Soil carbon pools, plant biomarkers and mean carbon residence time after afforestation of grassland with three tree species

Soil Biology & Biochemistry 43 (2011) 1341e1349

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Soil carbon pools, plant biomarkers and mean carbon residence time after afforestation of grassland with three tree species Zhiqun Huang a, *, Murray R. Davis b, Leo M. Condron c, Peter W. Clinton b a

College of Geographical Science, Fujian Normal University, Fuzhou 350007, China Scion, P.O Box 29237, Fendalton, Christchurch, New Zealand c Faculty of Agriculture and Life Sciences, Po Box 84, Lincoln University, Lincoln 7647, Christchurch, New Zealand b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 January 2011 Received in revised form 5 February 2011 Accepted 6 March 2011 Available online 22 March 2011

Afforestation of grassland has been globally identified as being an important means for creating a sink for atmospheric carbon (C). However, the impact of afforestation on soil C is still poorly understood, due to the paucity of well designed long-term experiments and the lack of investigation into the response of different soil C fractions to afforestation. In addition, little is known about the origins of soil C and soil organic matter (SOM) stability after afforestation. In a retrospective study, we measured C mass in the soil light and heavy fractions in the first 10 years after afforestation of grassland with Eucalyptus nitens, Pinus radiata and Cupressus macrocarpa. The results suggest that C mass in the soil heavy fraction remained stable, but the C mass in the light fraction decreased at year 5 under three species. Soil d13C analysis showed that the decrease in the light fraction may be due to reduced C inputs from grassland species litter and low inputs from the still young trees. After the initial reduction, the recovery of soil C in the light fraction depended on tree species. At year 10, an increase of 33% in light fraction soil C was observed at the 0e30 cm depth under E. nitens, compared to that under the original grassland (year 0). Planting P. radiata restored light fraction soil C to the original level under grassland, whereas planting C. macrocarpa led to a decrease of 33%. We concluded that the increase of light fraction soil C between year 5 and 10 is most likely due to C input from tree residues. Most of the increased C was derived from root turnover under pine and from both root and leaf turnover under E. nitens, as indicated by plant C biomarkers such as lignin-derived phenols and suberin and cutin-derived compounds in the 0e5 cm soil layer. Modelling of soil Δ14C& suggested that SOM had a greater mean residence time at year 10 than year 0 and 5 due to increased relative abundance of recalcitrant plant biopolymers. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Afforestation Carbon Grassland Mean residence time Plant biomarkers Radiocarbon Soil organic matter

1. Introduction Planting trees on grassland has occurred globally and is seen as a means to mitigate anthropogenic carbon (C) emissions (Berthrong et al., 2009; Buscardo et al., 2008; Davis and Condron, 2002; Hu et al., 2008). For example, in New Zealand alone, approximately 560,000 ha of grassland have been converted to plantation forests since 1989 (MfE, 2010). The afforestation of grassland can result in rapid C accumulation in the vegetation (Tate et al., 2003), but information on the effect of such afforestation on soil C stocks is not well understood. Soils may contain three times as much C as the above-ground vegetation, therefore even small

* Corresponding author. Tel.: þ86 591 83434802; fax: þ86 591 83465397. E-mail address: [email protected] (Z. Huang). 0038-0717/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2011.03.008

alterations in soil C storage resulting from changes in land use may significantly influence ecosystem C accumulation. Empirical studies have mostly suggested a significant decline in mineral soil C after afforestation of grassland at the initial stages (<10 years) (Davis and Condron, 2002; Guo and Gifford, 2002; Halliday et al., 2003; Laganiere et al., 2010; Paul et al., 2002). However, the data in these studies have been derived mainly from paired sites (Laganiere et al., 2010). In a meta-analysis of C changes after afforestation in agricultural soil, Laganiere et al. (2010) highlighted the importance of methodological approaches in such studies and suggested that a retrospective study with randomized block design is more powerful and less biased than paired sites studies. In addition, most studies available on the changes in soil C after afforestation of grassland have not separated soil organic C into labile and stable pools, which is essential to understanding the effects of land use on soil organic C dynamics as the stable pools have a much longer mean residence time than labile pools. Density

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fractionation using a high-density solution can separate soil C into “light” and “heavy” fractions. The heavy fraction is thought to be largely recalcitrant C associated with soil minerals and therefore is more crucial to soil C sequestration compared to the light fraction (Neff et al., 2002). The light fraction basically consists of partially decomposed plant, animal, and fungal residues (McLauchlan and Hobbie, 2004) and therefore it is referred to as being a labile fraction more sensitive to changes in soil management regime than the total soil C pool (Bremer et al., 1994). Tree species may affect soil organic C pools and their dynamics through variation in C inputs and by influencing soil organic matter (SOM) decomposition. For example, organic inputs via root turnover and exudation have been reported to affect labile soil C pool sizes (Neff and Asner, 2001). Hobbie et al. (2007) demonstrated that tree species affected SOM decomposition in surface mineral soils by causing divergence in the concentrations of cations (Al and Fe). Increased levels of these cations reduced the accessibility of SOM to decomposers. Although knowledge regarding the effect of tree species on SOM dynamics is crucial to predicting how soil C sequestration will respond to forest management activities and global environmental changes that alter plant species composition across the landscape, such studies in well designed experiments at the same site over decade time scale have not been undertaken. The initial decline in soil C following afforestation has been attributed to reduced litter inputs from the grassland species as they become shaded by the trees (Paul et al., 2003). Analyses of plant biomarkers (e.g. lignin, wax, cutin and suberin) in soil samples allow the tracing of the origin and transformation of certain organic molecules in soil. The relative abundance of these biomarkers is a measure of both living and decomposing biomass and can reflect the relative input from plant litter (Otto et al., 2005). The present study was undertaken to compare the effects of two tree species Eucalyptus nitens, and Cupressus macrocarpa, with that of New Zealand’s dominant plantation species, Pinus radiata, on soil C during the first 10 years after forest establishment in grassland. The objectives of this study were to: (1) determine the changes of soil C pools in the whole soil and its light and heavy fractions after afforestation of grassland; (2) compare species effects on soil C pools; (3) identify variation in the origin of soil C using key biomarker indicators; and (4) gauge the relative stability of SOM after afforestation of grassland.

