Organic Geochemistry 58 (2013) 56–68

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Organic Geochemistry

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The combined controls of land use legacy and earthworm activity on soilorganic matter chemistry and particle association during afforestation

Yini Ma a,b,⇑, Timothy R. Filley a,b, Cliff T. Johnston b,c, Susan E. Crow d, Katalin Szlavecz e,Melissa K. McCormick f

a Department of Earth, Atmospheric, and Planetary Sciences, Purdue University, IN 47907, USAb Purdue Climate Change Research Center, IN 47907, USAc Department of Agronomy, Purdue University, IN 47907, USAd Department of Natural Resources and Environmental Management, University of Hawaii, HI 96822, USAe Department of Earth and Planetary Sciences, The Johns Hopkins University, MD 21216, USAf Smithsonian Environmental Research Center, MD 21037, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 September 2012Received in revised form 21 February 2013Accepted 24 February 2013Available online 6 March 2013

0146-6380/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.orggeochem.2013.02.010

⇑ Corresponding author at: Department of Earth,Sciences, Purdue University, IN 47907, USA. Tel.: +1 7

E-mail address: ma25@purdue.edu (Y. Ma).

The chemistry and physical association of soil organic matter in the patchwork of successional foreststands in the eastern US is strongly controlled by past land use. Invasive earthworm activity in thesesame systems, however, may impart a chemical and physical disturbance exceeding that of land use leg-acy. We established eight plots within forests of the Smithsonian Environmental Research Center (SERC)(Edgewater, MD), to compare sites with no record of significant agricultural disturbance or earthwormactivity and successional mixed hardwood forests recovering from past agriculture (60–132 yr) that con-tained both native and non-native earthworms. Soils (0–15 cm) were separated into physical fractions bysize (microaggregates) and density (light and heavy particulate organic matter) and investigated fororganic carbon (C) and nitrogen (N) partitioning. In addition, molecular composition was analyzed usingFTIR spectroscopy and lignin phenol and substituted fatty acid (SFA) extraction.

Even after 132 yr of recovery, the successional forests were nearly devoid of Oa+e horizons; a conditionwe attribute to high activity of invasive earthworms. Additionally, soil organic carbon (SOC) concentrationprofiles, and 14C derived mean residence times indicated mixing of the surface soils and fresh input ofcarbon to 10 cm, distinct from the undisturbed, mature sites. The proportion of microaggregated particu-late organic matter (iPOM) and silt + clay (iSC) was significantly higher in successional than undisturbedforests, which we attribute to the combined influence of past agricultural land use and high earthwormactivity. Among the successional sites, older forests exhibited a significant decrease in the proportion ofC and N in iSC but an increase in their proportion in iPOM, suggesting selective incorporation of iPOM withearthworm activity over great periods of time. In addition, continual consumption and mixing activities ofthe earthworm population could also be a primary control of the higher concentration and less oxidizedlignin phenols as well as a higher proportion of lignin phenols to SFA in all soil fractions in the successionalsites. Using partial least squares (PLS) regression of FTIR spectra, we also demonstrated a strong correlationbetween soil C physical distribution (microaggregated vs. non-microaggregated) and chemical aspects ofspecific FTIR regions which confirmed our findings from the lignin and SFA and showed distinct chemicaldominance among the different sites. Our results indicated that while past agricultural practice may havebeen the primary initial influence on C and N stock and soil physical distribution in the successional sites,the prolonged legacy and trajectory of recovery from the past land disturbance can be controlled by thenature of the invasive and native earthworm activity during afforestation.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Land use change is one of the primary drivers of biogeochemicalchange in modern terrigenous ecosystems (Boutton et al., 1999;

ll rights reserved.

Atmospheric, and Planetary652372348.

Conant et al., 2004; Bondeau et al., 2007; Jiang et al., 2010). Theconversion of forest ecosystems to agricultural land causes dra-matic declines in soil organic matter (SOM) storage because of in-creased decomposition and erosion as well as a potential decreasein plant carbon input to the soil (Six et al., 2000; Murty et al.,2002; McLauchlan, 2006; Jandl et al., 2007). In contrast, afforesta-tion generally results in fast accumulation of an O-horizon and aslow increase in soil organic carbon (SOC) in shallow mineral

Y. Ma et al. / Organic Geochemistry 58 (2013) 56–68 57

horizons (Richter et al., 1999; Post and Kwon, 2000; Paul et al.,2002; Wang et al., 2006). The gradual increase in soil and forest floorC storage is a result of many factors including increased input fromfast growing plants in the early successional stage, changes inchemical composition of plant input and increasing aggregation ofparticulate carbon providing physical protection of SOC (Paulet al., 2002; Six et al., 2002b; Turner et al., 2005; Gamper et al.,2007).

Although not generally considered when studying ecosystem Cand N dynamics associated with land use change, the changing soilmacro-faunal community can significantly impact the nature ofSOM and litter dynamics (Filley et al., 2008b; Crow et al., 2009).Earthworms, in particular, can drive SOM stabilization or destabi-lization (Bohlen et al., 2004; Lyttle et al., 2011). For example, earth-worms, particularly when newly introduced, can change thevertical distribution of SOM by mixing the litter and Oa+e horizon,with mineral soil down to more than a meter in depth (Shusteret al., 2001; Hale et al., 2005), promote the formation and incorpo-ration of particulate organic matter (POM) into stable aggregates(Jegou et al., 2000; Bossuyt et al., 2006; Coq et al., 2007), andpotentially shift soil and litter microbial communities to more bac-teria dominated, influencing decay dynamics (Tiunov and Scheu,2000; Zhang et al., 2000; Dempsey et al., 2011). Additionally, earth-worms can have a large impact on the soil C and N cycles byenhancing denitrification rates (Wust et al., 2009; Nebert et al.,2011). Surface dwelling earthworms tend to preferentially feedon and digest the N- and aliphatic-rich leaf body parts, leaving lig-nin-rich petioles and midveins on the forest floor or mixed intosurface soil (Filley et al., 2008b; Crow et al., 2009). In this way,earthworm activity can increase lignin and decrease aliphatic bio-polymers in the surface litter residue and soil POM (Suarez et al.,2006; Filley et al., 2008b; Crow et al., 2009).

The analysis of soil fractions, separated by size and density intoparticles relating to theoretical physical and chemical protectionmechanisms of SOM, can provide a useful measurement of the soilC and N dynamics during land use change (Six et al., 2000; Chris-tensen, 2001; Billings, 2006; Sollins et al., 2006). These SOM poolsmay have different levels of resistance to disturbance and replace-ment rates and, as such, convey selective chemical and isotopic sig-natures of past and present land conditions (Krull et al., 2003;Filley et al., 2008a). It is known that the extensively cultivated soilsare dominated by smaller, microaggregate sized (53–250 lm),compared to larger particle dominancy in undisturbed forests orpastures ecosystems (Jastrow, 1996; Six et al., 2002b; DeGryzeet al., 2004). In all but very sandy systems, inclusion of particulateorganic matter (POM) into microaggregates (including free micro-aggregates and microaggregates within macroaggregates) is a keycomponent of long term stabilization of SOM and is a sensitiveand important pool of carbon during afforestation (Six et al.,2002a,b).

