e c o l o g i c a l e n g i n e e r i n g 3 4 ( 2 0 0 8 ) 300–310

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/ecoleng

The soil organic carbon in particle-size separates under

different regrowth forest stands of north eastern Costa Rica

Juan J. Jiménez a,∗ , Rattan Lal a , Ricardo O. Russo b , Humberto A. Leblanc b

a School of Environment and Natural Resources, The Ohio State University, 2021 Coffey Road, Columbus, OH 43210, USA
b EARTH University, Guácimo, Limón, Costa Rica

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

Article history: Despite the importance of the secondary forest (SF) in tropical areas, few studies have quan-
Received 19 March 2008 tified the soil organic carbon (SOC) pool in Costa Rica. Most of the studies conducted to date
Received in revised form 1 July 2008 in this country have focused mainly on changes in the soil C pool following conversion of
Accepted 3 July 2008 forests to pastures, which is the predominant land use in the tropics. The aim of this study
was to measure SOC concentration and pool in particle-size fractions down to 50 cm depth
in four SF stands regenerating from different intensities of prior land use in loamy sand
Keywords: and sandy loam soils of northeast Costa Rica: (i) a gallery forest (GF), (ii) a 15-year-old SF
Soil organic carbon enriched with commercially planted native trees (15SF), (iii) a 25-year-old SF (25SF), and (iv)
Secondary forests an abandoned Theobromma cacao plantation >60 years old (60SF). Additional objectives were
Particle-size fraction (1) to determine the relationship of SOC concentration with selected physical and chemical
Soil aggregation soil properties, and (2) to establish the key determinants of the depth distribution of SOC in
Tropical soils order to identify meaningful trends in the SOC pool. The SOC pool was highest under the
60SF (221.4 Mg C ha−1 ) followed by the 15SF (212.1 Mg C ha−1 ), the 25SF (195.9 Mg C ha−1 ) and
the lowest in the GF (183.5 Mg C ha−1 ). The SOC concentration decreased significantly from
59.7 to 94.1 g kg−1 in the 0–10 cm layer down to 31.0 to 45.5 g kg−1 in the 40–50 cm layer in all
forest stands. The fine silt + clay fraction contained the highest values of SOC concentration
in all forest stands. Soil texture and the age of the SF were identified as the main factors
that explained the variability in SOC. The age of SF stand influenced the distribution of size
class aggregates and SOC.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction lost following conversion of natural ecosystems to agro-

Carbon (C) sequestration is defined as the biotic process
whereby the atmospheric CO2 is transferred into a long-
lived C pool (Lal, 2004). The soil organic carbon (SOC)
pool is about 2.6 times the biotic pool (Post et al., 1990;
Eswaran et al., 1993) and twice the atmospheric pool. Hence,
SOC pool plays an important role in climate change pro-
cesses by acting either as source or sink of atmospheric
CO2 . As much as 75% of the antecedent SOC pool can be
ecosystems in tropical regions due to intensive tillage
practices that increase decomposition and litter removal
(Lal, 2005). This leads to decline in soil quality and plant
productivity and sometimes to important soil degradation
processes as loss of soil structure and aggregate stabil-
ity, fertility decline and beneficial soil organism depletion.
Thus, severe reduction in land productivity promotes tropi-
cal deforestation, contributing to continuous anthropogenic C
emissions.