2. Materials and methods 2.1. Site description and experimental design A unique replicated field experiment was intiated in 1999 at Orton Bradley Park on Banks Peninsula, New Zealand (S 43 390 5300 ; E 172 420 1700 ) to investigate and quantify temporal changes in soil properties and processes following afforestation of established

grazed pasture with three contrasting commercial tree species. The reference for the impact of the different tree species was comparison with the soil at tree planting, and it was neither feasible nor necessary to include a grazed pasture treatment in the trial design. The field trial was established on lower north-east facing slopes (70e150 m elevation) on Takahe silt loam soil (Mottled Fragic Pallic, NZ classification) formed in greywacke loess. Mean annual rainfall over the trial period at Living Springs (5 km distant, 38 m a.s.l.) was 900 mm. The trial included four replicates of the three species (P. radiata, E. nitens and C. macrocarpa) arranged in a randomized block design of twelve plots each measuring 30  30 m. The plots had been developed under grazed pasture with limited superphosphate fertilizer inputs at very low rates (<100 kg ha1) which is evident from the low level of Olsen phosphate (P) on the site before planting (Table 1). The plots were fenced off at trial establishment. The initial soil properties in the 0e5 cm layer before planting can be found in Table 1. The trees were planted at 1250 stems/ha, and no fertilizer was applied following establishment, the trees were pruned and thinned in accordance with commercial plantation management practices. In the nine years of tree growth, the radiata pine plots have a typical plantation floor with a substantial litter layer which ranged from 2 to 11 cm in thickness, depending on slope. In the Macrocarpa and Eucalypt plots, there was only a small amount of litter on the floor (Nall, 2010). 2.2. Soil sampling and bulk density determination In September 1999 (immediately prior to tree planting) (defined as year 0 hereafter), September 2004 (defined as year 5 hereafter) and November 2009 (defined as year 10 hereafter) soil samples were taken by soil Ellenkamp corer (2.5 cm diameter) at 4 depths (0e5, 5e10, 10e20, 20e30 cm) from each plot. Samples were taken from 5 sites randomly located midway between tree rows in the middle of each plot, vegetation was cleared prior to coring and duplicate cores taken from 0e5 and 5e10 cm depths at each sampling site to obtain equal quantities of soil from each depth. Soil samples were sieved (<2 mm) and air dried prior to analysis. The samples collected at year 0 and 5 were weighed to determine fine earth densities which were used to calculate soil C and N masses for those years. Mean fine earth densities for year 0 and 5 were used to calculate soil C and N masses for year 10 because (1) our data (Table 1) in year 0 and 5 suggest that soil bulk density changed little during the early stage of afforestation and (2) growing tree roots will not affect bulk density over time (Davis and Condron, 2002). 2.3. Density fractionation The soil was physically separated into two pools based on density fractionation. The soil light fraction was collected by

Table 1 Soil properties in the 0-5 layer before tree planting (Year 0), 5 and 10 years after tree planting. Means (standard deviation) are based on four replicates. P. radiata

pH Total C (%) Total N (%) Bulk density (t m3) Inorganic P (mg kg1) Organic P (mg kg1) Olsen P (mg kg1) CEC(cmol kg1) Base saturation (%)

E. nitens

C. macrocarpa

Year 0

Year 5

Year 10

Year 0

Year 5

Year 10

Year 0

Year 5

Year 10

5.1 5.0 0.46 0.86 137 496 13 18 43

5.2 4.2 0.39 0.88 e e e e e

5.2 (0.08) 5.0 (0.16) 0.39 (0.01) e e e e e e

5.1 4.7 0.42 0.87 127 474 12 17 42

5.2 4.4 0.41 0.87 e e e e e

5.4 (0.09) 5.4 (0.50) 0.43 (0.04) e e e e e e

5.2 4.9 0.44 0.87 141 517 15 18 44

5.3 4.6 0.40 0.83 e e e e e

5.5 (0.13) 5.2 (0.39) 0.41 (0.04) e e e e e e

(0.05) (0.34) (0.02) (0.06) (11.2) (31.5) (1.8) (1.0) (3.0)

(0.07) (0.07) (0.01) (0.03)

(0.04) (0.15) (0.01) (0.04) (21.4) (45.1) (2.6) (0.5) (1.8)

(0.09) (0.20) (0.02) (0.12)

(0.10) (0.14) (0.02) (0.03) (34.8) (46.5) (4.8) (0.60) (4.00)

(0.09) (0.14) (0.02) (0.14)

Z. Huang et al. / Soil Biology & Biochemistry 43 (2011) 1341e1349

a modified method of Carter and Gregorich (2008). In summary, 10 g of air-dried soil were placed in a centrifuge tube with 50 mL NaI (Fisher Chemical, UK) with a density of 1.70 g cm3. The tubes were shaken by hand for 3 min, then centrifuged at 1000 rpm for 15 min. The floating material was aspirated from the surface of tubes (about the top 20 mL) and then placed into a filter unit (in a funnel containing Whatman GF A/E filter paper). The shakingecentrifugationeaspiration process was repeated at least four times, until no floating material remained. The samples on the filter paper were rinsed thoroughly with deionized water and collected. The collected material, designated light fraction, was dried at 60  C for 24 h, and finely ground in a mortar and pestle before analysis. The material (heavy fraction) remaining at the bottom of the centrifuge tube was quantitatively washed onto a separate funnel. The heavy fraction was rinsed repeatedly with deionized water (about 300 mL) and dried at 60  C for 48 h. The heavy fraction material was then ground in a mortar and pestle for analysis. 2.4. The C and N contents in the whole soil, light and heavy fractions and d13C The C and N concentrations in the whole soil and heavy fractions were determined on finely ground (<0.20 mm) sub-samples using a LECO EPS-2000 CNS thermal combustion furnace (LECO Corp., St Jose, MI). The d13C of SOM in the light and heavy fractions in the 0e5 cm layer as well as the C and N contents in the light fraction samples were measured using a PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20e20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK). Isotope results are reported in the conventional d notation as per mil deviation relative to the d13C of the PDB standard. Soil C mass in the whole soil, light and heavy fractions were calculated conventionally as the product of concentration, bulk density and soil thickness (Davis et al., 2007). 2.5. Quantification of plant biopolymers in SOM in the 0e5 cm layer Owing to the labor-intensive nature of these analyses, the analyses of C biomarkers were only conducted for the soils from the 0e5 cm depth. Air-dried soils in the light (0.3 g) and heavy fractions (1.0 g) were subjected to base hydrolysis and alkaline cupric-oxide oxidation to isolate cutin- and suberin-derived compounds and lignin-derived phenols (Bull et al., 2000; Naafs and van Bergen, 2002; Otto et al., 2005). The soils were refluxed for 3 h with 50 mL of 1 M methanolic KOH. The suspension was centrifuged for 30 min at 2500 rpm with the supernatant kept in the refrigerator. The residue from base hydrolysis was extracted twice by sonication for 15 min with 30 mL dichloromethane:methanol (1:1; v/v). The combined extracts were acidified to pH 1 with 6 M HCl. Hydrolysable lipids were recovered by liquideliquid extraction in a separation funnel with diethyl ether, concentrated by rotary evaporation and dried under N2 in 2 mL glass vials. The base hydrolysis residues were air dried and weighed for alkaline cupric-oxide (CuO) oxidation to isolate lignin-derived phenols. The residues were extracted with 1 g CuO, 100 mg ammonium iron (II) sulfate hexahydrate [Fe (NH4)2(SO4)2.6H2O] and 10 mL of 2 M NaOH in 15 mL Teflonlined vessels under N2 for 3 h at 170  C. The vessels were then cooled and the extracts were acidified to pH 1 using 6 M HCl and left in the dark for 1 h to avoid reactions of cinnamic acids. After centrifugation (30 min at 2500 rpm), the supernatant was subject to liquideliquid extraction with diethyl ether. The ether extracts

