Journal for Nature Conservation 69 (2022) 126259

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Journal for Nature Conservation

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

Habitat fragmentation rather than habitat amount or habitat split reduces

the diversity and abundance of ground-dwelling anurans within forest
remnants of the Brazilian Cerrado
Werther Pereira Ramalho a, b, c, *, Kimberly A. With a, Gabryella de Sousa Mesquita b, d, Filipe
Viegas de Arruda e, Vinicius Guerra b, f, Denes Ferraz b, h, Murilo Sousa Andrade c, g, Vitor Hugo
Mendonça do Prado h
a
Laboratory for Landscape and Conservation Ecology, Division of Biology, Kansas State University, Manhattan, KS 66506, USA
b
Instituto Boitatá de Etnobiologia e Conservação da Fauna, Goiânia, GO, Brazil
c
Laboratório de Ecologia, Evolução e Sistemática de Vertebrados, Instituto Federal Goiano, Campus Rio Verde, Rio Verde, GO, Brazil
d
Programa de Pós-Graduação em Biodiversidade Animal, Universidade Federal de Goiás, Goiânia, GO, Brazil
e
Instituto de Pesquisa Ambiental da Amazônia, Asa Norte, Brasília, DF, Brazil
f
Programa de Pós-Graduação em Ciências Ambientais, Instituto de Ciências Naturais, Humanas e Sociais, Universidade Federal de Mato Grosso, Sinop, MT, Brazil
g
Programa de Pós-Graduação em Biodiversidade e Conservação, Instituto Federal Goiano, Rio Verde, GO, Brazil
h
Laboratório de Biogeografia e Ecologia Aquática, Universidade Estadual de Goiás, Anápolis, GO, Brazil

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

Keywords: Rampant deforestation has caused the loss and fragmentation of natural habitats, which has precipitated a global
Amphibians biodiversity crisis. Research on how land-use change contributes to a loss of biodiversity is urgently needed,
Deforestation especially in ecosystems that have undergone rapid anthropogenic changes. We sought to investigate the extent
Frogs
to which habitat loss, fragmentation, and habitat split (the separation of forest and aquatic habitats) negatively
Functional diversity
Habitat loss
influenced taxonomic diversity, functional diversity, total abundance, and the individual abundances of five
Land-use change anuran species in the Brazilian Cerrado. We sampled anurans between December 2017 and March 2018 using
Ponds pitfall traps at sites distributed along a gradient of habitat fragmentation/habitat split: unfragmented forest,
Taxonomic diversity forest fragments without habitat split, and forest fragments with habitat split. Forest cover was measured within
Toads a 1-km radius of each site. Sites within unfragmented forests had higher taxonomic and functional diversities
than either fragment type. Taxonomic diversity was highly correlated with functional diversity, but we did not
find a pattern to the loss of functional traits. Total anuran abundance and the abundances of Chiasmocleis
albopunctata, Physalaemus cuvieri, and Rhinella diptycha were higher in unfragmented forests compared to forest
fragments. No species was more abundant in fragments than in unfragmented forests. Our results indicate that
the fragmentation of forests by agricultural land use is directly and indirectly responsible for the loss of taxo­
nomic and functional diversity, as well as for reducing population sizes of ground-dwelling anurans. Although we
did not find a distinct effect of habitat split on ground-dwelling anurans, our study underscores the importance of
preserving continuous forest habitats for the maintenance of anuran diversity in the Cerrado.

1. Introduction
Deforestation and the modification of ecosystems for agricultural
production have altered landscapes worldwide, especially within
neotropical regions (Arroyo-Rodríguez, Melo, & Martínez-Ramos, 2017;
Püttker, Crouzeilles, & Almeida-Gomes, 2020; Rausch, Gibbs, & Schelly,
2019). Among the many negative impacts of landscape modification,
habitat loss and fragmentation are the ones that most harm biodiversity.
These two processes reduce the amount of native vegetation and
decrease connectivity between habitats (Fahrig, 2003; Fischer & Lin­
denmayer, 2007; Püttker et al., 2020), which in turn can reduce native
species diversity and disrupt community structure and ecosystem func­
tion (Dixo & Metzger, 2009; Martensen, Pimentel, & Metzger, 2008;
Riemann, Ndriantsoa, Rödel, & Glos, 2017; Silva & Rossa-Feres, 2011;

* Corresponding author.
E-mail address: werther@institutoboitata.org (W.P. Ramalho).

