Applied Soil Ecology 164 (2021) 103937

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Applied Soil Ecology

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

The impacts of a logging road on the soil microbial communities, and

carbon and nitrogen components in a Northern Zone Costa Rican forest
William D. Eaton a, *, Katie M. McGee b, Robert Donnelly c, Alex Lemenze c, Morgan Larimer d,
Mehrdad Hajibabaei b
a
Pace University Biology Department, One Pace Plaza, New York, NY 10038, USA
b
Biodiversity Institute of Ontario, Department of Integrative Biology, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada
c
NJMS-Molecular Resource Facility Rutgers Biomedical and Health Sciences, 185 South Orange Ave., MSB, F-503, Newark, NJ 07103, USA
d
Water: Science and Governance Candidate at King’s College London, King’s College London, Strand London, WC2R 2LS, United Kingdom

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

Keywords: Logging road development is considered as potentially more damaging to a tropical forest than the felling of the
Soil microbial ecology actual trees. However, little work has been conducted to determine how logging road development impacts the
Soil bacteria soil microbial communities and associated C and N cycle activities in tropical forests. This study was conducted
Soil fungi
within an upland tropical forest in the Northern Zone of Costa Rica that had a 2-year abandoned logging road
Microbial structure
Microbial diversity
system (used for harvesting trees felled during a tornado) to determine how development of logging roads
affected soil C and N cycle activities, efficiency of organic C use, and bacterial and fungal community compo­
sitions. Soil samples from a set of logging roads, the road edges, and adjacent primary forests were analyzed for
C, N metrics, the Microbial Quotients, and DNA-based microbial taxonomic community compositions; which
were tested for differences using multivariate statistical analyses. The logging road soils had significantly greater
bulk density and clay, and lower levels of sand, TN, NO−3 , NO−3 /NH+ 4 , TOC, C Biomass, and Microbial Quotients
compared to the road edge and forest soils. The composition of the total bacterial genera of the road edge and
forest soils were similar to one another and different from that of the logging road soils, and the composition of
the total fungal genera was unique within each of the three areas sampled. The relative abundance of DNA
sequences of N-cycle bacteria were greater, and lignin degrading bacteria and wood rot/lignin degrading fungi
were less in the logging roads compared to the edge and forest soils. These results suggest that the rate of re­
covery of both the C and N cycle activities and associated microbial groups in the soils from the road edges is
occurring more rapidly than in the abandoned logging road soils. Thus, we suggest that a new tropical forest
management practice should include the movement of the slash and debris from the road edge regions onto the
logging roads after abandonment, as it would enhance the rate of recovery of both the C and N cycle activities in
the soils, and perhaps begin to address the concern that logging roads add an additional 10–15 years to tropical
forest recovery following deforestation.

1. Introduction
Tropical forests are known to be critical for carbon (C) storage
(Jobbágy and Jackson, 2000), yet by 2009, more than 30% of all global
terrestrial land was agricultural land previously created via tropical
deforestation (Asner et al., 2009; Gibbs et al., 2010). In addition, by
2013, approximately 60% of the decrease in tropical forests was for
conversion to agricultural use (Johnson et al., 2014; Pendrill et al.,
2019). In the Northern Zone of Costa Rica alone, over 40 years of
deforestation activities in the second half of the 20th century caused the
loss of 70% of the country’s humid Atlantic lowland forests (Chassot
et al., 2005; Leopold et al., 2001; Sánchez-Azofeifa et al., 2001). This
type of land management has resulted in over 50% decreases in vege­
tation biomass C storage around the world (Erb et al., 2017; Zomer et al.,
2016).
The development and use of logging road systems during such
deforestation is considered to be one of the most damaging components
of this extraction-based land use practice, and is thought to cause far

* Corresponding author.
E-mail address: weaton@pace.edu (W.D. Eaton).

