68
Ciencias Agrarias/ Agricultural Sciences
Revista Ciencia y Tecnología (2025) 18(2) p 68 - 77 ISSN 1390-4051; e-ISSN 1390-4043 https://doi.org/10.18779/cyt.v18i2.846
Soil aggregate formation and carbon storage by endogeic earthworms in an Ultisol
Formación de agregados del suelo y almacenamiento de carbono por lombrices de tierra endogéicas en un Ultisol
Mauricio Renato Morejón Centeno
1
, Rocio Natividad Morejón Lucio
2
, José Nolberto Macías Veliz
3
1
University of Puerto Rico-Mayaguez; Universidad Técnica Estatal de Quevedo, Ecuador.
2
Universidad Técnica de Manabí; Universidad Técnica Estatal de Quevedo, Ecuador
3
Universidad Técnica Estatal de Quevedo, Ecuador
Autor de correspondencia: rocionluc.morejon@uteq.edu.ec
Recibido: 13/03/2024. Aceptado: 13/03/2025.
Publicado el 02
de julio de 2025.
Abstract
T
he role of soil micro and macro-organism in the
incorporation of carbon to soil aggregates of tropical soils
has been understudied. To test soil aggregate formation and
carbon incorporation by earthworm activity, we conducted a
eld experiment within a secondary forest and a microcosm
experiment at the University of Puerto Rico in Mayagüez. We
used 13C natural abundance in vegetation and the dierence
in δ13C between C3 and C4 plants to track carbon sources in
the soil. Maize leaves were utilized to trace the incorporation
of C4 carbon within soil aggregates, allowing for a clear
distinction between this carbon source and the C3 carbon
derived from forest vegetation. Earthworms and soil samples
(Typic Haplohumults) were collected at 0-10 cm soil depth.
Aggregates size classes were separated by the wet sieving
method. In the study site, two earthworm species were found
belonging to epigeic and endogeic ecological categories. In
a period of 6 months, our eld data suggests that endogeic
P. corethrurus can reorganize small macroaggregates to form
large macroaggregates. Our results suggest that P. corethrurus
shows a preference for consuming soil-derived carbon and
can translocate it from microaggregates to macroaggregates
by restructuring soil aggregates.
Keywords: Bioturbation, soil organic carbon, organic matter,
isotope, tropical soils.
Resumen
E
l rol de los micro y macroorganismos del suelo en la
incorporación de carbono a los agregados del suelo ha
sido poco estudiado en los suelos tropicales. Para evaluar
la formación de agregados del suelo y la incorporación de
carbono mediante la actividad de las lombrices de tierra,
realizamos un experimento de campo en un bosque secundario
en la Universidad de Puerto Rico en Mayagüez. Utilizamos la
abundancia natural de C13 en la vegetación y la diferencia del
isótopo de δ13C entre plantas C3 y C4 para rastrear las fuentes
de carbono dentro de los agregados del suelo. Se utilizaron
hojas de maíz para rastrear la incorporación de carbono C4
en los agregados del suelo, permitiendo una clara distinción
entre esta fuente de carbono y el carbono C3 derivado de la
vegetación del bosque. Se colectaron muestras de lombrices
de tierra y suelo (Typic Haplohumults) a una profundidad de
0-10 cm. Las clases de tamaño de los agregados se separaron
por el método de tamizado húmedo. En el sitio de estudio se
encontraron dos especies de lombrices de tierra pertenecientes
a las categorías ecológicas epigéicas y endogéicas. En un
período de 6 meses, nuestros resultados sugieren que la
especie endogéica P. corethrurus puede reorganizar pequeños
macroagregados para formar grandes macroagregados.
Nuestros resultados sugieren que P. corethrurus muestra una
preferencia por consumir carbono derivado del suelo y puede
translocarlo desde los microagregados a los macroagregados
mediante la reestructuración de los agregados del suelo.
Palabras clave: Bioturbación, carbono orgánico del suelo,
materia orgánica, isotopo, suelos tropicales.
Soil aggregate formation and carbon storage by endogeic earthworms in an Ultisol
2025. 18(2): 68-77 Ciencia y Tecnología. 69
Introduction
Earthworms are among the most abundant and important “soil
engineers” that impact the physical, chemical, and biological
characteristics of the humid tropical ecosystems (Blanchart
et al., 2004; González et al., 2007; Lavelle et al., 1992).
Earthworms are known to aect the uxes of organic matter
(OM) decomposition, soil organic carbon (SOC), and direct
incorporation of SOC into soil, thereby altering soil structure
and fertility (Aira et al., 2008; Blanchart et al., 1997; Fonte
et al., 2009; Le Couteulx et al., 2015; Pulleman et al., 2005;
Sánchez-de León et al., 2018; Whalen and Janzen, 2002).
Through their feeding and casting activities, earthworms
help to remove part of the plant litter from the soil surface
and inuence the SOM incorporation into soil aggregates
(Bossuyt et al., 2005) by ingest particulate organic matter
(POM) and mineral soil. This mixing of organic material with
mineral particles results in the formation of casts (Bossuyt et
al., 2004, 2005; Sánchez-de León et al., 2014). Earthworm-
formed aggregates provide physical protection to organic
carbon (OC) against rapid mineralization by microorganisms,
thus contributing to long-term carbon storage in soils (Bossuyt
et al., 2005; Pulleman et al., 2005; Sánchez-de León et al.,
2014; Six et al., 2002).
Although several studies show that earthworms species
can create new soil macroaggregates (Blanchart et al., 1997,
Bossuyt et al., 2004, 2005; Sánchez-de León et al., 2014),
it has also been shown that certain earthworm species can
fragment existing soil aggregates like small Eudrilidae
(Blanchart et al., 1997, 2004; Kamau et al., 2020) . Thus, the
eect of earthworms in soil aggregation seems to be species
dependent.