https://doi.org/10.1016/j.jnc.2022.126259
Received 4 March 2022; Received in revised form 8 July 2022; Accepted 4 August 2022
Available online 6 August 2022
1617-1381/© 2022 Elsevier GmbH. All rights reserved.
W.P. Ramalho et al.
Silva, Oliveira, Gibbs, & Rossa-Feres, 2012). Habitat loss and fragmen­
tation have been largely responsible for the loss of populations and
species taxonomic diversity (Dixo, Metzger, Morgante, & Zamudio,
2009; Hanski, 2011; Mendes, With, Signorelli, & De Marco, 2017; With,
2019; Püttker et al., 2020), as well as the diversity of ecological traits
(Farneda et al., 2015; Ribeiro, Colli, Batista, & Soares, 2017; Zambrano
et al., 2019; Bełcik, Lenda, Amano, & Skórka, 2020) that are important
for ecosystem functioning (i.e., functional diversity; Tilman, 2001;
Haddad, Brudvig, Clobert, Davies, & Gonzalez, 2015; Riemann et al.,
2017; Ouchi-Melo, Meynard, Gonçalves-Souza, & de Cerqueira, 2018;
With, 2019). Even though there is a consensus that landscape modifi­
cation caused by anthropogenic activities reduces biodiversity, some
organisms may face more drastic consequences than other.
Amphibians have undergone extreme global population declines,
with high extinction rates documented in many locations (Alroy, 2015).
These population declines and extinctions are due to inumerous factors,
such as habitat loss, fragmentation, habitat split, pollution, global
climate change, and diseases (Alroy, 2015; Becker, Fonseca, Haddad,
Batista, & Prado, 2007; Hopkins, 2007; Silvano & Segalla, 2005; Silvano,
Valdujo, & Colli, 2016). Indeed, anurans are ectothermic, have limited
dispersal ability, permeable skin, and specific reproductive modes (e.g.,
highly dependent on water) that make them especially sensitive to
environmental and land-use changes (Niemi & McDonald, 2004). The
negative effects of land-use change on anuran diversity are driven in
large part by the loss and fragmentation of natural habitat remnants,
which can cause a reduction of both taxonomic (Marques & Nomura,
2018; Marques, Rattis, & Nomura, 2019; Prado & Rossa-Feres, 2014;
Ramalho, Prado, Signorelli, & With, 2021; Signorelli, Bastos, De Marco,
& With, 2016) and functional diversity (Díaz-García, Pineda, López-
Barrera, & Moreno, 2017; Riemann et al., 2017), resulting in a loss of
ecosystem services (Colón-Gaud, Whiles, & Brenes, 2010; Hocking &
Babbitt, 2014; Valencia-Aguilar, Cortés-Gómez, & Ruiz-Agudelo, 2013).
During the fragmentation process, some fragments are also isolated from
adjacent ponds or streams, which in turn disrupts the connectivity be­
tween terrestrial and aquatic habitats required by diferent life-history
stages of amphibian species, in a process termed habitat split (Becker
et al., 2007; Becker, Fonseca, Haddad, & Prado, 2010; Fonseca, Becker,
Haddad, & Prado, 2008; Fonseca et al., 2013). As predicted by the
habitat-split concept, the forest fragments isolated from aquatic habitats
have lower amphibian diversity and population density than forest
fragments connected to a pond or stream (Becker et al., 2010; Fonseca
et al., 2013). Habitat split is thus an important environmental filter for
amphibians at the landscape scale (Ramalho et al., 2021) that can
reduce taxonomic diversity (Becker et al., 2007, 2010; Lion, Garda, &
Fonseca, 2014) and potentially reduce functional diversity via selection
of species with similar functional traits (Ramalho et al., 2021). There­
fore, habitat loss, fragmentation, and habitat split – as a specific case of
habitat fragmentation – are expected to cause local extinctions and
contribute to the loss of biodiversity, especially in ecosystems that have
undergone many anthropogenic changes, such as in the Cerrado biome
of South America.
Our goal in this study is to evaluate the relative effects of habitat loss,
fragmentation, and habitat split on different dimensions of diversity
(taxonomic and functional diversity) and on the abundance of ground-
dwelling anurans in the Brazilian Cerrado. The Cerrado biome is a
global biodiversity hotspot (Myers, Mittermeier, Mittermeier, da Fon­
seca, & Kent, 2000) owing to its high species richness, as well as the
widespread degradation of its natural habitats caused by the expansion
of agriculture in recent decades (Françoso et al., 2015; Grande, Aguiar,
& Machado, 2020; Rausch et al., 2019). Although around 52 % of the
Cerrado consists of remnants of natural vegetation (INPE, 2020), only 6
% are within legally protected areas (Françoso et al., 2015), which
means that most of the biome now occurs as highly fragmented land­
scapes located outside protected areas. To achieve our goals, we tested
the following hypotheses:
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Journal for Nature Conservation 69 (2022) 126259
(1) Habitat fragmentation is expected to reduce the richness and
abundance of anurans, especially for species most sensitive to
desiccation, owing to greater dispersal limitation between forest
fragments that are surrounded by agricultural land use, such as
pastures (Becker et al., 2010; Watling & Braga, 2015);
(2) Habitat fragmentation is expected to reduce the functional di­
versity of anurans. Forest fragments are expected to have fewer
resources and more adverse environmental conditions in relation
to continuous forests, which in conjunction with lower dispersal
success, should reduce functional diversity (Ribeiro, Colli, &
Soares, 2019). Moreover, the land-use matrix can act as an
environmental filter that selects species with specific traits,
causing a homogenization or reduction of functional diversity
(Ramalho et al., 2021; Riemann et al., 2017; Weideman, Slingsby,
Thomson, & Coetzee, 2020); and,
(3) Habitat split between forests and aquatic environments (e.g.,
ponds) is expected to result in lower diversity and abundance of
anurans within fragmented forests. Habitat split is expected to
exacerbate the effects of habitat fragmentation on anuran di­
versity and abundance in fragmented forests because it reduces
the success of dispersal between the reproductive environment
and the forested habitats, especially for pond-breeding species
(Becker et al., 2007, 2010).
2. Material and methods
2.1. Study area
Data collection was carried out in the Parque Estadual Altamiro de
Moura Pacheco (PEAMP) and Parque Estadual do João Leite (PEJL),
which are protected areas located between the municipalities of Goiânia
and Anápolis in the Central Region of the Cerrado biome, State of Goiás,
Brazil (Fig. 1). The vegetation formation in the region is mainly char­
acterized by remnants of forest vegetation, including semi-deciduous
seasonal forests and gallery forests (Ribeiro & Walter, 2008). The
climate is tropical (Köppen Aw) with two well-defined seasons, one
being rainy (October to March) and the other dry (April to September)
(Peel, Finlayson, & McMahon, 2007). The average monthly temperature
is 24.8 ◦ C, with the highest in September (26.9 ◦ C) and lowest in June
(22.8 ◦ C). The average monthly rainfall is 137.8 mm, with the highest
occurring in December (319.7 mm) and lowest in August (2.3 mm),
based on monthly averages obtained for the period 2008 to 2018 (Brasil
2018).
To tease apart the effects of fragmentation and habitat split on an­
urans, we selected study sites that were located either inside or in the
vicinity of PEAMP and PEJL. Like much of the Cerrado, this region has
been transformed by agriculture and urbanization, such that the largest
