Molecular Phylogenetics and Evolution 190 (2024) 107959

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Molecular Phylogenetics and Evolution

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

Integrative species delimitation uncovers hidden diversity within the

Pithecopus hypochondrialis species complex (Hylidae, Phyllomedusinae) and
its phylogeography reveals Plio-Pleistocene connectivity among
Neotropical savannas
Rafael F. Magalhães a, b, *, Elisa K. S. Ramos c, Lucas N. Bandeira d, Johnny S. Ferreira f,
Fernanda P. Werneck d, e, Marina Anciães d, e, Daniel P. Bruschi f, *
a
Department of Natural Sciences, Universidade Federal de São João del-Rei, Campus Dom Bosco, Praça Dom Helvécio, 70, São João del-Rei, MG 36301-160, Brazil
b
Postgraduate Programme in Zoology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, Belo Horizonte, MG 31270-
010, Brazil
c
Faculty of Philosophy and Natural Sciences, Department of Environmental Sciences, University of Basel, Bernoullistrasse 30, Basel 4056, Switzerland
d
Postgraduate Programme in Ecology, Instituto Nacional de Pesquisas da Amazônia, Avenida André Araújo, 2936, Manaus, AM 69067-375, Brazil
e
Scientific Biological Collections Program, Biodiversity Coordination, Instituto Nacional de Pesquisas da Amazônia, Avenida André Araújo, 2936, Manaus, AM 69067-
375, Brazil
f
Postgraduate Programme in Genetics, Department of Genetics, Biological Sciences Sector, Universidade Federal do Paraná, Caixa Postal 19071, Curitiba, PR 81531-
980, Brazil

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

Keywords: Despite their limited vagility and pronounced habitat heterogeneity in the tropics, many anuran species have
Amazonia unexpectedly extensive geographic ranges. One prominent example of this phenomenon is Pithecopus hypo­
Cerrado chondrialis, which is found in the Cerrado, Guianan savanna, and Llanos domains, as well as isolated tracts of
Ecological niche modelling
savanna and open habitat within the Amazon Forest. The present study employs an integrative species delimi­
Llanos
tation approach to test the hypothesis that P. hypochondrialis is in fact a species complex. We also reconstruct the
Guianan savannas
Phylogeographic diffusion relationships among the lineages delimited here and other Pithecopus species. In this study, we employ Ecological
Niche Modelling (ENM) and spatiotemporal phylogeographic reconstruction approaches to evaluate a multitude
of scenarios of connectivity across the Neotropical savannas. We identified three divergent lineages, two of which
have been described previously. The lineages were allocated to a lowland Pithecopus clade, although the re­
lationships among these lineages are weakly supported. Both the ENM and the phylogeographic reconstruction
highlight the occurrence of periods of connectivity among the Neotropical savannas over the course of the
Pliocene and Pleistocene epochs. These processes extended from eastern Amazonia to the northern coast of
Brazil. The findings of the present study highlight the presence of hidden diversity within P. hypochondrialis, and
reinforce the need for a comprehensive taxonomic review. These findings also indicate intricate and highly
dynamic patterns of connectivity across the Neotropical savannas that date back to the Pliocene.

1. Introduction isolation of lineages among distinct microhabitats (Guarnizo and Can­

Amphibians are generally considered to have a limited intrinsic
dispersal capacity (Smith and Green, 2005) and tend to have well-
defined habitat preferences. Surprisingly, however, some tropical spe­
cies are relatively widespread (Wynn & Heyer, 2001). The heterogeneity
of habitats in tropical regions tends to accentuate the specialisation and
natella, 2013; Rodríguez et al., 2015), and often leads to reduced gene
flow and increased lineage differentiation, which further reinforce these
patterns. However, philopatry is not a constant in the anurans, and some
species of the family Bufonidae may present a tendency for active, long-
distance dispersal (Fonte et al., 2019), and at least one species of the
family Leptodatylidae (Pseudopaludicola falcipes) appears to have high

* Corresponding authors.
E-mail addresses: rafaelfelixm@gmail.com (R.F. Magalhães), elisakramos@gmail.com (E. K. S. Ramos), azebandeira@gmail.com (L.N. Bandeira), johnny.sf@
gmail.com (J.S. Ferreira), fewerneck@gmail.com (F.P. Werneck), marina.anciaes@gmail.com (M. Anciães), danielpachecobruschi@gmail.com (D.P. Bruschi).

