Soil Biology and Biochemistry 150 (2020) 107998

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Soil Biology and Biochemistry

journal homepage: http://www.elsevier.com/locate/soilbio

Linking soil engineers, structural stability, and organic matter allocation to

unravel soil carbon responses to land-use change
André L.C. Franco a, b, *, Maurício R. Cherubin b, Carlos E.P. Cerri b, Johan Six c, Diana H. Wall d,
Carlos C. Cerri e, 1
a
Department of Biology, Colorado State University, Fort Collins, CO 80523, USA
b
Department of Soil Science, “Luiz de Queiroz” College of Agriculture, University of São Paulo, Piracicaba, SP 13418-900, Brazil
c
Department of Environmental Systems Science, Swiss Federal Institute of Technology, ETH Zurich, CH-8092 Zurich, Switzerland
d
Department of Biology & School of Global Environmental Sustainability, Colorado State University, Fort Collins, CO 80523, USA
e
Center for Nuclear Energy in Agriculture, University of São Paulo, Piracicaba, SP 13400-970, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Land use changes (LUC) for the expansion of bioenergy cropping have caused consistent reductions in soil
Land-use change organic carbon (SOC) stocks in tropical soils. This study addresses the mechanisms underlying such SOC losses by
Carbon stocks assessing LUC effects on, and relationships between, soil engineering invertebrate fauna, soil structural stability,
Soil aggregation
and C allocation and sequestration within soil aggregates. We sampled three sites with sets of land use types
Soil fauna
Soil organic matter
varying in the level of anthropogenic stress in sandy loam, sand clay loam, and clay soils along a 1000-km-long
Termites transect in central Brazil, where bioenergy cropping expands across pasturelands. We quantified the effects of
LUC on soil engineer fauna (i.e., termites, earthworms, coleopterans, and ants), soil structural stability, and C
allocation and fitted structural equation models (SEM) to elucidate mechanistic links between the measured
variables. We found that reductions in SOC stocks following LUC were concomitant with reductions in the
abundance of soil engineers (log abundance of soil engineers, − 1.17 ± 0.54; P = 0.0322), destabilization of soil
structure (normalized stability index, − 0.16 ± 0.04; P < 0.0001), and soil depth-dependent decreases in the
amounts and increases in the humification degree of aggregate-occluded SOC. Our SEMsupported the predicted
relationships among these responses, and indicated that soil engineering by invertebrates indirectly mediated
changes in SOC stocks across land uses by controlling the physical protection of low-humified, aggregate-
occluded SOC, a C fraction that accounted for over 90% of the change in total SOC stocks following LUC. When
analysing the influence of the different invertebrate groups separately, we found stronger support for the role of
termites in this process compared to that of earthworms, coleopterans, and ants. Hence, negative LUC effects on
populations of soil engineers weaken soil functioning and its C storage. These results highlight the need for land-
use strategies that maintain soil fauna in order to sustain key ecosystem processes like soil structural formation
and soil C stabilization.

1. Introduction
Land use changes (LUC) are among the most significant anthropo­
genic practices altering carbon (C) cycling and stocks in terrestrial
ecosystems (Sanderman et al., 2017). Historically, LUC have been net
sources of carbon dioxide (CO2) to the atmosphere (Foley et al., 2005),
with most of the recent emissions caused by LUC in the tropics
(Houghton and Nassikas, 2017; Palmer et al., 2019). Tropical LUC in wet
and dry forest ecosystems typically start with the conversion of native
forests into pasture for cattle ranching, followed by the conversion of
pasture into cropland (Dias et al., 2016). Areas cultivated with tradi­
tional staple crops or under cattle ranching in the tropics have increas­
ingly been converted into large-scale farming of commodity crops
(Goldemberg et al., 2014, Lapola et al., 2014; Strassburg et al., 2014).
Tropical deforestation and ecosystem conversion to arable land removes
plant biomass and disturbs soils, often resulting in a loss of soil organic
carbon (SOC), soil biodiversity, and soil chemical and physical quality
(Don et al., 2011; Franco et al., 2019; Mello et al., 2014; Smith et al.,

* Corresponding author. Department of Biology, Colorado State University, 200 West lake Street, 1878 Biology, Fort Collins, CO, 80523, USA.
E-mail address: andre.franco@colostate.edu (A.L.C. Franco).
1
In memorian.