∗
Corresponding author. Present address: Instituto Pirenaico de Ecología-CSIC, Avda. Regimiento Galicia s/n, E-22700 Jaca, Huesca, Spain.
E-mail addresses: jjimenez@ipe.csic.es, juanjo jimenezj@yahoo.com (J.J. Jiménez).
0925-8574/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.ecoleng.2008.07.001
 e c o l o g i c a l e n g i n e e r i n g 3 4 ( 2 0 0 8 ) 300–310
The forest area in Central America decreased at a rate of
350 × 103 ha year−1 during the period 1990–2005 (FAO, 2005).
Costa Rica, being once almost totally forested (Keogh, 1984),
has experienced severe deforestation between 1950 and 1984
(Hartshorn et al., 1982; Sader and Joyce, 1988). The forest
area of Costa Rica covers 46.8% of the total land area, and
comprises 180 × 103 ha of primary forest (no disturbance) and
1319 × 103 ha and 888 × 103 ha of modified natural and semi-
natural forest, respectively (FAO, 2005). Secondary forests (SF)
are becoming an extended land use in Costa Rica and many
other tropical countries in Central America, due mainly to
anthropogenic disturbances such as logging and conversion of
forests to agricultural lands (Skole and Tucker, 1993). The SFs
are important since they can act as important carbon sinks
and may offset C losses resulting from deforestation and land
use change.
The Neotropical SF varies in relation to the type, inten-
sity and duration of agricultural practices (Fearnside and
Guimaraes, 1996; Guariguata and Ostertag, 2001), which
affects plant community structure (Fernandes and Sanford,
1995). In Costa Rica, data on the SOC pool in SF following aban-
donment of the previous land use are lacking. In other tropical
sites these studies have been conducted by Hughes et al. (1999)
and Rhoades et al. (2000). In previously deforested areas the
SOC pool may either increase (Silver et al., 2000), decrease (Uhl
and Jordan, 1984) or be unaltered with forest age (Hughes et
al., 1999).
While some research information is available on the above-
ground biomass accumulation and productivity of commercial
tree plantations in Costa Rica and SF (Montagnini, 2000;
Russell et al., 2004), data on the SOC pool and its distribu-
tion within particle-size aggregates in SF are lacking. Recent
estimates of the total C pool (organic + inorganic) in forests
of Costa Rica are given at 112, 81 and 21 Pg (1 Pg = 1015 g) for
above, belowground and dead woody biomass, respectively
(FAO, 2005).
Unfortunately, no SOC pool estimates exist before land use
change occurred in the area, although studies conducted in
nearby areas as the Sarapiquí region in northeastern Costa
Rica have reported a SOC pool of 87.1 Mg C ha−1 (Powers, 2004).
Therefore our main objective was to quantify the SOC pool and
detect its variation in different processes of forest regenera-
tion, as it is expected that forest regrowth leads to positive
or negative variations both soil C concentration and pool (Uhl
and Jordan, 1984; Hughes et al., 1999; Silver et al., 2000).
Soil structure and distribution of size class aggregates can
be disrupted by disturbance (Wigginton et al., 2000). Carbon
accumulation can be facilitated through the formation of a
well soil macro-aggregated structure and it is expected that
the age of recovery has an influence in the distribution of soil
aggregates and SOC. A range of factors affects the formation of
stable aggregates as land use, management, soil mineralogy,
texture, quantity and quality of the organic matter (OM) incor-
porated, diversity and abundance of soil macrofauna (Jiménez
and Lal, 2006). The OM in the soil can be occluded within aggre-
gates and thus be protected against the mineralization process
as it is not readily available for micro-organism attack. We thus
assessed soil structure and particle-size aggregates in relation
to C concentration. This is important in the context of SOC
formation and stability, as C is more protected from mineral-
301
ization processes in large aggregates (Six et al., 1998; Jiménez
and Lal, 2006).
Our hypothesis was that under secondary forests estab-
lished in loamy sand and sandy loam soils both the
management and age of the SF contribute to differences in the
SOC pool. Therefore, our general objective in this study was
to assess the SOC pool and its distribution within size aggre-
gates and particle-size fractions down to 50 cm depth in TSF
of northeastern Costa Rica and elucidate key determinants of
the depth distribution of SOC related to some selected soil
properties by means of multivariate ordination methods.
2. Material and methods
2.1. Study site
The study was conducted at EARTH University (10◦ 10 N and
83◦ 37 W; 64 m a.s.l.) at the confluence of the “Parismina”
and “Destierro” rivers, in the Caribbean lowlands of Limón
Province, Costa Rica, during July 2005. Climate zone of the
region is classified as premontane, wet forest basal belt
transition (Bolaños and Watson, 1993). Topography is flat to
undulating with mean annual rainfall of 3464 mm, which
is evenly distributed with peaks observed during June, July,
August, November, and December. The mean annual temper-
ature is 25.1 ◦ C (iso-hyperthermy). The average annual relative
humidity is 87%. Soils at the study site, classified as Inceptisols
(Typic Dystropept), are moderate to very low in soil fertility.
Soil pH (H2 O, 1:1) ranged from 4.9 to 5.9 and texture from loamy
sand in the upper soil layers to sandy loam in the sub-soil
layers (Table 1).
Four forest stands with no thinning after establishment
and located in the same soil unit with no slopes were selected
for this study:
a) The “Tigre” reserve (60SF): This is a 10-ha SF derived from
an old abandoned cacao (Theobroma cacao L.) plantation
that has regenerated over >60 years (indicated by the pres-
ence of lianas of ca. 6–7 cm Ø). The current regeneration is
similar to the primary forest. Mean stand basal area (G) at
the study site was 10.9 + 3.5 m2 ha−1 and mean tree density
was 237 + 62 trees ha−1 >10 cm DBH (mean DBH = 24.2 cm).
Luehea seemanii, Castilla elastica, Hampea appendiculata, Burs-
era simarouba, Laetia procera, Simarouba amara, Vochysia
ferruginea, Vitex cooperi and Zanthoxylum kellermanii are part
of the floristic composition on this forest. Plant species that
are typical during the first succession stages, e.g., Cecropia
obtusifolia and Heliconia sp., are not abundant.
b) A 25-year-old regrowth forest (25SF): This is a 5-ha SF
characterized by the presence of pioneer plants, i.e., H.
appendiculata, C. obtusifolia, Ochroma pyramidale and abun-
dant understory vegetation (e.g., Heliconia spp.). Other
species in this forest were Cedrela odorata, Inga sp., L. see-
mannii, Rollinia microcephala, and Virola sebifera. Mean stand
basal area at the time of the study was 25.9 + 3.2 m2 ha−1
and mean tree density was 450 + 172 trees ha−1 >10 cm DBH
(mean DBH = 27.2 cm) (R. Russo data).
c) The “Charral” (15SF): This is a 29.5-ha 15-year-old SF that
was enriched 10 years ago with native trees such as H.
302 e c o l o g i c a l e n g i n e e r i n g 3 4 ( 2 0 0 8 ) 300–310

Table 1 – Average soil organic carbon (SOC) concentration and bulk density in the different soil layers analyzed in the SF
stands in north eastern Costa Rica
Depth (cm) Forest stand

GF 15SF 25SF 60SF

SOC (g kg−1 ) d (Mg m−3 ) SOC (g kg−1 ) d (Mg m−3 ) SOC (g kg−1 ) d (Mg m−3 ) SOC (g kg−1 ) d (Mg m−3 )