Soil aggregates formed by earthworm feeding and casting
activities can result in the physical storage of soil carbon
(Fonte et al., 2007; Sánchez-de León et al., 2014). Bossuyt et
al. (2004) found that soil aggregates formed by earthworm’s
activity contained 3,6 times larger macroaggregates (larger
than 2 000 μm) and more total carbon (4,26 g C kg-1 soil) than
treatment without earthworms. Similar results were reported
by Sánchez-de León et al. (2014) in an experiment where
the macroaggregate mass in treatments with earthworms
was two to three times greater than treatments without
earthworms. In addition, Bossuyt et al. (2004, 2006) found
that earthworm activity can increase the formation of water-
stable microaggregates inside large macroaggregates four-
fold compared to treatments without earthworms.
Despite these ndings, the role of earthworm aggregate
formation and their inuence on carbon storage in tropical
soils of Puerto Rico (i.e., Ultisols and Oxisols) has been little
studied. Ultisols and Oxisols are highly meteorized soils
characterized by low activity clays (1:1 clay minerals), and
low fertility (e.g., Soil Survey Sta, 2014). M
any
studies
related to earthworms in Puerto Rico have been focused on
feeding behavior, density, distribution, diversity, and their
inuence on soil physical properties (Amador et al., 2013;
Dechaine et al., 2005; González et al., 2007; González and
Zou, 1999; Hubers et al., 2003; Liu and Zou, 2002; Sánchez-
de León et al., 2003). However, studies using stable isotopes
to understand the aggregate formation, and incorporation of
carbon into aggregates by earthworms are few (Amador et al.,
2013; Hendrix et al., 1999; Lachnicht et al., 2002).
The aim of the experiment was to study the impact of
earthworms on an Ultisol found from the secondary forest
at Finca Alzamora in Mayaguez, Puerto Rico, focusing on
soil aggregates formation and its relationship with carbon
incorporation. Our objective was to measure the earthworm
abundance and aggregate distribution under natural eld
conditions in an Ultisol within a secondary forest vegetation
(C3-vegetation). In addition, we took advantage of the natural
13
C isotopic dierences between C3 and C4 plants (O’Leary
1981) to measure the incorporation of maize leaf-derived
carbon within aggregates under natural eld conditions.
Although, we did not expect that aggregate distribution would
be aected by C4 leaf litter addition, we hypothesized that
carbon added through the C4 leaf litter (maize) would be
readily incorporated into soil aggregates, and the
13
C isotopic
dierence will allow the tracking of this newly incorporated
carbon.
Materials and methods
The eld study was conducted within the secondary forest at
the Alzamora Farm, located in the University of Puerto Rico
in Mayagüez (near 18° 13’12,5” N, 67°08’49,0’’ W). The soil
was Consumo (clay ne, mixed, semiactive, isohyperthermic
Typic Haplohumults) (Soil Survey Sta, 2014). The area
has a tropical climate with a mean annual precipitation of
1.020 to 1.780 mm and mean temperature of 26 °C (Beinroth
et al., 2002; Harmsen et al., 2002; Ravalo et al., 1986).
The forest vegetation in the area in the past 45 years has
been dominated by C3 plant species such: Albizia procera,
Swietenia mahagoni, Castilla elastica, Tilipariti elatum,
Mangifera indica, Guarea guidonia, Ceiba petandra, Inga
fagifolia, Delonix regia, Peltophorum inerme, and Leucaena
leucocephala (Túa-Ayala, 2023). The eld experiment was
conducted from June 2017 through December 2017.
In September 2017, the island of Puerto Rico experienced
the eects of hurricanes Irma and Maria. The experimental
plots were checked on September 11, 2018, after the Irma
hurricane and on September 29, 2018 after the Maria hurricane.
In both cases, the experimental plots did not show addition or
loss of soil by erosion. Leaf litter and other vegetation debris
(e.g., mostly tree trunks) on top of plots were removed to
continue the eld experiment.
Morejón et al., 2025
2025. 18(2):68-77
Ciencia y Tecnología.70
Table 1. Latitude, longitude, aspect and gradient of experimental plots of Consumo soil series at Finca Alzamora
secondary forest
Plot Latitude Longitude Aspect (º) Slope (%)
Bulk density (g cm
‐3
)
1 18° 13’ 4,89’’ N 67° 08’ 38,46’’ W 227 1 1,02
2 18° 13’ 14,89’’ N 67° 08’ 37,55’’ W 295 1 1,09
3 18° 13’ 14,31’’ N 67° 08’ 38,42’’ W 320 2 1,21
4 18° 13’ 15,40’’ N 67’ 08’ 36,34’’ W 298 18 1,13
Plant material and eld application
Leaves from maize plants were collected from an organic
orchard located at the Alzamora Farm. leaves from the forest
oor (C3-leaves) at the study site were collected as other
treatment. All leaves were cut and dried in paper bags for
72 hours at 65 °C in an oven, followed by grinding to < 2
mm. For selecting the experimental plots, we used an aerial
photography of the study site, 16 sections of 20 × 20 m were
digitally delineated with ArcMap v.10.5 (Environmental
System Research Institute, Redlands, CA, USA) and of
these, four sections were randomly selected. In each of these
selected areas of the forest, one experimental plot of 1 × 1
m size was delimited using barrier landscape fabric without
altering the trees. Each plot was a replicate of the experiment.
Geographical information of the plots is included in Table 1.
The experimental plots were split in half (sub-plot of 0,5 × 1
m), with each half corresponding to one randomly assigned
treatment. The two treatments were: control forest oor leaves
(C3-leaves) and maize leaves (C4-leaves) with four repetitions
(n=4). On 5 June 2017, we applied 400 g m
-2
of maize leaves
(2,04 g C kg
-1
of soil) on each of the C4-leaves treatment
replicates and 400 g m
-2
of forest leaves (2,71 g C kg
-1
of soil)
on each of the C3-leaves treatment replicates.