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were concentrated by rotary evaporation, transferred to 2 mL glass vials and dried under N2. Aliquots of extracts from the previous extractions were converted to trimethylsilyl (TMS) derivatives by reaction with 90 mL N, O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) and 10 mL pyridine for 1 h at 70  C. The derivatized compounds were analyzed using an Agilent model 6890N GC coupled to an Agilent model 5973N quadrupole mass selective detector (MSD) (GCeMS). Individual compounds were identified by comparison of mass spectra from the literature, Wiley MS library data, and interpretation of mass spectrometric fragmentation patterns. Quantification was performed using external standards (C24D50 for hydrolysable lipids and vanillic acid-TMS for lignin-derived phenols) in the total ion current. The concentration of individual compounds was normalized to the sample organic C content (mg/100 mg soil C). In this study, the contributions of cutin and suberin to the light and heavy fractions of SOM were estimated by quantification of the major hydroxyalkanoic and alkanedioic acids (Otto and Simpson, 2006b). Cutin-derived compounds were the sum of mid-chain hydroxyalkanoic C14, C15, C17 acids, C16 mono- and dihydroxy acids, while suberin-derived compounds included hydroxyalkanoic and alkanedioic acids in the range of C20eC32, and 9, 10-epoxy-a, u C18 dioic acid (Otto and Simpson, 2006b). The weight sum (mg) of eight lignin phenols (three vanillyl phenols: vanillin, acetovanillone, vanillic acid; three syringyl phenols: syringaldehyde, acetosyringone, syringic acid; and two cinnamyl phenols: p-coumaric acid and ferulic acid) was also calculated. The ratios of syringyl to vanillyl and cinnamyl to vanillyl monomers were calculated to determine the origins of lignin (Otto and Simpson, 2006a). 2.6. Soil D14C analysis and mean residue time in the 0e5 cm layer Soil D14C values of light and heavy fraction SOM from the 0e5 cm depth are reported as defined in Stuiver and Polach (1977) and are the result of combusting soil samples to CO2 and subsequent D14C determination at the Rafter Radiocarbon Laboratory Accelerateor Mass Spectrometer Facility, New Zealand. To determine the mean residence time of SOM in the light and heavy fractions, we used the measured 14C content to constrain a flux model (Fontaine et al., 2007; Hsieh, 1993). The 14C content of soil organic C was modelled as:

Aa ¼

p X i¼b p X

exp½  ðp  iÞ=MRT  14 Ci exp½  ðp  iÞ=8268= exp½  ðp  iÞ=MRT

i¼b

where Aa is the specific 14C activity of an active soil organic C pool, p is the year when the soil sample was taken, MRT is the mean residence time of an active soil organic C pool, 14Ci is the specific 14C activity in the atmosphere of year i, and 8268 years is the MRT of 14 C. The atmospheric D14C record between 1950 and 2009 was derived from World Data Centre for Greenhouse Gases (http://gaw. kishou.go.jp/wdcgg/) which was submitted by Dr Kim Currie, NIWA, New Zealand. The chosen atmospheric D14C data reflect a growing season. 2.7. Data and statistical analyses Statistical analyses were performed using SPSS 11.5 for Windows and Microsoft Excel 2003. We used repeated-measures ANOVA to determine the effects of sampling time and tree species. The significance of differences between treatment means for each

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sampling year was determined using a Tukey post-hoc test and P < 0.05 used to indicate a significant difference. 3. Results 3.1. The C and N masses and C:N ratios in the whole soil ANOVA showed that sampling year significantly affected C mass in the whole soil at the 0e5 cm, 5e10 cm and 20e30 cm depths (Tables 2 and 3). At the 0e5 cm depth, the C mass significantly declined between years 0 and 5 under P. radiata, C. macrocarpa, and to a lesser extent (and non-significantly) under E. nitens, but then increased under all species between years 5 and 10. At the 5e10 cm depth, the C mass significantly decreased between years 0 and 5 under P. radiata only. At the 20e30 cm depth, mean C mass in the whole soil significantly declined over time under C. macrocarpa (Table 3). Cumulatively, for the full 0e30 cm depth, there was no significant effect of sampling year or tree species on soil C in the whole soil (Tables 2 and 3). Under P. radiata, soil N mass in the 0e5 cm and 5e10 cm layers declined significantly between year 0 and 5 and then showed no further change (Tables 2 and 3). There were no significant changes in these two layers under the other species. In the 10e20 and 20e30 cm layers, there were no signifcant changes for mean soil N mass under the three tree species across three sampling years. Cumulatively there was a significant decline of soil N mass between year 0 and 10 under C. macrocarpa for the full 0e30 cm depth (Tables 2 and 3). Soil C to N ratios generally increased progressively with time in the 0e5 (significantly under the three species) and 5e10 cm (significantly only under P. radiata) soil layers. In the 10e20 and 20e30 cm soil layers, no significant effect on soil C to N ratio due to sampling year or tree species was found (Tables 2 and 4). 3.2. The C mass in the light and heavy fractions The C masses in the heavy fraction SOM were not significantly affected by tree species or sampling year at any soil depth (Tables 2 and 5). However, ANOVA showed species and sampling year significantly impacted the C mass in the light fraction SOM in all soil layers and cumulatively at the 0e30 cm depth. In the 0e5 cm layer,

Table 2 P values from repeated-measures ANOVA for carbon (C) and nitrogen (N) mass in four soil layers and the 0e30 cm soil depth and for C:N ratios in four soil layers. Soil depth Effect (cm)

Nitrogen mass C:N (Mg ha1)

Carbon mass (Mg ha1)