https://doi.org/10.1016/j.apsoil.2021.103937
Received 2 August 2019; Received in revised form 11 February 2021; Accepted 14 February 2021
Available online 24 February 2021
0929-1393/© 2021 Elsevier B.V. All rights reserved.
W.D. Eaton et al.
greater forest ecosystem damage than the actual clearing of trees
(Alamgir et al., 2017; Gullison and Hardner, 1993; Sist et al., 2003;
Kleinschroth and Healey, 2017; Shirvani et al., 2020). Unfortunately, as
tropical deforestation is continuing to occur at alarming rates (Asner
et al., 2009; Giam, 2017; Hansen et al., 2013), so is the rate of devel­
opment and use of logging roads (Arima et al., 2008; Ahmed et al., 2013;
Gaveau et al., 2014; Kleinschroth et al., 2016a; Laporte et al., 2007),
with annual rates of road development as great as 40 km of new roads
per 10,000 km2 of forest area (Brandão and Souza, 2006).
Although studies have shown the impact of logging roads on above
ground tropical forest ecosystems (reviewed by Kleinschroth and Hea­
ley, 2017), the extent, intensity and mechanisms of damage these roads
might inflict on tropical soil ecosystems has been insufficiently studied
(Kleinschroth et al., 2016b; Kleinschroth and Healey, 2017; Takada
et al., 2015). Development of logging roads removes the forest topsoil
and exposes the subsoil, which when combined with truck traffic and
tractor/trailer use results in dramatic increases in soil compaction and
bulk density (Donagh et al., 2010; Guariguata and Dupuy, 1997;
Kleinschroth et al., 2016b). Subsequently, increased soil erosion also
occurs when the exposed subsoil gets directly subjected to the extensive
tropical rain (Clarke and Walsh, 2006; Kleinschroth et al., 2016b; Sidle
et al., 2004) and increased rates of surface water runoff and sediment
transport into streams (Kleinschroth et al., 2016b; Ziegler et al., 2007).
Some estimates suggest that the soil damage by logging roads inhibits
tropical forest recovery by an additional 10–15 years or more (Guar­
iguata and Dupuy, 1997; Kleinschroth et al., 2015; Kleinschroth et al.,
2016b).
The impacts of tropical deforestation and associated logging roads
have also been shown to negatively impact what are considered drivers
of soil biotic community structure. In particular, the increased soil
compaction and saturation, changes in soil pH, decreased levels of ox­
ygen, and decreased nutrient transfer capacity associated with logging
road development and use would likely impact soil microbial commu­
nities (Bradford and Crowther, 2013; Fierer et al., 2009). In addition, the
loss of vegetation, and reduced quantity and quality of the plant-based
resource inputs into the soils would also cause alterations in soil mi­
crobial populations, by changing the plant-soil feedback system (Ben­
nett and Klironomos, 2019; Fujii et al., 2018; Hobbie, 2015; Kulmatiski
et al., 2008; Pugnaire et al., 2019; Van der Putten et al., 2013). Many
studies have shown that such alterations to the plant and soil commu­
nities could have negative consequences for the C and N cycle dynamics,
such as selecting for less effective decomposition activities, shifts to­
wards the breakdown of more labile forms of organic C, increased rates
of CO2 release, and decreased efficiency of use of soil organic of C and N
for biomass (Anderson and Domsch, 2010; Banning et al., 2011; Bennett
and Klironomos, 2019; Bradford and Crowther, 2013; Bardgett and Van
der Putten, 2014; Fujii et al., 2018; Hobbie, 2015; Kardol and Wardle,
2010; Manzoni et al., 2012; Takada et al., 2015; Van der Putten et al.,
2013). These impacts on the tropical forest soils will negatively affect
the rates of recovery of the diversity and health of the vegetation com­
munities, and proper forest ecosystem functioning following disturbance
(Bardgett and van der Putten, 2014; Bossio et al., 2005; Bradford and
Crowther, 2013; Manzoni et al., 2012; Rodrigues et al., 2013; Pugnaire
et al., 2019). Consequently, determining the influence of logging road
development and use on the composition and functions of the soil mi­
crobial community and associated C and N components should be a
critical research focus for predicting future tropical ecosystem function
and recovery after deforestation, yet such studies are rare, receiving far
less attention compared to that of the aboveground biota (Loreau,
2010).
The assessment of the soil microbial community structure is now
being used to assess the impacts of different land management practices
on soil ecosystems (Banning et al., 2011; Bardgett and van der Putten,
2014; Eaton et al., 2019; McGee et al., 2018), in part due to the plant –
soil feedback system in which forest soil microbial communities influ­
ence and are influenced by the plant community composition (Bennett
2
Applied Soil Ecology 164 (2021) 103937
and Klironomos, 2019; Fujii et al., 2018; Van der Putten et al., 2013).
Moreover, it is known that these two communities collectively regulate
the types and rates of organic matter decomposition and soil organic
matter (SOM) development, C and N biomass development, C stabili­
zation and storage, and other C and N cycle processes important for
forest ecosystem health and recovery after disturbance (Bradford et al.,
2008; Madsen, 2011; Eaton et al., 2012; Eaton et al., 2017, 2019, 2020;
Hafich et al., 2012; Ibekwe et al., 2007; Kivlin and Hawkes, 2016;
Rodrigues et al., 2013; Pugnaire et al., 2019; Waring et al., 2013).
The soil bacterial populations have long been known to be involved
in the early stages of development of soil C and N processes (Lovell et al.,
1995; Moore and de Ruiter, 1991) such as decomposition of less complex
organic C, N-fixation, and ammonium oxidation that are critical in
managed, young, or recently damaged soils (Briones, 2014; Coleman
et al., 2017; Eaton et al., 2017, 2019, 2020; García-Orenes et al., 2013;
Madsen, 2011; Pajares and Bohannan, 2016; Tiwari et al., 2019). In
addition, due to their critical role in early recovery of soils, these bac­
terial groups would also be expected to be among the pioneer soil biota
to re-establish in the soils following abandonment of the roads.
The soil fungal communities are more important in later soil suc­
cessional stages, such as older forest soils or successfully restored soils,
due to their abilities to decompose the complex organic substrates more
efficiently than bacteria, leaving behind recalcitrant residues, thus,
enhancing the soil organic C matter (Briones, 2014; Coleman et al.,
2017; Eaton et al., 2017, 2019, 2020; García-Orenes et al., 2013; Luis
et al., 2004; Sinsabaugh, 2010; Talbot et al., 2008), and would be ex­
pected to occur in the later stages of soil recovery from logging road use.
It is assumed that both the bacterial and fungal populations would be
negatively impacted by the types of alterations in the quantity and
quality of plant nutrient input, the woody debris, the concentrations of
critical substrates, pH, temperature levels, and oxygen availability
typically associated with logging road development and use (Brienen
et al., 2015; Nottingham et al., 2015), thus, negatively affecting the soil
C and N cycle dynamics and diminishing the viability of the soils for
forest regeneration. However, there is still much work to be conducted
to understand the how logging roads impact the tropical forest soils after
deforestation.
Given that the impacts of logging roads on the tropical forest soil
biotic and abiotic ecosystems components are not well-established
(Loreau, 2010), the current study was an attempt to provide some
initial clarity on this issue for a specific region of tropical forests in the
Northern Zone of Costa Rica. Our goal was to identify potential biotic
and abiotic indicators of the soil ecosystem conditions along a distur­
bance gradient from a logging road to an intact Northern Zone primary
forest to provide a suite of tools for the future assessment of soil
ecosystem recovery post-abandonment of logging roads after defores­
tation in this region. To do this, our aim was to determine how differ­
ences in the C and N cycle metrics and efficient use of the soil organic C
were associated with changes in the bacterial and fungal community
compositions along a disturbance gradient from logging roads, their
edges, to the adjacent humid Atlantic lowland primary forest in a spe­
cific region of the Northern Zone of Costa Rica. Five broad questions
were asked: (1) How do the soil C and N-cycle metrics, C biomass
development, and the Microbial Quotient indicator of efficient use of
soil organic C (Anderson and Domsch, 1989; Maia et al., 2007) differ
between soils along the disturbance gradient? (2) Are there differences
in the overall bacterial and fungal community compositions between
these soils? (3) Are there differences in the composition of several crit­
ical functional groups important to the N and C-cycle activity and
development of the Microbial Quotient between the soils? (4) Do the C
and N cycle metrics, biomass and/or Microbial Quotients best explain
and predict the soil microbial community structures? (5) Which of these
metrics best serve as potential indicators of soil condition during re­
covery from damage?
We proposed that the compacted soil conditions and loss of soil ho­
rizons, trees, woody debris, and other vegetation during development
W.D. Eaton et al.
and use of the logging roads would obviously disturb the forest soil
microbial communities and C and N components. In particular, there
would be decreased levels of inorganic N, C biomass, and efficiency of C
utilization in the logging road soils, which would be associated with
reduced relative abundances of lignin-degrading microbes due to loss of
woody material, and greater relative abundances of N-cycle bacteria due
to the need to recover the soil N lost in the road areas. Further, we
predicted that the movement of the soil, vegetation, debris, etc. from the
forest to the edge regions of the logging roads would create raised areas
on the road edges that would have soil ecosystem conditions more
similar to the forest than the logging road.
2. Materials and methods
2.1. Site description and sample collection
The Maquenque National Wildlife Refuge (MNWR; 10◦ 27′ 05.7′′ N,
84◦ 1′ 24.32′′ W) was created in the Northern Zone of Costa Rica in 2005
to protect over 50,000 ha of humid Atlantic lowland primary and sec­
ondary tropical forests and other diverse ecosystems such as wetlands
(Chassot and Monge, 2006). Mean annual temperature within the
MNWR is 27 ◦ C and the mean annual rainfall is 4300 mm (Hartshorn and
Hammel, 1994). The dominant soil type in the Upland Forest areas used
in this study are also oxisols (Hartshorn and Hammel, 1994), and also
have been shown to have the same physical characteristics (McGee et al.,
2018, 2019).
In June 2010, a tornado hit the Uplands Forests on the lands of the
Laguna del Lagarto Lodge in the center of the MNWR, creating numerous
~50 m diameter circles of felled and broken trees, along approximately
2 km of forest. In order to harvest the trees, a network of logging roads
was installed and used between June 2011 and August 2013, at which
time the roads were then abandoned. The logging roads were approxi­
mately 10 m wide with 3 m edges on both sides of the roads that were
composed of the slash and debris scraped from the forest during road
construction. The current study was conducted in July of 2015, two
years after road abandonment. At that time, the logging roads were
mostly barren, with the exception of low densities of scattered 1–3 m tall
Vismia spp. and Cecropia spp. early successional trees and random
patches of 1–2 cm tall herbaceous growth. Soil samples were collected
from three regions associated with different logging roads (hereafter
called zones): the logging roads themselves (LR), the edges of the log­
ging roads (Edge), and the adjacent primary forest (Forest). Soils
sampled in the logging roads (LR samples) were not within 1.5 m of any
of the small trees, and not within any of the herbaceous patches.
Six logging roads were used for this study, all within the same Up­
lands Forest, and all roads had been abandoned for 2 years with no
restoration measures taken. However, during road development all
vegetation, woody debris, forest litter, O and A horizon layers had been
scraped away by tractors, leaving only the lower clay layer. The material
scraped from the forest had been placed to the sides of the logging roads,
and is the zone we are calling the Edge zones. The LR zone samples were
collected within approximately the middle of the roads, where two 2 m
× 2 m soil sample areas, about 1–2 m apart, were established within
each of the six logging roads, and within these sample areas two soil
profile cores (7.5 cm × 15 cm × 1.25 cm) were collected to a sampling
depth of 15 cm depth. Two 2 m × 2 m soil sample areas were similarly
established within each of the Edge and Forest zones. The Forest zone
sample areas were perpendicular to the LR and Edge locations, and