forest remnants are now found only within these two protected areas
(PEAMP and PEJL). Our unfragmented forest sites (n = 6) were therefore
all located within larger forested areas (or in large forest remnants
connected to those areas) that were at least 1,000 ha in size; all of these
sites were located within the parks’ boundaries and were adjacent to a
water body (unfragmented without habitat split). Habitat split is ulti­
mately a component of habitat fragmentation in this region, and thus
continuous forest areas tend not to exhibit habitat split as we define it
here (i.e., a minimum distance of 215 m; Fig. 1, Table S1). By contrast,
our fragmented forest sites were all located outside the parks’ bound­
aries in smaller forest fragments (<50 ha) surrounded by agricultural
land use, and were either adjacent to (fragmented without habitat split,
n = 6) or isolated from (fragmented with habitat split, n = 6) a pond. All
fragments without habitat split had streams inside or along the edge,
while all fragments with habitat split were not connected to or crossed
by streams. Sites without habitat split were connected to semipermanent
or permanent ponds with an average area of 1 ha that were created for
agricultural purposes.
W.P. Ramalho et al.
Fig. 1. Geographical location of the study area (A), showing the two protected areas and the distribution of the sampling sites (B) corresponding to the three
categories of fragmentation and habitat split. Protected areas: PE João Leite = Parque Estadual João Leite; PE Altamiro de Moura Pacheco = Parque Estadual
Altamiro de Moura Pacheco.
2.2. Sampling design and data collection
We surveyed anurans at each study site (n = 18) three times during
the rainy season: once at the beginning (December 2017), once at the
peak (January 2018), and once at the end (March 2018). We installed
pitfall traps (30-L buckets) along a transect located 15 m from the pond
or forest edge and extending 24 m parallel to the orientation of the
nearest pond. Each transect consisted of four pitfall traps that we
installed 8-m apart and buried so that the rim of the bucket was flush
with the ground. Pitfall traps were also connected by a 0.5-m-high
canvas “drift fence” to help guide anurans into the traps. Pitfall traps
remained open for seven consecutive days during each sampling period,
totaling 21 days of sampling effort at each site.
Because species diversity may be influenced more by the amount of
habitat in the surrounding landscape than by fragmentation (or habitat
split) per se (Fahrig, 2003), we also quantified the amount of forest
cover within a 1-km radius of each site (Table S1). Landscape variables
have been shown to affect amphibian abundance, richness, and species
composition, as well as community assembly, at a 1-km scale around
sample sites (Gagné & Fahrig, 2007; Ganci, Provete, Püttker, Linden­
mayer, & Almeida-Gomes, 2021; Ramalho et al., 2021; Signorelli et al.,
2016). Although some studies found significant influences beyond this
distance (Gagné & Fahrig, 2007; Signorelli et al., 2016), we did not
consider landscape scales larger than 1 km because this would have
resulted in significant overlap between some of our buffers, potentially
leading to pseudoreplication. To obtain the amount of forest surround­
ing each sample site, we used the latest land-cover maps provided by
MapBiomas (MapBiomas, 2021) and calculated the amount of forest
(class area, CA) within a 1-km buffer centered on each site using the
landscapemetrics package (Hesselbarth, Sciaini, With, Wiegand, &
Nowosad, 2019) in R (R Core Team, 2021).
2.3. Functional traits
We characterized functional diversity on the basis of seven traits
suggested by the global AmphiBIO database (Oliveira, São-Pedro,
3
Journal for Nature Conservation 69 (2022) 126259
Santos-Barrera, Penone, & Costa, 2017) related to the ecology,
morphology, and reproductive characteristics of amphibians. The
functional traits we selected were: (1) adult habit; (2) period of activity;
(3) seasonality; (4) toxicity; (5) body size; (6) main habitat; and (7)
reproductive mode (Table S2). For habit, we classified the adult stage of
each species as being either terrestrial (active mainly on the surface of
the soil or leaf litter) or fossorial (exhibits burying behavior and/or
reproductive activity within burrows or below the leaf litter). Activity
period was classified as diurnal, nocturnal, and/or crepuscular, whereas
season of activity was based on the combination of precipitation con­
ditions (wet or dry) and temperature (warm or cold) throughout the year
(wet/warm, wet/cold, dry/warm, and dry/cold). Defining season of
activity is important because it is related to the length of the repro­
ductive period of each species, which varies from “explosive” (all in­
dividuals reproduce at once) to prolonged (Prado, Uetanabaro, &
Haddad, 2005; Wells, 2007). To assign this particular trait, we used our
own field observations and previous research conducted in the same
area (Ramalho, França, Guerra, Marciano, Vale, & Silva, 2018; Ramalho
et al., 2021), as well as information from other studies in nearby areas
(Kopp, Signorelli, & Bastos, 2010; Oda, Bastos, & Lima, 2009). Toxicity
was characterized in relation to the species’ effect on predators: toxic
(causing death if eaten) or non-toxic (Wells, 2007). For body size, we
obtained the snout-vent length (SVL) of each species from either the
AmphiBIO database (Oliveira et al., 2017), the published literature (e.g.,
Brasileiro & Haddad, 2015), or from our own measurements during data
collection. We classified the main habitat of each species as those that
occur mainly in forest environments, in open environments, or in both.
Finally, we classified the reproductive mode of each species using the
classification proposed by Nunes-de-Almeida, Haddad, and Toledo
(2021). The species’ reproductive mode was characterized in terms of its
egg-laying site (e.g. directly in the water, in foam nests in the water, in
foam nests on the edge of the pond, or directly in the soil or leaf litter for
species with direct development). The reproductive mode is an impor­
tant functional trait because it can influence species’ responses to forest
fragmentation and habitat split, since each mode requires different
environmental conditions for reproductive success (Becker et al., 2007;
W.P. Ramalho et al.
Crump, 2015).
2.4. Abundance
To test hypotheses related to the effects of habitat fragmentation/
habitat split and habitat loss on species-specific abundances, we focused
on species that were most prevalent and well distributed in the areas
sampled. All species are terrestrial and require riparian environments or
their associated habitats (e.g., ponds) for reproduction (Kopp et al.,
2010; Oda et al., 2009; Ramalho et al., 2018). All collected specimens
were anesthetized and euthanized with 5 % Xylocaine, fixed with 10 %
formalin solution, and then preserved in 70 % alcohol and deposited in
the Coleção de Vertebrados Alípio de Miranda Ribeiro (CVAMR) of the
Instituto Federal Goiano, Campus Rio Verde, Rio Verde, Goiás, Brazil.
Licenses for sampling and specimen collection were granted by the State
Council of the Environment/State Secretariat of Environment and Sus­
tainable Development-CEMAm/SECIMA-GO (#022/2017) and Ministry
of the Environment/Chico Mendes Institute of Biodiversity Conservation
– MMA/ICMBio (#55420-3).
2.5. Data analysis