0–10 59.7 ± 18.0 a 0.79 ± 0.2 67.1 ± 16.0 a 0.83 ± 0.1 76.8 ± 19.4 a 0.74 ± 0.1 94.1 ± 12.9 a 0.63 ± 0.1
10–20 48.1 ± 21.8 b 0.99 ± 0.3 50.9 ± 10.1 b 0.89 ± 0.04 56.6 ± 4.4 b 0.70 ± 0.1 65.8 ± 8.1 b 0.71 ± 0.1
20–30 37.8 ± 13.5 c 0.93 ± 0.3 44.4 ± 9.3 c 0.90 ± 0.1 52.9 ± 5.4 c 0.68 ± 0.1 54.8 ± 7.1 c 0.76 ± 0.1
30–40 33.1 ± 11.2 d 0.96 ± 0.2 40.9 ± 11.2 d 1.01 ± 0.3 47.0 ± 4.7 d 0.70 ± 0.1 50.1 ± 9.8 d 0.77 ± 0.1
40–50 31.0 ± 10.0 e 0.98 ± 0.2 40.0 ± 10.0 d 0.91 ± 0.3 44.3 ± 4.5 d 0.74 ± 0.1 45.4 ± 10.8 e 0.78 ± 0.1

Different letters indicate significant differences at P < 0.05 (Tukey’s HSD test). Standard deviation within parentheses.

alchorneoides. Mean stand basal area at the time of the
study was 17.7 + 8.3 m2 ha−1 and mean tree density was
525 + 170 trees ha−1 >10 cm DBH (mean DBH = 18.3 cm). (R.
Russo data). C. obtusifolia, H. appendiculata, O. pyramidale and
Ficus weckleana are also common in this forest. Because of
the proximity of agricultural lands, cultivated plant species
were also present: Musa sp. and Erythrina costaricensis. The
presence of timber species, e.g., V. sebifera, H. alchorneoides,
Carapa guianensis, C. odorata, Chloroleucon mangense and Cor-
dia alliodora, indicates that enrichment in this area has
influenced the composition and structure of the vegetation
(Russo and Leblanc, unpubl.)
d) A gallery forest (GF): Close to the 15SF stand we
selected a gallery forest. Mean stand basal area (G) at
the time of the study was 11.8 + 5.1 m2 ha−1 and mean
tree density was 283 + 75 trees ha−1 >10 cm DBH (mean
DBH = 23.05 cm) (R. Russo data). Heliocarpus appendiculatus,
H. appendiculata, Zygia longifolia, C. alliodora, Ficus wer-
ckleana, Pentaclethra macroloba, Inga oerstediana, Nectandra
sinuata, R. microcephala, Trophis racemosa, Virola koschnyi, Pro-
tium costaricense, Sterculia apetala, Chloroleucon eurycyclum,
Cecropia spp., Ficus spp. and Guarea spp. are also common
in this forest.
Sites were carefully selected for comparisons based on the
time elapsed since perturbation and management, e.g., sow-
ing more productive trees that are normally used in the region.
gate separation and sieved through a 8 mm sieve to remove
roots, coarse debris and gravels. These samples were air-
dried for subsequent aggregate analyses, and carefully packed
for shipment to The Ohio State University for further lab
analyses.
Litter in the soil surface was collected manually from
0.5 m × 0.5 m metal frames prior to excavation of the pit. Litter
was oven-dried at 60 ◦ C for 72 h.
2.3. Soil physical properties
Soil bulk density (d ) was measured in all five layers by the
core method (Blake and Hartge, 1986) using metal cylinders
of 5 cm × 5 cm. The core sample was taken in the middle of
each layer, transferred to a labelled plastic bag and carried to
the lab for weight measurements. A small quantity of soil was
oven-dried at 105◦ C for 48 h to calculate moisture content and
report d on a dry basis.
Another sub-sample of 50 g air-dried soil was used for
aggregate size fractionation by dry sieving (Kemper and
Rosenau, 1986), and shaking mechanically the nest of sieves
for 30 min to obtain 6 aggregate size fractions: >4.75 mm,
4.75–2.0 mm, 2.0–1.0 mm, 1.0–0.5 mm, and 0.5–0.250 mm and
<0.250 mm. The data obtained were used to calculate the mean
weight diameter (MWD) and the aggregate size class distribu-
tion using the formula by Kemper and Rosenau (1986):

n

MWD = x̄i mi and the aggregate fraction

2.2. Soil sampling
i=1

At each selected forest stand four pits of 1 × 0.5 m2 were dug
out to collect soil samples down to 50 cm depth in 10 cm depth
increments. Although sampling could have been performed
down to 1 m depth, time and logistics constrained the col-
lection of soil samples to the top 50 cm. Due to the possible
spatial autocorrelation of C concentration in soil at short dis-
tances, i.e., <25 m as shown in some studies (Cerri et al., 2004;
Powers and Veldkamp, 2005), four soil pits were separated
100 m to reduce autocorrelation between samples. Precautions
were taken to minimize soil and site disturbance. Samples
were transferred into plastic bags and gently crumbled manu-
ally to break the aggregates along planes of weakness while
at field moisture content, and air-dried for several days.
Samples were dropped onto a hard surface to facilitate aggre-
Msieve i
(mi ) =
Mtotal sample
where x̄i is the mean diameter of each aggregate fraction;
Msieve i is the dry mass of the particles retained on the sieve i;
Mtotal sample is the dry mass of the initial total sample.
The mean diameter of each size class aggregate was con-
sidered as the average opening size of the two sieves retaining
the specific aggregate. Although dry sieving yields higher mass
of large aggregates compared to wet sievings, as reported in
other studies (Balabane and Plante, 2004), the former is also
a commonly used procedure in assessing the degree of soil
aggregation. The remaining soil was sieved at 2 mm and stored
for further analysis.
 e c o l o g i c a l e n g i n e e r i n g 3 4 ( 2 0 0 8 ) 300–310 303