https://doi.org/10.1016/j.soilbio.2020.107998
Received 4 June 2020; Received in revised form 31 August 2020; Accepted 3 September 2020
Available online 4 September 2020
0038-0717/© 2020 Elsevier Ltd. All rights reserved.
A.L.C. Franco et al.
2016). In addition, severe depletion of the SOC pool contributes to at­
mospheric CO2 enrichment that causes global warming (Lal, 2004).
Reductions of up to 59% in soil organic carbon (SOC) after conver­
sion of pastures to croplands have been consistently reported (Smith
et al., 2016). This SOC response to LUC has been attributed to (i)
reduced inputs of organic matter and high decomposability of crop
residues compared to native vegetation, and particularly to (ii) tillage
effects that destabilize soil structure and reduce the physical protection
of soil organic matter (SOM) (Guo and Gifford, 2002; Post and Kwon,
2000). The former impairs the stabilization of new SOM from crop
residues, and the latter promotes the loss of original SOM accumulated
from previous vegetation via disruption of soil aggregates. While soil
disaggregation has been identified as one of the main processes leading
to reductions in SOC after LUC, questions remain on the role of biotic
processes for soil aggregation (or disaggregation) after LUC in
highly-weathered tropical soils (Peng et al., 2015) which cover 60–70%
of tropical areas (Segalen, 1994).
Soil structure can be altered directly by LUC-induced soil distur­
bances (Cherubin et al., 2017), or indirectly through alterations on bi­
otic and abiotic factors known to affect soil structural stability (Barto
et al., 2010). While some studies have emphasized the key role of abiotic
factors related to soil texture and clay mineralogy in aggregating
weathered soils (Barto et al., 2010; Six et al., 2000a), others have shown
that the activity of certain soil macroinvertebrates such as termites,
earthworms, ants, and coleopterans can play an important role in the
structural stabilization of such soils (Ayuke et al., 2011; Fonte and Six,
2010; Franco et al., 2017; Lehmann et al., 2017; Six et al., 2002b). The
soil engineering concept makes reference to the ability of these in­
vertebrates to move through soil and to build biogenic structures with
distinct physical, chemical and microbiological properties (Jouquet
et al., 2006). Although the important role of soil engineers in controlling
soil structural dynamics has been demonstrated (Blouin et al., 2013;
Cammeraat and Risch, 2008; Jouquet et al., 2011), it remains relatively
neglected by soil scientists working on soil structure (Bottinelli et al.,
2015; Jouquet et al., 2016). Barros et al. (2001) demonstrated the role of
soil engineers in the transformation and reversibility of soil structure
across LUC. These organisms influence soil structure by coating their
nests and galleries with their saliva and by feeding on litter and organic
residues on the soil surface and incorporating the processed organic
matter into the soil profile within aggregates (Bottinelli et al., 2015;
Lavelle et al., 2020). Meta-analyses of experiments with various enclo­
sures have shown that soil engineer activity slows the rate of SOM
decomposition and has a significant impact on C cycling (de Graaff et al.,
2015; Filser et al., 2016; Frouz et al., 2015). The presence of earth­
worms, for example, can lead to larger amounts of microbial necromass
in stable fractions from macro- and microaggregates by which the
resilience to disturbances of that C is enhanced (Angst et al., 2019).
Frouz (2018) divided the faunal effect on SOM into three categories: i)
the (often small) direct effect on C mineralization; ii) the indirect effect
on mineralization, mobilization, or immobilization of nutrients via
changes on microbial activity; and iii) the effect of fauna on fluxes be­
tween various SOM pools.
In this study, we identified three sites with sets of land use types
varying in the level of anthropogenic stress: native vegetation (NV),
pasture (PA), and sugarcane crop (SC) along the Brazilian Cerrado and
Atlantic Forest biomes to assess land-use effects on, and relationships
between, soil engineers, soil structural stability, and C allocation and
sequestration. Transitions from PA to SC have added more than 6 Mha of
new sugarcane land in these Brazilian biomes during the last decade due
to the increasing demand for ethanol fuel (Dias et al., 2016; Goldemberg
et al., 2014). Our recent studies in the same areas showed that the
conversion of PA to SC produces a net soil C emission ranging from 1.0 to