The forests along the Atlantic coast of North America representa patchwork of land use and successional history spanning the lastfew hundred years. This is important, but often undocumented, forreconstructing how past agriculture, subsequent abandonmentand progressive afforestation influence SOM pools. The Smithso-nian Environmental Research Center (SERC, Edgewater, MD) in-cludes such a forest system with varied land use history and theadded complexity of containing sharp gradients in invasive earth-worm populations. SERC forests include sites recovered from pastagricultural activity over a span of �60–132 yrs as well as siteswith no record of significant disturbance (Parker, unpublisheddata; Higman, 1968). Previous work also has shown a dynamic na-tive and invasive earthworm population that generally has higherearthworm biomass and density in the young (�60–74 yr) succes-sional sites than the old (113–132 yr) successional sites (Filleyet al., 2008b; Szlavecz et al., 2011). Interestingly, there is no evi-

dence of the presence of earthworms in mature forest sites withno record of harvest (Szlavecz et al., 2011); possibly a due to acombination of edaphic/plant chemical inhibition and lack ofdisturbance.

The objective of the present study is to address the interactiveeffects of past agriculture land use, subsequent abandonmentand impacts of continuous earthworm activity on the changing dis-tribution and chemical characteristics of SOC and soil N in soilphysical fractions of purportedly different protection states duringforest development. Based upon previous studies we expected thatpast land use change and earthworm activity jointly affect soilcharacteristics. However, the relative importance of these interact-ing factors leads to different trajectories following abandonment. Ifearthworm activity was not a primary influence during the last130 yr of afforestation we would expect to observe the develop-ment of substantial O-horizon, a soil C and N depth profile withdistinct horizons and the increase of proportion of non-microag-gregated carbon. If earthworms were a major post abandonmentinfluence, however, we would expect to see a uniform C and Ndepth profile without significant O-horizon development, and soilbeing dominated by microaggregated POM. Additionally, in the la-ter scenario, the combined influence of physical mixing, selectivefeeding and suppression of fungal activity, would result in theaccumulation of lignin in mineral soil in afforested sites particu-larly with respect to cutin-derived substituted fatty acids.

2. Methods

2.1. Site description

SERC forests at the Smithsonian Environmental Research Centerlocated on the Chesapeake Bay (38�530N, 76�330W) contain morethan 100 species of woody plants (Higman, 1968) with a majorityof the forests classified as being in the tulip poplar (Liriodendrontulipifera) or chestnut oak–chestnut associations. The successionalsites are within the region of tulip poplar association where tulippoplar is the dominant tree species and Liquidambar styraciflua(sweet gum), Acer rubrum (red maple) Quercus alba (white oak),Quercus rubra (red oak), Fagus grandifolia (beech) are secondarytree species. The mature stands lie within the chestnut oak–chest-nut association with Q. alba (white oak), Q. rubra (red oak), F. gran-difolia (beech), Carya sp. (hickory) and Cornus florida (dogwood) asthe dominant tree species (Szlavecz et al., 2011).

Earthworm surveys at SERC have been conducted since 1999,identifying 12 earthworm species, three of which are native andnine are non-native, where non-native species were present at allsuccessional sites (Szlavecz and Csuzdi, 2007; Szlavecz et al.,2011). Lumbricus rubellus (invasive), Octolasion lacteum (invasive)and Eisenoides lonnbergi (native) were the dominant species.Although earthworm activities and abundances vary with seasonalweather controls, samplings prior to retrieval of soil cores for thepresent study generally found, higher earthworm biomass anddensity in young successional sites compared to old successionalsites and no earthworms in mature sites.

Soils at SERC are classified as Collington sandy loam (fine-loamymixed, active, mesic Typic Hapludult), Monmouth fine sandy loam(fine, mixed, active, mesic Typic Hapludult), or Donlonton finesandy loam (fine, mixed, active, mesic Typic Hapludults). The soilsamong all the sites had similar texture and mineralogy (Pierce,1974) with textural variations investigated in more detail herein.The mean precipitation in the region is 114.6 cm and the meanannual temperature is 13 �C (D. Correll, T. Jordan, and J. Duls,unpublished data). Leaf input rates at SERC are typically330–450 g m�2 yr�1 with low and high rates ranging from272–525 g m�2 yr�1 (Parker, unpublished data).

58 Y. Ma et al. / Organic Geochemistry 58 (2013) 56–68

Within SERC forests, eight 3 m � 3 m research plots containinga number of monitoring and litter manipulation experiments wereestablished in 2003 and 2008 within forest stands ranging from60 yr to > 200 yr. The determination of forest age for each sitewas based on a combination of historical records and tree coresof the largest trees in each forest area (Parker, unpublished data;Higman, 1968). For the purpose of this study, and consistent withprevious work at the site (Filley et al., 2008b; Crow et al., 2009), theplots were grouped into stands that ranged from 60–74 yr sinceabandonment (three young successional sites), 113–132 yr sinceabandonment (two old successional sites) and > 200 yr withoutrecord of cultivation (three undisturbed mature sites) (Table 1).All eight sites were within an area of 1.5 km2.

2.2. Soil sampling and physical fractionation

In October 2008, six soil cores (0–15 cm) were taken from eachof the eight plots and sectioned into 5 cm depth increments. Thesix cores were reduced to three composite cores by randomlychoosing two cores to combine within each plot. Field-moist soilswere passed through an 8 mm sieve and rocks, large roots andother debris were removed. The sieved soil samples were air drieduntil constant weight then stored in glass jars until processing.From each site, soil organic horizons, Oi and Oe + Oa were sampledin September 2011, where Oi was comprised of any visible leaves,twigs and wood fragments.

Sieved soils from 0–5 cm and 5–10 cm were further separated byboth size and density fractionation techniques using methods mod-ified from Six et al. (2002b) (Fig. 1) to obtain five soil fractions: (1)coarse particulate organic matter (cPOM), (2) fine particulateorganic matter (fPOM), (3) intra-microaggregate particulate organicmatter (iPOM), (4) free silt and clay (fSC), and (5) intra-microaggre-gate silt and clay (iSC). The mass of cPOM and iPOM was determinedby applying a sand content correction, McPOM(iPOM) = Mtotal �Msand,experimentally determined for each plot.

Table 1Land use history, age, and earthworm abundance of experimental forest stands at theSmithsonian Environmental Research Center.

Site Agegroup

Forestagea

Land usehistoryb

Earthwormabundancec

Soil pH(H2O)mean ± stddev

Plant typec

4 Young �60 Mixedcultivation,abandoned

High 5.05 ± 0.08 Tulip poplarassociation

5 Young 65 Mixedcultivation,abandoned

High 5.49 ± 0.04 Tulip poplarassociation

6 Young 74 Mixedcultivation,abandoned

High 5.36 ± 0.13 Tulip poplarassociation

2 Old 113 Mixedcultivation,abandoned

Low 4.31 ± 0.29 Tulip poplarassociation

3 Old �132 Mixedcultivation,abandoned

Low 4.18 ± 0.08 Tulip poplarassociation

BI Mature >200 Neverdisturbed

No 3.22 ± 0.02 Chestnut oak–chestnutassociation

FP Mature >200 Neverdisturbed

No 3.31 ± 0.03 Chestnut oak–chestnutassociation

HI Mature >200 Neverdisturbed

No 3.23 ± 0.06 Chestnut oak–chestnutassociation

a From unpublished tree ring data and historical land use map (Parker, unpub-lished; Higman, 1968).

b From documented land use history by Higman (1968).c Szlavecz et al. (2011).

