Global Change Biology (2010) 16, 427–438, doi: 10.1111/j.1365-2486.2009.01981.

Contribution of trees to carbon storage in soils of

silvopastoral systems in Florida, USA
S O L O M O N G . H A I L E *w , V I M A L A D . N A I R w and P. K . R A M A C H A N D R A N N A I R *
*Center for Subtropical Agroforestry, School of Forest Resources and Conservation, PO Box 110410, University of Florida,
Gainesville, FL 32611, USA, wSoil and Water Science Department, PO Box 110510; Institute of Food and Agricultural Sciences,
University of Florida, Gainesville, FL 32611, USA

Abstract
Silvopastoral systems that integrate trees in pasture production systems are likely to
enhance soil carbon (C) storage in lower soil layers due to the presence of deep tree roots.
To quantify the relative soil C contribution from trees (C3 plants) and warm season
grasses (C4 plants) in silvopastoral systems, soil samples were collected and analyzed
from silvopastures of slash pine (Pinus elliottii) 1 bahiagrass (Paspalum notatum), and
adjacent open pasture (OP), at six depths down to 125 cm, at four sites representing two
major soil orders (Spodosols and Ultisols) of Florida. The plant sources of C in whole
(nonfractionated) and three soil fraction sizes (250–2000, 53–250, and o53 lm) were traced
using stable C isotope signatures. The silvopasture sites contained higher amounts of C3-
derived soil organic carbon (SOC) compared with OP sites, at all soil depths. Slash pine
trees (C3 plants) seemed to have contributed more C in the silt 1 clay-sized (o53 lm)
fractions than bahiagrass (C4 plants), particularly deeper in the soil profile. Spodosols
sites contained more C in the o53 lm fraction at and below the spodic horizon (occurring
between 15 and 50 cm) in silvopasture compared with OP. The results indicate that most
of SOC in deeper soil profiles and the relatively stable o53 lm C fraction were derived
from tree components (C3 plants) in all the sites, suggesting that the tree-based pasture
system has greater potential to store more stable C in the soil compared with the treeless
system.
Nomenclature:
OP 5 open pasture
SOC 5 soil organic carbon
SP-A 5 center of the alley in a silvopasture
SP-T 5 in-between trees in a row of a silvopasture
Keywords: agroforestry, bahiagrass pasture, carbon sequestration, slash pine, stable carbon isotope

Received 10 December 2008; revised version received 21 April 2009 and accepted 7 May 2009

cultural) systems (Montagnini & Nair, 2004). Such

Introduction
Agroforestry systems (AFS) that combine trees and/or
shrubs with crops and/or livestock production are
planned and managed agroecosystems (Garrett et al.,
2000). Increasing the overall productivity and efficiency
of the land-use system are major goals of agroforestry
(Nair, 2005). AFS have the potential to enhance carbon
(C) sequestration in soil compared with treeless (agri-
Correspondence: Vimala D. Nair, tel. 1 352 392 1804, fax 1 352 392
3399, e-mail: vdn@ufl.edu
claims are based on the premise that the tree compo-
nents in AFS can be significant sinks of atmospheric C
due to their long-term storage of high amounts of C in
biomass, especially in the deep root systems. Further-
more, higher diversity of grassland species and specific
plant functional traits were reported to increase uptake
of C into the soil system through resource partitioning
(Steinbeiss et al., 2008). Similarly Saha et al. (2009) found
that homegardens with higher, compared with those
with lower, number of plant species, as well as higher
species richness and tree density had higher soil C,
especially in the top 50 cm of soil.