We use the FD index proposed by Petchey and Gaston (2002, 2006)
as a measure of functional diversity. The FD index is the sum of branch
lengths linking species belonging to the same group within a functional
dendrogram, which measures the distance of species in functional space
based on their complementarity in resource use (Petchey & Gaston,
2002). The index is calculated in three stages. First, we converted the
functional trait matrix into a distance matrix (the pair-to-pair distances
between species in the functional-trait space) using the Gower distance,
which is indicated for the analysis of mixed variables (continuous and
categorical), using the function dist.ktab (Pavoine, Vallet, Dufour,
Gachet, & Daniel, 2009) in the package ade4 (Dray, Dufour, & Chessel,
2007) in R (R Core Team, 2021). Second, we constructed a functional
dendrogram using the arithmetic means of unweighted pair groups
(UPGMA), which produced a higher correlation coefficient between the
original distances and the cophenetic distances (Mantel test based on
Pearson correlation, r = 0.81, P < 0.01, 999 randomizations). Finally,
we calculated the FD of anuran communities for each sampling site
separately, using the function pd in the R package picante (Kembel,
Cowan, & Helmus, 2010). A Pearson correlation test was used to test the
relationship between FD and taxonomic diversity (i.e., species richness).
The distribution of species’ abundances among the sampling sites and
fragmentation categories, highlighting functional groups in the func­
tional dendrogram, was graphically represented using a modification of
the poncho function (Dambros, 2020).
To assess how habitat fragmentation/habitat split and forest cover
can influence taxonomic diversity, functional diversity, total abundance,
and the abundances of select anuran species, we used generalized linear
models (GLMs; Crawley, 2007). Richness and abundance were log
transformed to reduce the effects of outliers and normalize the data. In
the analyses, predictor variables were the three categories of habitat
fragmentation (unfragmented forest, fragmented forest without habitat
split, and fragmented forest with habitat split), and the amount of forest
surrounding each site. The response variables were species richness,
functional diversity, total abundance, or the individual abundances of
the selected species. A model without interactions was created to test the
effect of the explanatory variables on each of the response variables
separately. Since the response variables were all continuous, we fit
models using a Gaussian distribution within the function glm of the R
package stats (R Core Team, 2021). In the case of a significant result for
the categorical variable of habitat fragmentation/habitat split, a
contrast analysis using the Tukey method was applied to the model to
test pairwise differences between the treatment groups, using the glht
function of the R package multcomp (Hothorn, Bretz, & Westfall, 2008).
We assessed data compliance with model assumptions of normality and
4
Journal for Nature Conservation 69 (2022) 126259
homogeneity of variances via visual inspection of the graphs according
to the protocol of Zuur, Ieno, and Elphick (2010) and using two specific
tests available within the functions shapiro.test and leveneTest of the
R packages stats (R Core Team, 2021) and car (Fox & Weisberg, 2019),
respectively. After analyzing the residuals from the models of the most
abundant species, five species were maintained to test the effect of
fragmentation and habitat loss on individual abundances: Chiasmocleis
albopunctata (Boettger, 1885), Physalaemus atim Brasileiro & Haddad,
2015, Physalaemus cuvieri Fitzinger, 1826, Physalaemus nattereri (Stein­
dachner, 1863), and Rhinella diptycha (Cope, 1862). Finally, we evalu­
ated the existence of spatial autocorrelation in the model residuals using
the Moran’s I test over six distance classes, using the R package pgirmess
(Giraudoux, 2022). As we did not find significant spatial autocorrela­
tion, it was not necessary to correct for this in the models (Figs. S1 and
S2).
3. Results
We collected 679 individuals of 17 species of ground-dwelling an­
urans, distributed in the families Leptodactylidae (10 species), Micro­
hylidae (3), Odontophrynidae (2), Bufonidae (1), and Craugastoridae
(1). The most abundant species were Physalaemus cuvieri (320 in­
dividuals), Chiasmocleis albopunctata (117), and Rhinella diptycha (50),
while the rarest species were Leptodactylus macrosternum Miranda-
Ribeiro, 1926 (2) and Dermatonotus muelleri (Boettger, 1885) (3)
(Table S3). Taxonomic diversity per site ranged from three to 12 (6.39
± 1.88 SD) species. Unfragmented forests had higher taxonomic di­
versity (total = 17 species; 8.33 ± 1.66 species/site) than fragmented
forests without habitat split (15 species; 5.33 ± 1.00 species/site) or
fragmented forests with habitat split (11 species; 5.50 ± 1.83 species/
site). The two fragmented-forest categories did not differ in taxonomic
diversity (Fig. 2A). Forest cover did not explain variation in taxonomic
diversity among sites (Table 1).
Functional diversity (FD) index values ranged from 1.08 to 2.91
(1.74 ± 0.46 SD) among sites. Functional diversity was significantly
higher for sites within unfragmented forest (FD = 2.19 ± 0.42)
compared to either fragmented forest without habitat split (1.49 ± 0.27)
or fragmented forests with habitat split (1.56 ± 0.45). The two cate­
gories of fragments did not differ in functional diversity (Fig. 2B,
Table 1). Taxonomic diversity was highly correlated with functional
diversity (r = 0.98, P < 0.01, df = 16). The loss of species within each
functional group did not indicate a clear pattern in the loss of functional
traits (Fig. 3). The amount of forest cover in the surrounding landscape
had no effect on anuran functional diversity within sites (Table 1).
Ground-dwelling anurans could be separated into two main func­
tional groups. The first functional group comprised predominantly
terrestrial species that reproduce during the warm and rainy period and
construct foam nests within water or soil, with the exception of Bar­
ycholos ternetzi, which has direct development. The second functional
group was composed of predominantly nocturnal species that are
fossorial and which reproduce in water mainly during the warm and
rainy period. One species, Rhinella diptycha, was distinct from these two
groups due to its unique characteristics involving large body size,
occurrence mainly within open habitats, and reproduction during the
warm/dry period of the year (Fig. 3).
The total abundance of anurans at sites ranged from eight to 160
(37.7 ± 25.90 SD) individuals. Total abundance was higher in unfrag­
mented forests (total = 435 individuals; 72.5 ± 47.00 individuals/site)
than in fragmented forests without habitat split (112; 18.7 ± 8.00 in­
dividuals/site) or fragmented forests with habitat split (132; 22.0 ±
10.67 individuals/site) (Fig. 4A, Table 1). We also found that Chiasmo­
cleis albopunctata exhibited significantly lower abundances in fragments
without habitat split than in unfragmented forests (Fig. 4B). We also
found a tendency (P < 0.1) for two other species to exhibit decreased
abundance in forest fragments: Physalaemus cuvieri tended to be less
abundant in fragmented forests with habitat split than in unfragmented
W.P. Ramalho et al. Journal for Nature Conservation 69 (2022) 126259