Earthworm sampling
During November 2 017; six months after treatment addition;
earthworms and cocoons were collected from a soil area of
25 × 25 cm to a depth of 10 cm by hand sorting and gently
breaking the soil. Earthworm samples were placed in plastic
bags with a moistened paper towel and transported in a cooler
to the Soil Chemistry Laboratory at the University of Puerto
Rico, Mayagüez, during the same day of collection.
Earthworms from each sub-plot were measured by their
abundance (individuals m
-2
), and fresh weight (grams of
fresh weight m
-2
). Earthworms were placed in petri dishes
with ber glass lter paper for 72 hours to let earthworms
empty their gut content (Schmidt, 1999; Whalen and Janzen,
2002). A sub-sample of adult earthworms was selected to be
preserved in 1:10 dilution of formaldehyde 37% for taxonomic
identication. Taxonomic identication was performed by
earthworm taxonomist Dr. Sonia Borges using (Borges, 1996)
taxonomic key.
Water stable aggregates
A soil core sampler (AMS Inc., USA) with 4,5 cm diameter
was used to collect the soil sample at a depth of 10 cm from
soil surface from each sub-plot. Soil samples were collected
between November 27 to December 15, 2017. Each soil
core was broken along its natural breaking points (Fonte et
al., 2009; Jastrow et al., 2005). The soil was gently passed
through an 8-mm sieve and roots were removed (Jastrow et
al., 1996, 2005; Six et al., 1998). Afterwards, soil samples
were air dried at room temperature. The soil samples were
fractionated in three 50 g soil sub-samples to be separated into
four aggregate size fractions by wet sieving method (Bossuyt
et al., 2005; Elliott, 1986; Sánchez-de León et al., 2014). For
this experiment, 12 sub-samples were analyzed per treatment,
for a total of 24 sifted sub-samples.
Three sieves were used to obtain four size fractions: 1)
large than 2.000 µm (large macroaggregates); 2) 250-2.000 µm
(small macroaggregates); 3) 53-250 µm (microaggregates);
and 4) less than 53 µm (silt+clay fraction). Aggregates
were manually sieved by vertical oscillation, moving the
sieve up and down 50 times by 3 cm, during a two-minute
period. Particles belonging to less than 53 µm (silt+clay
fractions) were released in the rinse water and collected via
centrifugation by 10 minutes at 4.500 rpm and 20 °C.
All fractions were placed into an oven to dry at 65 °C,
by 24 hours, and then weighed. Soil aggregate fraction was
corrected and expressed as sand-free aggregate fraction as
shown in Equation 1 (Elliott, 1986; Sánchez-de León et al.,
2014; Six et al., 2000). To express carbon concentration in
aggregates as a sand free basis, we used Equation 2 (Six et
al., 1998).
=
(
+
)
× 1
(
)
(1)
()
=
( )
1 ( )
(2)
Soil aggregate formation and carbon storage by endogeic earthworms in an Ultisol
2025. 18(2): 68-77 Ciencia y Tecnología. 71
Where C represents the carbon concentration within each
aggregate fraction. Sand content of macroaggregates and
microaggregates sizes were determined using the modied
method of particle size analysis described by Jackson (2005).
Isotope analysis
Samples of soil aggregates, maize leaves and leaf litter were
analyzed for δ
13
C, with an isotope ratio mass spectrometer
(Finnegan Delta Plus XL, Bremen, Germany) coupled with an
elemental analyzer (Costech Elemental Analyzer, California,
USA). The isotope analysis was performed at the University
of Illinois at Chicago, at the Geochemistry Stable Isotope
Laboratory. The result was expressed in delta notation using
part per thousand (δ ‰) as follows in Equation 3.
C =
R
sample
R
standard
1313
R
standard
13
13
× 1000
(3)
Where
13
R
sample
represents the ratio
13
C/
12
C in the sample
and
13
R
standard
is the ratio of the two isotopes in the standard
PDB (belemnite from Pee Dee Formation) (Bossuyt et al.,
2004; O’Leary, 1981). Equipment also provided data for
percent of soil carbon (%C = g
C
/100 g
soil
and %N=g
N
/100 g
soil
).
To determine the amount of the soil carbon and labeled maize
carbon that was incorporated into each soil aggregate size
class, we used the mixing model ISOERROR 1.04 spreadsheet
(Phillips and Gregg, 2001).
Data analysis
The experimental plots were established in the eld using a 2×4
factorial design, incorporating two types of leaf inputs (C4-
leaves and C3-leaves) and four aggregate size classes (large
macroaggregates, small macroaggregates, microaggregates
and silt+clay). We implemented a posteriori contrast analysis
to evaluate the eects of soil-derived carbon and leaf-derived
carbon on the aggregate size classes.The design includes
ve specic contrasts, each designed to evaluate particular
hypothesis or comparison among the treatments group. The
results were subjected to statistical analysis using a two-way
ANOVA. The relationship between earthworm abundance and
across dierent aggregate size classes was analyzed using a
linear regression model PROC REC analysis. Normality and
homogeneity of variances were tested using Shapiro-Wilks
and Levene’s tests. Transformation attempts of non-normal
data were unsuccessful for the mass of size fraction and carbon
concentration. Therefore, we used the PROC GLIMMIX of
SAS University Edition version 9.4 (SAS Institute Inc., 2015)
for analysis of variance. Separation of means were tested using
Tukey’s honestly signicant dierence at a level of p <0,05.