r 2009 Blackwell Publishing Ltd 427

428 S . G . H A I L E et al.
Silvopasture – the integration of trees into forage or/
and livestock – has been practiced in the southeastern
USA as ‘tree-pasture’ or ‘pine-pasture’ since the early
1950s (Nair et al., 2005). Indeed, silvopasture is the most
common form of agroforestry in North America (Gar-
rett et al., 2000; Nair et al., 2008). These agroecosystems
are usually established by incorporating trees in exist-
ing, managed pastures of bahiagrass (Paspalum notatum
Flueggé), common bermudagrass [Cynodon dactylon (L.)
Pers. var. dactylon] or other similar grasses (Workman
et al., 2003).
Functional consequences of integration of trees into
grass-dominated vegetation include changes of above-
and belowground productivity (Scholes & Hall, 1996;
Archer et al., 2001), modifications to rooting depth and
distribution (Gill & Burke, 1999), and changes in the
quantity and quality of litter inputs (Connin et al., 1997;
Jackson et al., 2000; Jobbágy & Jackson, 2000). Soil
organic matter (SOM) is extremely vulnerable to land-
use change (Paul et al., 1997; IPCC, 2001), as well as to
intensification of agricultural practices (Matson et al.,
1997). Thus, in order to quantify the strength and long-
evity of the C sink in tree-based pasture systems, it is
important to understand the mechanisms and processes
associated with C transformation and storage. SOM has
a very complex and heterogeneous composition, and is
associated with mineral soil constituents to form soil
aggregates. The nature and extent of turnover of soil
organic carbon (SOC) is intimately linked to organic
matter size fractions as well as to soil structure and
extent of aggregation (Martens, 2000). Different compo-
nents of SOC have different residence times, ranging
from labile to stable forms (Carter, 1996). Thus, to
evaluate changes in soil C and SOM dynamics correctly,
it is necessary to separate out functionally different
SOM fractions. Soil size fractionation helps to differ-
entiate these different SOC fractions, (Christensen,
1992); it is based on the premise that SOC associated
with sand-size aggregates (or macro-organic matter
4250 mm) is often more labile than SOC in the clay
and silt fractions (Tiessen & Stewart, 1983).
Stable C isotope-ratio analysis in SOC studies
emerged as a tool to trace the source of SOC to C3
and C4 components in vegetation (Stout et al., 1981).
Numerous studies (Bernoux et al., 1998; Ehleringer et al.,
2000; Accoe et al., 2002; Swap et al., 2004) have been
successful in applying d13C to understand plant–soil
SOM dynamics, making stable isotopic analysis a useful
technique. When one type of vegetation is replaced with
another, d13C values can be used to identify SOM
derived from residues in the native vegetation and the
new vegetation based on discrimination between C3
and C4 plants. The reported d13C values range from 9
to 19% for C4 plants and 20 to 35% for C3 plants
r 2009 Blackwell Publishing Ltd, Global Change Biology, 16, 427–438
(Nyberg et al., 2000; Biedenbender et al., 2004; Staddon,
2004). When a C4 plant is introduced to a system that
had previously been under a C3 plant (or vice versa), the
relative contribution of new vs. old soil organic C can be
quantified using the mass balance of stable isotope
contents based on the change in 13C signature of SOM
(Dawson et al., 2002; Del Galdo et al., 2003). In a
combined tree 1 grass land-use system, C3 inputs are
dominated by either woody shrubs or trees and C4
inputs are dominated by grass (McClaran & McPher-
son, 1995). In pine-based silvopastoral systems where
C3 and C4 plants are grown simultaneously, such
studies that use the natural abundance of d13C to
understand the C dynamics are rare.
The d13C isotope technique requires comparison be-
tween a site where the photosynthetic pathway of the
dominant vegetation (C3 or C4) has been changed and a
reference site where photosynthetic pathway of the
vegetation remains unchanged. The plant community
in silvopasture systems of the southeastern USA is
composed of slash pine (Pinus elliottii) (a C3 plant;
d13C  29.5%) and C4 plants dominated by bahia-
grass (P. notatum) (d13C  13.3%). The d13C value
ranges of C3 and C4 plants do not overlap. Therefore,
differences in isotope ratio can be used to quantify the
relative contribution of plants of each photosynthetic
pathway to SOM (Balesdent et al., 1988). Thus, combin-
ing SOM fractionation techniques with the 13C natural
abundance technique offers a compelling approach to
investigating small shifts in soil C stores that would be
significant in the long term, which might not be de-
tected by conventional methods. A change in land use
from open pasture (OP) to silvopasture presents a
unique opportunity to use the stable C isotope metho-
dology to study SOC dynamics following the integra-
tion of trees into OP. The present study was undertaken
in this context, with the objectives of assessing the
impact of integrating trees in pastures systems on the
SOC content and SOM fraction size compared with OP
systems, and quantifying the relative SOC contribution
of trees and warm-season grasses of silvopastoral sys-
tems using their natural C isotopic differences.
Materials and methods
Study area
Soil samples were collected from four sites, located in
Alachua (29145 0 N, 82133 0 W), Osceola (2819 0 N, 81110 0 W),
Hardee (27113 0 N, 8218 0 W), and Suwannee (30124 0 N,
8310 0 W) counties in Florida, USA. Hereafter, the sites
are designated by county names where the farms are
located. The sites represented two major soil orders
[Spodosols (Hardee and Osceola counties) and Ultisols
 CONTRIBUTION OF TREES TO CARBON STORAGE IN SOILS
(Alachua and Suwannee counties)] of Florida. The
Spodosol sites are on Ona (sandy siliceous, hyperther-
mic Typic Alaquods) or Immokalee fine sands (sandy
siliceous, hyperthermic Arenic Alaquods), whereas the
Ultisol sites are on Kendrick (loamy, siliceous, semiac-
tive, hyperthermic Arenic Paleudults) or Blanton sands
(loamy, siliceous, semiactive, thermic Grossarenic Pa-
leudults). Detailed description of the study sites can be
found in Haile et al. (2008). At each site, soils were
sampled in a silvopasture with slash pine (P. elliottii
Englem) and bahiagrass (P. notatum Flueggé) and ad-
jacent open (treeless) pasture with bahiagrass. The soil
particle-size distribution in these sites ranged from 87%
to 96% sand, 2% to 4% silt, and 3% to 9% clay (Haile
et al., 2008).
At each site, silvopasture and adjacent treeless pas-
ture plots were selected from which soil samples were
taken. Slope, aspect, and soil series were uniform across
plots within a site, thus ensuring that the land-use
system (pasture vs. silvopasture) was the primary factor
influencing the soil C content in plots. The OPs on
Suwannee, Osceola, Hardee, and Alachua farms, were
40, 22, 48, and 55 years old, respectively, since pasture
establishment. The respective ages (years) of silvopas-
tures since establishment at the four sites were: Suwan-
nee (40), Osceola (14), Hardee (12), and Alachua (8).
Sampling
Soil samples were collected from three different sam-
pling plot sets: two locations in the silvopasture sites
including between trees in a row at about 50 cm from a
tree trunk (SP-T) and at the center of an alley at 2–3 m
from tree trunk (SP-A), and one set of samples from OP.
Each of these sample sets had stratified grid sampling
points made by three parallel rows with four sampling
points in a row. The distance between samples was on
average 50 m. From each sampling point, soils were
collected using an auger, from six soil depths: 0–5, 5–15,
15–30, 30–50, 50–75, and 75–125 cm. While in the field, a
composite sample was created for each depth interval.
Equal amounts of soil from corresponding depth inter-