1.3 Mg ha− 1 yr− 1 in 20 years (Franco et al., 2015; Mello et al., 2014).
Thus, here, we tested the hypothesis that such depletion in SOC stocks
across LUC would occur concomitantly with reductions in the abun­
dance of soil engineers and destabilization of soil structure, which would
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Soil Biology and Biochemistry 150 (2020) 107998
in turn impair the ability of soils to physically protect SOM from
decomposition inside stable aggregates (Ayuke et al., 2011; Six et al.,
2002a). We applied structural equation modelling and tested for re­
lationships among individual soil engineer groups (i.e., termites,
earthworms, coleopterans, and ants), soil structural stability, and C
allocation in order to investigate the mechanistic pathway by which soil
engineers would mediate SOC stock responses to LUC.
2. Materials and methods
2.1. Site descriptions
The study was carried out in sites representing two major tropical
Brazil biomes, Cerrado and Atlantic Forest. Soils in these ecosystems are
primarily well-drained and highly weathered, typical of tropical wet
conditions. The portion of these biomes in Central-Southern Brazil has
experienced rapid and large-scale changes in land use mainly due to the
expansion of sugarcane crops into pastureland that has made this region
the world’s largest sugarcane producer (Lapola et al., 2014). Three study
sites were chosen along a transect of approximately 1000 km across this
region: Northern (Lat_17 S), Central (Lat_21 S), and Southern (Lat_23 S).
At each study site, we identified areas of land uses composed by native
vegetation (NV), pasture (PA), and sugarcane crop (SC). In order to
minimize the effects of climatic, topographic, and edaphic variations,
the three land uses were always adjacent within sites. Site coordinates,
climate information, and soil characteristics can be found in Table 1. A
complete soil chemical characterization as well as detailed information
on the past history of land use and soil management at the study sites is
provided by Cherubin et al. (2015). Briefly, LUC from NV to PA
happened in 1980 at Lat_17 S and Lat_21 S, and in 1979 at Lat_23 S. PA
areas are cropped with tropical grasses (Brachiaria and Cynodon genus)
and grazed at a low stocking rate (1–2 animal unit ha− 1). The SC was
established over part of PA in 2009 at Lat_17 S, 2010 at Lat_21 S, and
1990 at Lat_23 S.
2.2. Soil sampling and aggregate fractionation
Soil sampling was carried out during the rainy season (January).
Sampling for each of the 3 land-use types consisted of a grid with 9
sampling points (3 × 3 cell grid) spaced 50 m apart, composing 27
sampling points in each study site, or 81 sampling points in total. The
method for soil engineer sampling and counting is described in detail in
a previous paper (Franco et al., 2016). Briefly, from each sampling point,
one soil block of 25 × 25 cm–10 cm depth was collected from each of
three soil layers (0–10, 10–20, and 20–30 cm) for invertebrate extrac­
tion, and organisms were hand-sorted according to the standard Trop­
ical Soil Biology and Fertility Institute (TSBF) soil monolith method
(Anderson and Ingram, 1993; Moreira et al., 2008). Results are
expressed in terms of the absolute (individuals m− 2) and relative (%)
abundance of termites, earthworms, coleopterans, and ants.
For soil structure analyses, one soil block of 5 × 5 cm–10 cm depth
was collected from 0 to 10, 10–20, and 20–30 cm soil layers at each
sampling point and stored individually in plastic pots for transportation.
At the laboratory, field moist soil was gently passed through an 8-mm
sieve by manually breaking up the soil along natural planes of weak­
ness, and air dried for three days. Two pre-treatments were applied prior
to aggregate fractionation, according to the method of Six et al. (2000a):
(i) air-dried soil was rapidly immersed in water (slaked pre-treatment),
and (ii) air-dried soil was capillary rewetted to field capacity and
equilibrated overnight before fractionation (rewetted pre-treatment). By
using both pre-treatments we avoided physical induced variability due
to differences in the original water content, which are not related to soil
quality (Six et al., 2000a). In order to determine aggregate stability, we
adapted a method from Elliott (1986) that separates soil into four
fractions: (i) large macroaggregates (LM, ≥ 2000 μm), (ii) small mac­
roaggregates (SM, 250–2000 μm), (iii) microaggregates (Mi, 53–250
 A.L.C. Franco et al.