https://doi.org/10.1016/j.ympev.2023.107959
Received 12 May 2023; Received in revised form 25 October 2023; Accepted 27 October 2023
Available online 31 October 2023
1055-7903/© 2023 Elsevier Inc. All rights reserved.
R.F. Magalhães et al.
rates of passive dispersal (Langone et al., 2016). Even so, some recent
phylogeographic studies have shown that a number of Neotropical
anuran species previously thought to have extensive geographic ranges
may, in fact, be complexes of species, masked by cryptic diversity (e.g.,
Prado et al., 2012; Gehara et al., 2014; Nascimento et al., 2019; Vas­
concellos et al., 2019; Vacher et al., 2020; Abreu-Jardim et al., 2023).
Pithecopus hypochondrialis is a widespread, cis-Andean leaf frog,
which occurs in Suriname (the exact type locality is unspecified), Brazil,
Colombia, Guiana, French Guiana, and Venezuela (Frost, 2023). This
species thrives in a variety of ecological domains, including Amazonia,
the Cerrado, Guianan (or Roraima) savannas, and the Llanos of
Venezuela, where these frogs breed in swamps and ponds (Barrio-
Amorós, 2009; Bruschi et al., 2013; Guarnizo et al., 2015; Vaz-Silva
et al., 2020). The available evidence indicates that P. hypochondrialis
occupies open habitats, even within the Amazon rainforest (Barrio-
Amorós, 2009), where it is found primarily in savanna enclaves and
forest glades, clearings, and edges (Lescure et al., 1995; Bernarde, 2007;
Barrio-Amorós, 2009; Lima et al., 2017). Given the current disjunct
distribution of the savannas of South America, which include both
southern and northern blocks, as well as isolated fragments scattered
within the Amazonian rainforest (Silva and Bates, 2002; Werneck et al.,
2012; Azevedo et al., 2020), P. hypochondrialis has a discontinuous
distribution. This geographic discontinuity may contribute to the po­
tential isolation and eventual genetic structuring of its lineages.
Species of many other taxonomic groups have a similarly disjunct
distribution across the Neotropical savannas, including birds (e.g., Silva
and Bates, 2002; Lima-Rezende et al., 2019), lizards (e.g., Avila-Pires,
1995; Martins et al., 2021), and trees (e.g., Buzatti et al., 2017; Buzatti
et al., 2018). This recurring pattern may be related to the historical
connectivity among these regions, especially during the Quaternary
glacial cycles (Silva and Bates, 2002). Three primary corridors of con­
nectivity between the southern and northern Neotropical savannas have
been postulated (Webb, 1991; Silva and Bates, 2002): (i) an Andean
corridor through the cis-Andean slopes; (ii) a coastal Atlantic corridor,
connecting the blocks of savanna along the northern coast of Brazil; and
(iii) a central Amazonian corridor, which assumes that the retraction of
the forest during glacial periods favoured the expansion of savannas
through central Amazonia, forming a corridor of non-forest habitats that
connected previously isolated blocks of savanna. While most of the ev­
idence favours the Andean and Atlantic coast hypotheses (Silva and
Bates, 2002; Werneck et al., 2012), recent research has pointed to the
potential existence of central Amazonian corridors during the Quater­
nary (Bueno et al., 2017; Buzatti et al., 2018), although their timing and
configuration are not well understood, in part due to the absence of
macrofossils and palynological records that would validate the models
(Azevedo et al., 2020). Dowsett et al. (2016) also projected the potential
occurrence of savannas in present-day eastern Amazonia, during the
Pliocene (mid-Piacenzian, ~ 3 Ma), which represents an additional
hypothesis, on an older corridor (Azevedo et al., 2020). The phylo­
geography of P. hypochondrialis thus offers an opportunity to decipher
the possible centre of species diversification and test alternative hy­
potheses with regard to the ancient corridors connecting the Neotropical
savannas.
Few studies have investigated P. hypochondrialis from a broad
geographical perspective. Caramaschi (2006) examined the external
morphometry and colour patterns of specimens from Argentina, Bolivia,
Brazil, Colombia, and Suriname, which were all attributed to the nom­
inal species P. hypochondrialis at that time. This study identified two
colour morphotypes, which had previously been treated as two distinct
species – P. hypochondrialis and P. azureus – and revalidated the latter
taxon. In this analysis, P. hypochondrialis was distinguished from
P. azureus by the presence of a white stripe on the upper lip, which ex­
tends to the lower edge of the lower eyelid, and a narrow green stripe on
only 2/3 to 3/4 of the distal portion of the upper surface of the thigh (in
P. azureus, the white stripe on the upper lip does not reach the lower
edge of the lower eyelid, and the green stripe on the upper surface of the
2
Molecular Phylogenetics and Evolution 190 (2024) 107959
thigh is wide and covers its full length). Caramaschi (2006) showed that
these colour patterns were “congruent” (sic) within each species, which
suggests a lack of intraspecific variation. Pithecopus hypochondrialis was
assumed to have an exclusively Amazonia distribution, while the range
of the P. azureus morph extends across the Chaco, Pantanal, and Cerrado
domains.
Bruschi et al. (2013) revised comprehensively this taxonomic sce­
nario, re-examining the colour patterns and comparing them with DNA
sequences and chromosomal data. The results of this phylogenetic
analysis indicated that P. azureus is restricted to the Chaco and Pantanal
regions, and that it has a closer phylogenetic relationship with
P. nordestinus than P. hypochondrialis, a finding supported by other
studies (Faivovich et al., 2010; Haga et al., 2017; Andrade et al., 2020).
In contrast with the classification of Caramaschi (2006), the populations
of the Cerrado, together with their Amazonian counterparts, have been
attributed to P. hypochondrialis rather than P. azureus in Bruschi et al.
(2013). The authors identified three subclades (referred to here as A, B,
and C) within P. hypochondrialis. Subclade A included samples from
Belterra, in the Brazilian state of Pará, and Alta Floresta (Mato Grosso
state, Brazil). Subclade B encompassed samples from Chapada dos
Guimarães and Santa Teresinha (both in Mato Grosso), while subclade C
consisted of the remaining samples from the Brazilian states of Amapá,
Bahia, Maranhão, Minas Gerais, Pará, and Tocantins. The phylogenetic
relationship of these subclades was (A, (B, C)).
In particular, Bruschi et al. (2013) reported a broader variation in
colouration patterns than that found by Caramaschi (2006), and
described four distinct phenotypes. These phenotypes presented no
systematic geographical structuring, which contradicted the differenti­
ation of P. hypochondrialis and P. azureus by these traits. Bruschi et al.
(2013) also found variation in the distribution of the Nucleolar Orga­
niser Region (NOR) among the P. hypochondrialis populations examined
from subclades A, B, and C. In particular, they were able to discriminate
subclade A from the other subclades due to the presence of NORs on the
short arm of chromosome pair 8, although they interpreted this as
intraspecific variation linked to the population structure of the species.
The NORs are valuable chromosomal markers, which are pivotal for the
diagnosis of many anuran species (Schmid et al., 2014) and the identi­
fication of independent evolutionary lineages (Nascimento et al., 2019).
More recently, Haga et al. (2017) described the new species
P. araguaius, which is closely related to P. hypochondrialis. This species
corresponds to subclade B in the study of Bruschi et al. (2013). To
support their description, Haga et al. (2017) contrasted the adult
morphometry, bioacoustic traits, and mitochondrial DNA markers of
this new taxon with samples of P. hypochondrialis, classified as
“P. hypochondrialis North” and “P. hypochondrialis South” from Ama­
zonia and the Cerrado, respectively. Their findings indicate that
P. araguaius can be distinguished from P. hypochondrialis by statistically
significant differences in its morphometry and advertisement calls
(Haga et al., 2017). Given the overlap in the morphometric and acoustic
attributes of P. araguaius and P. hypochondrialis, however, these closely-
related species cannot be distinguished reliably based solely on their
phenotypic traits. As a result, they are considered to form a complex of
morphologically and acoustically cryptic species, referred to here as the
P. hypochondrialis species complex. An alternative position would be to
consider them as synonymous taxa.