2.3. Soil texture, elemental analysis and radiocarbon determination

Pooled soil samples (2 mm sieved) from six cores of each plotwere used for soil texture analysis by the hydrometer method (A& L Great Lakes Labs, Fort Wayne, IN) (Bouyoucos, 1962). Soil pHwas measured based on soil survey laboratory information manual(USDA, 1995).

Ground bulk soil and soil fractions were analyzed for C and Ncontent using a Sercon (Crewe, UK) GLS elemental analyzer inter-faced to a Sercon (Crewe, UK) Hydra 20/22 isotope ratio mass spec-trometer (IRMS) operating in continuous flow mode. The lowaverage soil pH (4.2–5.7) precluded soil carbonate accumulationbut several samples were checked for carbonate content usingthe acid fumigation method (Harris et al., 2001), and none wereidentified.

For 14C determination, subsamples of the ground bulk soilswere individually weighed into pre-combusted quartz tubes with60 mg of pre-combusted CuO and a strip of Ag foil then sealed un-der vacuum and combusted at 900 �C for 8 h. Purified CO2 from thecombusted soil was converted to graphite on an iron catalyst usingthe hydrogen reduction method (Vogel et al., 1984). The 14C/12Cand 13C/12C ratios were measured by accelerator mass spectrome-try at the 14CHRONO Centre for Chronology, Palaeoecology, and theEnvironment, Queen’s University Belfast. Mean residence time(MRT) of C in bulk soil was calculated using a one pool, time-dependent, steady-state model (Trumbore et al., 1995). The MRTwas determined by matching the measured and modeled F14C forthe year in which the soil was sampled. Assumptions of the modelwere (1) that C inputs equaled outputs in each year, and (2) theF14C value of the input equaled the previous year’s atmosphericvalue.

2.4. DR-FTIR analysis

Diffuse reflectance Fourier transform infrared spectroscopy(DR-FTIR) was used to analyze all bulk soil samples using a PerkinElmer Model 2000 GX FTIR spectrophotometer (Norwalk, CT)equipped with liquid nitrogen cooled MCT detector and a KBr beamsplitter. For each sample a total of 64 individual scans, which werethen signal averaged, were obtained using an optical resolution of4 cm�1 in the mid-infrared region from 4000–580 cm�1. The sam-ple absorption spectra were obtained using the Kubelka–Munktransformation to deduct the background absorption from KBr inthe Grams/32 AI Version 6.0 (Galactic software, Salem, NH).

2.5. Lignin and substituted fatty acid extraction and quantification

Bulk soil and soil fractions were extracted for lignin phenols andselected substituted fatty acids (SFA) derived from cutin and

Fig. 1. Flowchart of soil physical fractionation process on soils from the Smithso-nian Environmental Research Center. Microaggregates (mAG), free silt and clay(fSC) and coarse particulate organic matter (cPOM) were isolated using methodsmodified from Six et al. (2002b). The mAG fraction was further separated by bothsize and density fractionation using sieving and sodium polytungstate (SPT)flotation at 1.85 g/cm3 density. The mass of cPOM and iPOM was determined byapplying a sand content correction, McPOM(iPOM) = Mtotal �Msand, experimentallydetermined for each plot.

Y. Ma et al. / Organic Geochemistry 58 (2013) 56–68 59

suberin using alkaline cupric oxide (CuO) (Goñi and Hedges, 1990;Filley et al., 2008b) on the composited samples from each site. Thebiopolymer extractions utilized Monel reaction vessels (Prime Fo-cus, Inc. Seattle, WA, USA). A Shimazu QP2010 Plus Gas Chromato-graph–Mass Spectrometer (GC–MS) (Kyoto, Japan) withquantitation by extracted ion internal calibration curves was usedto assess compound concentrations (Filley et al., 2008b).

The trimethylsilyl (TMS) derivatives of vanillyl (V)-based (i.e.,vanillin, acetovanillone, vanillic acid), syringyl (S)-based (i.e.,syringealdehyde, acetosyringone, syringic acid), and cinnamyl(Ci)-based (i.e., p-hydroxycinnamic acid and ferulic acid) ligninwere quantified and the total yield of lignin phenols was denotedas ‘SVCi-lignin’. Additionally, the TMS derivatives of nine SFA peakswere quantified and included 16-hydroxyhexadecanoic acid, hexa-decanoic diacid, 18-hydroxyoctadec-9-enoic acid, a co-elution of9,16- + 10,16-dihydroxyhexadecanoic acid, 9-octadecene-1,18-dioic acid, 7- and 8-hydroxyhexadecane-1,16-dioic acid, 9,10,18-trihydroxyoctadec-12-enoic acid, and 9,10,18-trihydroxyoctanoicacid. Compound concentrations are given as mg compound/100 mg organic carbon (OC). Lignin-derived phenols and SFA werequantified by analysis of extracted ions of their trimethylsilane(TMS) derivatives based upon absolute (lignin) or proxy (SFA) stan-dard calibration curves relative to internal standard ethyl vanillin.Alkaline CuO provides a rapid means to asses SFA composition sim-ilar to other techniques that extract SFA from plants and soils usingbase hydrolysis and solvent extraction techniques (e.g., Riedereret al., 1993; Nierop et al., 2003; Otto and Simpson, 2006), althoughcompared to base hydrolysis the SFAs longer than C20 chain werepoorly recovered. Since the longer chain SFAs can comprise majorbuilding blocks of root suberin, our results from CuO extractionmay underestimate the contribution of roots to total SFAs as com-pared to base hydrolysis.

2.6. Data analysis

To study effect of stand age as well as earthworm disturbanceon soil C and N dynamics, one-way ANOVA with Tukey’s posthoc test was used for measured variables using SAS v.9.1 (SASInc., Cary, NC). The significance threshold was set at a = 0.05 unlessotherwise reported. The FTIR spectra for the bulk soil samples wereanalyzed using partial least square (PLS) regression in Grams/32 AIVersion 6.0 (Galactic software, Salem, NH) in a comparison withthe carbon storage within and outside of microaggregates. Multi-scatter-correction (MSC) method was used for the spectral pre-treatment process and the optimum number of factors in the PLScalibration models were determined by cross validation and de-fined by the prediction residual error sum of squares (PRESS) func-tion to avoid over-fitting the model. The PLS-derived correlationbetween the known vs. predicted concentration was obtainedand used to determine the spectral regions positively and nega-tively correlated to a given parameter.