vals of four sampling points in a row were mixed. Thus,
at each site, there were three composite samples for
each depth interval. The composite samples [each soil
depth interval in a row (grid line in OP)] constituted the
experimental unit of analysis and each unit had three
replications (rows), giving a total of 216 samples [six
depths  three replications  three ‘locations’ (SP-A, SP-
T, and OP)  four sites/counties] (Haile et al., 2008).
Needles, roots, wood (branch), bark, and cones were
collected from slash pine trees of silvopastures. The
samples were oven-dried at 60 1C for 72 h, and ground
to fine powder using a mill. A composite sample of the
r 2009 Blackwell Publishing Ltd, Global Change Biology, 16, 427–438
429
slash pine (C3) plant parts was prepared to determine
the d13C values. A similar procedure was adopted for
bahiagrass (C4 plant) by collecting leaves (shoots) and
roots of grass from the OPs.
Size fractionation
Soils were physically fractionated by wet-sieving fol-
lowing a procedure using the disruptive forces of slak-
ing and wet-sieving through a series of two sieve sizes
(250 and 53 mm) to obtain three soil fraction size classes,
as described by Elliott (1986) and Six et al. (1998). Briefly,
all field-moist composite samples were air dried and
passed through a 2 mm sieve. A subsample of 100 g of
the composite sample was submerged in deionized
water with disruptive forces of slaking for approxi-
mately 5 min before placing it on top of 250 mm sieve.
The sieving was done manually by moving the sieve
up and down approximately 50 times in 2 min. The
fraction remaining on the top of a 250 mm sieve was
collected in a hard plastic pan and allowed to dry in
oven at 65 1C and weighed. Water plus soil o250 mm
was poured through a 53 mm sieve and the same sieving
procedure was repeated. The overall wet sieving pro-
cedure yielded a water-stable macro-sized fraction, 250–
2000 mm; a micro-sized fraction, 53–250 mm, and a
silt 1 clay-sized fraction, o53 mm. The recovery of
mass soil fractions after overall wet sieving procedure
ranged from 96% to 99% of the initial soil mass. Taki-
moto et al. (2008) reported a recovery range from 97% to
99% mass soil fractions using a similar fractionation
procedure.
Chemical analysis
For chemical analysis, whole and fractionated soils
were oven-dried at 60 1C for 72 h, and ground to fine
powder using a ball mill (Cianflone Scientific Instru-
ments, Pittsburgh, PA, USA). Total SOC content was
determined for whole and fractionated soil samples by
dry combustion on an automated FLASH EA 1112 N C
elemental analyzer (Thermo Fisher Scientific Inc., Wal-
tham, MA, USA).
Stable C isotope analysis
Soil samples were analyzed for C concentrations and for
d13C values using a Carlo Erba EA-1108 (CE Elantech,
Lakewood, NJ, USA) interfaced with a Delta Plus
(Thermo Finnigan, San Jose, CA, USA) isotope ratio
mass spectrometer operating in continuous flow mode.
C isotope ratios are presented in d-notation:
d13 C ¼ ð½RSAMPLE  RSTD =RSTD Þ  103 ; ð1Þ
430 S . G . H A I L E et al.
where RSAMPLE is the 13C/12C ratio of the sample, and
RSTD is the 13C/12C ratio of the Vienna Pee Dee Belem-
nite (VPDB) standard (Coplen, 1996). Precision of du-
plicate measurements was 0.1%. None of the samples
contained CaCO3 or other forms of inorganic C. A
relative proportion of SOC derived from the bahiagrass,
a C4 plant, or from the slash pine, a C3 plant, was
estimated based on the following equations (Balesdent
& Mariotti, 1996):
% C4-derived SOC ¼ ðd  dT Þ=ðdG  dT Þ  100; ð2Þ
% C3-derived SOC ¼ 100  %C4-derived SOC; ð3Þ
13
where d is the d C of a given sample, dT a composite
sample of the C3 plant and dG is a composite sample of
pasture grass shoots and roots (C4).
Based on the percentages of C3- and C4-derived SOC
and the C content in each soil sample, the contributions
to total SOC by C3 and C4 plants were calculated as
follows:
C3-derived SOC ðkg m2 Þ ¼ð% C3-derived SOCÞ
 ðSOC content; kg m2 Þ;
ð4Þ
C4-derived SOC ðkg m2 Þ ¼ð% C4-derived SOCÞ
 ðSOC content; kg m2 Þ:
ð5Þ
Statistical analysis
Planned comparison analysis of variance (ANOVA) with
Tukey’s studentized range test (HSD) was used to
compare the mean differences between land-manage-
ment practices and plant sources on SOC in whole soil,
macro-sized, micro-sized and silt- and clay-sized frac-
tions at four sites. Statistical analyses were carried out
separately for all depth-classes. The composite sample
in a row (grid line in OP) within each soil depth
constituted the experimental unit of the analysis and
each unit had three replications (rows). All statistical
tests were performed with STATISTICAL ANALYSIS SYSTEM
(SAS Institute Inc., 2004) and differences were consid-
ered significant when Po0.05.
Results
13
Differences in the natural abundance of C SOC
13
Whole soil sample. The average d C value of whole soil
in OP for the four sites was 19.8%. This value was
significantly higher (Po0.001) than in SP-A or SP-T of
silvopasture with an overall average d13C value of
22.6% and 22.9%, respectively, but there was no
r 2009 Blackwell Publishing Ltd, Global Change Biology, 16, 427–438
difference between SP-A and SP-T locations. In general,
whole soil d13C values in silvopasture were strongly C3-
dominated signals with value ranging from 17.5% to
25.1% at all sites (Table 1). The d13C values of whole
soil had a maximum value of 15% at the surface (0–
5 cm) in OP, and a minimum value of 25.1% in the
deeper soil profile of the silvopasture.
Fractionated samples. The d13C values of the macro-sized
fraction (Fig. 1) showed a similar trend as the whole soil
sample with high values at the surface of the OP and
decreasing with depth in all four sites. At the upper
5 cm, the d13C values in the OP were higher by 38% in
Alachua (Fig. 1a, Po0.001), 33% in Suwannee (Fig. 1b,
Po0.001), 27% in Hardee (Fig. 1c, Po0.001), and 25% in
the Osceola sites (Fig. 1d, Po0.01) than the average
value at the silvopasture (SP-A and SP-T). Except at the
depth interval 3050 cm, the OP d13C values in macro-
sized fraction were significantly higher than values on
an adjacent silvopasture across Alachua, Suwannee,
and Osceola sites (Fig. 1a, b, and d).
The d13C values of the OP system in the micro-sized
fraction (53–250 mm) were significantly higher than
those at corresponding sampling locations in the
silvopasture in the Ultisol sites at any given depth
(Fig. 1e and f). Average values were 19.3% and
20.5% for OP in Alachua and Suwannee,
respectively, compared with average values 24.6%
and 24.7%, SP-T and SP-A, respectively, in Alachua
and 25.5% and 25.6% for SP-A and SP-T,
respectively, in Suwannee. Similarly, the d13C values
of OP system at surface in the Spodosol sites were
higher than those of locations in silvopasture. In the
silt 1 clay-sized fraction, a clear difference between d13C
values of OP and silvopasture was observed in Ultisol
sites as opposed to Spodosol sites that showed no
difference across depths (Fig. 1 g and h). In the upper
5 cm of the Alachua site, the values were 17.45% for
OP as compared with 23.8% and 23.5% for SP-A
and SP-T, respectively.
Plant sources of SOC in whole soil sample
In all the sites, except at depth 5–15 cm in Suwannee
and at depth 50–75 in Osceola, C3-derived SOC in the
whole soil sample was significantly higher at the two
silvopasture locations (SP-A and SP-T) than in the OP
system. A reverse trend was observed for the SOC
derived from C4 plants (Table 2), although the differ-
ence was not always significant for all depths. On
average, the amount of SOC derived from C3 plants
ranged from 9.4 kg m2 in OP, 25.6 kg m2 in SP-A, and
30.3 kg m2 in SP-T at the surface soil, compared with
corresponding values of 2.6, 4.1, and 6.4 kg m2, respec-
 CONTRIBUTION OF TREES TO CARBON STORAGE IN SOILS 431