Remarkably, Haga et al. (2017) did not sample any individuals from
Bruschi et al.’s (2013) subclade A, which raises the possibility of the
existence of at least one more candidate species within the
P. hypochondrialis species complex. The phenotypic variation found
among these lineages is somewhat confusing. While Bruschi et al. (2013)
demonstrated that conventional colouration patterns are not diagnostic
of P. hypochondrialis, they did identify differences in the frequency of the
morphotypes among the lineages, possibly along a longitudinal axis (see
Fig. 1 in Bruschi et al., 2013). By contrast, Haga et al. (2017) reported
morphometric differences along a latitudinal axis, with progressively
larger individuals moving northwards within the species’ range.
R.F. Magalhães et al.
These contrasting patterns in the distribution of the phenotypes, and
the lack of an integrative species delimitation (sensu Yeates et al., 2011),
raise a number of questions with regard to the taxonomic arrangement
of the species complex and the biogeographic history of its closely-
related lineages. In this context, the present study tests alternative hy­
potheses, i.e., whether P. hypochondrialis is a single, widespread species
or a cryptic species complex. For this, we employed coalescent and
integrative species delimitation methods to test the evolutionary inde­
pendence (sensu Simpson, 1951) of the different lineages. We also
investigated the extent to which the morphological variation observed
in the populations reflects the genetic structure found within the
P. hypochondrialis complex, which includes P. azureus in some compar­
isons, due to the apparent morphological similarities of this species
(Bruschi et al., 2013).
To address these questions, we investigated the intrinsic genetic
variation and genealogical history of the species complex using multi­
locus mitochondrial and nuclear datasets in a comprehensive integrative
approach. We generated novel sequence data and incorporated se­
quences available in public databases to cover the geographic distribu­
tion of the species complex as extensively as possible. We also re-
evaluated the colouration patterns examined by Bruschi et al. (2013)
and integrated the morphometric variation of the specimens into our
species delimitation analyses. Ultimately, the present study extends the
application of phylogeographic analyses and paleo-distribution model­
ling to a Plio-Pleistocene temporal scale. Through this approach, we aim
to decipher the historical connectivity of the savanna corridors of South
America, which may have played a fundamental role in the establish­
ment of the current distribution of the P. hypochondrialis species
complex.
2. Material and methods
2.1. DNA sampling and editing
We collected 131 tissue samples of P. hypochondrialis and P. araguaius
from 24 localities, together with 10 samples of P. azureus from four sites
(Table S1; see Results). We collected the latter samples due to their
morphological similarity and geographic proximity to our study species
(Faivovich et al., 2010; Bruschi et al., 2013; Haga et al., 2017). The
tissue samples were collected from euthanised specimens following the
application of an anaesthetic (5 % Lidocaine) to the skin, in accordance
with the recommendations of the Herpetological Animal Care and Use
Committee (HACC) of the American Society of Ichthyologists and Her­
petologists (available at: https://www.asih.org/publications). The
collection of samples was approved by SISBIO/Instituto Chico Mendes
de Conservação da Biodiversidade, through collecting permit number
14468-1/14468-4. Voucher specimens or tissue samples were deposited
in the Célio Fernando Baptista Haddad Amphibian Collection (CFBH) of
the Universidade Estadual Paulista in Rio Claro, São Paulo, Brazil; the
Prof. Adão José Cardoso Museum of Zoology (ZUEC) at Universidade
Estadual de Campinas in São Paulo, Brazil; the Shirlei Maria Recco-
Pimentel tissue collection (SMRP) at Universidade Estadual de Campi­
nas; the Zoological collection of Universidade Federal de Goiás (ZUFG)
in Goiás, Brazil; and the amphibian collection from Museu de Zoologia
da Universidade de São Paulo (MZUSP), in São Paulo, Brazil. We also
obtained sequences of at least one individual of each Pithecopus species
(except P. gonzagai Andrade et al., 2020) from GenBank (Table S2), as
well as one individual of Callimedusa tomopterna, and sequences of
P. azureus and P. hypochondrialis from locations not represented in our
sampling, including the P. hypochondrialis type locality (Table S3).
The genomic DNA was extracted from the samples of liver and
muscle tissue (preserved in 95 % ethanol) using the TNES method (see
Bruschi et al., 2012). We amplified and sequenced fragments of two
mitochondrial (mtDNA) genes – NADH dehydrogenase subunit 2 (ND2)
and the ribosomal 16S gene (16S) – and two nuclear (nDNA) loci –
Rhodopsin exon 1 (RHOD) and Seven in Absentia Homolog 1 (SIA-H1).
3
Molecular Phylogenetics and Evolution 190 (2024) 107959
Information on the primers used to amplify each fragment is provided in
Table S3. The Polymerase Chain Reactions (PCRs) were run in a total
volume of 25 µL containing 20–100 ng of the DNA template, 1X PCR
buffer, 1.5 mM MgCl2, 7 pmoles/µl of the forward and reverse primers,
1 mM of dNTPs, and 1U of Taq polymerase. The temperatures and
annealing times for each fragment are provided in Table S3. The
amplicons were purified with exonuclease I (10 units) and SAP (1 unit)
to remove the unincorporated dNTPs and primers. The amplified frag­
ments were sequenced in an ABI PRISM™ 3100 Genetic Analyzer
(Applied Biosystems, Foster City, CA, USA) in both directions, using the
original amplification primers and BigDye™ terminator v.3.1
sequencing chemistry according to the manufacturer’s protocol
(Applied Biosystems, Foster City, CA, USA). We employed CodonCode
Aligner 7.1.1 to verify the base calls and to correct the base-calling er­
rors found in the sequences.
We aligned the fragments with MAFFT v.7.427, which was imple­
mented in the online service (Katoh and Standley, 2013; Katoh et al.,
2019). We used the automatic strategy for coding fragments (i.e., ND2,
RHOD, and SIA-H1), while for the 16S gene, we used the Q-INS-i algo­
rithm (Katoh and Toh, 2008) set at the default parameters. Finally, we
reconstructed the haplotype phases of the nDNA fragments using PHASE
v.2.1.1 (Stephens et al., 2001) for some of the subsequent analyses,
considering a Posterior Probability (PP) threshold of 0.7 to accept the
estimated phases. The SeqPHASE webtool (Flot, 2010) was used to
interconvert the input/output of PHASE.
2.2. Morphometric and morphotyping data
We assessed 18 morphometric characters in 151 adult male speci­
mens of P. hypochondrialis and P. araguaius, which were obtained from
the herpetological collections mentioned above (Table S4). At least one
gene fragment was also sequenced in 79 % of these individuals. We
measured the Snout-Vent Length (SVL), First Finger Length (FFL), Head
Length (HL), Head Width (HW), Eye Diameter (ED), Tympanum Diam­
eter (TD), Eye-Nostril Distance (END), Nostril-Snout Distance (NSD),
Inter-Nostril Distance (IND), Inter-Orbital Distance (IOD), and Tibia
Length (TL) following the protocol of Duellman (1970). We also deter­
mined the Arm Length (AL), Forearm Length (FAL), Forearm Width
(FAW), Hand Length (HAL), Thigh Length (THL), and Foot Length (FL)
following Heyer et al. (1990), as well as the First Toe Length (FTL),
which we defined as the distance between the proximal margin of the
inner metatarsal tubercle and the distal apex of the first toe. We acquired
the measurements (in millimetres) using a needlepoint calliper with an
accuracy of 0.01 mm.
We followed the approach of Lleonart et al. (2000) to eliminate the
potential allometric effects in the data. This involves regressing the
log10-transformed variables against a size proxy determined by a Prin­
cipal Components Analysis (PCA). This proxy is the variable with the
highest loading value obtained by the first principal component. The
eigenvectors of the variance–covariance matrix were obtained using the
‘prcomp’ function in the vegan v.2.5–6 package of the R software
(Oksanen et al., 2016), with the variable FAL being selected as the proxy
for the analysis. We then ran pairwise linear regressions between the
FAL and the other variables using the stats package in R v.4.0.0 (R Core
Team, 2020). All the subsequent analyses were conducted using the
residuals of these regressions. Finally, we calculated the Variance
Inflation Factors (VIFs) among the corrected variables, to estimate the