Whole soil Light Heavy Whole soil fraction fraction 0e5

Year <0.01 Species 0.84 Interaction 0.24

<0.01 0.01 0.01

0.06 0.55 0.54

<0.01 0.84 0.06

<0.01 0.12 0.31

5e10

Year Species Interaction

0.04 0.42 0.22

<0.01 0.10 0.72

0.19 0.69 0.21

0.03 0.43 0.08

0.02 0.75 0.05

10e20

Year Species Interaction

0.95 0.84 0.14

0.01 0.01 0.01

0.56 0.61 0.14

0.61 0.62 0.11

0.08 0.62 0.46

20e30

Year Species Interaction

0.03 0.80 0.11

0.01 0.02 <0.01

0.23 0.48 0.17

0.33 0.77 0.11

0.06 0.89 0.09

0e30

Year Species Interaction

0.07 0.78 0.08

<0.01 0.01 0.01

0.82 0.54 0.14

0.11 0.70 0.05

e e e

light fraction soil C declined under all species between years 0 and 5 but then recovered between years 5 and 10, the recovery being especially marked under E. nitens (Table 5). In the 10e20 and 20e30 cm layers light fraction C mass showed an overall decline with no recovery in contrast to the upper layers, the decline being most marked under C. macrocarpa. Soil C mass in the light fraction SOM at the 0e30 cm depth significantly declined from year 0 to year 5 under the three tree species, but then increased under P. radiata and E. nitens between year 5 and 10. Planting C. macrocarpa led to a significantly lower C mass in the light SOM fraction compared to P. radiata and E. nitens. For example, at year 10, planting P. radiata and E. nitens had 3.7 and 5.5 Mg ha1 greater soil C mass in the light fraction SOM, respectively, compared to C. macrocarpa plots (Tables 2 and 5). 3.3. The C biomarkers in the 0e5 cm soil layer Tree species and sampling years significantly changed the relative abundance of vanillyl (P ¼ 0.01 for species and year) and cinnamyl (P ¼ 0.04 and 0.02 for species and year, respectively) phenols in the light fraction SOM, but did not markedly affect the relative abundance of syringyl phenols (P ¼ 0.11 and 0.19 for species and year, respectively) (Table 6). The relative abundance of vanillyl phenols in the light fraction SOM under P. radiata and C. macrocarpa increased with the increasing plantation age, which led to decreasing ratios of syringyl to vanillyl and cinnamyl to vanillyl phenols with the increasing plantation age. Under E. nitens, sampling years did not significantly change the relative abundance of vanillyl and springyl phenols, but the relative abundance of cinnamyl phenols decreased progressively with time, which led to the progressive decline of cinnamyl to vanillyl phenols ratio with age (Table 6). The relative abundance of cutin-derived compounds in the light fraction SOM was affected by sampling years (P ¼ 0.03), but not by species (P ¼ 0.09). The relative abundance of cutin-derived compounds tended to increase progressively with age under P. radiata and C. macrocarpa, but the differences are not statistically significant among sampling years. Under E. nitens, the relative abundance of cutin-derived compounds was greater at year 10 than year 5 and year 0. The light fraction SOM had significantly greater relative abundance of suberin-derived compounds under three tree species at year 10 than year 5 and year 0 (P ¼ 0.01) (Table 6). In the heavy fraction SOM, neither species (P ¼ 0.37) nor sampling years (P ¼ 0.29) significantly affected the relative abundance of lignin phenols. The ratios of syringyl to vanillyl and cinnamyl to vanillyl phenols in the heavy fraction SOM under P. radiata and C. macrocarpa were significantly lower at year 10 than year 0 (P ¼ 0.04). The cinnamyl to vanillyl phenols ratio under E. nitens was lower at year 5 and 10 than at year 0 (P ¼ 0.02). Afforestation also led to significant increases in the relative abundance of suberin-derived compounds in the heavy fraction SOM under all species at year 10 compared to that at year 0 and 5 (P ¼ 0.01) (Table 7). 3.4. d13C values of SOM in the 0e5 cm soil layer The ANOVA showed that the d13C values in the heavy fraction of SOM at the 0e5 cm depth remained constant under three tree species within 10 years after tree planting (P ¼ 0.18 and 0.56 for year and species, respectively). The tree species did not significantly affect the d13C values in the light fraction SOM either (P ¼ 0.50). However, the d13C values in the light fraction SOM under three tree species were significantly affected by sampling year (P ¼ 0.01) and were greater at year 5 than at year 10 and year 0. There were no

Z. Huang et al. / Soil Biology & Biochemistry 43 (2011) 1341e1349

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Table 3 Total carbon and nitrogen mass (Mg ha1) in the whole soil before tree planting (Year 0), 5 and 10 years after tree planting. Means (standard deviation) are based on four replicates. Separation of means was determined by a Tukey post-hoc test; different letters within a species and a row indicate that means differ significantly (P < 0.05) owing to sampling year effects. Depth (cm)