Table 1 d13C values of soil organic carbon (SOC) in whole (unfractionated) soil in different soil depths of silvopasture and open
pasture sites on two Ultisols (Alachua and Suwannee counties) and two Spodosols (Hardee and Osceola counties) in Florida, USA

d13C values of SOC, %

Ultisol sites Spodosol sites

Depth (cm) OP SP-A SP-T OP SP-A SP-T

Alachua Hardee
0–5 15.9a (0.9) 23.6b (0.5) 24.2b (0.4) 15.2a (0.4) 20.9b (0.0) 22.4c (0.2)
5–15 20.6 (1.0) 23.0 (0.6) 24.6 (1.1) 19.1a (0.1) 20.3ab (0.4) 21.7b (0.4)
15–30 20.6a (0.3) 24.9b (1.7) 23.2b (0.1) 21.0 (0.2) 21.7 (0.8) 21.2 (0.1)
30–50 19.8a (0.3) 25.0b (1.7) 23.2b (0.3) 22.2 (0.7) 21.1 (1.2) 21.8 (0.4)
50–75 19.5a (0.6) 23.1b (0.5) 23.3b (0.4) 23.7 (0.1) 23.6 (0.7) 24.4 (0.3)
75–125 20.3a (0.9) 23.3b (0.6) 23.2b (0.5) 23.3a (0.5) 24.7b (0.2) 24.7b (0.1)