multicollinearity of the data, in the car package v.3.0–7 (Fox & Weis­
berg, 2018) of the R software. Only traits with VIF values smaller than 5
(Akinwande et al., 2015) were retained for all subsequent analyses but
PERMANOVA (see section 2.4.3).
In addition to morphometric traits, colouration patterns have been
employed traditionally in taxonomic studies of the subfamily Phyllo­
medusinae (Caramaschi et al., 2006). While some studies (e.g., Bruschi
et al., 2013; Brunes et al., 2014) have raised doubts with regard to the
efficacy of these features for the diagnosis of taxa, they can provide
R.F. Magalhães et al.
additional insights into the differentiation of species, when an appro­
priate statistical approach is adopted. Given this, we re-evaluated the
four colour morphs defined by Bruschi et al. (2013) and assigned each of
the 179 specimens of P. hypochondrialis, P. araguaius, and P. azureus
analysed here (Table S4) to one of these morphotypes. Pithecopus azureus
was included in these comparisons specifically to assess the extent of the
differentiation between this species and P. hypochondrialis, considering
their complex taxonomic history. We classified the colour morphs
following the exact procedure outlined by Bruschi et al. (2013), as
follows:
Morphotype 1: Narrow white stripe on the upper lip extending to the
lower eyelid together with the presence of a discontinuous, wide green
stripe along 2/3 to 3/4 of the length of the upper surface of the thighs.
Morphotype 2: White stripe on the upper lip extending to the lower
eyelid together with the presence of a wide green stripe along the full
length of the upper surface of the thighs.
Morphotype 3: White stripe on the upper lip extending to the lower
eyelid together with a green stripe absent on the upper surface of the
thighs.
Morphotype 4: Thin white stripe on the upper lip that does not
extend as far as the lower eyelid. Wide green stripe along the full length
of the upper surface of the thighs.
2.3. Phylogenetic analyses
2.3.1. Concatenated phylogeny
Given the difficulty of distinguishing P. araguaius, P. azureus, and
P. hypochondrialis based on their external morphology (Bruschi et al.,
2013; Haga et al., 2017), we cannot rule out altogether the possibility
that some of the specimens obtained from the biological collections have
been misidentified. Given this, we constructed a tree concatenating all
the gene fragments of each species into a super-gene alignment before
running the lineage delimitation analyses, in order to verify the taxo­
nomic identity of each individual. To do this, we included specimens
from the type localities of P. araguaius (Pontal do Araguaia, Mato Grosso
state, Brazil) and P. hypochondrialis (Suriname), as well as specimens
that can be attributed unequivocally to P. azureus (from localities in
Argentina and Brazil, within the basins of the Paraná and Paraguay
rivers; Caramaschi, 2006), as taxonomic references.
To compile the phylogeny, we first excluded all the individuals that
lacked sequences for both mitochondrial fragments. We also included
representatives of all the remaining Pithecopus species except
P. gonzagai, and rooted the tree at Callimedusa tomopterna, given that
Callimedusa is the sister genus of Pithecopus (Faivovich et al., 2010;
Duellman et al., 2016). We ran the Bayesian inference in BEAST v. 2.6.2
(Bouckaert et al., 2019). We concatenated all the unphased fragments (i.
e., linking all the trees) while estimating the site and clock models of
each one independently, to increase the accuracy of the reconstruction
of the branch lengths. The site model was co-estimated with the
phylogenetic analyses using the bModelTest package v. 1.2.1 (Bouckaert
and Drummond, 2017), with the set of test models being defined as
‘allreversible’. The strict clock was set for all four fragments, with the
clock rate being fixed at 1.0 for the 16S gene, with all the others being
estimated relative to this gene. Finally, the tree prior was set to the Yule
Model. We ran the analysis twice, with 5 × 107 generations each,
thinning to 5 × 103. Convergence between the runs, the mixing of the
parameters, and the minimum Effective Sample Sizes (ESS > 200) were
verified in Tracer v. 1.7.1 (Rambaut et al., 2018). The results of both
runs were combined, with the first 10 % of the trees being discarded as
burn-in, and the Maximum Clade Credibility (MCC) tree being anno­
tated using the LogCombiner and TreeAnnotator post-processing tools,
respectively (Bouckaert et al., 2019).
2.3.2. Mitochondrial gene trees
Only the 16S sequences were used to generate the ultrametric
mtDNA gene trees, given that a large proportion (38.3 %) of the
4
Molecular Phylogenetics and Evolution 190 (2024) 107959
sequences were missing in the ND2 dataset (vs. 17 % in the case of 16S;
Tables S1-2). We identified the unique haplotypes in the haplotypes
v.1.1.2 package of the R software (Aktas, 2020) by using the haplotype()
function with the indels=“sic” option, and then removed the equal
haplotypes before a second run with the indels=“missing” option. This
step is important because the accumulation of short branches can lead to
an overestimate of candidate species by the GMYC algorithms (see
section 2.4.1) (Reid and Carstens, 2012; Talavera et al., 2013).
We generated a set of ultrametric gene trees from these filtered se­
quences using the BEAST 2 software (Bouckaert et al., 2019). For this
analysis, the substitution model was identified using bModelTest, as
above, with the Yule tree and log-normal clock models. This combina­
tion of priors resulted in a high percentage of correct hits in the GMYC
performance test, based on empirical data from butterflies (Talavera
et al., 2013). The analysis was run twice, with 1 × 107 generations in
each case, and thinning to 1 × 103. The convergence, mixing, and
minimum ESS were all verified as above. The results of these runs were
combined and MCC tree was annotated after a 5 % burn-in.
2.3.3. Nuclear haplotype networks
We constructed nuclear haplotype networks for comparative pur­
poses. For this, we generated Maximum Likelihood (ML) gene trees for
the RHOD and SIA-H1 fragments using MEGA X v.10.2.6 (Kumar et al.,
2018) under the GTR model. The initial trees for the heuristic search
were obtained automatically by applying the Neighbor-Joining and
BioNJ algorithms to a matrix of pairwise distances estimated using the
Maximum Composite Likelihood approach, with the topology with the
highest log-likelihood value being selected. The ML gene trees were
converted into haplotype genealogies using the HaploViewer program
(Salzburger et al., 2011).
2.3.4. Species tree dating
To estimate the relationships and divergence times among the spe­
cies identified in the P. hypochondrialis species complex (see Results), we
constructed a species tree in the StarBEAST2 v.0.15.5 package (Ogilvie
et al., 2017) of the BEAST2 software. For this, we linked the 16S and
ND2 fragments in the same mtDNA gene tree but estimated their sub­
stitution models and clocks independently. The trees and parameters of
the nDNA fragments were estimated independently. The substitution
model of each fragment was co-estimated using the bModelTest package
(Bouckaert and Drummond, 2017), with the set of test models being
defined as ‘allreversible’. The tree prior was set as the birth-and-death
process (Gernhard, 2008), and the species tree was rooted at
P. azureus. Given the lack of phylomedusine fossils with which to cali­
brate the species tree, we used the substitution rate of the ND2 gene
(0.957 % per lineage per million years; Crawford, 2003), which was
used as the reference to estimate the rates of the other fragments, with
an uncorrelated relaxed log-normal clock being assumed for all the
fragments (Drummond et al., 2006). We ran the analysis twice, with 1 ×
108 generations each, thinning to 1 × 104, and burnin 1 × 106.
Convergence between runs, mixing of the parameters, and ESS were
verified in Tracer. The results of both runs were merged and the MCC
tree was annotated using the LogCombiner and TreeAnnotator.
2.4. Species delimitation
2.4.1. Molecular species discovery and validation
We delimited the lineages within P. hypochondrialis by applying a
two-step iterative procedure, consisting of discovery and validation
approaches (Carstens et al., 2013), to the molecular data. In the dis­
covery step, we estimated the number of putative taxonomic units using
the General Mixed Yule-Coalescent (GMYC) model (Pons et al., 2006).
The GMYC analysis was implemented using two distinct algorithms. The
Maximum Likelihood version (ML-GMYC) was applied to the MCC gene