established at points 15 m and 20 m into the forest from the Edge zones.
All soil samples were collected between June 20–25, 2015, on days with
no rain. All soil samples were aseptically collected from each 2 m × 2 m
area within each of the three zones associated with the six different
logging roads, resulting in 12 replicate samples from each zone (6 roads
× 2 sample areas for each of the 3 zones per road).
To ensure the collection of true replicate and independent samples
from the zones within the different LR, Edge, and Forest zones, each
3
Applied Soil Ecology 164 (2021) 103937
logging road chosen was separated by 10–200 m. Due to the differences
in spatial scale associated with soil microbes as compared to “above-
ground” organisms, the samples collected from the different logging
roads were considered to be separated at the landscape-level separation
scale, resulting in 12 true replicate soil samples for each zone (Ettema
and Wardle, 2002; Bardgett and van der Putten, 2014). All soil samples
were passed through sterilize 4 mm sieve at field moist conditions prior
to all downstream analyses.
2.2. Carbon and nitrogen properties
Subsamples (200 g) of field moist soil from each soil sample were
delivered to the Center for Tropical Agriculture Research and Education
(CATIÉ) Laboratories in Turrialba, Costa Rica. The soil pH, density, the
% soil moisture, and the % sand, silt and clay were determined using
standard methodologies. As well, the total organic C (TOC) mass levels
were analyzed via dry combustion analysis of Anderson and Ingram
(1993); total N mass (TN) levels were determined by the Kjeldahl
method, and NH+ 4 and NO3 levels were measured from 2 M KCl extracts
−
using the spectrophotometric methods of Alef and Nannapieri (1995).
The ratio of NO−3 /NH+ 4 was calculated as a potential indicator of the
activities of the ammonium oxidizing bacteria in the soils. The soil
biomass (as Cmic) was determined by the substrate induced respiration
(SIR) method at the field site laboratory, following the methods of Höper
(2006), which measures only the living biomass, and does not include
dead cellular materials. The ratios of Cmic/TOC, or the Microbial Quo­
tient, were calculated as a potential indicator of the efficiency with
which the living (and not the living and dead) microbial communities
were utilizing organic C in biomass development (Anderson, 2003;
Anderson and Domsch, 1989).
2.3. DNA extraction, sequencing, and bioinformatics
Environmental microbial DNA (eDNA) was extracted from three
0.33 g replicate subsamples per independent soil zone sample, for a total
of 1 g for each soil sample, using the MoBio PowerSoil DNA Isolation Kit
(MO BIO Laboratories Inc., Carlsbad, CA, USA). The three replicate
eDNA extracts were then pooled to obtain a composite eDNA sample for
each of the 12 replicate soil samples from each 3 zones. The concen­
tration and purity (A260/A280 ratio) of the eDNA were determined prior
to downstream analyses using a NanoDrop 1000 spectrophotometer
(ThermoFisher Scientific, Waltham, MA). All downstream analyses are
described in detail by McGee et al. (2018). Briefly, eDNA extracts were
amplified via 2-step PCR, targeting the internal transcribed spacer (ITS)
rRNA gene region for fungi (Gardes and Bruns, 1993), and the v3 and v4
region of 16S rRNA gene region for bacteria and archaea (Caporaso
et al., 2011). All amplicons were dual indexed and sequenced in MiSeq
runs using a V3 MiSeq sequencing kit (600 cycles – 300 bp × 2; FC-131-
1002 and MS-102-3003). The subsequent sequences were analyzed
using a standard QIIME protocol (Caporaso et al., 2011) with in-house
Python data management scripts. Input reads were processed through
QIIME’s open reference clustering with standard parameters and 97%
similarity to the GreenGenes database or UNITE database for bacterial or
fungal analysis, respectively. The open reference clustering performed
all operational taxonomic unit (OTU) groupings compared to the data­
base as well as de novo OTU generation, and assigned known taxonomy
where possible using the UCLUST algorithm. All generated sequencing
data were submitted to the NCBI-Gene Expression Omnibus (GEO) re­
pository on August 1, 2019 (Submission: SUB6145149, BioProject:
PRJNA559202).
The soil fungal and bacterial DNA-based OTUs were organized
taxonomically at the genus rank. The variable library sizes were
normalized by converting read number to relative percent proportion
(Weiss et al., 2017), hereafter called the relative percent abundance (%
RA), as the number of sequences from each genus of bacteria or fungus
within each soil sample, divided by the total number of sequences per
W.D. Eaton et al.
that sample. The richness and diversity of the total fungal and bacterial
genera was determined by calculating Margalef’s Richness index (d = (S
− 1)/Log(N)), and the classical Shannon’s Diversity index H. Margalef’s
index is used to take into account the sample size, whereas traditional
species richness does not.
In addition to conducting an analysis of the differences in the
composition of the overall fungal and bacterial genera within each of the
3 logging road sample zones, we were interested in attempting to
approximate the differences between zones in the microbial commu­
nities with the potential to perform certain functions. The functional
groups of interest were the N-cycle associated bacteria (i.e., those
associated with N-fixation or ammonium oxidation), the lignin
degrading bacteria (Lignin) and the wood rot/lignin decomposer (WRT/
Lignin) fungi. Lists of microbial genera within these functional groups
were generated for use in this study by examination of 75 publications
(Table S1) that connected specific microbial genera to the certain
functional groups via either growth characteristics or the presence of
genes for specific enzymes (such as the nifH gene complex for N-fixing).
This method was not meant to indicate definitive representations of any
functional group activity in these soils. However, it is intended as a
component within a suite of possible indicators to suggest differences in
the soil biotic communities between the sample zones. The %RA of these
groups was also determined.
2.4. Data analysis
To address question 1 (i.e., Were there differences in C and N metrics
between the 3 zones?), we used a one-way analysis of variance (ANOVA)
followed by Tukey’s HSD post hoc tests in SPSS (v. 25, Armonk, NY,
USA) to determine if the mean values of the N and C-cycle metrics, the
Cmic, and/or Microbial Quotients were significantly different between
habitats. Prior to ANOVA, the Levene’s test was used in SPSS, which
showed that the variances of the data were heterogenous, supporting the
use of ANOVA testing.
To address question 2 (Were there differences in the bacterial and
fungal communities between the 3 zones?), the DNA sequence %RA data
of the total bacterial and fungal community compositions were fourth
root transformed, prior to establishment of the Bray-Curtis resemblance
matrices (Anderson et al., 2008; Clarke, 1993; Clarke and Gorley, 2006).
Then 1-way (with habitat as the factor) main and pairwise permuta­
tional multivariate analysis of variance (PERMANOVA) tests were con­
ducted on these Bray-Curtis resemblance matrices of the %RA data, and
also on the richness and diversity data using PRIMER-E v. 6 (Clarke and
Gorley, 2006) with its add-on PERMANOVA+ (Anderson et al., 2008).
All main and pair-wise PERMANOVA tests were based on 9999 unre­
stricted permutations. A Canonical Analysis of the Principal Coordinates
(CAP) was also used to determine the strength of the differences in %RA
of the different microbial taxa between the different habitats using the
PERMANOVA+ guidelines (Anderson and Willis, 2003; Anderson et al.,
2008). The CAP shows the strength of the differences in %RA between
habitats, which is based on how successfully the CAP model was able
correctly discriminate the different sample results and place them into
their appropriate groups Strong differences in the microbial commu­
nities between the different habitats are indicated by CAP axis squared
canonical correlations (R2) ≥ 0.7, and moderate differences are indi­
cated by squared canonical correlations (R2) ≥ 0.5 to 0.69 (Anderson
and Willis, 2003).
To address question 3 (Were there differences in the functional group
communities between zones?), the DNA sequence %RA data of the
different bacterial and fungal functional groups established into Bray-
Curtis resemblance matrices as above. The different matrices were
analyzed by PERMANOVA and CAP as described above. Mann-Whitney
tests were conducted in SPSS to determine if there were differences in
the %RA of any genera from the three functional groups between the soil
zones.
To address question 4 (Were there any C or N metrics that best
4
Applied Soil Ecology 164 (2021) 103937
predict and explain the differences in soil microbial community struc­
tures between the 3 zones?), a distance-based linear model permutation
test (DistLM) was implemented using PERMANOVA+ to identify which
soil environmental variables best predicted the multivariate patterns of
the soil bacterial and fungal communities across the three different soil
zones analyzed. We used the fourth root transformed resemblance
matrices of the taxonomic data as response variables, and the log (x + 1)
transformed the C, N, Cmic, and Microbial Quotient data as the predictor
variables (as recommended in the PERMANOVA+ guidelines). The
DistLM sequential tests are performed on the different environmental
predictor variables in a specific order, determining if the addition of a
particular environmental variable to the model significantly improves
the explanation of the variation in the biotic data cloud patterns. This
method takes into account the covariance between environmental pre­
dictor variables, and allows one to infer which environmental variables,
when combined, best explain, drive, or are structuring the differences in
the multivariate patterns of the microbial communities (Legendre, 2000;
Legendre and Fortin, 2010). The DistLM analyses were performed using
a “step-wise” selection process, 9999 permutations and the AICc
(Akaike’s information criterion corrected; Akaike, 1978) selection cri­
terion, as recommended for studies in which the number of samples (N)
is small relative to the number (v) of predictor variables (N/v < 40,
Anderson et al., 2008), as was the case in this study.
3. Results
3.1. Soil carbon and nitrogen properties
The ANOVA post hoc tests showed significant differences between
the environmental metric data in the soils from the 3 samples zones
studied, with the characteristics of the Edge and Forest soils being
similar, and the LR samples being more distinct from the other two soils
(Table 1). Specifically, the LR soils had the greatest soil density and %
clay, while both the Forest and the Edge soils had % sand, TN, NO−3 /
NH+ 4 , TOC, Cmic, and Microbial Quotient levels that were similar be­
tween these two habitats and greater than those from the LR samples (all
p values < 0.05). The levels of NO−3 were greatest in the Forest soils, least
in the LR soils, and intermediate in the Edge soils (all p values < 0.05).
3.2. DNA sequencing taxonomic data
3.2.1. Analyses of total bacterial and fungal genera
The NGS of the soil eDNA successfully identified 299 bacterial
sequence types, or OTU’s, as distinct genera across the 3 different hab­
itats. The results of the PERMANOVA (Table 2) showed there were
significant differences in the composition of the total bacterial genera
between the LR and Edge soils (Pseudo-F = 1.67, p = 0.074), and be­
tween the LR and Forest soils (Pseudo-F = 1.72, p = 0.018), but no
differences were evident in the bacterial community composition be­
tween the Forest and Edge soils (Pseudo-F = 0.81, p = 0.668). The mean
bacterial richness and diversity levels (Table 3a) were not different be­
tween any of the soil zones sampled (PERMANOVA results: Pseudo-F
range = 0.054 to 0.324, p range = 0.568 to 0.886; Table 3b). The CAP
analysis of the total bacterial community %RA data (Fig. 1a) indicated
there were mostly differences in the composition of the total bacterial
communities between the soil areas sampled. Strong separation was
shown between the soil bacterial communities of the LR and the Forest
and the LR and Edge zones (CAP 1 R2 = 0.71; p = 0.0001). However,
there was less separation of the communities between the Edge and the
Forest soil zones (CAP 2 R2 = 0.16; p = 0.0001). The CAP analysis model
correctly allocated 68.8% of the permutation samples to the appropriate
habitats (LR = 75.0%, Edge = 72.2%, Forest = 61.1%).
The NGS of the soil eDNA successfully identified 169 fungal sequence
types, or OTU’s, as distinct genera across the 3 different habitats. The
PERMANOVA results (Table 2) showed there were statistically signifi­
cant differences in the total fungal community composition between the
W.D. Eaton et al. Applied Soil Ecology 164 (2021) 103937