Suwannee Osceola
a b b
0–5 16.9 (0.3) 23.5 (0.5) 23.9 (0.3) 15.0a (0.1) 17.5b (0.3) 20.7c (0.6)
5–15 19.2a (0.4) 23.6b (0.1) 24.2b (0.3) 17.2a (0.5) 19.7b (0.4) 19.8b (0.2)
15–30 20.9a (0.3) 25.1b (0.9) 24.4b (0.4) 19.5 (0.4) 20.5 (0.3) 20.0 (0.1)
30–50 21.5a (0.1) 25.0b (1.0) 24.7b (0.4) 20.8 (0.1) 21.2 (0.2) 21.5 (0.5)
50–75 22.0a (0.3) 23.9b (0.3) 24.7b (0.1) 20.9 (0.3) 21.5 (0.5) 21.6 (0.5)
75–125 22.0a (0.4) 24.3b (0.2) 25.1b (0.1) 20.4a (0.7) 21.4b (0.3) 21.4b (0.8)

Lowercase letters indicate significant differences in SOC among pasture locations at a given depth and site. The values in
parentheses are the standard error of means.
OP, open pasture; SP-A, silvopasture, center of the alley; SP-T, silvopasture, in between trees in row.

tively, at the 75–125 cm depth. However, these C3 values
accounted for 16.7% in OP, 55.5% in SP-A, and 65.1%
in SP-T at the surface soil compared with the corre-
sponding values of 58.9%, 69.4%, and 70.6%, respec-
tively, at the 75–125 cm depth. The remaining
percentages of SOC were considered to be contributions
by C4 plants.
Plant sources of SOC in fraction sizes
The amounts of C3- and C4-derived SOC in the macro-
sized fraction were significantly different among the SP-
A, SP-T, and OP at the surface soil layers for Alachua
and Osceola sites, but not for Suwannee and Hardee
sites (Fig, 2a-d). Compared with the C4-derived SOC,
the proportion of C3-derived SOC generally increased
with soil depth in both land-use systems for all sites
(Fig. 2). For example, at the Alachua site (Fig. 2a, e, i, m,
q, and u) 11%, 68%, and 75% of SOC were C3-derived in
OP, SP-A, and SP-T, respectively, on surface soil
(05 cm). The corresponding values in the lowest pro-
file (75–125 cm) were 63%, 93%, and 82% at the same
site (Fig. 2a, e, i, m, q, and u).
The C4-derived SOC in the micro-sized (53–250 mm)
fraction was significantly higher in the OP surface soil
(0–5) than in SP-A or SP-T for Alachua, Suwannee, and
Osceola sites (Fig. 3a–d). The C3 component (slash
pine) contributed o20% of the total SOC in this frac-
tion size on surface soils (05 cm) at all four sites (Fig.
3a–d). The trend was similar to that of the larger
fraction in that there were significant differences be-
tween OP and the silvopasture locations. The greatest
difference between land uses in contribution of C3-
derived C was in the surface in all four sites, ranging
from 13% to 19% in OP and 30% to 76% in the
silvopasture (Fig. 3a–d). The SP-T generally had sig-
nificantly higher C3-derived SOC than that of OP or
SP-A, at the two lowest depths in all the sites except in
Alachua (Fig. 3q–x).
The silvopasture locations in general had accumu-
lated higher C3- and C4-derived SOC in the silt 1 clay
fractions than OP land use in the upper 15 cm depth
of the Spodosol sites. Generally, with an increase
in depth, proportion of C3-derived in SOC in the
smallest fraction size increased compared the propor-
tion of C4-derived SOC for all the sites. The C3-derived
content in the o53 mm fraction was also relatively high-
er at the spodic horizon (upper level of spodic was
between 15 and 50 cm) of the Spodosol sites (Fig. 4o
and p).

r 2009 Blackwell Publishing Ltd, Global Change Biology, 16, 427–438

432 S . G . H A I L E et al.

Fig. 1 Differences in d13C values of soil organic carbon (SOC) in macro-sized (250–2000 mm; a–d), micro-sized (53–250 mm; e–h), and
silt 1 clay (o53 mm; i–l) fractions with mid-points of sampled depths at three pasture locations [silvopasture center of the alley (SP-A)
and in-between tree rows (SP-T) and open pasture (OP)] for whole-soil of the Alachua (a, e, and i), Suwannee (b, f, and j), Hardee (c, g,
and k), and Osceola (d, h, and l) sites in Florida, USA. Lower and upper case letters indicate significant differences in C3- and C4-derived
SOC, respectively, among pasture locations at a given depth and site. Bars represent standard errors of the mean.