Results and discussion
In the study site, we found two exotic earthworm species:
Pontoscolex corethrurus (Müller, 1856) from South America
with endogeic behavior and Amynthas hawayanus (Rosa,
1981) from Asia with epigeic behavior. The most common
species was Pontoscolex sp. with 15 immature earthworms
compared to A. hawayanus with 2 adult earthworms of the
total earthworms taxonomically classied. Overall, the
earthworm abundance ranged from 64 individuals m
-2
to
336 individuals m
-2
and the biomass was from 9,46 to 53,1
grams of fresh weight m
-2
. The mean earthworm abundance in
treatments with C3-leaves was 200 (±62,82) individuals m
-2
and in treatment with C4-leaves was 192 (±50,6) individuals
m
-2
. The mean biomass in plots with C3 leaves was 23,59
(±5,96) grams of fresh weight m
-2
and in plots with C4-leaves
was 32,63 (±8,53) grams of fresh weight m
-2
.
As expected, there was no signicant dierence for the
factor leaves additions (Table 2). For the aggregate size class
factor, the proportion of aggregate mass was signicantly
higher for small macroaggregates (250-2.000 µm) than large
macroaggregates (> 2.000 µm), microaggregates (53-250 µm)
and silt+clay (< 53 µm) (Figura. 1, Table 2). Additionally, the
soil aggregates showed signicantly higher values of δ
13
C for
C4-leaves [-26,95 (±0,16) ‰] than C3-leaves [-27,58 (±0,16)
‰] (Table 2).
Figure 1. Aggregate size distribution in the eld experiment
in the Consumo soil. Letters with dierent lowercase are
signicantly dierent among aggregate size classes within
each treatment. Means (n=4) are signicantly dierent as
determined with Tukey’s Least Signicance Dierence test
(two-way ANOVA; p<0,05). Bars represent standard error
The distinct isotopic signal of the leaf litter enabled
the detection of carbon derived from the leaf litter that was
incorporated in the soil aggregate fractions (Table 2). Soil-
derived carbon concentrations were not signicantly dierent
for the aggregate size classes, and their interaction as shown
in Table 2. In our study, we observed dierences in leaf-
derived carbon concentrations, particularly within the soil’s
large macroaggregate and microaggregate fractions (Tables 3
and 4). In terms of the amount of carbon in the aggregates,
the contribution of carbon from C3-leaves was higher [4,24
(
±0,70) g C kg
-1
sand-free aggregates
] than C4-leaves [0,32 (±0,70) g C
kg
-1
sand free aggregates
] (Table 4). From the aggregate sizes compared,
only the large macroaggregates+C3 were signicantly higher
than large macroaggregates+C4, and the microaggregates+C3
Morejón et al, 2025
2025. 18(2):68-77
72 Ciencia y Tecnología.
were signicantly higher than microaggregates+C4 (Table 4).
Table 2. Summary of ANOVA to evaluate the aggregate proportion, carbon concentration, and δ13C signal in the eld study sites. Variables tested were treatments (C4-
vs. C3 leaf litter), aggregate size classes (large macroaggregates, small aggregates, microaggregates and silt+clay), and the interaction Treatments × Aggregate size classes
Leaves (C4 vs. C3) Aggregate size classes Leaves (C4 vs. C3) × Aggregate size classes
numDF
†
denDF
‡
F-value P-value numDF denDF F-value P-value numDF denDF F-value P-value
Aggregate mass
proportion
1 24 0,00 0,97 3 24 16,37
<0,0001
3 24 0,30 0,82
Carbon
Concentration
1 18 2,18 0,16 2 18 0,73 0,50 2 18 1,01 0,38
δ
13
C signal 1 18 7,72 0,01* 2 18 2,33 0,13 2 18 0,10 0,90
* Signicant at the 0,05 probability level.
†
Degrees of freedom for numerator (numDF).
‡
Degrees of freedom for denominator (denDF).
Table 3. Statistics of two-way ANOVA for soil derived-carbon and leaf derived-carbon sources in samples of Consumo soil under eld conditions. Variables tested were
leaves (C4 vs. C3), aggregate size classes [large macroaggregates (LM), small aggregates (SM), and microaggregates(M)], and the interaction Treatments × Aggregate size
classes
Soil derived-carbon Leaf derived-carbon
numDF
†
denDF
‡
F-value P-value F-value P-value
Aggregate size classes 2 18 0,63 0,55 0,57 0,57
Leaves (C4 vs. C3) × Aggregate size classes 2 18 0,55 0,58 0,26 0,77
LM+C4 vs. LM+C3 1 18 N/A
#
N/A 7,80 0,01*
SM+C4 vs. SM+C3 1 18 N/A N/A 3,14 0,09
M+C4 vs. M+C3 1 18 N/A N/A 5,39 0,03*
(LM vs. SM vs. M)+C4 2 18 N/A N/A 0,17 0,84
(LM vs. SM vs. M)+C3 2 18 N/A N/A 0,67 0,53
* Signicant at the 0.05 probability level.
†
Degrees of freedom for numerator (numDF).
‡
Degrees of freedom for denominator (denDF).
#
Not applicable.
Soil aggregate formation and carbon storage by endogeic earthworms in an Ultisol
2025. 18(2): 68-77 Ciencia y Tecnología. 73
Table 4. Carbon concentrations (g C kg-1 sand-free
aggregates) incorporated from C4-and C3-leaves sources
in samples of Consumo soil under eld conditions. Letters
with dierent lowercase among aggregate size class
within each carbon sources, are signicantly dierent as
determined with Tukey’s Least Signicance Dierence test
(two-way ANOVA; p<0,05). Values are means (n=4) with
standard error in parentheses
C4-leaves
source
C3-leaves
source
--- g C kg
-1
sand-free aggregates ----
Aggregate size
class
Large
macroaggregates
0,00 a*
(1,21)
4,76 a
(1,21)
Small
macroaggregates
0,08 a
(1,21)
3,10 a
(1,21)
Microaggregates
0,89 a
(1,21)
4,85 a
(1,21)
*Indicate a signicant dierent between carbon sources
(C4- and C3-leaves) within each aggregate size class.