2.4. Simplified particle-size fractionation

60SF

2.2
2.0
1.5
1.5
1.4
The use of physical fractionation methods has increased
in order to study the factors involved in the associations

25SF
MWD

2.4
1.9
1.8
1.7
1.8
between soil mineralogy and SOC differing in composition,
structure and function (Christensen, 1992, 2001; Cambardella
and Elliot, 1994). These methods expose OM that is phys-

15SF

2.4
2.3
1.7
1.9
2.2
ically protected in aggregates. However, no attempt has
been made in the study of SOC concentration in different

GF

3.1
2.8
2.1
2.6
1.9
particle-size fractions of SF. A composite sample of ca. 50 g
of the remaining <2 mm air-dried soil was dispersed in 50 ml
0.5 M Na-hexametaphosphate and 75 ml deionized water for

60SF

42.4
34.0
24.1
24.0
22.6
Table 2 – Distribution of particle-size aggregates after dry sieving and mean weight diameter (MWD) of aggregates in the four SF stands
18 h and stirred mechanically in a multi-mixer machine for
20 min. The mixture was then sieved through a nest of

25SF
sieves of 250, 105, 53, and 20 ␮m to retain the coarse sand

>2000 ␮m

46.0
33.7
31.5
28.1
30.2
(105–200 ␮m), fine sand (53–105 ␮m), coarse silt (20–53 ␮m)
and fine silt + clay (<20 ␮m) fractions in beakers that were

15SF
oven-dried at 60 ◦ C for 72 h. The <20 ␮m fraction was later floc-

43.0
41.1
28.6
31.8
38.9
culated with MgCl2 and allowed to settle before discarding the
supernatant.

63.0
53.4
36.9
43.7
31.8
GF
2.5. Aggregate-associated carbon and nitrogen

60SF
concentrations

21.2
18.4
17.1
14.8
15.1
Size class aggregates
Concentrations of C and N in soil were determined by the

1000–2000 ␮m

25SF

21.2
17.2
17.6
14.9
15.7
dry combustion method for each aggregate size fraction by
using a CN Analyzer (Elementar Vario EL, Germany). The HCl
test was performed to detect the presence of carbonate C in

15SF

21.2
23.5
20.1
17.7
18.7
the samples. Since all samples tested negatively, total C was
considered organic C. Sub-samples of aggregate size separates
that were obtained from the dry sieving method were ground

18.3
14.1
16.9
13.2
14.6
in a mortar and passed through a 200-␮m sieve. Any visible GF
roots and plant debris >1 mm were removed since we were
60SF

28.7
35.4
39.5
38.8
40.0
only interested in C and N concentration in the mineral soil
fraction.
The SOC pool, expressed as Mg ha−1 for a specific depth,
250–1000 ␮m

25SF

25.6
33.0
33.9
36.0
was computed by multiplying the SOC concentration (kg Mg−1 ) 34.9
with bulk density (Mg m−3 ), depth (m) and area (104 m2 ha−1 ).
The SOC pool was calculated with the formula (1) by summing
15SF

26.1
28.1
34.7
34.5
28.6

up the C pool in each soil layer (Batjes, 1996):

C poollayer (Mg ha−1 ) = Clayer (kg Mg−1 ) × d layer (Mg m−3 )

14.3
19.5
28.9
29.2
36.4
GF

×D(m) × 10−3 Mg kg−1 × 104 m2 ha−1

60SF

7.7
12.3
19.3
22.3
22.3

(1)
25SF

2.6. Statistical analyses

7.3
16.1
17.0
21.0
19.2
<250 ␮m

Data were log transformed (ln x + 1) to reduce asymmetry of

15SF

data before analysis when assumption of normality was not

9.6
7.3
16.6
16.0
13.7

achieved by the Kolmogorov–Smirnov test (Lilliefors, 1967). A

two-way ANOVA was used to determine significant differences
4.4
13.0
17.2
13.9
17.2
GF

with forest type and depth as the fixed main effects for d , size
class aggregates, MWD, C and N concentrations, and C:N ratio.
Soil depth (cm)

When significant differences appeared, the Tukey’s honestly

significant difference (HSD) test at ˛ = 0.05 was used for mean
comparisons between groups. The t-test was used to detect
significant changes in the total SOC pool between the TSFs.
0–10
10–20
20–30
30–40
40–50