Discussion as their composite sample (dG) in this study were not

The d13C value for composite sample of slash pine (dT)
measured in this study was 28.5%. Values for indivi-
dual plant parts such as needles, root, cones, bark, and
branches (wood) were within  2% of the dT. The
values were within the range of d13C values reported
in literature for a composite sample of plant parts or soil
underneath slash pine. Parasolova et al. (2003) reported
range between 25% and 29.5% for slash pine  Car-
ibbean pine (Pinus caribaea) hybrids. Similarly, Morta-
zavi & Chanton (2002) found value range between
27.3% and 28.5% for slash pine needles. The d13C
values range of terrestrial plants grown under natural
conditions are between 9% to 19% for C4 plants and
20% to 35% for C3 plants (Vogel, 1993; Nyberg et al.,
2000; Biedenbender et al., 2004; Staddon, 2004). The d13C
value for bahiagrass measured in this study was also
very close to the d13C value of 13.3% for shoots or
roots of bahiagrass reported by Nakano et al. (2001). The
d13C value of bahiagrass shoot and root samples as well
different, with a value of 12.9%.
Although the d13C values measured in the silvopas-
ture soil in this study (on average  24%) were
within the general range of values for terrestrial C3
plants as given in literatures, they were higher than
d13C value (28.5%) for a composite slash pine sample.
It is plausible that the increase in d13C values was
caused by SOC input from bahiagrass (C4 plants) in
the silvopasture.
The main source of C input to the soil is usually root
biomass, which is transformed to SOC by microorgan-
isms. Compared with trees, grass species generally
develop shallow root systems or alternatively allocate
the main root biomass to the uppermost soil layers,
even if single roots can reach depths of several meters.
The most negative d13C values ( 25%) were found in
deep soils of the silvopasture soils and the least nega-
tive values ( 15%) at the surface of soil layer of OP.
The d13C values showed a generally decreasing (become
more negative) trend with soil depth. The deep-rooted

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 Table 2 C3- and C4-derived soil organic carbon (SOC) in whole (unfractionated) soil in different land-use locations at four sites across soil depth on two Ultisols (Alachua and
Suwannee counties) and two Spodosols (Hardee and Osceola counties) in Florida, USA

SOC (kg m2)

Ultisol sites Spodosol sites

C3 C4 C3 C4

Depth (cm) OP SP-A SP-T OP SP-A SP-T OP SP-A SP-T OP SP-A SP-T

Alachua Hardee
0–5 11.78b (1.97) 34.25a (1.26) 30.85a (0.67) 43.63A (0.29) 21.32B (0.46) 10.67C (0.37) 6.89c (0.29) 16.49b (0.60) 23.65a (0.01) 35.08A (0.79) 15.81B (0.57) 17.29B (1.15)
5–15 4.42b (0.63) 12.17a (0.07) 11.96a (2.22) 7.06A (0.04) 7.80A (0.07) 2.82B (0.51) 5.54c (1.47) 44.77a (3.18) 24.2b (0.88) 8.55C (2.25) 48.34A (0.62) 18.97B (1.70)
15–30 3.43c (0.10) 10.00a (0.01) 4.62b (0.05) 3.59A (0.24) 4.26A (0.15) 2.16B (0.24) 6.16c (1.59) 25.23a (0.34) 15.76b (0.58) 4.21C (0.28) 27.02A (0.24) 14.21B (0.67)
30–50 1.58 (0.30) 2.32 (0.15) 1.92 (0.17) 2.33A (0.16) 1.17B (0.11) 1.01B (0.07) 11.68c (0.41) 13.4b (0.35) 19.37a (0.33) 7.96B (0.42) 9.15B (0.49) 14.85A (1.56)
50–75 2.20 (0.20) 2.53 (0.01) 2.05 (0.15) 2.37A (0.03) 1.13B (0.08) 1.04B (0.04) 4.15c (0.23) 13.75a (0.12) 11.08b (0.71) 2.52B (0.18) 9.51A (0.83) 3.50B (0.76)

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75–125 0.91b (0.05) 1.64a (0.10) 0.75b (0.04) 0.82A (0.04) 0.73A (0.06) 0.35B (0.02) 2.98c (0.61) 12.68a (1.18) 7.26b (0.99) 1.46C (0.10) 4.12A (0.18) 2.37B (0.27)