P. radiata

E. nitens

Year 0

Year 5

Year 10

Year 0

Year 5

Year 10

Year 0

Year 5

Year 10

Carbon

0e5 5e10 10e20 20e30 0e30

21.4 16.8 21.3 12.3 72.4

(1.4)a (1.0)a (4.6)a (2.9)a (7.3)a

18.5 15.3 23.2 13.2 70.1

(0.5)b (1.4)b (2.2)a (1.7)a (5.7)a

21.5 16.3 24.0 12.6 74.3

(0.9)a (1.2)a (2.4)a (2.0)a (6.3)a

20.4 16.3 23.0 14.4 74.0

19.1 15.9 23.3 13.2 71.6

(1.7)a (1.7)a (3.1)a (2.2)a (8.7)a

23.3 17.4 24.3 12.5 77.6

(2.8)b (1.4)a (3.3)a (0.6)a (6.3)a

21.3 16.6 24.6 15.8 78.4

(1.6)a (1.4)a (3.5)a (3)a (8.4)a

19.1 15.8 23.0 13.7 71.6

(1.4)b (1.2)a (3.1)a (1.8)ab (6.5)a

21.9 16.0 21.4 11.3 70.6

(1.8)a (1.0)a (2.8)a (1.3)b (5.5)a

Nitrogen

0e5 5e10 10e20 20e30 0e30

2.0 1.5 1.9 1.2 6.5

(0.1)a (0.1)a (0.5)a (0.4)a (0.9)a

1.7 1.3 2.1 1.3 6.4

(0.1)b (0.2)b (0.3)a (0.3)a (0.8)a

1.7 1.4 2.1 1.3 6.4

(0.1)b (0.2)ab (0.3)a (0.2)a (0.7)a

1.9 1.5 2.0 1.3 6.6

1.8 1.4 2.1 1.3 6.6

(0.3)a (0.2)a (0.4)a (0.3)a (1.1)a

1.9 1.5 2.1 1.2 6.8

(0.2)a (0.2)a (0.4)a (0.2)a (0.8)a

1.9 1.5 2.1 1.4 6.9

(0.2)a (0.2)a (0.4)a (0.4)a (1.1)a

1. 7 1.4 2.1 1.3 6.5

(0.2)a (0.2)a (0.4)a (0.3)a (1.1)ab

1.7 1.4 1.9 1.2 6.2

(0.2)a (0.2)a (0.4)a (0.2)a (1.0)b

significant differences in the d13C values in the light fraction SOM between year 10 and year 0 (Fig. 1). 3.5. The D14C, mean residence times and C:N ratios in light and heavy fraction SOM in the 0e5 cm soil layer The positive D14C values of SOM in the 0e5 cm soil layer indicate that SOM in both the light and heavy fractions was dominated by fast-cycling organic matter. The modelled mean residence time of SOM in the light fraction at the 0e5 cm depth averaged 3.2 years and was significantly (P ¼ 0.03) lower than that in the heavy fraction (4.7 years). The ANOVA showed that the mean residence time of SOM did not change significantly between year 5 and year 0 in either the light (P ¼ 0.23) or heavy (P ¼ 0.35) fractions. However, the SOM had a significantly greater mean residence time at year 10 than year 0 and year 5 in both the light (P ¼ 0.01) and heavy (P ¼ 0.01) fractions. There were no significant effects of tree species on the mean residence time of SOM in either the light (P ¼ 0.17) or heavy (P ¼ 0.08) fractions. The C:N ratios increased progressively in both the light and heavy (P ¼ 0.01) fractions with age under the three species (Table 8). 4. Discussion 4.1. The C mass in the whole soil As the grassland at our experimental site was established about 150 years ago, we assumed that soil C for this land use is at steady state (Saggar et al., 2001). We found no significant effect of sampling year on soil C mass at the full 0e30 cm depth in the first 10 years after afforestation. Our results for pine are consistent with those of Davis et al. (2007) who found no change in soil C to 30 cm depth under P. nigra in the first 10 years after afforestation on grassland, while more recently Parfitt and Ross (personal communication) found no change in soil C to 10 cm depth under P. radiata in the first 10 years. Modelling studies and meta-analyses of land use change indicate that C losses after afforestation of former

C. macrocarpa

(1.5)ab (1.5)a (1.8)a (2.5)a (6.2)a (0.1)a (0.2)a (0.3)a (0.3)a (0.9)a

grasslands depend on the amount of rainfall received (Guo and Gifford, 2002; Halliday et al., 2003; Kirschbaum et al., 2008). Where rainfall is not excessive (<1000 mm), N losses through nitrification and leaching are limited. The soil C:N ratio normally increases after afforestation of grassland, partly because of soil N transfer to vegetation in developing forests (Halliday et al., 2003). In this study, there was no significant effect of sampling year on soil N mass at the 0e30 cm depth under pine, but the soil C:N ratios increased in the 0e5 and 5e10 cm soil layers with age (Table 3). Therefore, the low-moderate rainfall (<1000 mm) of these retrospective study locations (900 mm, 600 mm and 995 mm in this study, those of Davis et al., 2007 and Parfitt and Ross, personal communication, respectively) may be the main reason why these studies have shown no overall loss in soil C at 10 years after planting. In contrast to the retrospective studies, the lack of change in soil C mass under P. radiata appears to be inconsistent with results from paired site studies for this species, which generally show soil C at the 0e30 cm depth to decline up to 20 years after grassland is planted with pine in the areas with a wide range of rainfall (Davis and Condron, 2002; Guo and Gifford, 2002; Paul et al., 2002; Scott et al., 1999). More retrospective studies in higher rainfall areas are needed to better understand the effects of afforestation of grassland on soil C. Short term decreases at the 0e5 cm depth under P. radiata and C. macrocarpa plantations and at 5e10 cm depth under P. radiata were observed in the present study. At year 5, the losses of 13.6% and 10.3% of soil C mass at the 0e5 cm depth under P. radiata and C. macrocarpa plantations, respectively, are comparable to the study of Guo and Gifford (2002) who, in a larger meta-analyses, suggested soil C content in the top 10 cm was about 10% lower under plantation forests of conifer species following afforestation than grasslands. 4.2. The soil C biomarkers, d13C and C mass in the light fraction SOM When the SOM was separated into light and heavy pools by density fractionation, soil C in the light fraction SOM at the 0e30 cm depth under forest plantations declined significantly

Table 4 The ratio of carbon to nitrogen in the whole soil before tree planting (Year 0) and 5 and 10 years after tree planting. Means (standard deviation) are based on four replicates. Separation of means was determined by a Tukey post-hoc test; different letters within a species and a row indicate that means differ significantly (P < 0.05) owing to sampling year effects. Depth (cm)

0e5 5e10 10e20 20e30

P. radiata

E. nitens

C. macrocarpa

Year 0

Year 5

Year 10

Year 0

Year 5

Year 10

Year 0

Year 5

Year 10

10.8 11.1 11.5 11.0

10.9 11.5 11.3 10.2

12.7 12.0 11.6 10.0

11.1 11.2 11.5 11.5

10.8 11.2 11.3 10.8

12.6 11.6 11.3 10.4

11.1 11.5 12.0 11.3

11.4 11.3 11.4 10.8

12.7 11.7 11.5 10.0

(0.1)a (0.3)a (0.8)a (1.5)a

(0.4)a (0.6)ab (0.7)a (1.1)a

(0.3)b (0.6)b (0.9)a (1.0)a

(0.4)a (0.8)a (1.0)a (1.2)a

(0.6)a (0.7)a (1.0)a (1.4)a

(0.4)b (0.8)a (1.0)a (1.6)a

(0.3)a (1.1)a (1.4)a (1.4)a

(0.7)a (1.0)a (1.2)a (1.9)a

(0.6)b (1.2)a (1.4)a (1.4)a

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Z. Huang et al. / Soil Biology & Biochemistry 43 (2011) 1341e1349

Table 5 Carbon masses (Mg ha1) in the light and heavy fractions of soil before tree planting (Year 0) and 5 and 10 years after tree planting. Means (standard deviation) are based on four replicates. Separation of means was determined by a Tukey post-hoc test; different letters within a species and a row indicate that means differ significantly (P < 0.05) owing to sampling year effects. Soil fraction

Depth (cm)