Table 1
Summary of the GLM model results examining the relationship between habitat
fragmentation/habitat split or forest cover and anuran taxonomic diversity,
functional diversity, total abundance, and individual species’ abundances within
forest sites. Post-hoc tests were performed for significant differences (P < 0.05)
or marginally significant differences (P < 0.1) and are based on Tukey analysis of
contrasts between the categories of habitat fragmentation/habitat split: U =
Unfragmented forests, F = Fragmented forests without habitat split, S = Frag­
mented forests with habitat split.
Variable Habitat fragmentation/habitat Forest cover
Response split
df LR P Post- df LR P
Chisq hoc test Chisq

All species
Taxonomic 2 9.06 0.01 U > F, 1 1.36 0.24
diversity U >S
Functional 2 8.87 0.01 U > F, 1 1.43 0.23
diversity U >S
Total abundance 2 10.43 0.01 U > F, 1 0.90 0.34
U >S
Abundance per
species
Chiasmocleis 2 7.36 0.03 U>F 1 0.24 0.62
albopunctata
Physalaemus atim 2 3.64 0.16 1 0.04 0.84
Physalaemus 2 5.00 0.08 U>S 1 2.99 0.08
cuvieri
Physalaemus 2 1.53 0.47 1 0.34 0.56
nattereri
Rhinella diptycha 2 6.76 0.03 U > F, 1 0.10 0.75
U>S