Or study uncovered a relationship between earthworm
abundance and macroaggregates, showing distinct patterns
based on the size of these aggregates (Figura 2). The
aggregate mass proportion demonstrated a positive quadratic
relationship between earthworm abundance with large
macroaggregates+C4-leaves (R2=0,95; P=0,001), as detailed
in Table 5 and illustrated in Figura 2A. The proportion of
large macroaggregates proportion peaked at an earthworm
abundance of 262 individuals m-2 (Figura 2A), beyond which
a slight decline was observed. While the relationship between
small macroaggregates+C4 leaves, and earthworm abundance
was negative (R2=0,80; P=0,001) as shown in Table 5 and
Figure 2B. For small macroaggregates+C4-leaves the lowest
proportion was observed at an earthworm abundance of 217
individuals m-2 (Figura 2B), after which there was a slight
increase.
Figure 2. Relationship between earthworm abundance
with mass proportion of site fraction. (A) large
macroaggregates+C4 and (B) small macroaggregates+C4
of Consumo soil. Each x-axis value corresponds to one
replicate for earthworm abundance and values in y-axis
correspond to three soil sub-samples per micro-plot
Table 5. Relationship between aggregate size classes with abundance and biomass of earthworms of Consumo soil
series in eld conditions
Aggregates
Earthworms abundance
(individuals m
-2
)
Earthworms biomass
(grams of fresh weight m
-2
)
R
2
F
P-value
R
2
F
P-value
Large macroaggregates+C4 0,95 21,31
0,001
§
0,68 7,29 0,02
Large macroaggregates+C3 0,91 5,61 0,04 0,34 0,1 0,76
Small macroaggregates+C4 0,80 11,64
0,01
§
0,68 11,45 0,01
Small macroaggregates+C3 0,95 53,8
<0,0001
§
0,67 0,13 0,72
Microaggregates+C4 0,98 35,57 0,0002 0,66 5,27 0,047
Microaggregates+C3 0,51 5,22 0,048 0,4 5,19 0,049
Morejón et al., 2025
2025. 18(2):68-77
Ciencia y Tecnología.74
Our results partially support the hypothesis that dierences
in isotopic composition between C4- and C3-leaves allow the
tracking and measure the carbon incorporation into aggregate
size classes under eld conditions. Six months after applying
the maize leaves, the change in δ
13
C signal was found only for
mean values between C4- and C3-leaves treatments. Only a
small carbon fraction from maize leaves-derived carbon (C4
leaves) was incorporated and stabilized into soil aggregates.
Most C4 leaves-derived carbon may have been incorporated
into silt + clay fraction or respired as CO
2
.
The lignin and aromatic compounds from plant debris
can be easily associated with silt and clay fractions or lost
from soil as respired CO
2
(Bossuyt et al., 2006; Oades and
Waters, 1991; Paul et al., 1999). While the C3 leaf litter-
derived carbon has been accumulated over a long time and
in higher amounts, showing a higher carbon concentration
than C4 leaf litter treatment. According to (Paul et al., 1999),
6% from total carbon can be lost in the form of CO
2
from
aboveground residues in conventional tillage treatments, 11%
in no-tillage treatments, 11% in low-chemical inputs, and
20% in zero-chemical inputs. In a study of mean residence
time conducted by Paul et al. (2008), in soils derived from
volcanic ash, the researchers could not detect the pasture-
derived carbon cultivated a few decades before that site
changed to a secondary forest. In another study conducted by
(Sánchez-de León et al., 2018) using free-air CO
2
enrichment
in a sweetgum (Liquidambar styraciua L.) plantation, the
researchers did not nd a change in the soil (Ultisol order)
carbon isotopic composition two years after CO
2
fumigation
ended.
We found that carbon incorporation follows the hierarchy
of soil aggregate formation (Oades and Waters, 1991; Six and
Paustian, 2014). This is because carbon from C4-leaf litter
source was incorporated rst into the microaggregates and
small macroaggregates, but not into large macroaggregates
(Table 4). The rst state of hierarchy of aggregate formation
is clay occulation with fresh plant material and microbial
products to form stable microaggregates (Jarvis et al., 2012;
Oades, 1993; Oades and Waters, 1991). We observed a
signicantly higher C4 leaf litter-derived carbon concentration
for microaggregates+C4 and small macroaggregates+C4
than large macroaggregates. Our results can be showing
the rst stages of aggregate formation and pathway of
carbon incorporation across aggregate size classes, where
microaggregates are formed by C4 leaf litter-derived carbon
encrusted in clay particles to form macroaggregates. Perhaps a
longer incubation would have permitted re-allocation of C4 leaf
litter into macroaggregates. While large macroaggregates+C3
showed a higher C3 leaf litter-derived carbon concentration
than small macroaggregates+C3 and microaggregates+C3
indicate the last level of the hierarchy of carbon incorporation
in the soil aggregates as shown in Table 4 (Oades and Waters,
1991; Six et al., 2000; Tisdall and Oades, 1982). Our results
suggest that carbon from maize leaves residue requires a
longer period for carbon stabilization and development of soil
aggregates structures in each aggregate size classes (Elliott,
1986; Oades and Waters, 1991; Six et al., 2002).