Suwannee Osceola
c b a A B B
0–5 8.01 (1.20) 25.73 (0.88) 32.28 (0.17) 18.83 (1.11) 13.98 (0.66) 14.14 (0.94) 10.93c (0.38) 26.13b (0.73) 35.23a (0.83) 74.23A (2.38) 57.70B (1.95) 42.79C (1.74)
5–15 9.19a (0.55) 6.98b (0.34) 7.17b (0.37) 12.45A (0.29) 3.28B (0.25) 3.76B (0.39) 7.40b (0.70) 9.64ab (0.56) 11.84a (0.11) 18.98A (0.35) 13.64B (0.03) 14.47B (0.47)
15–30 4.38c (0.19) 6.49b (0.00) 8.04a (0.02) 5.18A (0.04) 2.01C (0.38) 3.82B (0.32) 6.35b (0.70) 8.40b (0.68) 12.33a (0.82) 8.74AB (0.54) 7.52B (0.91) 11.33A (0.74)
30–50 3.57b (0.59) 2.71b (0.14) 6.92a (0.00) 3.40A (0.28) 1.13C (0.05) 1.98B (0.10) 5.66b (0.14) 6.09b (0.02) 9.73a (0.13) 5.67B (0.21) 6.10B (0.63) 8.86A (0.66)
b a B C A a b a
50–75 3.80b (0.13) 2.79 (0.03) 13.08 (1.66) 2.78 (0.16) 1.23 (0.10) 4.64 (0.06) 5.33 (0.83) 1.09 (0.46) 5.09 (0.08) 6.40A (0.62) 0.96B (0.41) 5.41A (0.17)
c a
75–125 2.56b (0.10) 1.03 (0.08) 11.53 (0.36) 1.83B (0.12) 0.38C (0.01) 3.31A (0.07) 4.11b (0.46) 1.06c (0.29) 6.03a (0.27) 4.50A (0.47) 1.22B (0.05) 5.77A (0.82)

Lower and upper case letters indicate significant differences in C3- and C4-derived SOC, respectively, among pasture locations at a given depth and site. The values in parentheses
are the standard error of means.
OP, open pasture; SP-A, silvopasture, center of the alley; SP-T, silvopasture, in between trees in row.
CONTRIBUTION OF TREES TO CARBON STORAGE IN SOILS
433
434 S . G . H A I L E et al.

Fig. 2 Differences in C3- and C4-derived soil organic carbon (SOC) in macro-sized (250–2000 mm), fractions with sampled depth
intervals at three pasture locations [silvopasture center of the alley (SP-A) and in-between tree rows (SP-T) and open pasture (OP)] for the
Alachua (a, e, i, m, q, and u), Suwannee (b, f, j, n, r, and v), Hardee (c, g, k, o, s, and w), and Osceola (d, h, l, p, t, and x) sites in Florida,
USA. Lower and upper case letters indicate significant differences in C3- and C4-derived SOC, respectively, among pasture locations at a
given site. Bars represent standard errors of the mean.

system of the tree component is most likely to increase
the C3-signature or decrease (more negative) d13C va-
lues with depth as reported by Kramer & Gleixner
(2008). By contrast, the least negative d13C values in
the surface soil layer (0–5 cm) of the OP suggests that
bahiagrass was the main plant source of the SOC. The
SOC beneath the silvopasture on the SP-T and SP-A
locations, including in the surface soils, were substan-
tially derived from the slash pine litter and fine roots
(Table 2).
Soil C decomposition is relatively fast in the surface
soil layer (Yang et al., 2009). Consequently, the SOC
residence time will be shorter at surface soils than at
lower depths. Evidence from radiocarbon studies also
indicate that the average age of SOC increases with
depth in the profile (Scharpenseel & Neue, 1984; Bales-
dent et al., 1990). The soil C at the surface is likely
composed of recent accumulation of SOM at a given site.
The d13C values combined with the possible varia-
tions in C turnover rate of the different soil fraction
sizes provide an indication of plant community history.
The d13C values of fraction sizes revealed that the
macro-sized fraction (Fig. 2) and micro-sized fraction
(Fig. 3) contributed most to the C derived from the C3
woody plant in the silvopasture compared with that in
OP across the soil depth, but particularly so in the lower
profiles (Figs 2o, p, s, w and 3m–x). Given that the soil
of the study sites is high in sand (94%, Haile et al., 2008),
soil C in the larger soil fraction is likely to be the C that
is newly incorporated into the soil. Thus, most of this
SOC, including that in the lower profile, could be
considered as contributed by the current C3 plant
component in the silvopasture, i.e. slash pine, which
has deeper root systems than the grass component.
When comparing d13C values of functionally distinct
SOC fractions, the silt 1 clay fraction showed a higher

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 CONTRIBUTION OF TREES TO CARBON STORAGE IN SOILS 435

Fig. 3 Differences in C3- and C4-derived soil organic carbon (SOC) in micro-sized (53–250 mm), fractions with sampled depth intervals
at three pasture locations [silvopasture center of the alley (SP-A) and in-between tree rows (SP-T) and open pasture (OP)] for the Alachua
(a, e, i, m, q, and u), Suwannee (b, f, j, n, r, and v), Hardee (c, g, k, o, s, and w), and Osceola (d, h, l, p, t, and x) sites in Florida, USA. Lower
and upper case letters indicate significant differences in C3- and C4-derived SOC, respectively, among pasture locations at a given site.
Bars represent standard errors of the mean.