P. radiata

E. nitens

Year 0

Year 5

Year 0

Year 5

Year 0

Year 5

Light

0e5 5e10 10e20 20e30 0e30

2.5 1.8 2.9 1.4 8.5

(0.7)a (0.4)a (0.1)a (0.1)a (1.3)a

1.2 1.2 2.2 1.3 5.9

(0.3)b (0.2)b (0.4)b (0.1)a (1.0)b

2.9 2.5 2.3 1.0 8.7

(0.8)a (0.5)c (0.2)b (0.2)b (1.4)a

2.2 1.6 2.6 1.4 7.9

(0.7)a (0.2)a (0.1)a (0.2)a (0.8)a

1.2 1.3 2.2 1.3 6.0

(0.3)b (0.4)a (0.3)b (0.1)a (1.0)b

4.9 2.5 2.1 1.0 10.5

(1.2)c (0.9)b (0.3)b (0.2)b (1.6)c

2.3 1.4 2.4 1.4 7.5

(0.5)a (0.2)a (0.1)a (0.1)a (0.8)a

1.1 1.1 2.1 1.3 5.6

(0.3)b (0.3)a (0.4)a (0.2)a (1.2)b

1.9 1.8 0.8 0.4 5.0

(0.2)a (0.8)a (0.1)b (0.1)b (1.0)b

Heavy

0e5 5e10 10e20 20e30 0e30

18.9 15.0 18.3 10.9 63.9

(1.5)a (1.3)a (4.7)a (3.0)a (8.2)a

17.3 14.0 21.0 11.9 64.2

(0.8)a (1.7)a (2.5)a (1.8)a (6.5)a

18.6 13.8 21.7 11.6 65.7

(1.3)a (1.7)a (2.4)a (1.9)a (6.7)a

18.2 14.7 20.5 12.9 66.2

(1.5)a (1.7)a (1.6)a (2.4)a (6.4)a

17.8 14.7 21.2 12.0 65.6

(2.0)a (2.1)a (3.5)a (2.2)a (9.7)a

18.5 14.9 22.2 11.6 67.1

(1.8)a (1.3)a (3.0)a (0.6)a (4.7)a

19.1 15.2 22.1 14.4 70.8

(2.1)a (1.1)a (3.7)a (2.9)a (8.9)a

18.0 14.7 20.9 12.4 66.0

(1.6)a (1.5)a (3.5)a (1.9)a (7.6)a

20.0 14.2 20.6 10.9 65.6

(1.7)a (1.6)a (2.9)a (1.3)a (6.3)a

Year 10

during the first 5 years compared to that under grassland (Table 5). We analyzed the composition of lignin-derived phenols in the light fraction SOM at the 0e5 cm depth. However, it should be noted that the data on the composition of lignin-derived phenols were derived from a shallow (0e5 cm) depth of mineral soil. There was possible contamination of these light and heavy fractions from the forest floor. These phenols are characteristic of major plant groups, with syringyl derivatives being unique to woody and nonwoody angiosperms, while cinnamyl groups are common to nonwoody plants (Hedges and Mann, 1979; Hedges and Parker, 1976). All gymnosperm and angiosperm woody and nonwoody tissues yield vanillyl phenols as oxidation products. Angiosperm lignin contains approximately equal proportions of both syringyl and vanillyl moieties, whereas the gymnosperm lignin contain a preponderance of vanillyl moieties. Nonwoody tissues should yield much greater cinnamyl/vanillyl ratio of lignin than woody residues (Lam et al., 2001). The ratios of syringyl to vanillyl and cinnamyl to vanillyl monomers are indicative of the botanical origin of the lignin and have been used to assess the source of lignin in soils (Otto and Simpson, 2006a). Our results showed that, under grassland at year 0, the ratios of syringyl to vanillyl monomers in the light fraction SOM are between 0.82 and 0.86 and the ratios of cinnamyl to vanillyl monomers range from 0.61 to 0.64 (Table 6). These ratios reflect the angiosperm origin of the lignin and are comparable to previously reported ratios of syringyl to vanillyl and cinnamyl to vanillyl monomers for grassland soils (Otto and Simpson, 2006a). At year 5, the syringyl/vanillyl and cinnamyl/ vanillyl ratios in the light fraction SOM under P. radiata and C. macrocarpa declined significantly compared to those ratios at year 0, reflecting the chemical signatures of increased soil C from gymnosperm and woody litter. Similarly, the decreased cinnamyl/ vanillyl ratio in the light fraction SOM under E. nitens at year 5

C. macrocarpa Year 10

Year 10

compared to that at year 0 suggests significantly increased soil C from woody litter. Despite the significant inputs from tree residues, soil C mass in the light fraction SOM under the three species significantly decreased from year 0 to year 5 (Table 5). The decrease of C mass in the light fraction SOM from year 0 to year 5 was associated with a significant increase of d13C values (Fig. 1A). There are two possible explanations for this significant increases of d13C values at year 5 compared to year 0 under the three species. First, soil micro-organisms discriminate against 13C and preferentially use 12C compounds during decomposition, hence residual SOM should become more enriched in 13C (Nadelhoffer and Fry, 1988). Increased decomposition due to disturbance during preparation of the plantation, as suggested by Laganiere et al. (2010), may lead to increased d13C values. However, this appears not to be the case for this study because: (1) the trees were hand planted and there would have not been much disturbance; (2) the soil samples were collected midway between rows to minimize any disturbance effect; and (3) the lack of significant differences in Δ14C& and mean residence time were shown between year 0 and 5 in the light fraction SOM (Table 8), which may suggest no significant difference in microbial decomposition rates and very low C inputs from the small trees. Second, the C-mixing hypothesis suggests that d13C values in SOM are influenced by the mixing of new C inputs with existing and older soil organic C pools (Billings and Richter, 2006). In terrestrial ecosystems, plant above-ground litter and belowground root inputs have lower d13C relative to existing SOM in mineral soil and may contribute to a shift in 13C:12C ratios in SOM (Garten et al., 2000). The reduced input from above- and belowground fresh grass litter led to less depleted d13C values. We therefore believe that lower C mass in the light fraction SOM at year 5, compared to year 0, may be due to the reduced input from grass litter (Scott et al., 1999).