Table 1
The mean values of the soil environmental metrics from the 3 zones examined associated with logging roads (Forest = primary forest; LR = logging road; Edge = edge
between the forest and the LR) within the same Uplands Forested area in the Maquenque National Wildlife Refuge in the Northern Zone of Costa Rica. The metrics
analyzed were the % sand, % silt, % clay, soil density, total nitrogen (TN), total organic carbon (TOC), N-NH+
4 , N-NO3 , the ratio of NO3 /NH4 , Cmic, and the microbial
− − +

quotients (as Cmic/TOC). The patterns of the significant differences in mean values between habitats, and ANOVA F Stat and p values are given.
Mean values (±S.D.) ANOVA main ANOVA post hoc

Logging road Edge Forest F stat p Value Mean value patterns p Value range

% Sand 44.84 (±16.95) 57.54 (±9.85) 62.39 (±5.21) 8.9 0.002 Forest and Edge > LR 0.0016–0.0001
% Silt 12.45 (±2.55) 15.13 (±5.81) 12.06 (±2.22) 2.36 0.1067 Not done Not done
% Clay 42.71 (±18.41) 27.32 (±8.98) 25.55 (±4.33) 9.52 0.0006 LR > Forest and edge 0.0019–0.0004
Density (g/cc) 2.62 (±0.07) 2.57 (±0.10) 2.54 (±0.09) 4.7 0.0134 LR > Forest and Edge 0.0320–0.0027
TN (μg N/g) 25.67 (±6.68) 50.25 (±9.53) 50.42 (±4.74) 13.29 0.0001 Forest and Edge > LR 0.0002–0.0003
ToC (μg C/g) 462.58 (±88.5) 642.75 (±172.0) 611.58 (±88.8) 10.6 0.0002 Forest and Edge > LR 0.0009–0.0001
N-NH+ 4 (μg N/g) 2.30 (±1.34) 1.61 (±0.44) 1.70 (±0.71) 0.76 0.5031 Not done Not done
N-NO−3 (μg N/g) 12.13 (±3.93) 18.86 (±3.29) 22.04 (±3.70) 5.3 0.0085 Forest > Edge > LR 0.0494–0.0042
NO3/NH4 7.20 (±3.93) 11.68 (±2.84) 16.84 (±2.17) 2.69 0.0663 Forest and Edge > LR 0.0495–0.0382
Cmic (μg C/g) 621.40 (±183.2) 1104 (±182.2) 1347.46 (±254.5) 4.15 0.0187 Forest and Edge > LR 0.0122–0.0160
Microbial quotient 1.38 (±0.44) 1.79 (±0.45) 2.2 (±0.35) 5.13 0.0.0002 Forest and Edge > LR 0.047–0.0010
Soil pH 5.73 (±0.51) 5.88 (±0.47) 5.56 (±0.26) 1.23 0.2919 Not done Not done
% Moisture 57.57 (±14.41) 52.92 (±9.13) 57.93 (±7.58) 0.87 0.425 Not done Not done

Table 2
The PERMANOVA results showing differences in the composition of the total
bacterial and total fungal communities, and the bacterial N-cycle and lignin
degrading, and the fungal wood rot/lignin decomposing communities from the 3
soil zones examined (Forest = primary forest; LR = logging road; Edge = edge
between the forest and the LR) within the same Uplands Forested area in the
Maquenque National Wildlife Refuge in the Northern Zone of Costa Rica. The
main and pairwise PERMANOVA pseudo-F stat and p values are giving for each
comparison, as well as the trends in the %RA values of the 3 functional groups by
habitat.
PERMANOVA main and pairwise tests Pseudo-F p Value
Table 3
Analyses of the richness and diversity of the communities of the total bacterial
genera and the total fungal genera in soils from the 3 zones examined associated
with logging roads (Forest = primary forest; LR = logging road; Edge = edge
between the Forest and the LR) within the same Uplands Forested area in the
Maquenque National Wildlife Refuge in the Northern Zone of Costa Rica. a)
Mean values of the Richness and diversity of the total fungal genera and total
bacterial genera across the 3 zones examined; b) Results of the PERMANOVA
tests for differences in the richness or diversity values, where pseudo-F stat and p
values are provided to demonstrate the relative strength of the differences.
a)

Main test on total bacterial genera 1.58 0.028 Bacterial Bacterial Fungal Fungal
Pairwise test: LR to Edge 1.67 0.074 richness diversity richness diversity
Pairwise test: LR to Forest 1.72 0.018
LR 21.04 (±7.62) 4.16 (±0.40) 11.56 (±4.93) 3.51 (±0.62)
Pairwise test: Edge to Forest 0.81 0.668
Edge 21.78 (±7.00) 4.18 (±0.34) 12.08 (±4.78) 3.52 (±0.71)
Main test on total fungi 7.08 0.001
Forest 22.08 (±5.72) 4.21 (±0.29) 7.358 (±0.32) 2.58 (±1.27)
Pairwise test: LR to Edge 3.91 <0.001
Pairwise test: LR to Forest 10.19 <0.001
Pairwise test: Edge to Forest 8.92 0.002 b)