tree (see section 2.3.2), using the single-threshold method implemented
in the splits package v.1.0–19 of the R software (Fujisawa and
R.F. Magalhães et al.
Barraclough, 2013; R Core Team, 2020). In addition, to account for the
uncertainties in the topology and branch lengths estimation, we applied
the Bayesian version of the model (bGMYC) to a sample of 2.5 × 103
random trees generated by BEAST 2 (see section 2.3.2). This analysis
was conducted in the bGMYC v.1.0.2 package of the R software (Reid
and Carstens, 2012; R Core Team, 2020), using the single-threshold
method and limiting the boundaries for the number of putative species
to a threshold between one and the maximum value of the credibility
interval of the entities delimited by the ML-GMYC analysis.
Talavera et al. (2013) reported an improvement in precision when
the taxonomic operational units are represented by only a single or a few
haplotypes. Given this, we ran the discovery step twice. In the first step,
we constructed the mtDNA gene tree using all the unique haplotypes. In
the second iteration, we randomly selected one of the most complete
sequences delimited per lineage among the sequences with the greatest
length (Table S5), estimated new gene trees, and ran the ML-GMYC and
bGMYC analyses once again. The results of this second iteration were
used in the validation step. To compare the results of the GMYC itera­
tions, we estimated the uncorrected p-distances within and between the
16S lineages delimited by each step. These distances were generated
using MEGA X, with all the ambiguous positions (i.e., missing data and
gaps) being removed from each sequence pair (pairwise deletion
option).
The validity of the lineages discovered during this analysis was tested
using the mtDNA and nDNA datasets. For this, we used the STACEY
v.1.2.5 package (Jones, 2017) of the BEAST 2 software, grouping in­
dividuals that were discovered in the previous step as putative species.
Each locus was now set to have its own independent substitution model,
estimated by bModelTest. The clock rates were set as strict and inde­
pendent for each fragment. The 16S and ND2 fragments were linked in
the same mtDNA gene tree. The inheritance scalars were set at 0.5 for
the mtDNA, and at 2 for the nDNA genes. We set the collapse height of
the priors as 1 × 10-3, their collapse weight as ~ uniform (0.1), the
population prior scale as ~ lognormal (M = -7, S = 2), the growth rate as
~ lognormal (M = 5, S = 2), and the relative death rate as ~ β (α = 1, β
= 8). The analysis was run twice, with 1 × 108 generations in each case,
and thinning to 1 × 104. The convergence, mixing, and minimum ESS
were all verified as above. The results of these runs were combined and
MCC tree was annotated after a 10 % burn-in. The best number of
candidate species was determined using the speciesDA program (Jones,
2017) with a burn-in of 10 %, and collapse heights of 1 × 10-4, 5 × 10-4,
7.5 × 10-4, and 1 × 10-3. This set of heights was adopted to verify the
robustness of the delimitation.
2.4.2. Isolation-by-distance test
As Isolation By Distance (IBD) may lead to an overestimate of the
number of candidate species in molecular delimitation methods
(Chambers and Hillis, 2020; Mason et al., 2020), we applied the Mantel
test (Mantel, 1967) to the pairwise FST and geographical distance
matrices of the 16S sequence data. This analysis was implemented in the
Arlequin v.3.5.2.2 software (Excoffier and Lischer, 2010). We selected a
specific subset of the 16S sequences for the IBD analysis (Table S1) as a
strategy to reduce the impact of incomplete sequences on our analysis.
We estimated the linear distances between sampling sites using the
Geographic Distance Matrix Generator v.1.2.3 (GEODIS) platform of the
American Museum of Natural History (Posada et al., 2000), using the
sample coordinates. The runs consisted of 1 × 104 permutations and
were applied to the sequences representing the entire distribution of the
species complex and separately for each lineage discovered during the
analysis.
2.4.3. Morphological analyses
We used morphometric data to estimate separately the optimal
number of clusters using the phenotypic model implemented in the
Geneland v.4.0.6 package of the R (Guillot et al., 2012; R Core Team,
2020), without incorporating the spatial model or making any a priori
5
Molecular Phylogenetics and Evolution 190 (2024) 107959
assumptions about the taxonomic identity of the individuals. We ran the
first analysis with Kmin = 1 and Kmax = 12 (i.e., the optimal number of
genetic clusters identified in the first iteration of the GMYC: see Results).
We conducted 10 independent runs, each with 1 × 107 generations, 1 ×
103 thinning, and a burn-in period of 150 first results before thinning.
We checked for mixing and convergence by inspecting the posterior
density of each run and comparing the results across the runs. Once we
had determined the best K value, we conducted an additional run, for
which we kept the K fixed at its optimal estimate. We used this step to
generate more precise morphometric membership probabilities for the
individual samples (Guillot et al., 2005). We then generated the Q-plot
for this final run using DISTRUCT version 1.1 (Rosenberg, 2004). To
determine whether the morphometric data could discriminate inde­
pendently the lineages validated by the genetic species delimitation
analysis (see above), we applied a Permutational Analysis of Variance
(PERMANOVA) to the morphometric traits in Euclidian distance, using
the vegan v. 2.5–7 package of the R software (Oksanen et al., 2016).
Finally, to test for patterns of variation in the frequency of the
different colour morphotypes among the candidate species, we applied
Pearson’s χ2 test in R stats v. 3.5.0. We included the P. azureus specimens
in this analysis to test Caramaschi’s (2006) hypothesis that individuals
of this species can be differentiated from P. hypochondrialis based on
their colouration patterns (i.e., colour morphotypes 4 and 1,
respectively).
2.4.4. Integrative species validation
We employed an integrative validation analysis, based on the inte­
grated Bayesian Phylogenetics and Phylogeography model, which we
implemented in iBPP v.2.1.3 (Solís-Lemus et al., 2015). We used both
the molecular and the morphometric data, following the VIF correction,
to satisfy the assumptions of the analysis. As the species tree retrieved
low support for the relationships among the lineages (see Results), we
ran distinct analyses using all the three possible species trees, and tested
the divergence scenario of the putative species recovered by the second
iteration of the GMYC. Four distinct gamma prior combinations were
used for the theta (θ) and tau (τ) parameters, with two replicates each,
including (i) large ancestral populations and deep divergence (θ ~ G (2,
100), τ ~ G (2, 200)), (ii) small ancestral populations and shallow
divergence (θ ~ G (2, 1000), τ ~ G (2, 2000)), (iii) small ancestral
populations and deep divergence (θ ~ G (2, 1000), τ ~ G (2, 200)), and
(iv) large ancestral populations and shallow divergence (θ ~ G (2, 100),
τ ~ G (2, 2000)). The priors for the continuous morphometric trait data
were set as in Solís-Lemus et al. (2015). We used the Dirichlet prior
(Yang and Rannala, 2010) for the locus-rate parameters. We chose an
automatic adjustment in the MCMC step length, and ran all the analyses
with 2 × 105 generations and a burn-in of 2 × 104 generations.
2.4.5. Heuristic species delimitation
To assess the strength of the divergence among the lineages validated
by the iBPP, we conducted a heuristic analysis using the genetic diver­
gence index (gdi) of Jackson et al. (2017). We initially ran the A00
analysis in the iBPP framework to obtain the posterior distributions for
the τ and θ parameters across the three different tree topologies with the
prior combinations of θ and τ used in the iBPP. For this, we applied the
same MCMC settings as those used in the integrative species validation.
We subsequently used these distributions to calculate the gdi for all the
potential species divergence events based on our guide trees. We
calculated the gdi values using the R function available at https://github.
com/melisaolave/mafalda. In this approach, the study populations are
classified as distinct species if their gdi values exceed 0.7, while gdi
values below 0.2 indicate that they should be considered to belong to the
same species. Intermediate gdi values, ranging from 0.2 to 0.7, indicate
an ambiguous species status (Jackson et al., 2017).
R.F. Magalhães et al. Molecular Phylogenetics and Evolution 190 (2024) 107959