This study found that earthworm abundance with endogeic
dominance had a relationship with large macroaggregates+C4
leaves, and small macroaggregates+C4 leaves. These trends
also suggest that the large macroaggregates were formed at the
expense and reorganization of small macroaggregates during
burrowing and casting activities. Our results are consistent
with previous studies. For example, Barois et al. (1993)
observed with a transmission electron microscopy that P.
corethrurus destroyed microaggregates during the gut transit
and new microaggregates were formed. Sánchez-de León et al.
(2014) observed that microaggregates and silt+clay fractions
were reduced in quantity in the presence of Diplocardia spp.
under microcosm conditions. Barois et al. (1993) found that
aggregates were restructured during the transit by the gut of
P. corethrurus.
Our results show that earthworm abundance could
aect the proportion of large macroaggregates and small
macroaggregates as well as earthworm abundance inuences
the restructuration of small macroaggregates to form large
macroaggregates. In addition, it is possible to nd other
interactions between earthworms with aggregate fractions
according their feeding behavior and size. Blanchart et al.
(1997) reported that earthworm Milsonia anomala may ingest
microaggregates and create new macroaggregates larger than
5 mm. Dierences in earthworm abundance and earthworm
biological stages observed across the study site could also
inuence the dynamics of aggregate formation and could
create the observed variations.
Conclusion
The eld experiment showed that leaf-derived carbon follows
the aggregate hierarchy process. Carbon from C4-leaf source
was incorporated rst into the microaggregates and small
macroaggregates, but not in the large macroaggregates. The
relationship between earthworm abundance and aggregate
proportion indicates that earthworms (with dominance of P.
corethrurus) consumed small macroaggregates and created
large macroaggregates. In conclusion, our results suggest
that P. corethrurus shows a preference for consuming soil-
derived carbon and may translocate it from microaggregates
to macroaggregates by restructuring soil aggregates. The
endogeic P. corethrurus plays a crucial role in soil bioturbation
by restructuring soil aggregates, nutrient cycling, and organic
soil carbon. Its widespread presence and adaptability make
it vital for soil ecology research, providing insights into soil
health and future studies on ecosystem resilience.
Soil aggregate formation and carbon storage by endogeic earthworms in an Ultisol
2025. 18(2): 68-77 Ciencia y Tecnología. 75
Acknowledgment
The authors gratefully acknowledge the support of the
University of Puerto Rico at Mayagüez and the U.S.
Department of Agriculture’s National Institute of Food and
Agriculture (NIFA) Program H-461 project. Special thanks
to Dr. Yaniria Sánchez de León, Dr. David Sotomayor, and
Dr. Mario Flores. To Dr. Sonia Borges for her collaboration
in earthworm taxonomic identication and to Dr. Raúl
Macchiavelli for his assistance with statistical analysis.
Bibliographic references
Aira, M., Sampedro, L., Monroy, F. y Domínguez, J. (2008).
Detritivorous earthworms directly modify the structure,
thus altering the functioning of a microdecomposer food
web. Soil Biology and Biochemistry, 40(10), 2511–2516.
https://doi.org/10.1016/j.soilbio.2008.06.010
Amador, J. A., Winiarski, K. y Sotomayor-Ramírez, D.
(2013). Earthworm communities along a forest-
coee agroecosystem gradient: Preliminary evidence
supporting the habitat-dependent feeding hypothesis.
Tropical Ecology, 54(3), 365–374.
Barois, I., Villemin, G., Lavelle, P. y Toutain, F. (1993).
Transformation of the soil structure through Pontoscolex
corethrurus (Oligochaeta) intestinal tract. Geoderma,
56(1–4), 57–66. https://doi.org/10.1016/0016-
7061(93)90100-Y
Beinroth, F. H., Engel, R., Lugo, J., Santiago, C., Ríos, S. y
Brannon, G. (2002). Updated taxonomic classication of
soils of Puerto Rico (Bull. 303). Agricultural Experiment
Station.
Blanchart, E., Albrecht, A., Brown, G., Decaens, T.,
Duboisset, A., Lavelle, P., Mariani, L. y Roose, E.
(2004). Eects of tropical endogeic earthworms on soil
erosion. Agriculture, Ecosystems y Environment, 104(2),
303–315. https://doi.org/10.1016/j.agee.2004.01.031
Blanchart, E., Lavelle, P., Braudeau, E., le Bissonnais, Y.
y Valentin, C. (1997). Regulation of soil structure by
geophagous earthworm activities in humid savannas
of Cote d’Ivoire. Soil Biology and Biochemistry,
29(3–4), 431–439. https://doi.org/10.1016/S0038-
0717(96)00042-9
Borges, S. (1996). The Terrestrial Oligochaetes of Puerto
Rico. Annals of the New York Academy of Sciences, 776,
239–248. https://doi.org/10.1111/j.1749-6632.1996.
tb17423.x
Bossuyt, H., Six, J. y Hendrix, P. F. (2004). Rapid incorporation
of carbon from fresh residues into newly formed stable
microaggregates within earthworm casts. European
Journal of Soil Science, 55(2), 393–399. https://doi.
org/10.1111/j.1351-0754.2004.00603.x
Bossuyt, H., Six, J. y Hendrix, P. F. (2005). Protection of soil
carbon by microaggregates within earthworm casts. Soil
Biology y Biochemistry, 37(2), 251–258. https://doi.
org/10.1016/j.soilbio.2004.07.035
Bossuyt, H., Six, J. y Hendrix, P. F. (2006). Interactive
eects of functionally dierent earthworm species on
aggregation and incorporation and decomposition of
newly added residue carbon. Geoderma, 130(1–2), 14–
25. https://doi.org/10.1016/j.geoderma.2005.01.005
Dechaine, J., Ruan, H., Sánchez-de León, Y. y Zou, X.
(2005). Correlation between earthworms and plant litter
decomposition in a tropical wet forest of Puerto Rico.