The Sigmastat package was used to perform all analyses and

Sigmaplot software for graphics.
304 e c o l o g i c a l e n g i n e e r i n g 3 4 ( 2 0 0 8 ) 300–310
Since four soil samples were collected in each forest
stand, thus pseudo-replicates, a specific multivariate ordina-
tion technique was employed to identify the main sources of
variation and significance between land uses and ecosystems,
the between-within principal component analysis (PCA). A
first PCA identifies the sources of variability based on the
groups (forest stands) that are responsible in the definition of
axes. This between-class PCA focuses on the between groups’
differences (see Dolédec and Chessel, 1989 for more details).
Statistical significance was tested with a Monte Carlo ran-
domisation (10,000 simulations) procedure (Manly, 1991).
This analysis was later followed by a within-class PCA in
order to focus on the remaining variability after the group
effect (forest stand) is removed. All centers of groups are
placed at the origin of the factorial plane while sampling
units are scattered with the maximal variance around the
origin (Dolédec and Chessel, 1991). The discriminant analy-
sis module included in the ADE4 software package was used
(Thioulouse et al., 1997).
3. Results
3.1. Soil physical properties
The soil bulk density in the GF and in the 15SF was significantly
higher than that obtained in the 25 and 60SF. Average values
for soil d ranged from 0.6 to 1.0 Mg m−3 in the 60SF and 15SF,
respectively (Table 1). Significant differences were found for
the main factor SF stand (ANOVA, P < 0.001), but no significant
differences were observed either for depth (ANOVA, P = 0.346)
or the interaction (ANOVA, P = 0.970). There were no significant
differences in the d obtained at the different soil layers for all
treatments (HSD Tukey test).
The distribution of size class aggregates and MWD is indi-
cated in Table 2. The percentage of aggregates <0.250 mm
increased with depth with values ranging from 5 to 20%
(Table 2). On the contrary, for aggregates larger than 1 mm
and especially for those >2 mm the opposite was found. In
general, when aggregate distribution was examined by depth,
40–60% of aggregates were larger than 2 mm in the 0–10 cm
layer in all SF stands, and thus average MWD decreased with
depth. Significant differences appeared in the MWD of aggre-
Table 3 – Two-way ANOVA for aggregate size class distribution and MWD (mm) in the four SF stands evaluated, with
forest type and sampling depth as main fixed factors
Source of variation d.f. MWD
<0.25 0.25–0.50
**
SF type (A) 3 5.218 1.286 NS
Depth (B) 4 0.144 NS 0.232 NS
A×B 12 0.583 NS 0.172 NS
MS residual 60 0.41 67.51 35.64
MS total 79 0.43 57.13 35.30
The F-ratio is indicated for each variable. NS, not significant.
∗
P < 0.05.
∗∗
P < 0.01.
∗∗∗
P < 0.001.
gates for the main fixed factor SF type (ANOVA, P < 0.01),
while no significant differences were observed either for depth
or the interaction (Table 3). However, differences were not
statistically significant (Table 3). Differences in size class
aggregate distribution were statistically significant for aggre-
gates in the size range of 0.25–0.50 mm (P < 0.05) and in the size
range of 0.50–1 mm (P < 0.001), 1–2 mm (P < 0.01) and >4.75 mm
(P < 0.001) (Table 3). The distribution of aggregates >4.75 mm
was significantly different between the GF and the rest of SF
stands (P < 0.05).
3.2. SOC concentration (ground soil)
The highest SOC concentration was measured under the 60SF
and the lowest in the GF (P < 0.001; Fig. 1). The SOC concen-
tration decreased significantly with increase in depth in all
forest stands and ranged from 59.7 to 94.1 g kg−1 in the 0–10 cm
layer up to 31.0–45.5 g kg−1 in the 40–50 cm layer under the
GF and the 60SF, respectively (Fig. 1). Significant differences
in SOC concentration were observed for all the main factors
and their interaction (ANOVA, P < 0.001). The SOC concentra-
tion was not significantly different among the aggregate size
fractions collected after dry sieving in each SF stand (Fig. 1).
3.3. SOC concentration in particle-size fractions (wet
sieving)
The SOC concentration within particle-size fractions of wet-
sieved aggregates <250 ␮m decreased with increasing soil
depth (Fig. 2). The highest SOC concentrations were observed
in the fine silt + clay fraction (<20 ␮m) in all SF evaluated,
whereas the coarse-sand (105–200 ␮m) and fine-sand fractions
(53–105 ␮m) contained less SOC concentration. In the GF the
SOC concentration was higher in the fine silt + clay fraction
than in the rest of particle-size fractions, probably due to an
effect of fine silt + clay enrichment from alluvial depositions,
although differences were not statistically significant.
Compared to the average values of SOC obtained by grind-
ing the soil, the <20 ␮m fraction of <250 ␮m aggregates yielded
the highest percentage (30–35% of C), whereas the coarse-
sand fraction contributed 17–20% of the SOC concentrations
(Table 4). The SOC concentration comprises both the light
fraction and the particulate organic matter (POM), since no
Size class aggregates
0.50–1.00 1.00–2.00 2.00–4.75 >4.75
* *** **
3.844 7.261 4.636 1.268 NS 6.177***
0.364 NS 0.210 NS 0.716 NS 0.557 NS 0.535 NS
0.438 NS 1.033 NS 0.349 NS 0.243 NS 0.793 NS
14.57 17.01 106.65 66.09
17.53 17.43 93.08 75.45
 e c o l o g i c a l e n g i n e e r i n g 3 4 ( 2 0 0 8 ) 300–310 305

Fig. 1 – The SOC concentration (g kg−1 ) in the different aggregate size fractions (ground soil) in four forest systems. Different
letters indicate significant differences for average SOC concentration (ANOVA, P < 0.001); for each soil layer differences
between size class aggregates within the same SF stand were not significant (HSD Tukey test, P < 0.05).