PERMANOVA results of the richness and diversity of the total microbial genera
PERMANOVA main and pairwise Pseudo- p Value Trends of %RA of
Bacterial Pseudo- p Bacterial Pseudo- p
tests F microbial groups
richness F Value diversity F Value
Main test on lignin degrading 1.89 0.024 Forest > Edge and LR
Main test 0.165 0.847 Main test 0.165 0.847
bacteria
Pairwise tests Pairwise tests
Pairwise test LR to Edge 0.97 0.446 No difference observed
LR to Edge 0.11 0.738 LR to Edge 0.039 0.837
in %RA
LR to Forest 0.324 0.568 LR to Forest 0.206 0.651
Pairwise Test LR to Forest 2.93 0.009 Forest > LR
Edge to 0.054 0.886 Edge to Forest 0.072 0.788
Pairwise test Edge to Forest 1.88 0.065 Forest > edge
Forest
Main test on N-Cycle Bacteria 1.49 0.072 LR > Edge and forest
Pairwise test: LR to Edge 1.79 0.059 LR > Edge
Pairwise test LR to Forest 2.06 0.031 LR > Forest b)
Pairwise test Edge to Forest 0.86 0.615 No difference observed
in %RA PERMANOVA results of the richness and diversity of the total microbial genera
Main test on wood rot/lignin 3.41 <0.001 Forest and Edge > LR Fungal richness Pseudo- p Value Fungal diversity Pseudo- p Value
decomposer fungi F F
Pairwise test LR to Edge 3.78 0.002 Edge > LR
Pairwise test LR to Forest 5.65 0.003 Forest > LR Main test 3.95 0.027 Main test 4.83 0.013
Pairwise test Edge to Forest 1.09 0.226 No difference observed Pairwise tests Pairwise tests
in %RA LR to Edge 0.027 0.896 LR to Edge 0.005 0.963
LR to Forest 4.745 0.04 LR to Forest 5.804 0.023
Edge to 4.893 0.036 Edge to 5.234 0.034
LR and Edge (Pseudo-F = 3.91, p ≤ 0.001), LR and Forest (Pseudo-F = Forest Forest

10.19, p ≤ 0.001) and the Edge and the Forest (Pseudo-F = 8.92, p =
0.002) soils. The mean richness and diversity of the total fungal com­ fungal genera communities between all three habitat soil communities
munity (Table 3a) were found to be not different between the LR and (CAP 1 R2 = 0.92 and CAP 2 R2 = 0.75; p = 0.0001). The CAP model
Edge soils by PERMANOVA tests (Pseudo-F = 0.027, p = 0.896; and correctly allocated 93.7% of the permutation samples to the appropriate
Pseudo-F = 0.005, p = 0.963; respectively; Table 3b), but were different habitat (LR = 91.7%; Edge = 94.4%; Forest = 94.1%).
between the LR and Forest soils (Pseudo-F = 4.745, p = 0.040), and the
Edge and Forest soils (Pseudo-F = 5.804, p = 0.023). The CAP analysis
(Fig. 1b) showed was strong separation in the composition of the total

5
W.D. Eaton et al.
a)
b)
Fig. 1. Canonical Analysis of Principal Coordinates (CAP) showing the strength
of the dissimilarity of the overall bacterial and fungal community composition
within the soil ecosystems from the 3 zones examined (Forest = primary forest;
LR = logging road; Edge = edge between the forest and the LR) within the same
Uplands Forested area in the Maquenque National Wildlife Refuge in the
Northern Zone of Costa Rica. a) CAP showing strong dissimilarity in the overall
bacterial community composition between the LR and Forest soils and the LR
and Edge soils (CAP 1 R2 = 0.71, p = 0.0001), but not between the Forest and
the Edge soils (CAP 2 R2 = 0.16, p = 0.0001). b) CAP showing strong dissim­
ilarity in the overall fungal community composition between the soils of all
three sample zones (CAP 1 R2 = 0.92 and CAP 2 R2 = 0.75; and p = 0.0001).
3.2.2. Analyses of proposed bacterial and fungal functional group genera
There were 25 bacterial genera proposed to be associated with lignin
degradation found across the three sample areas (Fig. 2a). The %RA of
the genera from this group were greater in the Forest soils compared to
either the Edge or LR soils (Table 2), as shown by the PERMANOVA tests
(Pseudo-F = 1.88, p = 0.065; Pseudo-F = 2.93, p = 0.009, respectively).
The most common lignin degrading bacteria were Solibacter, Bradyrhi­
zobium, Burkholderia, Bacillus, Flavobacterium, Pandoraea, and Sphingo­
monas, which were present in all three sample zone types (Fig. 2b).
Notably, the %RA of the genus Burkholderia was greatest in the Forest
soils, and the %RA of Bradyrhizobium was greatest in the LR soils
(Fig. 2b). Members of the genus Solibacter had the greatest %RA in all 3
habitats (41.73–49.46%RA), which were greater in the Forest soil
samples in comparison to the other sites (Fig. 2c). The CAP analysis of
this group of genera (Fig. 3a) showed some separation between the LR
and Forest soil communities (CAP 1 R2 = 0.31, p = 0.014), but not be­
tween the Edge and Forest or the LR and Edge soils (CAP 2 R2 = 0.07; p
= 0.014). The CAP analysis model allocated 58.3% of the permutation
samples to the correct habitat, with the Forest samples being more
6
Applied Soil Ecology 164 (2021) 103937
consistently allocated correctly than the samples from the other two
habitats (LR = 50.0%, Edge = 40.4%, Forest = 77.8%).
There were differences found in the %RA of 23 bacterial genera
identified across the habitats proposed to be associated with N-cycle
activities (Fig. 2a). The PERMANOVA (Table 2) showed that the %RA of
the genera from this group were significantly greater in the LR than the
Edge soils (Pseudo-F = 1.79, p = 0.059) and LR than the Forest soils
(Pseudo-F = 2.06, p = 0.031). Four genera appeared to dominate from
this group in all three sample zones (Fig. 2d). These were Burkholderia
(ammonium oxidation and N-fixation groups), Rhodoplanes (N-fixation),
Nitrospira (ammonium oxidation group), and Bradyrhizobium (N-fixa­
tion). Although the %RA of Rhodoplanes was fairly constant across
habitats, the %RA of Nitrospira and Bradyrhizobium was greatest in the
LR soils, and Burkholderia was greatest in the Forest soils (Fig. 2d). The
CAP analysis of the %RA data for this group (Fig. 3b) showed nearly
strong separation between the LR and Forest soil habitat communities
(CAP 1 R2 = 0.69 p = 0.0001), but very little difference in the compo­
sition of this group occurring between the Edge and the other two soil
zones (CAP 2 R2 = 0.11; p = 0.0001). The CAP analysis model allocated
67.6% of the permutation samples to the correct habitat, with the Forest
samples being more consistently allocated correctly than the samples
from the other two habitats (LR = 77.8%, Edge = 66.7%, Forest =
61.5%).
There were 41 genera proposed to be WRT/Lignin fungi (Fig. 2a)
found across the different sample zones. The PERMANOVA results
(Table 2) showed that the %RA of this group of genera were greater in
the Edge and Forest soils than found in the LR soil samples (Pseudo-F =
3.78, p = 0.002; Pseudo-F = 5.65, p = 0.003). The most common genera
present in all zones were Scedosporium, Podospora, Penicillifer, Hypocrea,
Gliocladiopsis, Fusarium, Cylindrocladium, and Calonectria (Fig. 2e). The
Forest and Edge soils had significantly greater %RA of Hypocrea, Cylin­
drocladium, and Calonectria than found in the LR soils, while the LR soils
had significantly greater %RA of Scedosporium, Podospora, Penicillifer,
Gliocladiopsis, and Fusarium than found in the Forest, and in some cases,
the Edge soils (Fig. 2e). The CAP results on the WRT/Lignin fungal
genera (Fig. 3c) indicated moderate separation existed between the LR
and both the Forest and Edge soil zones (CAP 1 R2 = 0.51, p = 0.0002),
but less separation existed between the Edge and the Forest community
compositions (CAP 2 R2 = 0.18; p = 0.0002). The CAP analysis model
allocated 62.5% of the permutation samples to the correct habitat (LR =
75.0%, Edge = 61.1%%, Forest = 55.6%).
3.2.3. DistLM modeling
The DistLM (Table 4) results showed that some of the soil environ­
mental data were good predictors of the variations observed in the
multivariate data cloud of the composition of both the total fungal and
bacterial genera communities and the communities of the proposed
functional groups. The soil TN, % Clay and % Moisture were the best
predictors of the total bacterial community patterns across the soil zones
(Pseudo-F = 2.63, p = 0.0108, AICc = 293.47), explaining 21.8% of the
variation observed in the total bacterial community composition. The
levels of the Biomass (or Cmic) and % silt in the soils were the best
predictor of the total fungal community patterns observed, explaining
12.2% of the variation within this group (Pseudo-F = 2.99, p = 0.0156,
AICc = 344.58). The TN, % Sand, and % Moisture were the best pre­
dictors of the structure of the Lignin Degrading bacterial communities
(Pseudo-F = 2.50, p = 0.0151, AICc = 220.23), explaining 20.6% of the
variation within the structure of this community. The % Moisture, and
levels of TN and NO−3 explained 20.8% of the variation in the N-cycle
bacterial communities (Pseudo-F = 2.74, p = 0.0109, AICc = 217.95),
while TOC levels and %Sand explained 13.0% of the WRT fungal com­
munity composition (Pseudo-F = 2.89, p = 0.0097, AICc = 242.39).
4. Discussion
The development of extensive systems of logging roads resulting
W.D. Eaton et al. Applied Soil Ecology 164 (2021) 103937