Table 1
Climate and soil characteristics for the sites used in this study.
Lat_17 S Lat_21 S Lat_23 S

General
Latitude 17◦ 56′ S 21◦ 14′ S 23◦ 05′ S
Longitude 51◦ 38′ W 50◦ 47′ W 49◦ 37′ W
Climate
MAT (◦ C)a 24 23.4 21.7
MAP (mm)a 1600 1240 1470
Native Sclerophyllous and xeromorphic species. Denser Atlantic forest–Cerrado transition. Seasonal semideciduous forest with more Atlantic forest–Cerrado transition. Seasonal semideciduous forest with more
Vegetation vegetation compared to Cerrado stricto sensu- xeromorphic species than wetter areas of Atlantic forest, and less xeromorphic xeromorphic species than wetter areas of Atlantic forest, and less xeromorphic
savanna species than Cerrado species than Cerrado
Soil
description
Native Anionic Acrudox Typic Rhodudalf Rhodic Hapludox

3
Vegetation
Pasture Typic Hapludox Typic Kandiudult Rhodic Kandiudox
Sugarcane Anionic Acrudox Typic Hapludalf Rhodic Hapludox
Soil textureb
Native Sand clay loam Sandy loam Clay
Vegetation
Pasture Sandy loam Sandy loam Clay
Sugarcane Sand clay loam Sandy loam Clay
Total SOC
stocksc
Native 38.63 ± 3.83 56.84 ± 9.43 78.64 ± 8.97
Vegetation
Pasture 25.77 ± 2.86 35.24 ± 2.68 72.26 ± 7.26
Sugarcane 30.89 ± 3.56 32.96 ± 2.76 42.94 ± 6.15
a
Obtained from CIIAGRO and CEPAGRI climate data from Jataí, GO, Valparaíso, SP, and Ipaussu, SP.
b
According to US Soil Survey Staff (2014).
c
Mg C ha− 1 in 0–30 cm soil layer. Previously published in Franco et al. (2015).
Soil Biology and Biochemistry 150 (2020) 107998
A.L.C. Franco et al.
μm), and (iv) silt + clay sized particles (S + C, ≤ 53 μm). A subsample of
50 g of soil (slaked or rewetted) for each sample was submerged in
deionized water on the top of a set of three sieves (2000, 250, and 53
μm) and oscillated with a displacement of 4 cm at 30 cycles per minute
for 10 min. After that time, aggregates remaining on the sieves were
rinsed into a pre-weighted plastic pan for oven-drying at 50 ◦ C for two
days, and weighed. The sum of the weights of LM, SM, and Mi was
subtracted from the initial weight of the sample (50 g) to obtain the total
amount of the S + C fraction (≤53 μm). To sand-correct the aggregate
fraction masses, the sand size distribution for LM, SM and Mi of rewetted
and slaked soils was determined by sieving after dispersing aggregates
with sodium hexametaphosphate (5 g L− 1), and the aggregate-sized sand
fraction was subtracted from the whole fraction masses (Six et al.,
2000a).
The large and small macroaggregate fractions for each sample were
then combined and used for the isolation of microaggregates within
macroaggregates (mM; 53–250 μm). The macroaggregate fractionation
was carried out with a self-built device based on Six et al. (2000b) that
breaks up macroaggregates and minimizes disruption of micro­
aggregates. About 10 g of oven-dried macroaggregates were pre-slaked
overnight in deionized water in order to slake the strongly stable mac­
roaggregates of the tropical weathered soils (Zotarelli et al., 2007). The
macroaggregates were then transferred to the device which held a
250-μm mesh sieve and shaken with 50 glass beads (4 mm diameter)
until all macroaggregates were broken up. The microaggregates released
from macroaggregates were immediately flushed through the 250-μm
screen and deposited onto a 53-μm screen by a continuous flow of
deionized water through the device. The microaggregates on the 53-μm
sieve were then wet-sieved to separate the stable microaggregates from
dispersed silt and clay particles. All microaggregates were backwashed
into a pre-weighed container, oven-dried at 50 ◦ C for 48 h, weighed and
stored.
The organic C contents of microaggregates within macroaggregates
were determined by dry combustion on elemental analyzer – LECO®
CN-2000 (furnace at 1350 ◦ C in pure oxygen). This fraction was also
used for the assessment of SOM humification degree through Laser-
Induced Fluorescence Spectroscopy (LIF), using the methods described
by Milori et al. (2006). Approximately 0.5 g of mM, after being grinded
to pass a 250-μm mesh, were pressed into pellets of 1-cm diameter and
2-mm thickness, which were inserted into a bench custom-made appa­
ratus in order to run LIF measurements. Samples were excited with 458
nm blue radiation, emitted by argon laser equipment with power of
around 300 mW. The ratio LIF spectrum’s area for each sample over the
corresponding C concentration was defined as the SOM humification
index (HLIF) and is expressed in arbitrary units (a.u.).
2.3. Calculations of soil structure indices and C allocation
We calculated the mean weight diameter (MWD) for slaked and
rewetted soils as a preliminary index of aggregate stability because
MWD is the most commonly used measurement of soil aggregate sta­
bility and, hence, enables comparison with previous studies. The
normalized stability index (NSI) was calculated and used as the main
indicator of soil structural stability because it eliminates the confound­
ing effects of original soil water content, corrects for differences in sand
size distribution among soils, aggregates size classes and pre-treatments,
and normalizes the level of disruption imposed by slaking by using a
maximum level of disruption (Six et al., 2000a). The NSI was measured
according to the calculation procedures described by Six et al. (2000a).
However, we used no weighting factors in the calculation of both
disruption level and maximum disruption level, as done by Six et al.
(2000b), because the arbitrary weighting factors for the disruption in
different aggregate size classes made the index insensitive to the pref­
erential disaggregation of larger size classes in our weathered tropical
soils.
Soil organic carbon (SOC) stocks associated with mM (mM-C) were
4
Soil Biology and Biochemistry 150 (2020) 107998
calculated for each sample by multiplying its C content by the propor­
tion of this aggregate fraction relative to the bulk soil density and by
layer thickness (10 cm) of each sample. To account for the effect of
differing soil bulk densities on stock comparisons, mM-C within pasture