3. Results

3.1. Soil profile bulk elemental, pH, radiocarbon dating and texturalcharacteristics

The soil texture of the plots was primarily loam/sandy loam(Table 2) with site 3 deviating with slightly higher clay content(3% in 0–5 cm) as a loam/silt loam. Percent sand ranged from32–53%, while clay ranged from 3–19%. There was no significantrelationship between soil texture and stand age. Soil pH variedwith stage age where young successional sites ranged from5.05–5.49, old successional sites ranged from 4.18–4.31, andmature sites from 3.22–3.31 (Table 1).

The thickness of Oa + Oe horizons in mature forests, whereearthworms were absent, ranged between 3 and 4.5 cm. In allbut one successional site, where earthworms were present, min-eral soil was exposed below fresh litter without an intermediatehumus layer. One old successional site (site 2, 132 yr) containeda sporadic Oa + Oe horizon, but it was never more than 3 mm thick.The fresh litter layer (Oi) in mature forests was also much thickerthan successional forests (visual observation).

Soils in each of the age categories exhibited a different depthtrend for wt% C (P = 0.005) but not wt% N (P = 0.13) (Table 3). At0–5 cm young successional sites had significantly lower averagewt% C (3.08%) than the mature and old successional forest sites(4.49% and 4.39%, respectively). Percent C decreased more steeplywith depth in the mature sites (from �4.5% to 1.55%) than in thesuccessional sites (4.39–2.50% for old forests and 3.08–1.89% foryoung forests). For each depth, the concentration of %N generallytrended as old successional > young successional > undisturbedmature forests, with successional sites and mature sites being sig-nificantly different. The exception was the 0–5 cm depth range,where old sites had significantly higher %N than mature sites,but neither was different from young sites. The mature forest soilhad the highest C/N ratio at all depths with differences amongage groupings driven by N concentration.

The mature sites contained significant stocks of OC in the Oe +Oa horizons (�2388 g C/m2), which accounted more than 2/3 ofthe total soil C stored in the 0–15 cm mineral soil (�3617 g C/m2) (Table 3). However, in successional forests, carbon stored atOe + Oa horizons accounted for less than 1% of total carbon(0–15 cm) in old successional sites and was absent in youngsuccessional sites. Excluding the O-horizons, the total carbonstorage within the upper 15 cm of mineral soil was distinctbetween old (4067 ± 493 g C/m2) and young (3383 ± 204 g C/m2)successional forest stands (Table 3). The mature forests exhibitedintermediate values but with a wide range (3617 ± 1335 g C/m2).The total N storage in the mineral soil was significantly less inmature forests than successional forests. However, significantlymore N was stored in the O horizon in mature sites (101 g N/m2)compared to successional sites (3 g N/m2 and 8 g N/m2 for youngand old successional sites, respectively). The total storage of N,when considering the combined mineral soil (0–15 cm) andO-horizon, in the mature forests was similar to successional sites.

The 14C derived mean residence time (MRT) of SOC among thesites generally increased with depth (Table 4) but to different ex-tents. In mature forests, the MRT of SOC in 0–5 cm soil(139 ± 64 yr) was, on average, significantly shorter than 5–10 cm(425 ± 150 yr) and 10–15 cm soil (671 ± 216 yr) in comparisonwith successional sites with past disturbance. In successional for-ests with past agricultural land use and current earthworm distur-bance the MRT of SOC with depth exhibits, on average, less of ashift to longer MRT. In fact, one successional site (site 6 with amaximum forest age of 74 yr), exhibits an inverted MRT profile,as SOC in the 5–10 cm had a lower MRT than in 0–5 cm.

3.2. Soil physical fraction proportion and C and N content

Both 0–5 and 5–10 cm depths in the mature sites were domi-nated by fSC (>60% of total mass) with iSC being the next mostdominant (20–25% of total) (Fig. 2A). Mass proportions of thesetwo fractions were nearly equal (�40%) in the young successionalforests, while old successional forests were intermediate betweenyoung successional and mature sites. The mass of cPOM and fPOMincreased from young to mature forest sites in the 0–5 cm depthbut remained constant at 5–10 cm. The iPOM fraction was indistin-guishable between age groups. The mass proportion of soil in iSCdecreased progressively from the young to mature sites while theinverse relationship existed for fSC.

Table 2Soil texture of study plots at the Smithsonian Environmental Research Center.

0–5 cm 5–10 cm 10–15 cm

% Sand % Silt % Clay Category % Sand % Silt % Clay Category % Sand % Silt % Clay Category

YoungSite 4 52 37 11 Loam 52 34 14 Loam 52 30 18 LoamSite 5 48 37 15 Loam 44 37 19 Loam 44 37 19 LoamSite 6 40 45 15 Loam 36 49 15 Loam 43 42 15 Loam

OldSite 2 36 49 15 Loam 40 45 15 Loam 40 45 15 LoamSite 3 48 49 3 Sandy loam 44 49 7 Sandy loam 44 45 11 Loam

MatureSite FP 56 33 11 Sandy loam 52 37 11 Loam 53 33 16 Sandy loamSite HI 40 45 15 Loam 36 45 19 Loam 32 49 19 LoamSite BI 36 53 11 Silt loam 36 45 19 Loam 36 45 19 Loam

Table 3Percent C, percent N, C/N ratio, and C and N storage in the bulk soil and O-horizon of the experimental plots at the Smithsonian Environmental Research Center, Edgewater,Maryland. Young successional (sites 4, 5, 6), old successional (sites 2, 3), and mature (sites FP, HI, BI) are compared. Data are grouped by stand age and depth. The mean ±1standard deviation is shown. Different letters indicate significant differences among stand ages within the same depth.

Thickness C% N% C/N C storage (g/m2) N storage (g/m2)

YoungOi �1 cm 31.89 ± 2.63 a 0.90 ± 0.08 a 35.65 ± 1.40 a 116 ± 51 a 3 ± 1 aOe + Oa NA NA NA NA 0 a 0 a0–5 cm 3.08 ± 0.36 a 0.22 ± 0.02 ab 14.02 ± 0.95 a 1583 ± 190 a 117 ± 10 a5–10 cm 1.89 ± 0.29 b 0.16 ± 0.02 a 11.96 ± 1.14 a 1077 ± 142 a 94 ± 8 a10–15 cm 1.03 ± 0.16 b 0.10 ± 0.01 a 10.35 ± 0.63 a 724 ± 94 b 70 ± 7 a

Total 3499 ± 204 a 284 ± 13 a

OldOi �1–1.5 cm 38.9 ± 2.69 a 1.06 ± 0.07 a 36.62 ± 0.97 a 186 ± 23 a 5 ± 1 aOe + Oa <0.3 cm 14.95 ± 0.85 b 0.64 ± 0.02 b 23.26 ± 1.71 b 66 ± 33 b 3 ± 1 b0–5 cm 4.39 ± 0.87 b 0.29 ± 0.06 b 15.26 ± 1.41 a 1855 ± 311 a 119 ± 23 a5–10 cm 2.50 ± 0.38 a 0.18 ± 0.04 a 13.96 ± 1.04 b 1264 ± 111 a 87 ± 15 a10–15 cm 1.67 ± 0.36 a 0.12 ± 0.04 a 14.00 ± 1.08 b 949 ± 174 a 63 ± 19 a