Table 6 Summary of lignin-derived phenol, cutin and suberin-derived compounds (mg/100 mg OC) and parameters for the light fraction SOM at the 0e5 cm depth before tree planting (Year 0) and 5 and 10 years after tree planting. Means (standard deviation) are based on four replicates. Separation of means was determined by a Tukey post-hoc test; different letters within a species and a row indicate that means differ significantly (P < 0.05) owing to sampling year effects. P. radiata

V S C VþSþC S/V C/V Cutin Suberin

E. nitens

C. macrocarpa

Year 0

Year 5

Year 10

Year 0

Year 5

Year 10

Year 0

Year 5

Year 10

2.26 1.85 1.40 5.51 0.82 0.62 2.12 1.78

3.26 1.89 1.52 6.67 0.58 0.47 2.20 2.19

4.02 1.73 1.26 7.01 0.43 0.31 2.27 2.74

2.42 1.98 1.47 5.57 0.82 0.61 1.98 1.89

2.69 2.21 1.29 5.99 0.76 0.48 2.21 2.27

2.82 2.43 1.08 6.33 0.86 0.38 2.49 2.76

2.25 1.94 1.46 5.65 0.86 0.64 2.09 1.71

3.58 1.90 1.76 7.24 0.53 0.49 2.17 1.93

3.99 1.81 1.34 7.14 0.44 0.34 2.20 2.68

(0.45)a (0.33)a (0.29)a (1.08)a (0.10)a (0.08)a (0.30)a (0.29)a

(0.60)b (0.40)a (0.20)a (1.19)a (0.07)b (0.06)b (0.28)a (0.40)a

(0.61)c (0.29)a (0.31)a (1.21)b (0.05)c (0.05)c (0.22)a (0.31)b

(0.53)a (0.29)a (0.20)a (1.01)a (0.10)a (0.07)a (0.25)a (0.27)a

V, vanillyl; S, syringyl; C, cinnamyl; S/V, syringyl/vanillyl; C/V, cinnamyl/vanillyl.

(0.47)a (0.33)a (0.18)ab (0.99)a (0.13)a (0.06)ab (0.19)a (0.35)a

(0.49)a (0.51)a (0.27)b (1.26)a (0.09)a (0.07)b (0.20)b (0.22)b

(0.40)a (0.27)a (0.29)a (0.95)a (0.18)a (0.11)a (0.33)a (0.20)a

(0.71)b (0.30)a (0.33)a (1.34)b (0.07)b (0.08)b (0.23)a (0.25)a

(0.83)b (0.22)a (0.15)a (1.19)b (0.06)c (0.05)c (0.27)a (0.19)b

Z. Huang et al. / Soil Biology & Biochemistry 43 (2011) 1341e1349

1347

Table 7 Summary of lignin-derived phenol, cutin and suberin-derived compounds (mg/100 mg OC) and parameters for the heavy fraction SOM at the 0e5 cm depth before tree planting (Year 0) and 5 and 10 years after tree planting. Means (standard deviation) are based on four replicates. Separation of means was determined by a Tukey post-hoc test; different letters within a species and a row indicate that means differ significantly (P < 0.05) owing to sampling year effects. P. radiata

V S C VþSþC S/V C/V Cutin Suberin

E. nitens

C. macrocarpa

Year 0

Year 5

Year 10

Year 0

Year 5

Year 10

Year 0

Year 5

Year 10

0.85 0.64 0.30 1.79 0.75 0.35 0.91 0.65

0.87 0.68 0.26 1.81 0.78 0.30 0.82 0.70

0.98 0.62 0.26 1.86 0.63 0.27 0.98 0.83

0.85 0.62 0.29 1.75 0.72 0.34 0.85 0.60

0.86 0.67 0.24 1.78 0.77 0.28 0.99 0.67

0.89 0.64 0.25 1.79 0.80 0.28 0.93 0.79

0.79 0.63 0.28 1.70 0.80 0.35 0.77 0.68

0.85 0.70 0.28 1.82 0.82 0.32 0.84 0.71

1.06 0.72 0.24 2.03 0.67 0.23 0.91 0.87

(0.15)a (0.12)a (0.05)a (0.32)a (0.09)a (0.05)a (0.13)a (0.08)a

(0.18)a (0.09)a (0.04)a (0.30)a (0.07)a (0.04)ab (0.15)a (0.05)a

(0.20)a (0.15)a (0.06)a (0.42)a (0.06)b (0.02)b (0.07)a (0.10)b

(0.21)a (0.11)a (0.04)a (0.35)a (0.10)a (0.03)a (0.11)a (0.08)a

(0.17)a (0.14)a (0.06)a (0.37)a (0.15)a (0.03)b (0.15)a (0.07)a

(0.10)a (0.19)a (0.03)a (0.31)a (0.09)a (0.04)b (0.08)a (0.10)b

(0.20)a (0.09)a (0.06)a (0.34)a (0.07)a (0.05)a (0.10)a (0.05)a

(0.15)a (0.08)a (0.02)a (0.26)a (0.09)a (0.06)a (0.09)a (0.10)a

(0.29)a (0.15)a (0.05)a (0.48)a (0.07)b (0.03)b (0.12)a (0.09)b

V, vanillyl; S, syringyl; C, cinnamyl; S/V, syringyl/vanillyl; C/V, cinnamyl/vanillyl.

4.3. Recovery of soil C mass in the light fraction SOM at year 10 and tree species planted

compared to that under grassland, while planting P. radiata restored the light fraction to the original level under grassland, whereas planting C. macrocarpa led to a decrease of 33%. The increases of soil C in the light fraction SOM between year 5 and 10 under P. radiata and E. nitens could be associated with enhanced C input from the developing forest species as suggested by the significantly decreased syringyl/vanillyl and cinnamyl/vanillyl ratios in the light fraction SOM under P. radiata and the decreased cinnamyl/vanillyl ratio under E. nitens at the 0e5 cm depth (Table 6). Under P. radiata, the relative abundances of suberinderived compounds in the light fraction SOM increased significantly, but the relative abundance of cutin-derived compounds

A number of studies have indicated that the tree species planted can have a significant impact on the restoration of the soil C pool following afforestation because of the variability in their C inputs (Lemma et al., 2006; Paul et al., 2003). Planting broadleaf trees is often associated with a quicker and a greater recovery of soil C compared to conifer species (Laganiere et al., 2010). Our analysis of soil C in the light fraction SOM at the 0e30 cm depth supports this (Table 5). At year 10, an increase of 33% soil C in the light fraction SOM at the 0e30 cm depth is observed when E. nitens is used,

A

P. radiata

E. nitens

C. macrocarpa

-26

δ13C (‰)

-26.5 -27

b

b

-27.5

a

a

b

a

a

a

a

-28 -28.5

B

P. radiata

E. nitens

C. macrocarpa

-26

δ13C (‰)

-26.5 -27

a

a

a

a

a

a

a

a

a

-27.5 -28 Year 0

year 5

year 10

-28.5 Fig. 1. The d13C (&) of light (A) and heavy (B) fraction SOM at the 0e5 cm depth before tree planting (Year 0) and 5 and 10 years after tree planting. Means and standard deviations are based on four replicates. For the same species, means with the same letter are not significantly different owing to sampling year effects. Separation of means was determined by a Tukey post-hoc test (P > 0.05).