a) c)

N-Cycle Bacteria LR

Habitats
WRT/Lignin Fungi
Edge

Lignin Bacteria
Forest

0 10 20 30 40 50 60 70 80 90
0 10 20 30 40 50 60
% Relave Abundance of Microbial Funconal Groups
% Relave Abundance of Solibacter
Forest Edge LR
Solibacter Forest > LR (p = 0.053); Forest > Edge (p = 0.060)

Nitrogen Cycle Lignin-Degrading WRT/Lignin-Degrading

Bacterial Genera Bacterial Genera Fungal Genera
LR = Edge; p = 0.977 Edge = LR; p = 0.671 Edge > LR; p = 0.055
LR > Forest; p = 0.041 Forest > LR; p= 0.011 Forest > LR; p = 0.049
Edge > Forest; p = 0.058 Forest > Edge; p = 0.057 Edge = Forest; p = 0.525

d)

b) Burkholderia

Genus of N-Cycle Bacteria

Sphingomonas Rhodoplanes

Flavobacterium
Nitrospira
Pandoraea
Burkholderia Paenibacillus
Bradyrhizobium
35
Bacillus Bradyrhizobium

-1 0 1 2 3 4 5 6 7 8 -5 0 5 10 15 20 25 30
% Relave Abundance of the Most Common Lignin % Relave Abundance of the Most Common N-Cycle Bacteria
Degrading Bacteria,Not Including Solibacter
Forest Edge LR
Forest Edge LR

Bradyrhizobium LR > Edge (p = 0.060); LR > Forest (p = 0.041)

Bradyrhizobium LR > Edge (p = 0.060); LR > Forest (p = 0.041) Burkholderia Forest > Edge (p = 0.038); Forest > LR (p = 0.045)
Burkholderia Forest > Edge (p = 0.038); Forest > LR (p = 0.045) Nitrospira LR > Edge (p = 0.052); LR > Forest (p = 0.045)

e)
Scedosporium
Podospora
Penicillifer
Hypocrea
Gliocladiopsis
Fusarium
Cylindrocladium
Calonectria

0 5 10 15 20 25 30
% Relave Abundance of Most Common WRT/Lignin Fungi
Forest Edge LR

Scedospoium LR > Edge (p = 0.028); LR > Forest (p = 0.002)

Podospora LR > Edge (p = 0.045)
Penicillifer LR > Edge (p = 0.053)
Hypocrea Forest > LR (p = 0.020); Edge > LR (p = 0.032)
Gliocladiopsis LR > Edge (p = 0.041)
Fusarium LR > Edge (p = 0.024); LR > Forest (p = 0.001)
Cylindrocladium Forest > LR (p = 0.033); Edge > LR (p = 0.041)
Calonectria Forest > LR (p = 0.030); Edge > LR (p = 0.040)

Fig. 2. A comparison of the % Relative Abundance (%RA) of the DNA sequences from the most common genera of N-cycle bacteria, lignin degrading bacteria, and
wood rot/lignin degrading fungi (WRT/Lignin) in the soil ecosystems from the 3 zones examined (Forest = primary forest; LR = logging road; Edge = edge between
the forest and the LR) within the same Uplands Forested area in the Maquenque National Wildlife Refuge in the Northern Zone of Costa Rica. The %RA values, the
patterns of the distribution of the %RA, and the Mann-Whitney p values for the mean differences are presented. a) %RA values of the 3 different microbial functional
groups in the 3 soil zones; b) %RA values of the most common lignin degrading bacteria in soils of the 3 soil zones, excluding Solibacter; c) %RA of Solibacter in the 3
soil zones; d) %RA of the most common N-cycle bacteria in the 3 soil zones; and e) %RA of the most common WRT/Lignin fungi in the 3 soil zones.

from the on-going clearing of tropical forests is likely creating more Putten et al., 2013), with changes to the structure of the soil microbial
ecosystem damage than the actual clearing of trees in these forests communities and operation of the biogeochemical cycles such as
through the long-term consequences of the removal and compaction of decomposition, N-cycle activity, CO2 flux, efficiency of the use of soil
the topsoil and loss of plant-based resource inputs to the forest floor organic C, and the ability of soils to serve as C-sinks (Anderson and
(Gullison and Hardner, 1993; Kleinschroth and Healey, 2017; Sist et al., Domsch, 2010; Banning et al., 2011; Bardgett and Van der Putten, 2014;
2003). These disturbances will result in a negative influence on the plant Kardol and Wardle, 2010; Manzoni et al., 2012; Lee-Cruz et al., 2013;
– soil feedback system (Bennett and Klironomos, 2019; Fujii et al., 2018; Rodrigues et al., 2013; Takada et al., 2015; Tiwari et al., 2019). More­
Hobbie, 2015; Kulmatiski et al., 2008; Pugnaire et al., 2019; Van der over, the consequences of such impacts would be the alteration of the

7
W.D. Eaton et al. Applied Soil Ecology 164 (2021) 103937

a) Table 4
The distance-based linear modeling (DistLM) sequential tests describing the soil
environmental variables that were the best predictors for the variation in the
patterns in the composition of the (a) total bacterial genera, (b) total fungal
genera, (c) lignin degrading bacterial genera, (d) wood rot/lignin decomposer
fungal genera, and (e) N-cycle bacterial genera, from the 3 soil zones examined
associated with logging roads within the same Uplands Forested area in the
Maquenque National Wildlife Refuge in the Northern Zone of Costa Rica, using
stepwise sequential tests following AICc criteria. (Cv prop. = the cumulative
proportion of the total variation in the microbial group composition).
Sequential test AICc Pseudo-F p Value Cv. prop.