and sugarcane soils were adjusted to an equivalent soil mass basis in the
corresponding NV reference site (Lee et al., 2009). Finally, we calculated
the proportion of total SOC changes caused by LUC that is accounted for
by changes in mM-C. Total SOC stocks for these study sites have been
previously and similarly computed (Franco et al., 2015), and are pre­
sented in Table 1.
2.4. Statistical analyses
We generated statistical models for the log-transformed total abun­
dance of soil engineers, soil structure indices (MWD and NSI), mM-C,
and log-transformed HLIF by using land-use type, soil depth, and their
interaction as explanatory variables. We used linear mixed effects
models with a site-level random effect term to account for the interde­
pendency that stems from having repeated measurements per site. For
each model, both the marginal r2 (proportion of variance explained by
the moderator variables) and the conditional r2 (that of the whole
model, including the random effect) were calculated following Naka­
gawa and Schielzeth (2013). We also tested for the effects of LUC and
soil depth on soil engineer community composition and on soil aggre­
gate size distribution by running permutational multivariate analysis of
variance (Permanova) on Bray-Curtis dissimilarity matrix. The
aggregate-size distribution for slaked rather than rewetted soil is pre­
sented because the capillary-wetted aggregate distribution is less suit­
able to assess land-use effects (Six et al., 2000a). All analyses were
conducted using the software R, version 3.2.2 (R Core Team, 2014), and
packages nlme (Pinheiro et al., 2014), piecewiseSEM (Lefcheck, 2016),
vegan (Oksanen et al., 2016), and ggplot2 (Wickham, 2009).
We used structural equation models (SEM) to analyse the dataset as a
system of interrelated variables. SEM uses two or more cause-effect
equations to model multivariate relationships (Grace and Kelley,
2006) and allows for an intuitive graphical representation of complex
networks of causal relationships (Eisenhauer et al., 2015). Structural
equation modelling is useful to represent and test mediation and direct
versus indirect relationships, and the partial contributions of correlated
explanatory variables (Eisenhauer et al., 2015). We applied SEM to
investigate how the different soil engineer taxa influence SOC stocks by
considering variation in soil structural stability (NSI) and C allocation in
stable aggregate fractions (mM-C). We combined data from the three
sites (Lat_17 S, Lat_21 S, and Lat_23 S), three land uses (NV, PA, and SC),
and three soil depths (0–10, 10–20, and 20–30 cm) to fit the models,
yielding a total of 243 observations. Five separate models used un­
transformed engineer abundances as exogenous explanatory variables to
test control effects of the different taxa: termites, earthworms, co­
leopterans, ants, and total soil engineer abundances. In addition, five
other similar models were implemented where soil structure stability
(NSI) was included as the exogenous and soil engineer taxa as endoge­
nous explanatory variables to test for the opposite causality that soil
structure may drive engineer abundance and SOC stocks (Table S1). All
models were implemented using lavaan package in R (Rosseel, 2012).
Chi-square statistics and four others fitting functions (Comparative Fit
Index (CFI); Tucker–Lewis Index (TLI); Root Mean Square Error of
Approximation (RMSEA); Root Mean Square Residual (SRMR)) were
measured in order to test the goodness-of-fit of the models. Desired
values for acceptable fit are P-values greater than 0.05, CFI and TLI
greater than 0.95, and close to 0 for RMSEA and SRMR. Only those
models showing acceptable fitting to the data are graphically presented.
A.L.C. Franco et al.
3. Results
3.1. Soil engineer abundance
Land use and soil depth significantly affected the total abundance of
soil engineers (Land use P < 0.0001; Depth P < 0.0001; r2marginal = 0.35,
r2conditional = 0.37; Fig. 1A). Compared to NV, the total (log+1) abun­
dance of soil engineers was higher in PA (1.72 ± 0.55; P = 0.0019) and
lower in SC (− 1.17 ± 0.54; P = 0.0322), while soil depth effects were
negative at both 10–20 cm (− 1.81 ± 0.54; P = 0.0010) and 20–30 cm
(− 1.91 ± 0.54; P = 0.0005) compared to the 0–10 cm surface layer
(Fig. 1A). Effects of LUC (Permanova; F = 8.81, df = 2, P = 0.001, r2 =
0.10) and site (F = 14.36, df = 2, P = 0.001, r2 = 0.16) altered com­
munity composition of soil engineer taxa (Fig. 1B). Highest relative
abundance of coleopterans and lowest relative abundance of termites
occurred under sugarcane across all sites (Fig. 1B), whereas LUC effects
Fig. 1. Soil engineer abundances as a function of land-use type in three locations (Lat_17 S, Lat_21 S, Lat_23 S). (A) Absolute abundance of organisms plotted as
median values (thick lines in the boxes), interquartile ranges, and outliers (circles) for n = 9. (B) Percent community composition (averaged for 0–30 cm soil depth),
n = 27. NV = native vegetation; PA = pasture; SC = sugarcane.
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Soil Biology and Biochemistry 150 (2020) 107998
on ants and earthworms were site-dependent.
3.2. Soil structural stability
Land use explained a much larger portion of the variation on the
distribution of aggregate classes (F = 174.46, df = 2, P = 0.001, r2 =
0.51) if compared to either site (F = 42.30, df = 2, P = 0.001, r2 = 0.12)
or soil depth (F = 11.8, df = 2, P = 0.001, r2 = 0.03). In fact, LUC effects
on the distributions of soil aggregate-size classes were consistent across
all sites (Fig. 2). Soils were highly aggregated, with water-stable mac­
roaggregates (SM plus LM) dominating the aggregate-size distribution at
all field sites and land uses. This fraction on average accounted for 87,
91 and 77% of the soil in NV, PA and SC soils, respectively, with the
greatest proportions found in the clay soils at Lat_23 S, and lowest in the
sandy clay loam soils at Lat_21 S. Large macroaggregates comprised the
highest proportion of aggregates under NV and PA, while SM and Mi
A.L.C. Franco et al. Soil Biology and Biochemistry 150 (2020) 107998