chemical treatment or flotation technique was employed to concentration between the 25SF and the 60SF for 20–30 cm
remove the organic debris. depth.
Regarding the C:N ratio, significant differences were
3.4. N concentrations and C:N ratio observed for the fixed main factors and also for the interaction
(ANOVA, P < 0.001). The C:N ratio ranged between 10 and 11 in
Total N concentration (%) decreased significantly with increase all secondary forest stands (Table 5). Within particle-size frac-
in depth in all SF stands (Table 5). Significant differences tions, the highest C:N ratio was measured in the fine silt + clay
were observed in the mean N concentration among forest fraction, i.e., 7.5 in the first soil layer and increased with depth
stands (ANOVA, P < 0.001), depth (ANOVA, P < 0.001) and the (data not shown).
interaction (ANOVA, P < 0.001). Except in the GF and the CH-
SF, total N concentration differed significantly among forest
3.5. Litter and SOC pool
stands for 0–10, 10–20 and 30–40 cm soil depths (HSD Tukey
test, P < 0.05). There were no significant differences in N
The highest litter accumulation was observed in the 15SF
(Table 6) with 1.6 ± 0.2 kg m−2 and decreased in the order
60SF (1.08 ± 0.05 kg m−2 ), 25SF (1.0 ± 0.08 kg m−2 ), and the GF
Table 4 – Percentage of C in particle-size class aggregates (0.41 ± 0.01 kg m−2 ). The amount of litter collected (expressed
<250 ␮m after fractionation of the mineral soil (average in Mg C ha−1 ) in the four forest stands was significantly differ-
of all depths) ent (t-test, P < 0.05).
Secondary forest Particle-size class fraction (␮m) The highest SOC pool was measured under the 60SF and the
<20 20–53 53–105 105–200 lowest in the GF, similar to that obtained in the 25SF (Table 6).
Gallery SF 34.7 23.2 23.8 18.3 Differences were significant at P < 0.05 (t-test). The SOC pool
15SF 29.2 26.0 24.4 20.4 was not evenly distributed through the soil profile. In all SF
25SF 30.5 25.7 23.8 19.9 stands 50% of the total SOC pool (50 cm depth) was in the top
60SF 34.0 27.1 21.6 17.2 20 cm (Table 6).
306 e c o l o g i c a l e n g i n e e r i n g 3 4 ( 2 0 0 8 ) 300–310

Fig. 2 – The distribution of SOC concentration (g kg−1 ) with soil depth in the particle-size class fractions <200 ␮m collected
from wet sieving in the four SF stands.

3.6. Multivariate analysis
The first two axes of the between-class PCA explained 84.0%
of the total variability (Fig. 3a). Axis I explained 66.1% of vari-
ability and discriminated soil samples with high values of C in
all fractions, although to a lesser extent the <20 ␮m fraction,
and the distribution of aggregates <0.5 mm and percentage
of sand were high in opposition to those variables related
to physical soil properties, i.e., MWD, aggregates >4.75 mm
and bulk density. This axis showed differences in soil char-
acteristics among the four forest stands. The second axis
retained from the between-class PCA explained 17.9% of varia-
tion. It clearly separated percentage of clay, 1–2 mm aggregates
and C:N ratio from pH. Axis II was interpreted as the man-
agement effect of the SF stands. The projection of objects
(ecosystems) onto the factorial plane defined by the first two
axes (Fig. 3c) was significant (P = 0.0026; Montecarlo permu-
tation test). The GF was characterized by a high proportion

Table 5 – Average N concentration (%) and C:N ratio in the four SF studied
Depth (cm) GF 15SF 25SF 60SF

N (%) C:N N (%) C:N N (%) C:N N (%) C:N

0–10 0.64 a, A 9.8 a, A 0.65 a, A 10.4 b, B 0.74 a, B 10.4 c, B 0.95 a, C 9.9 c, A

10–20 0.52 b, A 9.1 c, A 0.51 b, A 9.9 c, B 0.59 b, B 9.6 e, C 0.68 b, C 9.7 d, C
20–30 0.40 c, A 9.3 bc, A 0.43 c, B 10.4 b, B 0.54 c, C 9.9 d, C 0.52 c, C 10.5 b, B
30–40 0.34 d, A 9.8 a, A 0.36 d, A 11.2 a, B 0.42 d, B 11.1 b, BC 0.46 d, C 10.9 a, C
40–50 0.32 d, A 9.9 a, A 0.35 d, B 11.3 a, B 0.38 e, C 11.5 a, C 0.41 e, D 10.9 a, D

Values followed by the same letter within a column or a row are not statistically different at P < 0.05 (HSD Tukey test). Lower letters indicate
significant differences between soil depth within the same site, and capital letters between sites within the same soil layer.
 e c o l o g i c a l e n g i n e e r i n g 3 4 ( 2 0 0 8 ) 300–310 307

Table 6 – The SOC pool and litter (Mg C ha−1 ) in the four
SF stands
SOC pool (Mg C ha−1 )

GF 15SF 25SF 60SF

Depth (cm)
Litter (Mg C ha−1 ) 2.1 8.0 5.0 5.4
0–10 44.7 a 54.3 a 55.5 a 59.6 a
10–20 44.9 a 45.3 b 39.5 b 46.1 b
20–30 33.3 b 39.2 c 35.5 c 41.8 b
30–40 30.4 b 39.0 c 32.8 d 38.5 c
40–50 30.2 b 34.3 d 32.6 d 35.4 c

Total in soil 183.5 a 212.1 ab 195.9 a 221.4 b

Fig. 4 – Within-class PCA showing (a) ordination of soil

variables in the factorial plane, (b) “eigenvalues” diagram
and (c) ordination of soil samples with all centroids (groups
or ecosystems) at the origin of the factorial plane;
ordination of samples by depth (i) and by soil texture (ii).

of >4.75 mm aggregates and MWD, and the 60SF by high SOC

concentration.
Regarding the within-class PCA, the first and second axes of
the within-class PCA (Fig. 4a) accounted for 81.4% of the iner-
tia. Axis I (66.1% of variability explained) showed an opposition
between those soil samples with a large proportion of small
aggregates, high bulk density and pH in opposition to those
samples with high C concentrations in all fractions and MWD.
Axis II (15.3%) separated samples with high percentage of sand
in contrast to those with high C:N ratio and percentage of clay
and silt. The projection of SF stands onto the factorial plane
defined by both axes in the within-class PCA is given in Fig. 4a.
The ecosystem effect is removed in the within-class PCA by
placing all centers of gravity in the axes origin of the facto-
rial maps, while the sampling units (soil from each land use
at different depth) are scattered, with the maximal variance
around the origin (Fig. 4c, i and ii).