Furthermore, habitat split did not appear to exacerbate these fragmen­

Fig. 2. Variation in taxonomic (A) and functional (B) diversities of terrestrial
anurans recorded in areas with different habitat fragmentation/habitat split
categories in the Brazilian Cerrado. Different letters denote significant differ­
ences (P < 0.05) found in a contrast analysis among different categories of
habitat fragmentation/habitat split.
forests (Fig. 4C) and Rhinella diptycha tended to be less abundant in all
forest fragments, regardless of habitat split (Fig. 4D). In no case did the
abundance differ significantly between fragments with or without
habitat split (Fig. 4, Table 1). Four species (Leptodactylus podicipinus,
Leptodactylus mystaceus, Leptodactylus natalensis, and Elachistocleis
cesarii) did not occur in fragmented forests with habitat split, whereas
two species (Leptodactylus macrosternum and Proceratophrys goyana) did
not occur in fragmented forests at all (Table 1, Fig. 3). The amount of
forest cover in the surrounding landscape had no effect on either total
anuran abundance or on individual species’ abundances within these
sites (Table 1).
4. Discussion
In this study, we found that habitat fragmentation, regardless of
habitat split or amount of forest cover in the surrounding landscape, was
an important predictor of taxonomic diversity, functional diversity, and
abundance of ground-dwelling anurans in the Cerrado. Our hypotheses
were thus partially corroborated in that habitat fragmentation nega­
tively influenced anuran assemblages, leading to a loss of both taxo­
nomic and functional diversities as well as a reduction in species
abundance within forest fragments compared to unfragmented forests.
tation effects, given that there was no significant difference between
fragment types.
The agricultural conversion of the Cerrado (e.g., to non-native grass
pastures) is largely responsible for the fragmentation of forest habitats in
this region, which has altered the intervening matrix and increased the
amount of forest-edge habitat within these landscapes. We posit that
agricultural land use reduces the permeability of the matrix to dispersal
among forest fragments for many ground-dwelling anurans, particularly
those that are prone to desiccation owing to higher temperatures, lower
humidity, and greater exposure to UV-B radiation in a more exposed
habitat matrix (Watling & Braga, 2015). Other factors may also increase
the resistance of the agricultural matrix to dispersal, such as greater
predation risk or exposure to toxic agrochemicals in the matrix (Becker
et al., 2010; Watling & Braga, 2015). Furthermore, the sort of high-
contrast forest edges created by agricultural land use is known to in­
crease the severity of negative edge effects, such as increased wind
disturbance, higher temperatures, and lower humidity within forest
fragments (With, 2019). These altered environmental conditions within
forest remnants can in turn reduce habitat suitability and resource
availability for some species of anurans (Barbosa, Marquet, & Baciga­
lupe, 2010; Ewers, Thorpe, & Didham, 2007; Harper, Macdonald, &
Burton, 2005; Laurance, Lovejoy, & Vasconcelos, 2002). Indeed, some
studies have found that species richness is better explained by the type of
matrix than by the size or isolation of habitat fragments (Prevedello &
Vieira, 2010; Reider, Donnelly, & Watling, 2018), whereas others have
failed to detect an effect of either fragment size or isolation on the
richness of anurans in landscapes where matrix permeability is high
(Dixo & Martins, 2008; Silva et al., 2012).
For example, Dixo and Martins (2008) did not find a significant effect
of habitat fragmentation on the richness of anurans and lizards in the
Atlantic Forest, which they attributed to the fact that these forest frag­
ments were embedded in a highly permeable matrix that contained some
secondary forest and shaded plantations, such as cocoa and rubber tree
plantations. In other words, high matrix permeability not only facilitates
dispersal, but can also reduce negative edge effects on forest anurans
(Dixo & Martins, 2008; Faria, Paciencia, Dixo, Laps, & Baumgarten,

5
W.P. Ramalho et al. Journal for Nature Conservation 69 (2022) 126259

Fig. 3. Functional dendrogram of terrestrial anurans and their relative abundance within different categories of habitat fragmentation/habitat split. Histograms
represent the logarithmic species abundance at each site. The shaded areas of the dendrogram highlight the two main functional groups identified by this analysis.
Asterisks denote species that showed significant difference in abundance (** = P < 0.05) or that had a tendency to decrease in abundance (* = P < 0.1) within
fragmented forests according to the results of the GLM and Tukey pairwise analysis. Species abbreviations follow Table S1.