Fig. 2. Soil aggregate size distribution (averaged for 0–30 cm soil depth) as a function of land-use type in three locations (Lat_17 S, Lat_21 S, Lat_23 S). Aggregate
fractions include large macroaggregates (LM; >2000 μm), small macroaggregates (SM; 250–2000 μm), microaggregates (Mi; 53–250 μm) and silt and clay (S + C;
<53 μm). NV = native vegetation; PA = pasture; SC = sugarcane.

Fig. 3. Soil structural stability indices as a function of land-use type in three locations (Lat_17 S, Lat_21 S, Lat_23 S). MWD = Mean weight diameter (MWD) of slaked
and rewetted soils. Plotted are median values (thick lines in the boxes), interquartile ranges, and outliers (circles) for n = 9. NV = native vegetation; PA = pasture; SC
= sugarcane.

6
A.L.C. Franco et al. Soil Biology and Biochemistry 150 (2020) 107998

dominated SC soils (Fig. 2). 3.3. Carbon allocation

NSI was lower in SC compared to PA and NV and also lower in sur­
face compared to deeper soil layers (Land use P < 0.0001; Soil depth P =
0.0041; r2marginal = 0.27, r2conditional = 0.37). The lowest NSI occurred in
SC compared to NV (− 0.16 ± 0.04; P < 0.0001) and at 20–30 cm soil
layer compared to 0–10 cm (− 0.14 ± 0.03; P = 0.0004) (Fig. 3). Dif­
ferences in NSI between PA and NV were non-significant (− 0.02 ± 0.04;
P = 0.6270). The effects of LUC on MWD for both slaked and rewetted
soils were dependent on depth (significant Land use*Depth interaction;
MWD slaked P = 0.0003, r2marginal = 0.56, r2conditional = 0.78; MWD
rewetted P < 0.0001, r2marginal = 0.79, r2conditional = 0.92). For all sites,
SC soils had the lowest MWD values, but the difference with other land
uses was larger in the surface (0–10 cm) than deep soil layers (10–20 and
20–30 cm) (Fig. 3). Overall, capillary-wetted soil MWD showed similar
trends across land uses compared to MWD upon slaking (Fig. 3).
Lower SOC stocks associated with microaggregates within macro­
aggregates (mM-C) were found in SC compared to PA and NV, but this
land-use effect was dependent on soil depth with larger effects at 0–10
cm depth compared to deeper layers (Land use*Depth significant
interaction; P < 0.0001, r2marginal = 0.29, r2conditional = 0.87) (Fig. 4A).
These effects on mM-C accounted for over 90% of the difference in total
SOC stock between NV and PA in both Lat_17 S and Lat_23 S (Fig. 4B). In
those same sites, mM-C contributions to total SOC stock changes were
<20% from PA to SC. This measure was much more variable in the sandy
loam soils of Lat_21 S (Fig. 4B).
The humification index for SOM occluded in microaggregates-
within-macroaggregates (HLIF) increased from NV to PA and SC, but
this land-use effect was also dependent on depth with larger effects at
0–10 cm compared to both 10–20 and 20–30 cm (Land use*Depth sig­
nificant interaction; P = 0.0203, r2marginal = 0.13, r2conditional = 0.86)

Fig. 4. Carbon allocation within macroaggregate-occluded microaggregates (mM) as a function of land-use type in three locations (Lat_17 S, Lat_21 S, Lat_23 S). (A)
mM-associated soil organic carbon (SOC) stocks. (B) The difference in total SOC among land-use types that is accounted for by mM-associated C. Plotted are median
values (thick lines in the boxes), interquartile ranges, and outliers (circles) for (A) n = 9 and (B) n = 27. NV = native vegetation; PA = pasture; SC = sugarcane.

7
A.L.C. Franco et al. Soil Biology and Biochemistry 150 (2020) 107998

Fig. 5. Humification degree of soil organic matter occluded in microaggregates within macroaggregates as a function of land-use type in three locations (Lat_17 S,
Lat_21 S, Lat_23 S). Humification index (HFIL) obtained through laser-induced fluorescence spectroscopy and expressed as arbitrary units (a.u.). Plotted are median
values (thick lines in the boxes), interquartile ranges, and outliers (circles) for n = 9. NV = native vegetation; PA = pasture; SC = sugarcane.

(Fig. 5). Overall, higher HLIF occurred in PA and SC soils compared to NV
soils.
3.4. Structural equation models
Among the five models which included soil engineer abundances as
exogenous variables, all but the model for ants could not be rejected as
potential explanation of the observed covariance matrix (chi-square test;
P > 0.05; Fig. 6). The models for termites, earthworms, coleopterans,
and total engineer abundance explained between 56% and 57% of the
variation in total SOC stock (Fig. 6) and gave good fits to the data
(CFI>0.95; TLI>0.95; RMSEA<0.05; and SRMR<0.08). The SOC stor­
age was indirectly mediated by both total soil engineer (Fig. 6A) and
termite (Fig. 6B) abundance through their controls on soil structural

Fig. 6. Structural equation models showing relationships among the abundance of soil engineers, the normalized soil stability index, SOC allocation in micro­
aggregates within macroaggregates (mM), and total SOC stocks. Light gray box indicates exogenous variables (soil engineer abundance), and endogenous explanatory
variables are given with white boxes. Values associated with arrows (and line width) relate to path strength; the higher the value, the stronger the path strength.
Percentages close to endogenous variables indicate the proportion of variation explained for the given variable (r2). Solid arrows indicate significant standardized
coefficients (***p < 0.001, **p < 0.01). Dashed arrows indicate non-significant coefficients. Data did not significantly deviate from the models, except the model
with ants, which is not shown here. See main text for model fits, and the significance and strength of the indirect effects.