Total 4319 ± 493 b 276 ± 56 a

MatureOi �2–3 cm 46.75 ± 0.44 b 1.32 ± 0.10 a 35.58 ± 2.47 a 428 ± 45 b 12 ± 2 bOe + Oa �3–4.5 cm 35.28 ± 6.23 c 1.32 ± 0.26 c 26.96 ± 1.63 b 2388 ± 819 c 89 ± 31 c0–5 cm 4.49 ± 1.21 b 0.18 ± 0.05 a 25.46 ± 1.74 b 2041 ± 708 a 80 ± 29 b5–10 cm 1.55 ± 0.78 b 0.07 ± 0.03 b 21.88 ± 1.44 c 932 ± 508 a 42 ± 22 b10–15 cm 0.96 ± 0.34 b 0.05 ± 0.02 b 18.50 ± 1.96 c 644 ± 241 b 37 ± 12 b

Total 6432 ± 1335 c 260 ± 58 a

Table 4Fraction modern (F14C ± analytical error) and modeled mean residence time (MRT in years) for the bulk soil among plots investigated at the Smithsonian EnvironmentalResearch Center, Edgewater, Maryland. Young successional (sites 4, 5, 6), old successional (sites 2, 3), and mature (sites FP, HI, BI) are compared. Data are grouped by stand ageand depth and the mean MRT ± 1 standard deviation is shown.

0–5 cm 5–10 cm 10–15 cm

F14C MRT (yr) Mean MRT (yr) F14C MRT (yr) Mean MRT (yr) F14C MRT (yr) Mean MRT (yr)

Young Site 4 1.0841 ± 0.0022 101 116 ± 14 1.0510 ± 0.0026 154 157 ± 70 0.9806 ± 0.0022 413 436 ± 308Site 5 1.0660 ± 0.0028 127 1.0220 ± 0.0028 230 0.9102 ± 0.0048 756Site 6 1.0691 ± 0.0024 122 1.0942 ± 0.0022 89 1.0583 ± 0.0021 140

Old Site 2 1.0708 ± 0.0025 119 105 ± 20 1.0555 ± 0.0028 145 140 ± 8 0.9760 ± 0.0025 439 321 ± 167Site 3 1.0919 ± 0.0023 92 1.0614 ± 0.0028 135 1.0310 ± 0.0023 203

Mature Site FP 1.0993 ± 0.0026 84 139 ± 64 0.9646 ± 0.0024 509 425 ± 150 0.9497 ± 0.0026 611 671 ± 216Site HI 1.0667 ± 0.0022 126 1.0226 ± 0.0025 229 0.9675 ± 0.0040 491Site BI 1.0291 ± 0.0041 209 0.9606 ± 0.0029 536 0.9122 ± 0.0028 909

60 Y. Ma et al. / Organic Geochemistry 58 (2013) 56–68

There was a significant effect of forest age on the C and N pro-portions among soil fractions (Table 5). Additionally, C and N incPOM and iSC fractions showed a significant interaction betweenforest age and depth, where the difference of C and N proportionsbetween age groups tended to diminish with depth (Fig. 2B and C).Overall, when fSC and iSC were combined as total silt and clay, theyrepresented from 45–60% of C and 50–70% of N (Fig. 2B and C),

making this fraction the dominant reservoir for carbon and nitro-gen. The iPOM fraction was the largest contributor to the total Cin POM in the successional forests, while iPOM and cPOM domi-nated and were more evenly distributed in the mature sites. Theproportion of C and N present in POM decreased from 0–5 cm to5–10 cm depths in each age group. Similar to mass distribution,the proportion of C and N in iSC was highest in young successional

Y. Ma et al. / Organic Geochemistry 58 (2013) 56–68 61

sites and decreased progressively from the young to mature sitesfor both depths. Mature sites exhibited the highest proportionsof C in fSC and cPOM. The proportion of C and N in iPOM was con-sistently lower in mature sites than successional sites. Althoughwith different masses for different fractions, the proportional dis-tributions of C and N among fractions and forest ages (Fig. 2Band C, respectively) showed the same pattern as the mass propor-tions of fractions (Fig. 2A).

3.3. Soil carbon storage in physical fractions

Microaggregated and non-microaggregated C storage over 0–10 cm, calculated from values from 0–5 cm and 5–10 cm depthintervals, showed that forest age, and thus generally earthwormabundance, influenced the C distribution (P = 0.0025 andP = 0.0148 for microaggregated and non-microaggregated, respec-

Fig. 2. (A) Mass, (B) proportion of C, and (C) proportion of N in soil fractions for differenCenter, Edgewater, Maryland. Young successional (sites 4, 5, 6), old successional (sites 2, 3separately. Different letters signify significant differences (p < 0.05) between different st

tively) (Fig 3). Microaggregated C (iSC + iPOM) in the mature for-ests (1073 g C/m2) was significantly lower than both young(1533 g C/m2) and old (1670 g C/m2) successional forests. Whilethe overall non-microaggregated C (cPOM + fPOM + fSC) increasedfrom young (1127 g C/m2) to old (1449 g C/m2) successionalforests on a trajectory toward the undisturbed mature forests(1882 g C/m2), the C proportion of individual non-microaggregatedfractions only showed modest increase. Additionally, the propor-tion of the overall microaggregated C was significantly higher insuccessional forests.

3.4. Biopolymer characteristics in soil and soil fractions

Within each age group the concentration (mg/100 mg OC) ofSVCi lignin and SFA varied among fractions (Fig. 4A and B). Ingeneral, for each depth, lignin concentrations were higher

t stand ages among plots investigated at the Smithsonian Environmental Research), and mature (sites FP, HI, BI) are compared. 0–5 cm and 5–10 cm depths are shownand ages for each fraction within the same depth.

Table 5ANOVA for mass proportion and C and N distribution among plots investigated at theSmithsonian Environmental Research Center, Edgewater, Maryland. P values show thesignificance of age, depth and age � depth interaction of mass, C and N proportions foreach fraction. Bolded numbers indicate significant effects (a < 0.05).

cPOM fPOM iPOM fSC iSC

Mass proportionAge 0.0308 0.0300 0.0578 <0.0001 <0.0001Depth <0.0001 <0.0001 0.0388 0.008 0.3706Age � depth 0.4743 0.0483 0.6311 0.9411 0.8830

C proportionAge <0.0001 0.0125 <0.0001 <0.0001 <0.0001Depth <0.0001 0.0006 0.0188 <0.0001 <0.0001Age � depth <0.0001 0.2605 0.9270 0.1950 0.0231

N proportionAge <0.0001 0.0024 <0.0001 <0.0001 <0.0001Depth <0.0001 <0.0001 0.0083 <0.0001 <0.0001Age � depth <0.0001 0.0842 0.9391 0.0677 0.0412

62 Y. Ma et al. / Organic Geochemistry 58 (2013) 56–68

in POM fractions than in SC fractions, with cPOM having the high-est concentration. The difference in concentration betweenfSC + iSC associated lignin and that contained in POM was the larg-est in young successional sites and decreased with forest age forboth depths and was driven by gains in the cPOM lignin. Also, allfractions exhibited higher concentration of SVCi lignin in 0–5 than5–10 cm depth. Across forest age groupings SVCi lignin tended todecrease in bulk soil as well as all fractions with increasing age(Fig. 4A). The Ad/Alv ratio of all fractions increased following theorder of cPOM < fPOM < iPOM < fSC < iSC in successional forests,while in mature forests, iPOM had higher Ad/Alv than fSC, so themicroaggregated fractions (iPOM and iSC) had the highest Ad/Alv

ratio. Across age groups, Ad/Alv ratio of bulk soil and all fractionsin mature sites was higher than successional sites (Fig. 4D).