(a) Total bacterial genera

b) TN 296.63 3.6781 0.0077 0.07556
% Clay 293.87 4.989 0.0044 0.16970
+% Moisture 293.47 2.6344 0.0108 0.21764

(b) Total fungal genera

Biomass C 345.39 2.9795 0.0137 0.06210
% Silt 344.58 2.9888 0.0156 0.12176

(c) Lignin degrading bacterial genera

% Moisture 222.47 3.9263 0.0008 0.08025
TN 220.49 4.1805 0.0001 0.16005
% Sand 220.23 2.4946 0.0151 0.20611

(d) Wood rot/lignin decomposer fungal genera

TOC 243.09 3.5435 0.0012 7.30E− 02
% Sand 242.39 2.8859 0.0097 0.13005

(e) N-cycle bacterial genera

% Moisture 220.21 4.0396 0.0015 0.08237
TN 218.47 3.9375 0.0014 0.15775
c)
N-NO−3 217.95 2.744 0.0109 0.20827

Fig. 3. Canonical Analysis of Principal Coordinates (CAP) showing the strength
of the dissimilarity of the composition of the functional group genera within the
soil ecosystems from the 3 zones examined (Forest = primary forest; LR =
logging road; Edge = edge between the forest and the LR) within the same
Uplands Forested area in the Maquenque National Wildlife Refuge in the
Northern Zone of Costa Rica. a) CAP showing some dissimilarity in the
composition of the Lignin Degrading bacterial community between the LR and
Forest soils (CAP 1 R2 = 0.31, p = 0.014), but not between the Edge and Forest
or LR and Edge soil communities (CAP 2 R2 = 0.07, p = 0.014). b) CAP showing
strong dissimilarity in the composition of the N-cycle bacterial community
between the LR and Forest soils (CAP 1 R2 = 0.69, p = 0.0001), but not between
the Edge and Forest or LR and Edge soils (CAP 2 R2 = 0.11, p = 0.0001). c) CAP
showing moderate dissimilarity in the composition of the WRT/Lignin fungal
community between the LR and both the Forest and Edge soils (CAP 1 R2 =
0.51, p = 0.0002), but not between the Edge and Forest or LR and Edge soil
communities (CAP 2 R2 = 0.18, p = 0.0002).
overall health of an ecosystem and/or the changing of the trajectory of
recovery within damaged lands undergoing restoration (Guariguata and
Dupuy, 1997; Eaton et al., 2019; Kleinschroth et al., 2016a, 2016b;
McCalley et al., 2014; Nemergut et al., 2013). Here, we provide the first
evidence that a set of logging roads used in the Costa Rican Northern
Zone were associated with significant changes in the soil ecosystems.
The results show clear separation of the soil environmental metrics be­
tween the logging road soils and those of the edge and forest, and of the
total fungal and bacterial community compositions, and the composi­
tions of the proposed N-cycle bacterial genera, Lignin Degrading bac­
terial genera, and WRT/Lignin degrading fungal genera between all
three soil zones (i.e., the logging roads, the edges of the roads and the
adjacent primary forest soils). We also provide evidence that these dif­
ferences are being driven by the impacts of the damage occurring due to
the logging roads. More importantly, these data provide preliminary
evidence that the movement of the debris from the edge regions onto the
logging roads after abandonment should be a management practice to
enhance the rate of recovery of both the C and N cycle activities in the
soils, and perhaps begin to address the concern that logging roads add an
additional 10–15 years to tropical forest recovery following deforesta­
tion (Guariguata and Dupuy, 1997; Kleinschroth et al., 2016a, 2016b;
Pugnaire et al., 2019).
The loss of topsoil and the soil compaction that occurred in the LR
soils in this study were associated with a decrease in the % sand, and an
increase in % clay and soil bulk density in the LR soils compared to the
others. Such changes in soil systems have been known to negatively
impact soil microbial communities, which require certain ranges of soil
texture (and other physicochemical conditions) to provide the limits of
the “environmental envelope” within which different microbial groups
can survive (Fierer et al., 2007; Fierer et al., 2009). In addition, the loss
of vegetation and litter fall resources due to such extraction-based land
use practices have been shown to result in the types of differences in soil
abiotic components and microbial community composition we observed
in this study (Banning et al., 2011; Eaton et al., 2012, 2019, 2020;
Takada et al., 2015; Tiwari et al., 2019; Waring et al., 2013).
The greater %RA of the N-cycle bacteria found within the LR and
Edge than in the Forest soils represents patterns one would expect to
observe in the microbial community from soils of older, more estab­
lished forests in comparison to those of damaged and recovering soils.
Specifically, it has been shown that during the early stages of succes­
sional development in damaged tropical forest soils, the N deficiencies in
the soils resulting from damaging management practices result in a high
demand for N-cycle activity, in order for the development of soil quality
and regrowth of the vegetation (Amazonas et al., 2011; Batterman et al.,
2013; Davidson et al., 2007; Erickson et al., 2001; Groppo et al., 2015;
Pajares and Bohannan, 2016; Powers, 2004). This could be associated
with an increase in the abundance of bacteria for N-fixation and
ammonium oxidation, but also reduced standing pools of soil inorganic