8
A.L.C. Franco et al.
stability and mM-C. This indirect mediation of total soil engineer and
termite abundances on SOC stock was statistically significant (P < 0.01
in both cases), while neither abundances nor soil structural stability had
a significant direct path to SOC stocks (Fig. 6A and B). In contrast to total
soil engineers and termites, and despite the overall models fitting the
data, the abundance of earthworms (Fig. 6C) and coleopterans (Fig. 6D)
showed no direct or indirect mediation over SOC storage. In addition, all
five models which included soil structure as exogenous and engineer
abundances as endogenous explanatory variables could be rejected as
potential explanation of the observed covariance matrix according to the
chi-square test (P < 0.05).
4. Discussion
Our previous studies provided evidence that sugarcane expansion
over pasturelands yield net increases in C emissions from the soil
(Franco et al., 2015; Mello et al., 2014). Here, we confirmed the hy­
pothesis that such SOC depletion following pasture to sugarcane LUC is
linked to reductions in the abundance of soil engineers, destabilization
of soil structure, and reduced C allocation within aggregates. Fitting
structural equation models to our dataset revealed that total soil engi­
neer abundance indirectly mediates total SOC stocks across land uses
through the stabilization of soil structure and promotion of C allocation
within mM (Fig. 6A). The direct effects within the SEM provided further
support to the widely described macrofauna–soil aggregation and soil
aggregation–SOC positive interactions (Ayuke et al., 2011; Bossuyt
et al., 2006; Ketterings et al., 1997; Pulleman et al., 2005a, 2005b).
Previous studies also pointed to the abundance and activity of soil en­
gineers as strong predictors of C accumulation in the soil (Frouz et al.,
2013; Józefowska et al., 2017).
4.1. Which soil engineers mediate structural stability and C storage across
land uses?
As soil engineer groups differ substantially in the type and magnitude
of engineering effect, we tested individual groups in separate models
and found evidence pointing to a more prominent role of termites than
any of the other groups in mediating SOC stocks through controls on soil
structure and C allocation in these tropical soils (Fig. 6). Soil-feeding
termites enhance aggregation stability either by passing soil material
through their intestinal system and depositing it as faecal pellets or by
mixing the soil with saliva using their mandibles (Six et al., 2004). They
build their nests and line their galleries with soil particles glued together
with fecal matter (Jouquet et al., 2006). The influence of termites on soil
structure is dynamic and varies in scale from mineral-weathering at the
clay particle scale to the internal reorganization of soil aggregates and
bioturbation at the soil profile scale (Bottinelli et al., 2015; Jouquet
et al., 2016).
This improvement of soil structural stability is critical for termites to
protect their colonies against water flux and the intrusion of predators
into nests (Jouquet et al., 2006) and has ecosystem-scale benefits to the
climate resilience of tropical landscapes, with termite mounds leading to
islands of fertility and plant production in tropical forests and grasslands
(Ashton et al., 2019; Bonachela et al., 2015; Duran-Bautista et al., 2020).
The lack of statistical support for the role of earthworms, coleopterans,
and ants controlling soil structural stability and C allocation was
somewhat surprising given the abundant literature evidencing that these
groups promote the formation of C-rich stable aggregates as they
burrow, nest, and forage on litter and soil (e.g., Ayuke et al., 2011;
Blouin et al., 2013; Brussaard et al., 2006; Lavelle et al., 2014). Never­
theless, these results corroborate the important role of termites in the
dynamics of soil aggregation and SOM stabilization in highly-weathered
soils (Reatto et al., 2009).
Importantly, we tested the alternative hypothesis that soil structure
would mediate soil engineer abundance in separate SEM for each
invertebrate group. The obvious example would be a compactly
9
Soil Biology and Biochemistry 150 (2020) 107998
compressed soil deprived of porous micro-habitats for large in­
vertebrates (e.g., Canisares et al., 2020), and thus unlikely to house
abundant soil engineer communities. Contrary to the SEM presented in
Fig. 6, all models which tested for this alternative hypothesis did not fit
the dataset (chi-square test P < 0.05), thus providing no evidence of soil
structure controls on soil engineer communities across our study sites.
We speculate that a complementary approach using the soil pore
network in addition to the aggregate size distributions used here could
potentially unravel effects of soil structure on invertebrates not detected
in our analyses (Six and Paustian, 2014).
4.2. Mechanisms of LUC effects on soil engineers, structural stability, and
SOM protection
Land-use effects on soil engineer communities included overall in­
creases in the relative abundances of ants and coleopterans and de­
creases in earthworms and termites in sugarcane sites (Fig. 1B). The
absence of frequent tillage in our pasture sites and high biomass inputs
from tropical grasses provide suitable conditions that often support high
abundances of termites and earthworms (Freymann et al., 2008; Schon
et al., 2015). This habitat suitability is greatly disrupted following LUC
to sugarcane cropping that involves ploughing and chemical inputs.
Earthworms and termites have been pointed out as the most important
soil engineers in tropical ecosystems (Lavelle et al., 1997), and therefore
this shift in community composition was expected to cause negative
effects on soil structure.
Even with contrasting soil textures among sites (Table 1), the effects
of LUC on size distribution of soil aggregates were broadly consistent
across the regional transect (Fig. 2). Pasture soils tended to have the
highest proportion of water-stable macroaggregates (Fig. 2) and similar
structural stability as soils under native vegetation (Fig. 3). The abun­
dant soil engineer community and root biomass of C4 tropical grasses in
combination with the absence of frequent tillage on pasturelands pro­
vide favourable conditions for macroaggregate formation and stabili­
zation via biological activity (Bach et al., 2010; Jastrow, 1987; Pinheiro
et al., 2004). Soil engineers such as termites and earthworms, dominant
in our pasture sites (Fig. 1B), can substantially enhance nutrient uptake
and biomass production in tropical pastures, and this positive interac­
tion between soil fauna and grasses enhances soil structural stability
through increased accumulation of SOM from plant roots within ag­
gregates (Fonte et al., 2012). The conversion of pastures to sugarcane
cropping destabilized soil structure (Figs. 2 and 3) even after only 3 and
4 years of cultivation, as observed respectively at Lat_17 S and Lat_21 S,
indicating a short-term increase in erosion susceptibility following LUC
(Cherubin et al., 2016). Silva et al. (2007) reported a reduction in
aggregate stability after 2 years of sugarcane cultivation in Northeast
Brazil, followed by a recovery of aggregate stability after 18 years of
cultivation. Our results at Lat_23 S, however, showed persistent re­
ductions in LM, MWD, and NSI after more than 20 years of sugarcane
cropping. Marked reductions in macroaggregates and MWD in sugar­
cane soils compared to uncropped soils were also reported by Blair
(2000). Every five years a cultivation cycle is implemented in sugarcane
fields with ploughing and fertilization for planting of new stem cuttings.
These tillage operations break soil aggregates (Cavalcanti et al., 2020),
thus destroying the habitats of soil engineers. Furthermore, successive
machine traffic for fertilization, weed control, harvest and transport
operations, impose compressive stress that may exceed the soil’s bearing
capacity, causing the breakup of macroaggregates and soil compaction
(Barbosa et al., 2019; Cavalcanti et al., 2019; Cherubin et al., 2016;
Esteban et al., 2020).
Our results also supported the hypothesis that LUC effects impair the
capacity of soils to physically protect SOM from decomposition within
stable aggregates. Land-use changes not only tended to reduce mM-C
(Fig. 4A), but it also increased the humification degree of this C frac­
tion (Fig. 5), with the magnitude of both effects being larger near the
surface than in deeper soil layers (Fig. 5). These results indicate that LUC
A.L.C. Franco et al. Soil Biology and Biochemistry 150 (2020) 107998