In contrast to SVCi lignin, SFA concentration (mg/100 mg OC)exhibited no trend among fractions within an age group, althoughthe variation in measured values was higher among sites. In addi-tion, SFA tended to increase with increasing forest age (Fig. 4B),with changes in fSC being the most significant (e.g. 1.17, 1.77,6.04 mg/100 mg OC for young successional, old successional andmature sites, respectively, at 0–5 cm depth).

Because of both higher lignin and lower SFA concentration insuccessional sites, the lignin/SFA ratio was significantly higher in

Fig. 3. O-horizon (Oa, Oe, Oi), microaggregated (mAG) and non-microaggregated (non-mnon-mAG C ratio (dotted line, right axis) among plots investigated at the Smithsonian Envold successional (sites 2, 3), and mature (sites FP, HI, BI) are compared. Different letters fomethod. Error bars of mAG/non-mAG ratio represent field variation, and error bars for c

successional sites compared to mature sites for all fractions exceptfor cPOM (Fig. 4C). Generally, cPOM had the highest lignin/SFA ra-tio, while iPOM or iSC had the lowest. Although not significant, thelignin/SFA ratio for bulk soil was also higher in successional sitesthan mature sites (e.g. 0.47, 0.72, 0.68 for mature, old successionaland young successional sites, respectively).

3.5. PLS modeling and FTIR

FTIR analysis (48 samples) of bulk soil was used to provide anassessment of chemical differences among sites. A representativeDR-FTIR spectrum of a bulk soil sample is shown in Fig. 5 alongwith the main diagnostic spectral regions. The well resolved sharpm(CH) bands are particularly diagnostic in studies of soil- andplant-derived organic matter to quantify and characterize waxesand related alkyl-carbon containing moieties (Madari et al., 2006;Lehmann et al., 2007; Filley et al., 2008b; Verchot et al., 2011). Con-sistent with the SFA results (Fig 4), the integrated intensity of thisregion increased with forest age in the 0–5 cm depth, while the 5–10 cm depth remained the same (Fig. 6).

The PLS model was used to examine the relationship betweenthe DR-FTIR spectral regions and the amount of microaggregatedand non-microaggregated C (Fig. 2). The observed correlations inthe ‘‘leave-one-out analysis’’ were with adjusted r2 values of 0.81and 0.88, respectively, indicating useful predictive power (datanot shown). The correlation between the FTIR spectra and the loca-tion of C with respect to microaggregated structure also provideddetailed information related to which chemical functional groupsbest explained the relationship (Fig. 7). In the 3800–2600 cm�1 re-gion (Fig. 7A), the amount of non-microaggregated C gave a strongpositive correlation in the regions of m(CH) bands at 2925 cm�1 and2854 cm�1, and the m(OH) feature of externally bound water at3444 cm�1. In contrast, the amount of microaggregated C exhibitednegative correlation with m(CH) bands at 2925 cm�1 and2854 cm�1, and strong positive correlation at hydrogen bondedwater with a m(OH) frequency of 3275 cm�1. In the lower frequencyregion from 1800–1000 cm�1, non-microaggregated C was mostpositively correlated to the bands at 1730 cm�1, and at1611 cm�1 and 1449 cm�1, representing the C@O stretch and ali-phatic CAH deformation (Tatzber et al., 2010). The amount of mic-roaggregated C was the most positively correlated to 1640 cm�1,

AG) carbon storage (g/m2) in in 0–10 cm mineral soil (bar graph) as well as mAG C/ironmental Research Center, Edgewater, Maryland. Young successional (sites 4, 5, 6),r each section show significant difference groupings among stand age using Tukey’sarbon storage are not shown.

Fig. 4. Extracted lignin phenol and substituted fatty acid in bulk soil and physical fractions in 0–5 cm and 5–10 cm depth among different age forest plots investigated at theSmithsonian Environmental Research Center, Edgewater, Maryland. Young successional (sites 4, 5, 6), old successional (sites 2, 3), and mature (sites FP, HI, BI) are compared.(A) shows total extracted syringyl (S), vanillyl (V), and cinnamyl (Ci) lignin phenols, (B) shows substituted fatty acid (SFA) composition, (C) depicts the SVCi/SFA ratio, and (D)depicts the Ad/Alv (vanillic acid/vanillin) ratio. Error bars designate field variation. Different letters signify significant differences among stand ages for each fraction withinthe same depth.

Y. Ma et al. / Organic Geochemistry 58 (2013) 56–68 63

1537 cm�1 and 1430 cm�1, representing C@O; C@N stretch(amide) and aromatic C@C or aromatic skeletal, respectively.

4. Discussion

4.1. Earthworm activity limits O-horizon development during therecovery from past agriculture

Plant succession after agriculture abandonment is accompaniedby accumulation of litter layer, rebuilding of O-horizon and in-crease of carbon in the A horizon. The rate of these changes con-trolled primarily by a combination of local climate, ecology andedaphic properties (Richter et al., 1999; Paul et al., 2002). Forexample, during 88 yr of afforestation in Massachusetts, forestfloor C (Oi + Oe + Oa) accumulated to �2500 g m�2 in conifer forestsand �1000 g m�2 in hardwood forest (Compton and Boone, 2000).Poeplau et al. (2011) predicted forest floor accumulation rate to be38 ± 4 g C m�2 yr�1 for afforestation systems in temperate zones,

with conifer forests (71.5 ± 7.7 g C m�2 yr�1 after 20 yr) beingslightly but not significantly higher than broadleaf forests(54.4 ± 8.4 g C m�2 yr�1 after 20 yr).

While rates of O-horizon accumulation are expected to varywith species and climate (Bautista-Cruz and del Castillo, 2005;Kalinina et al., 2009), it is significant that no consistent and quan-tifiable Oe + Oa horizon had accumulated even after 132 yr of affor-estation in our successional sites, let alone a significant litter layer(Oi) (116–186 g m�2). Similar to our findings, a study in southeast-ern Ontario, Canada demonstrated that only three out of fifteen ofmature forest sites (dominated by sugar maple and red oak) exhib-ited recovery of Oe + Oa layer and no surface litter accumulation(Foote and Grogan, 2010). Foote and Grogan (2010) documentedhigh earthworm activity in their study sites, which they attributedto the loss of annual litter and mixing and possibly to an impededO-horizon development.