8
W.D. Eaton et al.
N due to the great need for N required for vegetation and soil ecosystem
recovery following disturbance (Booth et al., 2005; Gehring et al., 2005;
Lovell et al., 1995; Menge and Chazdon, 2016; Moore and de Ruiter,
1991; Nasto et al., 2014; Russell and Raich, 2012; Sahrawat, 2008). This
pattern was observed in our study as both the %RA of the N-cycle bac­
terial DNA and the inorganic N metrics were generally lower in the LR
and Edge soils than in the Forest soils, suggesting that differences in
these biotic and abiotic metrics could serve as indicators of soil recovery
after disturbance. Moreover, the results indicate that moving the edge
debris onto the logging roads could be a beneficial and inexpensive
practice to enhance soil N-cycle recovery.
The greater %RA of the Lignin-Degrading bacteria and the WRT/
Lignin fungi in the Forest and/or Edge soils were similar to patterns
observed in more established forest soils as compared to soils under­
going more primary soil succession (Bradford et al., 2008; Eaton et al.,
2012, 2019, 2020; Hafich et al., 2012; Kivlin and Hawkes, 2016; Sin­
sabaugh, 2010; Talbot et al., 2008). These studies showed that differ­
ences in the community structure of the fungal functional groups
associated with wood rot, and the fungal and bacterial groups associated
with lignin degradation were found to correlate with increases in soil
TOC, C-biomass and C-use efficiency along soil and vegetation succes­
sional gradients. As this is consistent with our results, it may be that
using the %RA of these functional groups in combination with the soil
TOC, C-biomass and C-use efficiency (as Microbial Quotients) should be
further studied as potential indictors of soil recovery. Similar to the N
cycle data, these data also suggest that moving the edge debris back onto
the logging road should be considered as a practice to enhance soil C-
cycle recovery.
In the current study, changes in the composition of the soil microbial
communities associated with wood rot, lignin degradation, degradation
of other complex C compounds, and C-use efficiency were observed
along the disturbance gradient, consistent with findings from other
studies (Anderson and Domsch, 2010; Bailey et al., 2002; Bradford et al.,
2008; Eaton et al., 2012, 2019, 2020; Hafich et al., 2012; Kivlin and
Hawkes, 2016; Talbot et al., 2008). In fact, the changes we found in
certain genera associated with these functions could indicate some level
of soil ecosystem damage or recovery. For example, the WRT/Lignin-
associated fungal genera Hypocrea, Calonectria, and Cylindrocladium,
were the most abundant in the Forest and Edge soils, while the genera
Scedosporium, Podospora, Penicillifer, Gliocladiopsis, and Fusarium were
most abundant in the LR soils. Members of the genus Hypocrea are
known to enhance seed germination in flowering plants (Celar and Valic,
2005), phosphorus uptake by plants (Rudresh et al., 2005), and pro­
duction of enzymes that degrade plant debris and litter in healthy soils
(Baig et al., 2004; Nobe et al., 2004; Osono, 2005), and are considered
normal inhabitant of forest soils (Hagn et al., 2003; Roiger et al., 1991).
Members of the genus Calonectria are more common in a forest habitat
that has a wider range and abundance of plant species (Horst, 2008),
such as a healthy forest as compared to a logging road. The greater %RA
of Hypocrea and Calonectria in the Forest and Edge compared to the LR
soils may suggest these genera might serve as potential fungal indicators
of soil ecosystem recovery following damage to in these and other
Northern Zone habitats. Members of the genus Fusarium are known
pathogens of agricultural and forest plants, causing different types of
root rot, the severity of which is enhanced with increased soil compac­
tion and other conditions that reduce the rate of root growth (Dzha­
barova et al., 2018; Elmer, 2015; Gordon et al., 2015). The greater levels
of Fusarium in the LR soils in suggests that the enhanced soil compaction
and the decreased root health due to the vegetation loss may have
selected for possible Fusarium-induced rot of dead and damaged roots in
the logging roads, and -may be a potential indicator of damaged soil
ecosystems. The detrimental conditions of the LR soils would logically
select for fungal groups that are more resistant to harsher environmental
conditions. For example, the WRT/Lignin degrading fungi Scedosporium
and Podospora, with the greatest %RA in the LR soils, are known to grow
well in soils that are poorly aerated and impacted by human activities,
9
Applied Soil Ecology 164 (2021) 103937
and, as such, are considered important in degradation of organic C in
early stages of soil ecosystem recovery (Rougeron et al., 2017; Tayyab
et al., 2019). These findings suggest that the %RA of these fungi could be
used as part of a suite of indicators of soil ecosystem condition.
The Lignin degrading-associated bacteria Burkholderia and Solibacter
were at a greater %RA in the Forest than in the other two soils, while
Bradyrhizobium was at a greater %RA in the LR soils. Although all three
bacteria are known to decompose lignin, Bradyrhizobium has been found
to be an important N-fixing bacteria, whose presence in young or
recovering soils is likely critical for an influx of reduced forms of N to
stimulate soil recuperation (Amazonas et al., 2011; Batterman et al.,
2013; Booth et al., 2005; Gehring et al., 2005; Moore et al., 2005;
Davidson et al., 2007; Lojka et al., 2010; Zhalnina et al., 2013). Some
member of the genus Burkholderia have also been associated with
nitrification and N-fixation activity. Additionally, the Acidobacteria
genus Solibacter, has been associated with enhanced ammonium pro­
duction in soils through reduction of nitrite (Kielak et al., 2016). Thus, it
appears that all three of these bacteria may be critical in both the C and
N cycle activities in these soils. The greater %RA of the nitrifying bac­
teria Nitrospira in the LR soils was expected due to their role in
enhancing the NO−3 levels, which are needed for soil biomass develop­
ment and recovery damaged soils (Booth, 2005; Cleveland et al., 2003;
Hayatsu et al., 2008; Isobe et al., 2011; Reiners et al., 1994; Sahrawat,
2008). These results suggest that assessing the %RA of all four of these
genera could serve as indicators of soil ecosystem condition after
disturbance.
The DistLM results from this study provide preliminary evidence that
there are some abiotic and biotic metrics that could serve as predictors of
the variation within the microbial communities along the disturbance
gradient. Changes in the % sand, silt or clay, and the % moisture were
some of the best predictors of the differences in the microbial commu­
nity compositions, while different C and N cycle components were also
closely associated with differences observed in some of the microbial
communities. For example, total N or total N and NO−3 were good pre­
dictors of the variation within the total bacteria, N-cycle bacteria, and
lignin degrading bacterial communities, all of which are important in
the recovery of damaged forests and soils (Briones, 2014; Coleman et al.,
2017; Eaton et al., 2017, 2019, 2020; García-Orenes et al., 2013; Mad­
sen, 2011; Pajares and Bohannan, 2016; Tiwari et al., 2019). The N-cycle
bacterial groups replace the inorganic N lost during conversion to
agricultural and other land uses, and which is also needed for the high
demand of N required for the rapid growth rates in recovering forests
(Amazonas et al., 2011; Batterman et al., 2013; Booth et al., 2005;
Davidson et al., 2007; Erickson et al., 2001; Gehring et al., 2005; Groppo
et al., 2015; Menge and Chazdon, 2016; Nasto et al., 2014; Russell and
Raich, 2012).
The DistLM analyses also showed that the C Biomass or TOC were the
best predictors of the differences in the composition of the total fungal
and the wood rot/lignin decomposing fungal community compositions
across the disturbance gradient. These findings are consistent with those
of previous studies which have shown that changes in soil fungal com­
munity composition were associated with changes in the soil TOC and C
Biomass (Bradford et al., 2008; Eaton et al., 2012, 2019; Hafich et al.,
2012; Kivlin and Hawkes, 2016; Luis et al., 2004; Sinsabaugh, 2010;
Talbot et al., 2008). Further analyses of the connections between some
of these soil microbial communities, the inherent soil factors, and the
specific C and N metrics could lead to more clear predictors and models
of the impact that logging roads have on the biotic and abiotic compo­
nents in the soils.
It is believed that soil microbial communities undergo changes over
time along disturbance gradients that are consistent with the classical
ecology “Competitive Exclusion” model (Hardin, 1960). This suggests
that competition would lead to more stable microbial populations of
increasing dominance developing along these gradients over time, as the
quantity and quality of nutritional resources change during recovery
(Boddy, 2000; Cherif and Loreau, 2007; Frąc et al., 2018; Ghoul and
W.D. Eaton et al.
Mitri, 2016; Morón-Ríos et al., 2017). We showed this may be occurring
within the soil fungal populations (but not the bacterial) along the
disturbance gradient as the richness and diversity of the total fungal
genera were less in the Forest than the other two soils. This was asso­
ciated with the Forest soils having a greater %RA of the fungal genera
proposed to be linked to wood rot and lignin decomposition than the
other soils, and potentially resulting in a greater efficiency of organic C
use, as suggested by the greater levels of Microbial Quotients. These
results could suggest greater dominance of these important fungal
genera might be occurring in the forest, while in earlier stages of soil and
vegetation successional stages there is still greater competition for re­
sources and niche space associated with the fungal communities
developing within the LR soils (Eldridge et al., 2017; Lekberg et al.,
2007; Louca and Doebeli, 2016; Maherli and Klironomos, 2012). It
would be interesting to further study the role that Competitive Exclusion
may be playing in driving changes in these microbial communities and
in the C-use efficiency as the soils recover from damage due to the
development of the logging roads.
Hurricanes Otto, Nate and Maria hit Costa Rica in 2016 and 2017,
causing extensive damage to the Northern Zone forests and the Cloud
Forests of Monteverde, while in 2020 there were several hurricanes that
hit Nicaragua with tropical storm winds reaching Costa Rica. It is to be
expected that more hurricanes and tropical storms will surely hit Costa
Rica and other tropical forested areas in the future. These extreme
weather events have already and will continue to stimulate more
removal of felled and damaged trees, necessitating the development of
networks of logging roads to clear the felled trees. This current study
provides important evidence to suggest that movement of the slash and
debris from the edge regions that resulted during road development
back onto the logging roads at abandonment will result in enhanced
rates of recovery of the soil ecosystems. This will be associated with
changes in the soil microbial communities that increase efficiency of soil
C use, resulting in a recovery trend more towards the characteristics
found in intact primary forest soils. It would be worth further study to
use the suite of indicators suggested by our results to assess whether this
change in management practice actually increases the soil ecosystem
recovery rate on logging roads. We would suggest a project to move the
slash and debris from the edges onto the logging roads after abandon­
ment and monitoring the C metrics, the Microbial Quotients, and % RA
of the fungal genera Hypocrea, Calonectria, Fusarium, Scedosporium and
Podospora, and the bacterial genera Burkholderia, Solibacter, and Bra­
dyrhizobium along logging road disturbance gradients over time to
determine if these potential indicators show trends in the development
of soil ecosystems, their biota, and vegetation communities that
approach the patterns in adjacent and intact primary forest.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.apsoil.2021.103937.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
We would like to thank Vinzenz and Kurt Schmack, and all the staff
members at the Laguna del Lagarto Lodge. This study was supported by a
grant to WDE from the National Science Foundation (DBI-1262907), the
Costa Rican Government original Permit #063-2008-SINAC, and the
Government of Canada via Environment and Climate Change Canada,
and NSERC to MH.
10
Applied Soil Ecology 164 (2021) 103937
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