weaken SOM physical protection mechanisms and promote decompo­ Table S1 (continued )
sition of low-humified SOM occluded within aggregates (Six et al., Exogenous Endogenous variables P chi- CFI TLI RMSEA SRMR
2000b). These processes occur mainly at 0–10 cm soil depth, the most variables square
biologically active and tillage affected soil layer. Our results also provide Structural Earthworms – mM- <0.001 0.825 − 0.052 0.315 0.112
further evidence for mM-C being a robust indicator for LUC-induced stability carbon – SOC stock
SOC changes (Denef et al., 2007; Six and Paustian, 2014), especially Structural Coleopterans – mM- 0.035 0.980 0.882 0.106 0.040
stability carbon – SOC stock
following the conversion of native ecosystems on fine-textured soils
Structural Ants – mM-carbon – <0.001 0.827 0.259 0.287 0.098
(Fig. 4B). stability SOC stock

5. Conclusions

Our study links the negative effects of LUC on soil engineer abun­
dance, soil structural stability, and C allocation to explain reductions in Acknowledgements
total SOC stocks across a regional scale. We found that, over a range of
soil textures (16–66% clay), soil engineering by invertebrates indirectly A.L.C.F. and M.R.C thanks the São Paulo Research Foundation –
mediates changes in SOC storage by controlling the physical protection FAPESP (2012/22510-8, 2013/24982-7, 2013/17581-6, 2018/
of low-humified SOM occluded in microaggregates within macroaggre­ 09845–7) for the research and fellowship grants. We appreciate the
gates, a C fraction that accounted for most of the change in total SOC work of Bruno Castigioni, Marie Bartz, Evandro Schonell, Eduardo
stocks following the conversion of native forests. Therefore, negative Lucianer, Edpool Rocha, Manuela Testa, and Lucas Canisares during the
LUC effects on populations of soil engineers weaken soil functioning and field works and soil fauna sorting. We also thank Felipe Bonini da Luz for
its C storage. When analyzing the influence of the different invertebrate support with aggregates fractionation and microaggregates isolation,
groups separately, we found stronger evidence for the role of termites in and Débora Milori for support with the spectroscopy analyses.
this process compared to that of earthworms, coleopterans, and ants.
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