–soybean–winter wheat with red clover, respectively.

Within the same fraction class, a few solvent extractable compound concentrations in relatively high diversified (more than two crop species) crop rotations were significantly different compared to low diversified (monoculture or two crop species) crop rotations (P < 0.05, Table 2). In light fractions, concentrations of long-chain aliphatic lipids, n-alkanes, and n-alkanols in highly diversified crop rotations (CCSW and CCSW_RC) were significantly lower than in low diversified crop rotations (AAAA, CCCC, CCAA, and CCSS; P < 0.05, Table 2). The plant-derived steroids and cyclic lipids concentrations in relatively high diversified crop rotations (CCSW and CCSW_RC) were higher than the concentrations in low diversified crop rotations (AAAA, CCCC, CCAA, and CCSS) in F_{53–2000 µm} and F_{2–53 µm} (Table 2). AAAA exhibited the most distinctive results compared to other crop rotations within the same fraction class or in soil samples (Fig. 1). For example, AAAA exhibited higher concentrations of long-chain aliphatic lipids (≥C₂₀) in the light fraction and F_{53–2000 µm} than other crop rotations (Table A2). Significantly higher concentrations of total n-alkane and long-chain n-alkanes (≥C₂₀) of AAAA in F_{53–2000 µm} (P < 0.05) but lower in F_{2–53 µm} or F_{<2 µm} than in other crop rotations were observed (Table A2). The concentration of short-chain n-alkanols (<C₂₀) of AAAA in the light fraction was higher than other crop rotations (P < 0.05, Table A2), and long-chain n-alkanols (≥C₂₀) and total n-alkanols concentrations of AAAA in F_{53–2000 µm} were higher than other crop rotations (P < 0.05, Table A2).

Higher concentrations of cutin- and suberin-derived compounds were observed in F_{<2 µm} than in other fractions or whole soil among different crop rotations (Figs. 2a and 2b). Positive correlations were observed between suberin-derived and suberin- and cutin-derived compounds concentrations in F_<2 µm and in whole soil (Fig. A2). Samples from F_{<2 µm} had a relatively lower cutin degradation state (ω-C₁₆/ΣC₁₆) compared to other fractions for the majority of crop rotations, but this fraction was more degraded than whole soil (Fig. 2c). The suberin/cutin ratios varied in fractions across crop rotations but were mostly lower than the ratios of soil (Fig. 2d). Lower concentrations of suberin- and cutin-derived compounds with significance (P < 0.05) were observed in relatively high diversified crop rotations (CCSW and CCSWRC) compared to low diversity (AAAA, CCCC, CCAA and CCSS) only in the light fractions and F_{53–2000 µm} (Table 2). The concentrations of suberin- and cutin-derived compounds also varied with crop species in the F_{<2 µm}. Crops containing alfalfa (AAAA and CCAA) had relatively lower suberin-derived compounds and higher cutin-derived compounds than soybean-containing treatments (CCSS, CCSW and CCSW_RC; Table A3) in the F_{<2 µm}.

Identified and quantified lignin-derived phenols (Table A4) included: cinnamyl (p-coumaric acid and ferulic acid, Fig. 3a), syringyl (syringic acid, syringaldehyde and acetosyringone, Fig. 3b), and vanillyl (vanillic acid, vanillin, acetovanillone, Fig. 3c) classes (Goñi and Hedges 1990a;Otto and Simpson 2006b). The total concentration of lignin-derived phenols (sum of vanillyl, syringyl and cinnamyl compounds) decreased from light fraction, F_{2–53 µm}, F_{53–2000 µm}, to F_{<2 µm} (Fig. 3d). The total concentrations of lignin-derived phenols in the light fraction were similar to that of the whole soil (Fig. 3d). Positive correlations were observed between the total concentration of lignin-derived phenols in the unfractionated soil and light fraction, as well as in the soil and F_{2–53 µm} (Fig. A2). The distribution of phenols in different fractions across crop rotations was further examined using plots of syringyl/vanillyl versus cinnamyl/vanillyl phenols (Fig. 4). The ratios of syringyl/vanillyl and cinnamyl/vanillyl phenols have been used to assess the origin of lignin (i.e., gymnosperm or angiosperm wood, non-woody vascular plant tissues) (Ertel and Hedges 1984; Goñi et al. 2000; Otto and Simpson 2006b) and exhibit a range of lignin-derived phenol ratios (Fig. 4) that are consistent with non-woody angiosperm sources. Compared to the unfractionated soil, there was a greater separation of lignin-derived phenol distributions in fractions across crop rotations, especially in F_{2–53 µm} and F_{<2 µm} (Fig. 4). Relatively higher cinnamyl/vanillyl phenols ratios of samples from light fraction and F_{53–2000 µm} than the other two fractions were observed, but the syringyl/vanillyl phenols ratios were similar among fractions and with the whole soil (Fig. 4). Both oxidation state proxies ((Ad/Al)_v and (Ad/Al)_s) decreased from F_{<2 µm}, F_{2–53 µm}, F_{53–2000 µm} to light fraction (Figs. 3e and 3f), except for the F_{2–53 µm} sample of CCSS. The degradation state of lignin-derived phenols of the unfractionated soil was at a similar level with F_{2–53 µm} for the majority of crop rotations (Figs. 3e and 3f). Within the same soil fraction, lignin phenol distributions varied across crop rotations, especially between monoculture and diversified crop rotations (Fig. 4). Compared to monoculture treatments (AAAA and CCCC), the distributions of phenols (syringyl/vanillyl versus cinnamyl/vanillyl phenol ratios) from diversified crop rotations were less distinct from each other (Fig. 4). The total lignin-derived phenol concentrations were higher in relatively high diversified crop rotations (CCSW and CCSW_RC) than in low diversified crop rotations (AAAA, CCCC, CCAA, and CCSS) in light fraction, F_{53–2000 µm} and F_{2–53 µm} (Table 2). Both oxidation state proxies ((Ad/Al)_v and (Ad/Al)_s) in F_{2–53 µm} were higher in low diversified crop rotations (Table 2).