Pedobiologia, 49(6), 601–607. https://doi.org/10.1016/j.
pedobi.2005.07.006
Elliott, E. T. (1986). Aggregate Structure and Carbon,
Nitrogen, and Phosphorus in Native and Cultivated
Soils. Soil Sci .Soc.Am.J., 50(3), 627–633. https://
acsess.onlinelibrary.wiley.com/doi/abs/10.2136/
sssaj1986.03615995005000030017x
Fonte, S. J., Kong, A. Y. Y., van Kessel, C., Hendrix, P. F.
y Six, J. (2007). Inuence of earthworm activity on
aggregate-associated carbon and nitrogen dynamics
diers with agroecosystem management. Soil Biology
and Biochemistry, 39(5), 1014–1022. https://doi.
org/10.1016/j.soilbio.2006.11.011
Fonte, S. J., Winsome, T. y Six, J. (2009). Earthworm
populations in relation to soil organic matter dynamics
and management in California tomato cropping systems.
Applied Soil Ecology, 41(2), 206–214. https://doi.
org/10.1016/j.apsoil.2008.10.010
González, G., García, E., Cruz, V., Borges, S., Zalamea, M.
y Rivera, M. M. (2007). Earthworm communities along
an elevation gradient in Northeastern Puerto Rico.
European Journal of Soil Biology, 43(SUPPL. 1). https://
doi.org/10.1016/j.ejsobi.2007.08.044
González, G. y Zou, X. (1999). Plant and Litter Inuences
on Earthworm Abundance and Community Structure in a
Tropical Wet Forest. Biotropica, 31(3), 486–493. https://
www.jstor.org/stable/2663944
Harmsen, E. W., Goyal, M. R. y Torres-Justiniano, S. (2002).
Estimating evapotranspiration in Puerto Rico. Journal
of Agriculture of the University of Puerto Rico, 86(1–2),
35–54. https://revistas.upr.edu/index.php/jaupr/article/
view/3182
Hendrix, P. F., Lachnicht, S. L., Callaham, M. A. y Zou,
X. (1999). Stable isotopic studies of earthworm
feeding ecology in tropical ecosystems of Puerto
Rico. Rapid Communications in Mass Spectrometry,
13(13), 1295–1299. https://doi.org/10.1002/
(SICI)1097-0231(19990715)13:13<1295::AID-
RCM605>3.0.CO;2-9
Morejón et al., 2025
2025. 18(2):68-77
Ciencia y Tecnología.76
Hubers, H., Borges, S. y Alfaro, M. (2003). The
oligochaetofauna of the Nipe soils in the Maricao
State Forest, Puerto Rico : The 7th international
symposium on earthworm ecology · Cardi · Wales
· 2002. Pedobiologia, 47(5–6), 475–478. https://doi.
org/10.1078/0031-4056-00216
Jackson, M. L. (2005). Soil Chemical Analysis –
Advance Course. https://books.google.com.ec/
books?id=VcEOK9QCkVEC&hl=es-419
Jarvis, S., Tisdall, J., Oades, J. M., Six, J., Gregorich, E. y
Kögel-Knabner, I. (2012). Landmark papers. European
Journal of Soil Science, 63(1), 1–21. https://doi.
org/10.1111/j.1365-2389.2011.01408.x
Jastrow, J. D., Boutton, T. y Miller, R. M. (1996). Carbon
dynamics of aggregate – associated organic matter
estimated by carbon – 13 natural abundance. Soil Sci.
Soc. Am. J., 60(3), 801–807. https://doi.org/10.2136/
sssaj1996.03615995006000030017x
Jastrow, J. D., Miller, R. M., Matamala, R., Norby, R. J.,
Boutton, T. W., Rice, C. W. y Owensby, C. E. (2005).
Elevated atmospheric carbon dioxide increases soil
carbon. Global Change Biology, 11(12), 2057–2064.
https://doi.org/10.1111/j.1365-2486.2005.01077.x
Kamau, S., Barrios, E., Karanja, N. K., Ayuke, F. O. y
Lehmann, J. (2020). Dominant tree species and
earthworms aect soil aggregation and carbon content
along a soil degradation gradient in an agricultural
landscape. Geoderma, 359. https://doi.org/10.1016/j.
geoderma.2019.113983
Lachnicht, S. L., Hendrix, P. F. y Zou, X. (2002). Interactive
eects of native and exotic earthworms on resource use
and nutrient mineralization in a tropical wet forest soil of
Puerto Rico. Biology and Fertility of Soils, 36(1), 43–52.
https://doi.org/10.1007/s00374-002-0501-5
Lavelle, P., Blanchart, E., Martin, A., Spain, A. V. y Martin, S.
(1992). Impact of Soil Fauna on the properties of Soils
in the Humid Tropics. In Myths and Science of Soils of
the Tropics. SSSA Spe- cial Publication. (Vol. 29, pp. 29).
https://doi.org/10.2136/sssaspecpub29.c9
Le Couteulx, A., Wolf, C., Hallaire, V. y Pérès, G. (2015).
Burrowing and casting activities of three endogeic
earthworm species aected by organic matter
location. Pedobiologia, 58(2–3), 97–103. https://doi.
org/10.1016/j.pedobi.2015.04.004
Liu, Z. y Zou, X. (2002). Exotic earthworms accelerate
plant litter decomposition in a wet forest. Ecological
Applications, 12(5), 1406–1417. https://doi.org/https://
doi.org/10.1890/1051-0761(2002)012[1406:EEAPLD]2
.0.CO;2
Oades, J. M. (1993). The role of biology in the formation,
stabilization and degradation of soil structure. Geoderma,
56(1–4), 377–400. https://doi.org/10.1016/0016-
7061(93)90123-3
Oades, J. M. y Waters, A. G. (1991). Aggregate hierarchy in
soils. Australian Journal of Soil Research, 29(6), 815–
825. https://doi.org/10.1071/SR9910815
O’Leary, M. H. (1981). Carbon isotope fractionation in
plants. Phytochemistry, 20(4), 553–567. https://doi.
org/10.1016/0031-9422(81)85134-5
Paul, E. A., Harris, D., Collins, H. P., Schulthess, U. y
Robertson, G. P. (1999). Evolution of CO2 and soil
carbon dynamics in biologically managed, row-crop
agroecosystems. Applied Soil Ecology, 11(1), 53–65.
https://doi.org/10.1016/S0929-1393(98)00130-9
Paul, S., Flessa, H., Veldkamp, E. y López-Ulloa, M.