Fig. 3 – Results of the between-class principal component

analysis (PCA) showing (a) ordination of variables in the
factorial plane, (b) “eigenvalues” diagram and (c) projection
of the soil samples onto the factorial plane defined by axes
I and II. The circles represent the centroids of each cloud of
points for each ecosystem. Variables are defined as follows:
Bd = bulk density; MWD = mean weight diameter of
aggregates; Ccosa = C in coarse-sand fraction; Cfisa = C in
fine-sand fraction; Ccosi = C in coarse silt fraction; Cs + c = C
in the fine silt and clay fraction.
4. Discussion and conclusions
4.1. SOC pool and comparison with other studies
The SOC pool (0–30 cm) under forest vegetation in humid
northeastern Costa Rica was estimated at 87.1 and
80.5 Mg ha−1 (Powers, 2004; Powers and Veldkamp, 2005).
In the southern area of Costa Rica, Krishnaswamy and Richter
(2002) reported a SOC pool under forest of 79.5 Mg C ha−1 in the
308 e c o l o g i c a l e n g i n e e r i n g 3 4 ( 2 0 0 8 ) 300–310
top 30 cm. These values are much lower than those reported in
the present study, i.e., from 130.5 to 147.5 Mg C ha−1 (Table 6).
In Costa Rica, the SOC pool in SF of 10–15 years old was
21 Mg C ha−1 compared to 16 Mg C ha−1 in the 0–10 cm depth
under pastures (Reiners et al., 1994). In our study, the SOC
pool was higher and increased with the age of the SF, from
56.1 to 60.1 Mg C ha−1 (0–10 cm) in the 15SF and 60SF, respec-
tively (Table 5). The SOC pool in the 15SF was higher (Table 5)
than that reported by Russell et al. (2004) in soils from La Selva
Biological station with a maximum of 40.5 Mg C ha−1 . The SOC
pool in a T. cacao and Erythrina poeppigiana shade tree system
ranged from 115 to 140 Mg C ha−1 (0–45 cm) over a 9-year period
(Fassbender, 1998). In our study the SOC pool in the 60SF (old
T. cacao plantation) has likely increased prior to the abandon-
ment of this land use.
In other tropical areas as in the Brazilian Amazon basin,
the SOC pool (0–30 cm depth) in agricultural land of less than
10 years old was reported at 27.4–62.0 and 36.8–39.1 Mg C ha−1
by Neill et al. (1997) and Fearnside and Barbosa (1998), respec-
tively. These values are lower than that obtained in our study
(Table 6). In Puerto Rico, the SOC pool (0–50 cm) under forest
and reforested sites derived from pastures was estimated at
99.0 and 95.2 Mg C ha−1 , respectively (Silver et al., 2004). In Los
Tuxtlas, Mexico, the total SOC pool (1 m depth) reported in
SF from 6 months to 50 years old remained stable along the
succession and averaged 207 Mg C ha−1 (Hughes et al., 1999).
In Central Amazonia, the total SOC pool (0–10 cm) in SF was
estimated at 43.3 Mg C ha−1 (Hughes et al., 2000). Rhoades et
al. (2000) observed SOC pool increased by 1.9 Mg ha−1 year−1 ,
and the total SOC pool attained the antecedent level within
20 years that under TSF of lower montane Ecuador (South
America). The review of literature revealed that reforestation
of abandoned agricultural land, mainly pastures, results in a
slow increase of the SOC pool.
Regarding the gallery forests in our study, the SOC pool
in the GF was higher, i.e., 183.5 Mg C ha−1 , than that reported
in other tropical sites. For example, in a study conducted by
Veneklass (pers. commun.) in the eastern plains of Colombia,
the average SOC down to 1 m depth in the Yucao watershed
was estimated at 133 Mg C ha−1 . In the dry tropical forest
of Guanacaste (Costa Rica), the SOC under a gallery forest
has been reported at 66.1 Mg C ha−1 (Jiménez et al., 2008, this
issue).
4.2. Determinants of SOC accumulation in SF
Forest type, soil texture, litter production, root inputs, the
time elapsed since the perturbation and intensity of the pre-
vious land use are factors responsible for variability in soil’s
response to perturbation (Allen, 1985; Brown and Lugo, 1990;
Neill et al., 1997; Russell et al., 2004). In our study, plant diver-
sity, i.e., understory vegetation and trees, which is highest
in the 60SF (Russo, unpubl. results), and litter production are
important factors affecting the SOC concentration. Data from
temperate agroforestry systems have shown that the SOC pool
was not increased from the tree row compared with the center
after 13 years of alley cropping, although a positive trend was
observed (Oelbermann and Voroney, 2007).
Several studies have revealed that soil type and clay con-
tent are strong determinants of the SOC concentration and
pool. Desjardins et al. (2004) observed higher SOC concen-
trations in clayey Ferralsols (more than 80% of kaolinite)
in Central Amazonia (ca. 6.0 kg C m−2 ) than in sandy–clayey
Acrisols from Eastern Amazonia (ca. 3.0 kg C m−2 ). Several
authors reported similar values of SOC pool for clayey Oxisols
and sandy Ultisols in the Amazonia (Choné et al., 1991;
Desjardins et al., 1994; Koutika et al., 1997; Neill et al., 1997).
The SOC pool (0–20 cm) estimated in volcanic soils of Mar-
tinique was 80.3 and 109.3 Mg C ha−1 in soils with 27 and 36%
silt + clay fraction, and 69.7 to 82.9 Mg C ha−1 in soils with ca.
65% of silt + clay (Feller et al., 2001). In our study there is an
inverse relationship, i.e., while clay content increases, the
SOC concentration decreases with soil depth (Fig. 4a). Textural