2007). Habitat fragmentation likely had a greater effect on the taxo­
nomic diversity of anurans in the Cerrado than in the Atlantic Forest
study owing to the significant modification of the landscape for agri­
cultural land use that has resulted in a less-permeable matrix (e.g.,
pastures) and increased the potential for negative edge effects within
forest fragments. Thus, only the most opportunistic and mobile species
may be able to persist in these fragmented forest landscapes (Ficetola &
De Bernardi, 2004). Forest fragmentation in the Cerrado may therefore
act as an environmental filter, selecting a small subset of species that are
able to traverse the hostile matrix and survive within the less hospitable
environments found within forest fragments.
The loss of functional diversity mirrored that of taxonomic diversity:
functional diversity was greater in continuous forests than in forest
fragments, and the species lost from forest fragments tended to have
complementary functional traits, resulting in communities with more
similar traits (Petchey & Gaston, 2002). These results corroborate pre­
vious studies that found greater functional diversity of anurans in
landscapes with large forest patches (Ribeiro, Colli, Batista, et al., 2017)
and a loss of functional diversity within agricultural areas in the Cerrado
(Ribeiro, Colli, Caldwell, et al., 2017; Ribeiro et al., 2019). The decrease
in functional diversity may be a consequence of habitat simplification,
such as reducing the area and quality of forest environments and
increasing negative edge effects, which can have a direct effect on the
functioning of the ecosystem (Batalha, Cianciaruso, & Motta-Junior,
2010; Riemann et al., 2017). For example, the loss of certain func­
tional traits (e.g., occurring predominantly in forest habitats, having a
fossorial habit, or reproducing in water or in foam nests at the edges of
ponds) present in species that were rare (e.g., Elachistocleis cesarii
(Miranda-Ribeiro, 1920), Leptodactylus natalensis Lutz, 1930, Lep­
todactylus mystaceus (Spix, 1824) and Leptodactylus podicipinus (Cope,
1862)) or absent (e.g., L. macrosternum and Proceratophrys goyana
(Miranda-Ribeiro, 1937)) from forest fragments could lead to the loss or
alteration of certain ecosystem processes, such as trophic transfer,
nutrient cycling, soil bioturbation, and energy flow (Lourenço-de-Mo­
raes, Campos, & Ferreira, 2020; Riemann et al., 2017). Colón-Gaud et al.
(2010) have warned that the global decline of amphibians will cause
significant changes to the structure and function of freshwater habitats,
which will have consequences for adjacent terrestrial environments as
well. Our results suggest that continuous forest environments, with their
higher functional diversity, are likely to be more important for
ecosystem functioning than forest fragments that have become isolated
by a pasture matrix, including those affected by habitat split (not adja­
cent to a pond or other aquatic breeding habitat). However, the loss of
functional traits between the two species groups identified by our
analysis did not exhibit a clear pattern with regard to habitat fragmen­
tation and/or habitat split. We thus have much to learn about how the
loss or replacement of functional traits among species occurs within
these human-modified landscapes.
Habitat fragmentation negatively influenced total anuran abun­
dance, as well as the abundances of three species, regardless of habitat
split: a forest specialist (Chiasmocleis albopunctata) and two open-habitat
species (Physalaemus cuvieri and Rhinella diptycha). In general, we would
expect the greater severity of environmental conditions within the ma­
trix (Becker et al., 2010; Watling & Braga, 2015) and/or forest fragments
(Barbosa et al., 2010; Ewers et al., 2007; Harper et al., 2005; Laurance
et al., 2002) to limit the establishment of populations in fragmented
habitats (Cushman, 2006). However, species will respond in different
ways to landscape change based on their individual life-history char­
acteristics (Gagné & Fahrig, 2007; Suárez, Zaccagnini, & Babbitt, 2016;
Van Buskirk, 2005). This means that amphibian population-
management strategies must take into account regional differences in
agricultural land use that may contribute to different patterns of habitat
fragmentation and matrix types (Johansson, Primmer, Sahlsten, &
Merilä, 2004). Even in the Cerrado, the response of individual anuran