Using estimates of earthworm biomass and density at SERC(Szlavecz and Csuzdi, 2007; Szlavecz et al., 2011) and assumingconsumption rates of 20–40 mg dry litter/day per g fresh animal

Fig. 5. A representative FTIR spectra of soil from 0–5 cm in plots investigated at the Smithsonian Environmental Research Center, Edgewater, Maryland to show differentspectrum regions.

Fig. 6. Integrated area of the alkyl m(CH) bands in the 2800–3000 cm�1 region as afunction of afforestation age in bulk soil samples (0–5 cm and 5–10 cm depths)investigated at the Smithsonian Environmental Research Center, Edgewater,Maryland.

64 Y. Ma et al. / Organic Geochemistry 58 (2013) 56–68

weight (Szlavecz, unpublished data), up to 1–1.5 g m�2 dry littermass should be consumed daily during the late spring through fall;a rate that is sufficient to consume nearly all the annual fallen leaflitter at SERC plots (Curry and Schmidt, 2006; Szlavecz et al., 2007,2011). With such litter feeding rates and concomitant physicalmixing, the lack of O-horizon development is consistent withearthworm presence. In addition, the more uniform 14C basedMRT in the upper 10 cm and shorter MRT of 5–10 cm in succes-sional sites supports our supposition of greater mixing of recentC into mineral soil, although the values in 10–15 cm depth varied(Table 4). Based on these findings we predict that these secondarysuccessional forests will continue to maintain well mixed A-hori-zons and no O-horizon development.

4.2. Past land use and earthworm activity interact to change soil Nprofiles

Unlike carbon, nitrogen concentration in young successionalforests, where agriculture was abandoned �60 yr ago, had alreadyreached or even exceeded N% in mature forests, indicating eitherincreased input or retention of N during past agricultural activityor recent input and stabilization because of annual translocationby earthworm activity.

A variety of crops were grown on these sites including tobacco,legumes, and corn but no detailed records of fertilizer type or usewere available and thus, we could not determine specific effects ofagricultural practices on N stocks in the successional sites. In gen-eral, tillage could cause dramatic loss of soil N by as much as 65%(Davis et al., 2003). On the other hand, input through inorganic andorganic N fertilizer could increase soil N (Edmeades, 2003). Addi-tionally, the leguminous, N-fixing, crops planted previously onthe successional sites at SERC, such as alfalfa (Medicago sativa L.)could counteract the N loss rate (Tiessen et al., 1982). Similar toagricultural practices, earthworm activity can either increase ordecrease the N content in mineral soil. Earthworms may cause Nloss by increasing N mineralization and denitrification rates(Willems et al., 1996; Wust et al., 2009; Nebert et al., 2011). Atthe same time, earthworms relocate forest floor N into mineral soil,perhaps promoting N stability through binding to mineral particlesand aggregation.

If N addition from past agriculture was the main driving factorfor N vertical distribution in our sites, we might expect decreasesin N with afforestation due to intensive plant uptake of N and slowdegradation of organic N from litter layer (Smal and Olszewska,2008). This would lead to a state similar to the mature forests atSERC with high N stocks in the O horizon and low N stocks inthe mineral soil. However, old successional forests had a higherN% than young successional forests, which indicates that transloca-tion of litter N into mineral soil by earthworm activity is driving Naccumulation in mineral soil horizons.

It is known that the decay of litter with a high initial C/N ratiocan result in movement of N from soil to litter by fungal hyphae

Fig. 7. Correlation coefficient along the FTIR spectral region from (A) 3800–2500 cm�1 and (B) 1800–1000 cm�1 between non-microaggregated C storage (non-mAG) (top)and microaggregated C storage (mAG) (bottom) among soil investigated at the Smithsonian Environmental Research Center, Edgewater, Maryland. Spectral regions above 0.0indicate positive correlations with C storage.

Y. Ma et al. / Organic Geochemistry 58 (2013) 56–68 65

(Wessen and Berg, 1986; Hart and Firestone, 1991). This upwardstransportation could lead to the observed N accumulation in theO horizon and loss in the mineral soil of our mature forest sites.In the successional sites, the limited accumulation of the O-hori-zon, caused by continuous removal and mixing through earth-worm activity, could sharply decrease fungal biomass in theforest floor (Dempsey et al., 2011). Thus instead of N ‘‘flowing’’ up-wards by soil–litter decomposing fungi, N ‘‘flowed’’ downwardsinto mineral soil with the impact of earthworm activity (Dempseyet al., 2011; Fahey et al., 2011).

4.3. Soil microaggregation as a legacy of past agriculture andearthworm activity

SOM-particle dynamics have a strong control on soil C recoveryfollowing land use conversion (Jastrow and Miller, 1998; Six et al.,1998, 2000). Smaller mineral-organic aggregate size classes aremost resistant to physical disturbance and, thus, the SOC associ-ated within smaller aggregates is protected to a greater degreeafter disturbance (Elliott, 1986; Beare et al., 1994; Six et al.,2000, 2002a). For example, cultivated soil is enriched in microag-gregated (53–250 lm) C relative to macroaggregated (>250 lm)C as compared to native forest or pasture ecosystems. Additionally,coarse macroaggregated POM, which is dominant in forest sys-tems, was preferentially lost compared to microaggregated POM,as a result of disturbance (Jastrow 1996; Six et al., 2002b; DeGryzeet al., 2004). After the cultivated land is abandoned and allowed toreturn to forest, the exposed microaggregated mineral surfacesfrom the past agriculture disturbance become favorable loci toquickly sequester new carbon input. Thus, it is possible that inthe successional forests at SERC, the greater proportion of iPOMand iSC mass as well as C and N within microaggregated structuresrelative to the mature forests could be still a primary legacy of thedisturbance from past farming, even after 132 yr.

Earthworms have been shown to promote the formation oflarge macroaggregates and increase the rate of macroaggregate

turnover, but also to incorporate plant residue into microaggre-gates within macroaggregates during digestion (Barois et al.,1993; Bossuyt et al., 2006; Snyder et al., 2009). Studies have alsodemonstrated that microaggregate-sized fractions within earth-worm casts were enriched in C and N by a factor of two comparedto the surrounding soil (Zhang et al., 2003). The results from SERCare consistent with their findings, even after 132 yr of recovery, theproportion of non-microaggregated C was still much lower com-pared to mature sites driven by the large proportion of microaggre-gated C (Figs. 2 and 3). In addition, the proportion of iPOMincreased from young to old successional sites at SERC while theiSC proportion decreased. This is expected, as earthworm activityselectively promotes POM vs. SC incorporation into microaggre-gates (Bossuyt et al., 2006).

4.4. Controls on the lignin and SFA trajectory during afforestation

Earthworm activity is suggested to increase lignin content insurface soil, especially POM. This is due to a combination of factors:(1) inability of earthworms to degrade lignin, (2) their indirect sup-pression of fungal activity by earthworms, and (3) the selectiveaccumulation of leaf structural tissue (Lawrence et al., 2003;Brown and Doube, 2004; Filley et al., 2008b; Jayasinghe andParkinson, 2009). As high soil N is thought to suppress microbiallignin decomposition, it is possible that our successional sites, withhigher N stocks and lower C/N ratios in mineral soil, will slow lig-nin decay and contribute to its accumulation. However, recentfindings from an N deposition gradient in Michigan did not find aselective accumulation of lignin in the mineral soil even as totalsoil C increased (Thomas et al., 2012).