2.5. Paleodistribution models and climatic stability across geological
periods
We modelled the Ecological Niche (ENM) of the species complex to
estimate both its current potential distribution (i.e., the Species Distri­
bution Model, SDM) and its historical distribution during nine periods in
the past (0.3 ka–3.3 Ma, see below), as well as mapping the areas of
suitable climate available over time (areas of stability).
To this end, the totality of the occurrence records of
P. hypochondrialis species complex was used to construct the niche
models using the subsequent modelling routines. The occurrence re­
cords were compiled from the herpetological collections consulted
during the study Bandeira et al. (2021) and are listed in Table S6. We
also verified the potential existence of complementary records in the
relevant published papers to include data points that could be assigned
unambiguously to a precise locality via the GPS coordinates provided in
the publication. After obtaining, verifying, and validating each occur­
rence record, our final set of unique geo-referenced localities included
296 records of P. hypochondrialis species complex, which represents a
satisfactory coverage its known geographic range (Table S6).
To compile the current and past climate patterns, we assembled 17
layers of climatic variables with continuous data on temperature and
precipitation that are closely related to the ecological and physiological
tolerances of anurans (Duellman & Trueb, 1994; Wells, 2007). All the
variables were obtained from the Paleoclim online bioclimatic dataset
(https://paleoclim.org/, Otto-Bliesner et al., 2006; Dolan et al., 2015;
Hill, 2015; Fordham et al., 2017; Brown et al., 2018) at a resolution of
2.5′ (~5 km). For the present day, bioclimatic parameters were derived
from the monthly min, max, mean temperature, and mean precipitation
values, to determine annual trends (e.g., mean annual temperature,
annual precipitation), seasonality (e.g., the annual range in temperature
and precipitation), and extreme or limiting environmental factors (e.g.,
temperature of the coldest and warmest months, and the precipitation of
the rainiest and driest quarters), for the 1967–2013 time-series. For past
periods, we decided to assemble paleo-climatic simulations representing
nine periods ranging from the Pliocene to the Holocene: (i) the Pliocene,
M2 (ca. 3.3 Million years ago – Ma), (ii) the mid-Pliocene warm period
(3.205 Ma), (iii) MIS19 (ca. 787 thousand years ago – ka), (iv) the Last
Interglacial – LIG (ca. 130 ka), (v) the Last Glacial Maximum – LGM (ca.
21 ka), (vi) Heinrich Stadial 1 (11–14.7 ka), (vii) the early Holocene
(11.7–8.326 ka), (viii) the mid Holocene (8.326–4.2 ka), and (ix) the late
Holocene (4.2–0.3 ka).
The ENMs were constructed using the Maximum Entropy algorithm,
MaxEnt (Phillips et al., 2006), which was implemented in the KUENM
package v.1.1.7 (Cobos et al., 2019) in R v.4.0.0. The MaxEnt algorithm
generates predictions from presence-only data, based on the principle of
maximum entropy, which assumes that the best approximation for an
unknown distribution is the one that satisfies any constraint on its dis­
tribution (Phillips et al., 2006; Elith et al., 2011). This algorithm is
considered to be extremely efficient and is widely used to model species
distributions.
The final models were generated using 10 independent replicates,
while 50 % of the points were used for testing (Fig. S1), using the
Bootstrap method (Pearson, 2010). We chose the logistic output to
represent the ENMs in geographic space (SDM), with the suitability of
each pixel (geographic cell) ranging from 0 (totally inadequate condi­
tions) to 1, i.e., maximum suitability (Phillips et al., 2006). The per­
formance of the candidate models was evaluated hierarchically using
three independent, but complementary criteria: significance, predictive
ability, and complexity. The models were then filtered to detect the
statistically significant ones, and the “low-omission criterion” (the Area
Under the Curve [AUC] method; Phillips et al., 2006) was applied to
further reduce the set of models. Finally, the significant models with the
lowest omission rate whose delta AICc values were lower than two were
selected as the best models (Cobos et al., 2019).
To project the SDM of the P. hypochondrialis species complex into the
past, we converted the suitability maps (with values ranging from 0 to 1)
to binary presence-absence maps using a “cut-off” threshold value. As
recommended by Liu et al. (2013), we converted the SDMs using the
“Maximum Test Sensitivity plus Specificity – MTS + S” criterion as the
threshold value. This minimizes the areas identified erroneously as un­
suitable (false negatives) in the candidate models, as well as those