6
W.P. Ramalho et al.
Fig. 4. Variation in total anuran abundance (A) and the relative abundance of Chiasmocleis albopunctata (B), Physalaemus cuvieri (C), and Rhinella diptycha (D) among
different categories of habitat fragmentation/habitat split in the Brazilian Cerrado. Asterisks and different letters denote significant difference in abundance (** = P
< 0.05) or that had a tendency to decrease in abundance (* = P < 0.1) among different categories of habitat fragmentation/habitat split according to the results of the
GLM and Tukey pairwise analysis.
species to a matrix comprising non-native grasses (pasture) is likely to be
very different from one comprising row crop agriculture (e.g., soy­
beans). Finally, we agree with Silva et al. (2012) that the preservation of
larger connected forest fragments in agricultural areas can be an effi­
cient means to conserve amphibian populations, as this can help protect
larger populations and maintain greater population viability that re­
duces extinction risk in landscapes fragmented by agricultural land use.
Although the differences between the two categories of habitat
fragmentation were not statistically significant, the finding of lower
taxonomic and functional diversity, reduced abundance of some species
(Physalaemus cuvieri and Rhinella diptycha), and the complete absence of
at least six species (e.g., Leptodactylus podicipinus, L. mystaceus,
L. natalensis, Elachistocleis cesarii, L. macrosternum and Proceratophrys
goyana) from within forest fragments that were isolated from ponds in
relation to unfragmented forests provides some support for our hy­
potheses regarding the expected negative effects of habitat split on
anuran abundance and diversity. The isolation of terrestrial environ­
ments from nearby aquatic habitats is a major factor known to influence
anuran communities in tropical forests (e.g., Atlantic Forest), contrib­
uting to lower richness, abundance, and probability of occurrence for
many species (Becker et al., 2007, 2010; Fonseca et al., 2008; Lion et al.,
2014; Silva & Rossa-Feres, 2011; Silva, Gibbs, & Rossa-Feres, 2011). An
increase in habitat split between forests and ponds would force adult
anurans that have aquatic larvae to undertake risky migrations through
7
Journal for Nature Conservation 69 (2022) 126259
the intervening land-use matrix to locate suitable breeding habitat
(Becker et al., 2007, 2010). In the Cerrado or in the Cerrado-Atlantic
Forest transition zone, studies have shown that forest fragments in
close proximity to breeding habitats (e.g., ponds) have a positive effect
on anuran abundance and diversity (Silva et al., 2012; Ribeiro, Colli,
Batista, et al., 2017). In experimental work, Silva et al. (2012) found a
greater abundance and richness of ground-dwelling anuran species in
small pools established near forest edges.
Although our results appear consistent with these studies, we found
no significant differences in anuran abundance or diversity between the
two fragment types, and so the effect of habitat split may be far weaker
than that of fragmentation in this part of the Cerrado. Alternatively, the
lack of a strong effect of habitat split could be due to the constraints of
our study design. In an effort to standardize habitat split in our study, we
sampled anurans within forest fragments that were located at a mini­
mum of 215 m from the nearest pond, such that the anuran communities
we surveyed represented a mix of species associated with both open and
forest habitats (Ramalho et al., 2018). It is thus possible that this dis­
tance was not a barrier for some species, demonstrating that Cerrado
anurans may be more resistant to desiccation than those that strictly
occur within forest ecosystems, such as the Atlantic Forest (Becker et al.,
2010). Furthermore, our focus here is solely on ground-dwelling an­
urans, which tend to have greater dispersal ability within open areas
than arboreal species (Basham, Seidl, Andriamahohatra, Oliveira, &
W.P. Ramalho et al.
Scheffers, 2019; Cayuela, Valenzuela-Sanchez, & Teulier, 2020; Ram­
alho et al., 2021). We suggest that future studies consider habitat split as
a continuous variable, perhaps encompassing distances considerably
greater than 215 m, and compare the response of species that use
different vegetation strata (e.g., terrestrial vs arboreal) or that occur
within other types of Cerrado habitats (e.g., cerrado sensu stricto).
In sum, the results of our study demonstrate that habitat fragmen­
tation can negatively affect the assemblage of terrestrial anurans in
several ways. The fragmentation of habitat by a non-native pasture
matrix appears to function as an environmental filter, selecting a
reduced number of species with similar functional characteristics that
are capable of traversing this relatively inhospitable matrix. We have
also shown that unfragmented forests are important for maintaining a
greater richness and overall abundance of ground-dwelling anurans, and
possibly ecosystem function as well, through the preservation of greater
functional diversity. We thus conclude that the fragmentation of forest
habitat by a matrix of non-native grasses (pasture) is largely responsible
for the loss of diversity and reduction in ground-dwelling anuran pop­
ulations within this region of the Cerrado.
Funding
This study was financed in part by the Coordenação de Aperfeiçoa­
mento de Pessoal de Nível Superior - Brasil (CAPES), which granted
scholarships to WPR (proc. 88882.448043/2019-01). VHMP was
financed by the Support Program for University Research and Scientific
Production of the Universidade Estadual de Goiás (PROBIP/UEG). WPR
is regional development researcher supported by the Fundação de
Amparo à Pesquisa do Estado de Goiás (FAPEG) and Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq) (process n◦
317724/2021-5). VG is supported by the Fundação de Amparo à Pes­
quisa do Estado de Mato Grosso (FAPEMAT.0000054/2022) and re­
ceives scholarship from CNPq (PDCTR - process n◦ 300094/2022-1).
This paper was also developed in the context of the National Institutes
for Science and Technology (INCT) in Ecology, Evolution and Biodi­
versity Conservation, supported by MCTIC/CNpq (proc. 465610/2014-
5) and FAPEG (proc. 201810267000023).
6. Permits
Permission to handle and collect animals was given by the Ministério
do Meio Ambiente / Instituto Chico Mendes de Conservação da Bio­
diversidade (#55420-1) and Conselho Estadual do Meio Ambiente /
Secretaria de Estado de Meio Ambiente e Desenvolvimento Sustentável
(#015/2016).
CRediT authorship contribution statement
Werther Pereira Ramalho: Conceptualization, Data curation,
Formal analysis, Funding acquisition, Investigation, Methodology,
Project administration, Resources, Supervision, Validation, Visualiza­
tion, Writing – original draft, Writing – review & editing. Kimberly A.
With: Conceptualization, Formal analysis, Investigation, Supervision,
Validation, Visualization, Writing – original draft, Writing – review &
editing. Gabryella de Sousa Mesquita: Investigation, Methodology,
Validation, Visualization, Writing – original draft, Writing – review &
editing. Filipe Viegas de Arruda: Investigation, Methodology, Valida­
tion, Visualization, Writing – original draft, Writing – review & editing.
Vinicius Guerra: Investigation, Methodology, Validation, Visualiza­
tion, Writing – original draft, Writing – review & editing. Denes Ferraz:
Investigation, Methodology. Murilo Sousa Andrade: Investigation,
Methodology. Vitor Hugo Mendonça do Prado: Conceptualization,
Formal analysis, Investigation, Supervision, Validation, Visualization,
Writing – original draft, Writing – review & editing.
8
Journal for Nature Conservation 69 (2022) 126259
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.
Data availability
Will be made available upon reasonable request.
Acknowledgments
We thank CAPES, CNPq, FAPEG, and FAPEMAT for the scholarships
granted to the authors; to professors and researchers Hélida Cunha,
Fabrício Teresa, Ibere Machado, Adriano Melo, Fernando Silva, and
João Carlos Nabout for providing helpful comments during the writing
of the manuscript; Edgar Luiz de Lima and Rony Peterson Almeida for
help in data analysis and editing the graphics; and to Ádamo Barros,
Alex Miranda, Ana Paula Pio, Arthur Sobral, Kalliman Lira, Gabrielly
Rodrigues, Jéssyca Morais, Jhonnathan Silva, José Ulisses Araújo,
Journan Calil, Lucas Gaehwiler, Lhayza Arantes, Mateus Cruz, Nathane
Costa, and Nicole Mendes for their assistance in the field work.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jnc.2022.126259.
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