analysis also showed that the percentage of sand in the soils
studied was higher than 80%. In other words, even if the high-
est SOC concentrations were associated with the fine silt + clay
fractions in the present study, the SOC in the sand-size frac-
tion play an important role in the area and merits further
research.
Higher fine root biomass has been observed in SFs com-
pared with tree plantations of similar age (Cuevas et al.,
1991), and sometimes similar to old-growth forests (Cavelier
et al., 1996). Plant diversity provides different qualities of OM
that may increase the SOC pool. Studies on signatures of
OM in order to identify their origin with near infrared spec-
troscopy (NIRS) (Joffre et al., 2001) can be relevant to address
these questions and elucidate the contribution of plant lit-
ter and roots to the SOC pool. Further studies are needed
to assess litter and fine roots contribution to the total SOC
pool in addition to the contribution of black carbon or char-
coal. The study area was previously occupied by humans
before the arrival of Europeans and evidence of fire use
exists.
4.3. SOC concentration and aggregate size distribution
The distribution of size class aggregates is an indicator of soil
quality, i.e., soil structure. In general, the topsoil was more
aggregated than the deeper layers. Aggregates larger than
2 mm were more abundant in the topsoil than in the deeper
soil layers and thus average MWD decreased with increase in
depth (Table 2). It seems that aggregate distribution is affected
by forest regeneration since there was an increase in the pro-
portion of aggregates <0.250 mm with increasing age of the SF,
whereas the opposite was observed for aggregates >2 mm. The
consequence of this pattern is that an important proportion of
SOC was occluded within large aggregates in SFs of less than
20 years old, while aggregates of <1 mm size are important for
SOC accumulation in the more aged SF. A higher aggregation in
the topsoil can be the result of higher root density and soil bio-
logical activity that has been favored by the time elapsed since
the recovery of the SF stands. In fact, diplopods and earth-
worms were normally observed in high numbers in the older
SFs contributing to aggregate stability in this ecosystem.
Carbon in the soil can be stabilized within aggregates (Six
et al., 1998). In the present study the pattern observed in
SOC concentration indicated uniformity between size class
aggregates (dry sieving) and a slight increase in aggregates
<250 ␮m (Fig. 1). Therefore, in the soils studied under different
SF stands, the increase of SOC concentration with decrease in
 e c o l o g i c a l e n g i n e e r i n g 3 4 ( 2 0 0 8 ) 300–310
particle-size fraction is an indication of relative C enrichment
in the <250 ␮m size aggregates. These results are in accordance
with those reported by Lehmann et al. (1998) and Neufeldt
et al. (1999). Conversely, data provided by Angers and Giroux
(1996) indicated that C was uniformly distributed regardless of
the separation of primary particles. On the contrary, increases
in SOC concentration with increase in aggregate size have
been reported by several authors (Gijsman, 1996; Wigginton
et al., 2000; Conant et al., 2004). In our study, the fact that
fine silt + clay fraction (<20 ␮m) in the <250 ␮m aggregates con-
tained the highest concentration of SOC (Fig. 2) revealed that
this C is physically protected and occluded within larger aggre-
gates. The SOC concentration in this fraction was much higher
compared to values obtained by grinding the soil for all depths
(Fig. 1).
In the GF, a higher enrichment in SOC concentration was
observed in the fine silt + clay fraction than in the rest of
particle-size fractions, although no significant differences
were observed. The fact that the soil is of alluvial nature could
have influenced deposition of clay and sand within the soil
profile, and thus affect SOC accumulation in particle-size frac-
tions.
Finally, no estimates of SOC sequestration are provided in
this study due to differences regarding land use history and
intensity of previous land use in the 15SF, 25SF and 60SF stands
and because they do not constitute a true chrono-sequence.
However, a clear trend was observed, the older the TSF the
higher the SOC pool (Table 6). It is possible that C accumu-
lation has been favored by the well aggregated soil structure
observed in the older SF stands. This confirmed that the age
of recovery influenced the distribution of size class aggregates
and SOC pool. Although reforested areas may differ in plant
community composition from mature forests (Lugo, 1992),
they constitute an important ecosystem for soil C sequestra-
tion, especially when the total area of SF currently exceeds the
area of primary forest (FAO, 2005).
Acknowledgements
This work was funded by the US Department of Energy. We are
grateful to Juan Orlando for his assistance in the field, and to
C. Hernández and H. Arrieta for kindly permitting the use of
lab facilities at EARTH during field work, and Y. Raut for his
help at OSU lab. The first author thanks “Fundación ARAID”
(Spain) for financial support during manuscript preparation.
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