proportion of C3-derived SOC than C4-derived SOC at
the lower soil profile (Fig. 4n–x). However, in the
silt 1 clay fraction, no difference was observed between
silvopasture and OP in the lower depths for most sites
(Fig. 4v–x), except the Alachua site where the land was
under agriculture (corn, Zea mays) before the current OP
(Fig. 4u). The silt 1 clay fraction was less enriched with
13
C than the whole soil and appeared to contain SOC
largely derived from C3 (o75%). In fact, the C asso-
ciated with the silt 1 clay fraction tends to be ‘older’ C
(Anderson & Paul, 1984), indicating that prior land-use
history of the site has a substantial effect on the current
status of C storage in soil fraction sizes. Forty-five years
ago, the current study sites were under forest vegeta-
tion, popularly called Florida Flatwoods and it is logical
to surmise that the current C status of the site is a
reflection of that land-use history.
At the Spodosol sites, at and below the spodic hor-
izon (Bh), which occurred between 15 and 50 cm depth,
the C3-derived C in silt- and clay-sized fraction was
relatively high for both the pasture and the silvopasture
(Fig. 4k, l, o, t). The spodic horizons differ from other
soil profile materials due to the prevalence of organi-
cally associated aluminum (Al) and have very high
surface area for the retention of SOC (Eswaran et al.,
2003). Percival et al. (2000) found that the pyropho-
sphate-extractable Al was correlated strongly with both
soil C content and soil C concentration across different
types of soils. Schwartz et al. (1986) showed from an
analysis of the depth distribution of d13C in podzols
(equivalent name for Spodosols, according to the FAO
system of soil classification) developed under C4 sa-
vanna in the Congo that the Bh horizons exhibited
typical C3 values because the podzolic morphology
was inherited from past forest phases 430 000 years
old. Using long-term incubation experiments and nat-
ural 13C-labelling of C3- and C4-derived SOC collected
from across major environmental gradients in Australia,

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436 S . G . H A I L E et al.

Fig. 4 Differences in C3- and C4-derived soil organic carbon (SOC) in silt + clay-sized (o53 mm), fractions with sampled depth intervals
at three pasture locations [silvopasture center of the alley (SP-A) and in-between tree rows (SP-T) and open pasture (OP)] for the Alachua
(a, e, i, m, q, and u), Suwannee (b, f, j, n, r, and v), Hardee (c, g, k, o, s, and w), and Osceola (d, h, l, p, t, and x) sites in Florida, USA. Lower
and upper case letters indicate significant differences in C3- and C4-derived SOC, respectively, among pasture locations at a given site.
Bars represent standard errors of the mean.

Wynn & Bird (2007) found that the active pool of SOC
derived from C4 plants decomposed at more than twice
the rate of total pool of active SOC, and concluded that
C4-derived SOC decomposed faster than C3-derived
SOC in mixed C3/C4 soils. This would suggest that
the higher amount of C3-derived SOC at lower depths
in silvopastoral systems could also be a result of lower
decomposition rate of C3-derived SOC and not only of
its higher input.
The silvopasture system in the present study, which
represents a change from a C4-dominated herbaceous
community (bahiagrass) to a mixed plant community of
C3 woody slash pine and bahiagrass, provides an ideal
situation for the use of the natural 13C-labeling techni-
que (Nitschelm et al., 1997). The quantification of the
progressive incorporation of new C into soil organic
fractions provides a powerful means to elucidating the
pathways of C transformations and stabilizations. How-
ever, the 13C method is unable to distinguish between
residual ‘primary forest’ C and new C derived from tree
component in the silvopasture (Gillson et al., 2004). The
use of 14C and bomb C models (Trumbore, 2000) is
relevant to ‘date’ soil fractions and to distinguish newly
incorporated C derived from tree component in the
silvopasture from old residual C retained from primary
forest. Such methods could also reduce uncertainties in
turnover rates.
Conclusions
The study showed a consistent trend of increase in C3-
signature dominated the d13C values with soil depth.
Evidently, this has translated to a slight increase in C3-
derived SOC pools following tree integration into pas-
tures in this study. The fractionated soil revealed that a
substantial proportion of C3-derived SOC was found in
macro- and micro-sized fractions at the surface and
deeper soil profiles. At all sites, we found accumula-
tions of larger proportion of C3-derived than C4-de-
rived SOC associated with silt 1 clay, particularly in the

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 CONTRIBUTION OF TREES TO CARBON STORAGE IN SOILS
deeper soil layer. These results suggest that, in the long
term, silvopasture may help sequester more SOC in the
soil. Obviously, such long-term sequestration of C in the
soil has promising environmental implications. How-
ever, more information on several key issues is needed
before the suggested potential of silvopasture can be
convincingly established.
Acknowledgements
This research was supported in part by a grant from the USDA-
Initiative for Future Agriculture and Food Systems (IFAFS)
through the Center for Subtropical Agroforestry. We thank all
the individuals who participated in this research project espe-
cially, Fred Clark (Alachua), Bruce Goff (Suwannee), and Harris
Hill (Osceola) who allowed us to collect soil samples and other
field data from their farms. We also thank Sam Allen, Alain
Michel and Subhrajit Saha (SFRC, UF) for their help during soil
sampling.
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