(2008). Stabilization of recent soil carbon in the
humid tropics following land use changes: Evidence
from aggregate fractionation and stable isotope
analyses. Biogeochemistry, 87(3), 247–263. https://doi.
org/10.1007/s10533-008-9182-y
Phillips, D. L. y Gregg, J. W. (2001). Uncertainty in source
partitioning using stable isotopes. Oecologia, 127(2),
171–179. https://doi.org/10.1007/s004420000578
Pulleman, M. M., Six, J., Uyl, A., Marinissen, J. C. Y. y
Jongmans, A. G. (2005). Earthworms and management
aect organic matter incorporation and microaggregate
formation in agricultural soils. Applied Soil Ecology,
29(1), 1–15. https://doi.org/10.1016/j.apsoil.2004.10.003
Ravalo, E. J., Goyal, M. R. y Almodóvar, C. R. (1986).
Average Monthly and Annual Rainfall Distribution in
Puerto Rico. The Journal of Agriculture of the University
of Puerto Rico, 70(4), 267–275. https://revistas.upr.edu/
index.php/jaupr/article/view/7103
Sánchez-de León, Y., Lugo-Pérez, J., Wise, D. H., Jastrow, J.
D. y González-Meler, M. A. (2014). Aggregate formation
and carbon sequestration by earthworms in soil from
a temperate forest exposed to elevated atmospheric
CO2: A microcosm experiment. Soil Biology and
Biochemistry, 68, 223–230. https://doi.org/10.1016/j.
soilbio.2013.09.023
Sánchez-de León, Y., Wise, D. H., Lugo-Pérez, J., Norby, R. J.,
James, S. W. y Gonzalez-Meler, M. A. (2018). Endogeic
earthworm densities increase in response to higher ne-
root production in a forest exposed to elevated CO2.
Soil Biology and Biochemistry, 122, 31–38. https://doi.
org/10.1016/j.soilbio.2018.03.027
Sánchez-de León, Y., Zou, X. y Borges, S. (2003). Recovery
of Native Earthworms in Abandoned Tropical Pastures.
17(4), 999–1006.
SAS Institute Inc. (2015). Introduction to Mixed Modeling
Procedures. SAS/STAT 9.2 User’s Guide, Chapter 6,
116–125.
Schmidt, O. (1999). Intrapopulation variation in carbon
and nitrogen stable isotope ratios in the earthworm
Aporrectodea longa. Ecological Research, 14(4), 317–
328. https://doi.org/10.1046/j.1440-1703.1999.00310.x
Soil aggregate formation and carbon storage by endogeic earthworms in an Ultisol
2025. 18(2): 68-77 Ciencia y Tecnología. 77
Six, J., Conant, R. T. y Paustian, K. (2002). Stabilization
mechanisms of soil organic matter: Implications for
C-saturation of soils. Plant and Soil, 241, 155–176.
https://doi.org/10.1023/A:1016125726789
Six, J., Elliott, E. T. y Paustian, K. (2000). Soil macroaggregate
turnover and microaggregate formation: A mechanism
for C sequestration under no-tillage agriculture. Soil
Biology and Biochemistry, 32(14), 2099–2103. https://
doi.org/10.1016/S0038-0717(00)00179-6
Six, J., Elliott, E. T., Paustian, K. y Doran, J. W. (1998).
Aggregation and soil organic matter accumulation
in cultivated and native grassland soils. Soil Science
Society of America Journal, 62(5), 1367-1377. https://
doi.org/10.2136/sssaj1998.03615995006200050032x
Six, J. y Paustian, K. (2014). Aggregate-associated soil organic
matter as an ecosystem property and a measurement tool.
Soil Biology and Biochemistry, 68, A4-A9. https://doi.
org/10.1016/j.soilbio.2013.06.014
Soil Survey Sta. (2014). Keys to soil taxonomy. Soil
Conservation Service, 12, 410.
Tisdall, J. M. y Oades, J. M. (1982). Organic matter and water-
stable aggregates in soils. Journal of Soil Science, 33(2),
141–163. https://doi.org/10.1111/j.1365-2389.1982.
tb01755.x
Túa-Ayala, G. Z. (2013). Transformación de bosques noveles
a sistemas agroforestales en la región húmeda de Puerto
Rico: Pasos iniciales. [Master’s Thesis, University of
Puerto Rico-Mayagüez]. Retrieved from https://hdl.
handle.net/20.500.11801/3558
Whalen, J. K. y Janzen, H. H. (2002). Labeling earthworms
uniformly with C-13 and N-15: implications for
monitoring nutrient uxes. Soil Biology and Biochemistry,
34(12), 1913–1918. https://doi.org/10.1016/S0038-
0717(02)00207-9
Copyright (2025) © Mauricio Morejón Centeno, Rocio Morejón Lucio y José Macías Veliz.
Este texto está protegido bajo una licencia internacional Creative Commons 4.0. Usted es libre para compartir, copiar y redistribuir el material
en cualquier medio o formato. También podrá adaptar: remezclar, transformar y construir sobre el material. Ver resumen de la licencia.