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Z. Huang et al. / Soil Biology & Biochemistry 43 (2011) 1341e1349

Table 8 The D14C, modelled mean residence time (MRT) and carbon (C) : nitrogen (N) ratio of SOM fractions at the 0e5 cm depth before tree planting (Year 0) and 5 and 10 years after tree planting. Means (standard deviation) of C:N ratios are based on four replicates. Separation of means was determined by a Tukey post-hoc test; different letters within a species and a row indicate that means differ significantly (P < 0.05) owing to sampling year effects. Soil fraction

Light

P. radiata

D14C (&) MRT (yr) C:N ratio

Heavy

D14C (&) MRT (yr) C:N ratio

E. nitens

C. macrocarpa

Year 0

Year 5

Year 10

Year 0

Year 5

Year 10

Year 0

Year 5

Year 10

111.3 2.9 15.2 (0.5)a

84.1 2.8 17.1 (1.0)b

71.5 5 18.2 (1.1)b

105.6 2.1 15.5 (0.5)a

84.3 2.9 15.5 (0.3)a

68.8 4.5 17.7 (1.2)b

102.9 1.7 15.7 (0.5)a

78.1 2 16.9 (0.8)b

70.4 4.8 20.2 (1.4)c

115.5 4.4 10.4 (0.1)a

96.0 4.8 10.7 (0.4)a

82.9 6.8 12.2 (0.4)b

114.5 3 10.7 (0.4)a

90.1 3.8 10.6 (0.6)a

81.4 6.6 11.7 (0.7)b

113.5 2.8 10.8 (0.3)a

88.0 3.6 11.2 (0.7)a

82.2 6.7 12.3 (0.6)b

remained stable (Table 6). Suberin-derived compounds mainly originate from roots and bark (Feng et al., 2008, 2010), however, inputs from above-ground bark up to age 10 are likely to be quite small for pine trees (Romanyà et al., 2000). Cutin-derived compounds originate from the waxy coating of leaves (Goñi and Hedges, 1990; Otto and Simpson, 2006b). We therefore believe that the increase of soil C mass in the upper soil layer between year 5 and 10 should be mainly attributed to the inputs from tree roots despite a significant needle layer in radiata pine plots. However, under E. nitens, the relative abundance of both suberin and cutinderived compounds in the light fraction SOM increased from year 5e10, which suggests that inputs from both roots and aboveground leaves contributed to the significantly increased soil C. The earlier input of tree leaf derived compounds to light fraction SOM under E. nitens than under conifers may in part explain the quicker and greater recovery of soil C under E. nitens at year 10. The quicker decomposition of above-ground E. nitens litter and C return to mineral soil can also be evidenced by the small amount of litter buildup on the ground observed in this study although up to 10 Mg ha1 of above-ground litter may be deposited annually in eucalypt forests of this age (Guo and Sims, 1999). Soil C in the light fraction SOM under C. macrocarpa was significantly recovered at the 0e5 cm depth at year 10 as the result of C input from gymnosperm and woody litter which can be shown from significantly decreased syringyl/vanillyl and cinnamyl/vanillyl ratios (Table 6). However, the soil C in the light fraction SOM at the 10e30 cm depth decreased substantially from year 5e10. The decrease in soil C mass in the light fraction SOM in the deeper layers led to a trend of decreased soil C in the light fraction SOM at the 0e30 cm depth. The decreases in soil C in the light fraction SOM at the 10e30 cm depth may be associated with the greater reduction in soil N that occurred under C. macrocarpa (Kirschbaum et al., 2008). In addition, lower C inputs into the soil between year 5 and 10 under C. macrocarpa is consistent with the lower productivity of this species relative to P. radiata and E. nitens (data not shown). 4.4. Stability of SOM and plant biopolymers The soil C pool in the heavy fraction did not change significantly at the 0e30 cm depth over the first 10 years under any of the three tree species. However, the significantly lower cinnamyl/vanillyl ratios of lignin phenols at year 10 under all species and the lower syringyl/vanillyl ratios under the two conifers show a significant amount of soil C from woody litter entered into the soil heavy fraction at the 0e5 cm depth between year 5 and 10 (Table 7). The C inputs from woody residues into heavy fraction SOM at the 0e5 cm depth between year 5 and 10 was associated with significantly increased C:N ratios in this fraction and the increased mean residence time of SOM (Table 8). The observed increase of mean residence time in soil C at year 10 compared to year 5 and year 0 in both the light and heavy fraction SOM components may be due to the

increased relative abundance of recalcitrant plant biopolymers (e.g. greater relative abundances of suberin and cutin-derived compounds) (Tables 6 and 7). Suberin and cutin-derived compounds are thought to be highly recalcitrant (Gleixner et al., 2001) and woody litter has higher concentrations of these compounds than grass residues (Otto and Simpson, 2006b). 5. Conclusions Our retrospective study suggests that within the first 10 years after afforestation of grassland the soil C in the upper layer and in light fraction SOM is much more sensitive than that at the 0e30 cm depth and in the heavy fraction. The decrease in soil C mass in light fraction SOM at year 5 occured under three tree species and at all soil depths in this subhumid site (900 mm rainfall annually), which is in agreement with many paired site and chronosequence studies showing that afforestation of grassland leads to significant organic C loss at the initial stage due to reduced C inputs from the grassland. The data also support the assertion that tree species can have a significant impact on the restoration of the soil C pool following afforestation and planting E. nitens appears to give a quicker and greater recovery of soil C compared to P. radiata and C. macrocarpa. We conclude that the restoration of soil C in the light fraction SOM at year 10 is most likely due to C input from residues of the developing trees, with the main sources from tree root turnover under conifers and from both root and leaves under E. nitens, based on our analysis of soil C biomarkers. The analysis and modelling of soil Δ14C& suggested that afforestation increased the stability of SOM in both the light and heavy fractions at year 10 compared to its stability under grassland due to the increased relative abundance of recalcitrant plant biopolymers. Acknowledgements The field trial establishment and maintenance were carried out in collaboration with the Orton Bradley Park Trust Board, with funding provided by the Brian Mason Scientific and Technical Trust and the C Alma Baker Trust. This study was financed by a Scion (New Zealand Forest Research Institute Limited) Post-doctoral grant. We are especially grateful to Mr Hank Krose, Mr Murray Robinson and Dr Bernadette Nanayakkara for their help in sample preparation and GC/MS analyses. References Berthrong, S.T., Schadt, C.W., Piñeiro, G., Jackson, R.B., 2009. Afforestation alters the composition of functional genes in soil and biogeochemical processes in South American grasslands. Applied and Environmental Microbiology 75, 6240e6248. Billings, S.A., Richter, D.D., 2006. Changes in stable isotopic signatures of soil nitrogen and carbon during 40 years of forest development. Oecologia 148, 325e333.

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