Fig. 1. Maximum clade credibility tree obtained from the Bayesian phylogenetic inference of the concatenated mtDNA (16S, ND2) and nDNA (RHOD, SIA-H1)
fragments, showing the relationships among the Pithecopus species. The information contained within the parentheses includes the acronym and the registration
number of the collection in which the specimen is deposited, a two-letter ISO (International Organisation for Standardisation) 3166 standard country code, the state
or department, and the municipality of registration. The circles at the branch nodes show their respective posterior probabilities (PP), as indicated in the legend.
Nodes with PP > 0.95 (strong support) are denoted by black circles. Please see Fig. 2 for further insights into the relationships within the P. hypochondrialis
species complex.

6
R.F. Magalhães et al. Molecular Phylogenetics and Evolution 190 (2024) 107959

(caption on next page)

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R.F. Magalhães et al. Molecular Phylogenetics and Evolution 190 (2024) 107959

Fig. 2. Maximum clade credibility tree obtained from the Bayesian phylogenetic inference of concatenated mtDNA (16S, ND2) and nDNA (RHOD, SIA-H1) frag­
ments, showing the relationships among the specimens in the Pithecopus hypochondrialis species complex. The information contained within the parentheses includes
the acronym and the registration number of the collection in which the specimen is deposited, a two-letter ISO (International Organisation for Standardisation) 3166
standard country code, the state or department, and the municipality of registration. Branches with highlited information in bold represent topotypic individuals of
P. araguaius and P. hypochondrialis. The circles at the branch nodes show their respective posterior probabilities (PP), as indicated in the legend. Nodes with PP > 0.95
(strong support) are denoted by black circles. Nodes with PP < 5 % are not highlighted with circles. The numbers within the white vertical bars with black borders on
the right side of the tree refer to the lineages identified during the initial iteration of the general mixed Yule-coalescent (GMYC) analysis (see Results; Fig. S3). The
colour-coded vertical bars on the right indicate the lineages validated by the molecular delimitation analyses (see Results). The geographical distribution of these
lineages is depicted using colour-coded minimal convex polygons in the map in the lower left corner. The circles outlined in black on this map represent the type
localities of P. araguaius (orange) and P. hypochondrialis (blue). For the relationships within the outgroups, see Fig. 1. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)