The higher Ad/Alv values of microaggregated fractions (iPOMand iSC) than the non-microaggregated fractions (cPOM, fPOM,fSC) (Fig. 4D) may indicate a selective role of oxidized lignin frag-ments in the microaggregation process through strong binding tomineral surfaces and promotion of cross linked structures duringthe decay of SOM (Hernes et al., 2007). This is particularly evident

66 Y. Ma et al. / Organic Geochemistry 58 (2013) 56–68

in the mature soils. In successional sites, however, there was nolarge increase of Ad/Alv ratio in microaggregated fractions (iSCand iPOM), which we attribute to frequent influx of fresh lignininto microaggregates. Additionally, the Ad/Alv ratios for all frac-tions were much lower in successional sites indicating a fresherlignin input, or the suppression of lignin oxidation caused by thedisruption of fungal hyphae.

In contrast to lignin, the SFAs may be degraded or rapidlyhydrolyzed to monomers by earthworms through the action of en-zymes such as serine-proteases, which have been shown to haveesterase activity (Nakajima et al., 2005). In an early open chamberleaf decay study at SERC, we demonstrated the selective losses ofordered aliphatic compounds and esters and an increase in cross-linked lignin with increased earthworm activity (Filley et al.,2008b). Our present work is consistent with that study as SFAand FTIR CAH bands increased with forest age and decrease ofearthworm activity in the 0–5 cm depth, as did the lignin/SFA ratio(Figs. 4C and 6).

Other factors associated with forest succession, however, couldalso influence lignin/SFA chemistry at SERC forests. For instancesoil pH, which usually decreases as forests age, is suggested to re-sult in a selective accumulation of free and macromolecular lipids,including those from cutin and suberin, relative to lignin (Dinelet al., 1990; Nierop and Verstraten, 2003). The mechanism for thischange is thought to be driven by lower cutinase/suberinaseactivity or more cross-linked structure formed at lower pH.(Kögel-Knabner et al., 1992; Kolattukudy, 2001; Nierop et al.,2003). As soil pH decreases with the age of the SERC forests(Table 1) this phenomenon may also play out in our study. It isinteresting, also, that earthworm abundance and soil pH arecoupled, as earthworm activity drives soil pH higher through theformation of soil carbonates which would result in lower soil lipidconcentration in successional sites (Wiecek and Messenger, 1972;Canti and Piearce, 2003). Additionally, our previous work at SERCdemonstrated that the average leaf litter input chemistry of ligninand SFA shifted among many of the dominant species in thesuccessional and mature forests, although the total lignin andSFA concentrations were similar (Crow et al., 2009). The relativedominance of 9,10,18-trihydroxyoctanoic acid in oak leaves, foundin greater abundance in older sites, relative to mono- and dihydr-oxyl SFA dominant in leaves from younger sites could potentiallyresult in the accumulation of this more cross-linked SFA. In addi-tion, the high Ca concentration in tulip poplar leaves could leadto higher soil pH by changing cation exchange capacity (Chandler,1941) and, more importantly, could influence earthworm abun-dance and thus significantly alter litter decomposition patterns(Hobbie et al., 2006).

4.5. FTIR-PLS modeling successfully links soil aggregation and chemicalcharacteristics

FTIR of the bulk soils and subsequent analysis by PLS modelingprovided an important chemical insight into the formation of mic-roaggregated C and overall SOC chemistry. As shown in Fig. 6, thecorrelation between the non-microaggregated C stock and the bulksoil FTIR spectra demonstrated the importance of m(CH) bands at2925 cm�1 and 2854 cm�1, free H2O band at 3444 cm�1, and car-boxylate R-COOH features at 1730 cm�1. However, these signifi-cant correlations were not found for microaggregated C, whichinstead, peaked at 1662 cm�1, 1537 cm�1 and 1430 cm�1, consis-tent with amide and aromatic like features (Fig. 4B). The differentpattern of correlation indicated that young successional sites withhigh intra-microaggregate C (iPOM and iSC) also had more aromat-ics, amides and less aliphatic C, while the mature sites with lowestintra microaggregate C had higher aliphatic C and low aromaticsand amides. In addition, the water in young forest soils was more

likely to be mineral bound while in mature forests the soil watertended to be more free/externally bound. These findings werestrongly supported by the bulk C/N data and alkaline CuO extrac-tion data and provide confirmation of this spectral approach toscreening soils (Table 3, Figs. 3 and 4).

5. Conclusion

Overall, past agriculture practice has greatly influenced C and Nstock and physical distribution, but its extended legacy in the soilis most likely controlled by continuous invasive earthworm activ-ity during the afforestation. In the presence of earthworms, therecovery of the O horizon and the development of a distinct depthprofile after agriculture abandonment has been significantly im-peded, even after 132 yr of forest growth. In addition, the propor-tion of microaggregated SOC (iPOM + iSC) remained higher insuccessional forests but exhibited shifts from C and N being pri-marily in the iSC fraction to being in iPOM in old successional sites,a condition we attributed to known earthworm driven increases iniPOM. It is known that invasive earthworms can shift soil microbialcommunity from fungal to bacterial dominance and that theyselectively consume soft, lignin poor, body parts of litter leavingthe lignin rich structural tissue. At SERC, the concentration and oxi-dation state of lignin phenols, their distribution among soil physi-cal fractions, as well as the relative proportion of lignin tosubstituted fatty acids among the sites are consistent with an ex-pected influence of earthworms on the soil chemistry. Overall,the interactive effects of aging forests and shifting tree species, inthe context of high invasive earthworm activity, created a situationwhere the trajectory of the organic geochemistry of the forest soilwas altered.

The overprint of invasive earthworm activity on a recoveringforest could result in a trajectory that may act to either stabilizeor destabilize SOM pools. In the current system, overall microag-gregated C and N stocks increased with successional age butimportantly; the proportion of lignin also increased. This, in thecontext of a system that might be shifting from lignin degradingfungal to bacterial dominated, could act to slow the overall decayrates of soil C. However, the introduction of fresh SOC and N intodeeper depths may act increase microbial activity and cause apriming effect in these soils. More studies, especially long termfield experiments, and laboratory incubations, will be needed tostudy the overall impact on the stability of SOM during afforesta-tion stages.

Acknowledgements

This research was supported by Grant EAR-0748746 from theNational Science Foundation. We would like to thank Seth L. Kingand Grace Conyers for helping physical separation of soil samples;David Gamblin and Ian Schaller for helping lignin and SFA extrac-tion and analysis; Kira Albright and Gnanasiri S. Premachandra forperforming the FTIR analysis of samples. We thank two anonymousreviewers for valuable comments, which led to a considerableimprovement of the manuscript.

Associate Editor—Ingrid Kögel-Knabner

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