lacking predicted suitable habitat (false positives), which has a higher
risk of overestimating distributions rather than missing important
habitats.
Finally, each of the SDMs was intersected to obtain a final model
representing the distribution of climatically stable areas for the occur­
rence of the species complex over time. In geographical terms, this
model represents all the sites or regions that potentially remained
occupied by P. hypochondrialis throughout all the periods analysed, i.e.,
that remained suitable for the species and can be identified as a climatic
refugium (e.g., Carnaval and Moritz, 2008; Werneck et al., 2012).
2.6. Spatiotemporal phylogeographic dynamics
We used a spatiotemporal species tree diffusion approach (Nylinder
et al., 2014) to determine which Neotropical savanna corridor hypoth­
esis best fits our data. This approach is based on the log-normal Relaxed
Random Walk (RRW) algorithm (Lemey et al., 2010), which was
implemented in BEAST v.1.8.4 (Drummond et al., 2012). The species
trees used as the input were produced by StarBEAST2, as described
above. However, we initially estimated the relationships among the 12
lineages discovered in the first iteration of the GMYC rather than the
three validated species (see Results, Fig. 2). In this step, we merged the
individuals from Santarém with those of lineage 1.2 (see Results). We
used the ND2 substitution rate (see section 2.3.4) to calibrate the species
trees, and the substitution rates estimated in the first StarBEAST2
analysis for the other genes, with a normal distribution (mean = 4.82 ×
10-3, sigma = 5.70 × 10-4 for 16S; mean = 3.06 × 10-4, sigma = 9.20 ×
10-5 for RHOD; mean = 5.84 × 10-4, sigma = 1.55 × 10-4 for SIA-H1),
setting the clocks as uncorrelated relaxed log-normal (Drummond et al.,
2006). Each analysis was run twice, with 1 × 108 generations, 1 × 104
thinning, and 10 % burn-in. The ESS and convergence between runs
were checked in Tracer, as above. The log and tree files of each gene tree
were combined using LogCombiner v.1.8.4, and the MCC gene trees
were computed with TreeAnnotator v.1.8.4 (Drummond et al., 2012).
When three or more geographic records were available for an
inferred lineage (i.e., lineages 1.3, 3.1, 3.4, 3.5, and 3.6; see Results), the
geographic range was represented as a minimum convex polygon, with a
0.5 decimal degree buffer. In the case of the lineages with only one re­
cord (i.e., lineages 1.1 and 3.2), the range was drawn as a circle with a
radius of 0.5 decimal degrees around the point. Finally, the geographic
ranges of the lineages with two records (i.e., lineages 1.2, 2.1, 2.2, 2.3,
and 3.3) were represented as an ellipse formed by a 0.5 decimal degree
buffer around a line connecting the two points. We used the tutorial
provided by Nylinder et al. (2014) to configure the input analysis from
the posterior sample generated by the StarBEAST2 analysis (see section
2.3.4) and the geographic distribution of the lineages. We ran the
analysis twice, with different starting points for each lineage. Each
replicate was run over 5 × 107 generations, with 5 × 104 thinning, and
10 % burn-in. We combined the results of the two runs in LogCombiner
and annotated the MCC tree using TreeAnnotator. Finally, we used the
MCC tree to compile the spatiotemporal dynamics using Spread v.1.0.7
(Bielejec et al., 2011), and summarised the variation in diffusion rates
over time using the TimeSlicer tool (Lemey et al., 2010).
3. Results
3.1. Concatenated phylogenetic analysis and haplotype networks
The concatenated tree has two main Pithecopus clades (Fig. 1). One
clade includes P. palliatus, P. rohdei, P. megacephalus, P. rusticus,
P. oreades, P. centralis, and P. ayeaye. While the inclusion of P. palliatus in
this clade is poorly supported (PP = 0.25; Fig. 1), the remaining species
form a highly supported (PP = 1) monophyletic group. The other clade is
strongly supported (PP = 0.99), and includes P. nordestinus, P. azureus,
P. araguaius, and P. hypochondrialis, with P. araguaius + P. hypochon­
drialis (or the P. hypochondrialis species complex) as the sister of a clade
composed by P. azureus + P. nordestinus (Fig. 1).
The monophyly of P. azureus is confirmed and well supported
(Fig. 1), including individuals from the Pantanal, and the Chaco and
Beni savannas. Three major clades with strong nodal support (PP > 97
%) can be observed within the P. hypochondrialis complex (Fig. 2). The
largest of these clades includes a P. hypochondrialis sample from the type
locality (i.e., Suriname) and specimens collected from north-eastern
Amazonia and the core Cerrado domains, which we identify as
P. hypochondrialis here (lineage 3; Fig. 2). This clade is the sister group of
the P. araguaius (lineage 2) + Pithecopus sp. (lineage 1) clades (Fig. 2).
The P. araguaius clade (lineage 2; Fig. 2) includes a subclade composed
of topotypes of P. araguaius (Haga et al., 2019) from Pontal do Araguaia
(Mato Grosso) plus samples from Chapada dos Guimarães, in Mato
Grosso (PP = 1), and Colombia, whereas the sample from Santa Ter­
ezinha (Mato Grosso) groups with those from Marabá (Pará) in a
strongly-supported subclade (PP = 0.99; Fig. 2). It is important to note
here that the samples from Colombia and Marabá were identified pre­
viously as P. hypochondrialis (Fig. 2), and that our findings support the
revision of the species allocation of these specimens. Finally, the samples
from the northwestern border between Cerrado and Amazon (Paranaíta,
Apiacás, Nova Bandeirantes, and Juína – Mato Grosso) plus Santarém
(Pará), which were also identified previously as P. hypochondrialis, were
recovered together in the same clade (lineage 1; Fig. 2).
We obtained sequences of RHOD for 63.8 % of the individuals and
SIA-HI for 44.7 % (Tables S1–2). Although we did not find any exclusive
haplogroups for the RHOD fragment, some structuring in the nDNA of
these lineages is indicated by the exclusive haplotypes identified in the
SIA-H1 (Fig. S2).
3.2. Molecular species delimitation
The first iteration of the ML-GMYC analysis delimits 12 distinct en­
tities (Fig. S3a), with a confidence interval of 9–15. The null hypothesis,
that no distinct species exist in the gene tree, can be rejected, based on
the Likelihood Ratio (LR) test = 9.09, p = 0.01. All these lineages are
well supported by the bGMYC, although there is some uncertainty on the
differentiation of lineages 3.3 and 3.4 as independent candidate lineages
(Fig. S3a). The second iteration of the ML-GMYC delimits two entities
(Fig. S3b), with a confidence interval of 1–11. In this case, however, the
null hypothesis of no distinct species in the gene tree cannot be rejected
(LR test = 2.41, p = 0.30). On the other hand, the scenarios of one and
two candidate species are both rejected by the bGMYC analysis, in

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R.F. Magalhães et al. Molecular Phylogenetics and Evolution 190 (2024) 107959

favour of three well-supported candidate species (Fig. S3b). Table 1

While the “12 candidate species” scenario results in lineages with
low pairwise genetic distances (range: 0.9–5.6 %) and some overlap
between intra- and inter-lineage distances (Appendix A), the “3 candi­
date species” scenario results in lineages with higher pairwise genetic
distances (range: 4.2–4.5 %), and a clear gap between the intra- and
inter-lineage distances (Appendix A). Given this, we proceeded with the
“3 candidate species” scenario for validation. These three candidate
species are validated by STACEY, with a probability of more than 99 %,
regardless of the collapse height used. We assign the three lineages as
follows: Lineage 1: Pithecopus sp., Lineage 2: P. araguaius, and Lineage 3:
P. hypochondrialis (Fig. 2).
We found isolation by distance (IBD) in the 16S dataset, with a
moderate correlation between the pairwise FST values and the
geographic distances between the sampling points (R2 = 0.3781, p <
0.001). We also found moderate IBDs in lineages 1 (R2 = 0.5150, p <
0.05) and 3 (R2 = 0.3065, p < 0.01), and a strong IBD in lineage 2 (R2 =
0.8329, P < 0.05).
3.3. Morphological delimitation
The independent variables (i.e., those with VIF < 5) used in the
Geneland analysis were FAL, SVL, IOD, END, AL, THL, TL, FL, HL, HW
and FTL. This analysis identified three morphometric clusters, with PP
= 0.99. However, these clusters did not coincide with the candidate
species validated by STACEY (Fig. 3). The results of the PERMANOVA
further confirm this, showing no significant morphometric divergence
among the three lineages validated in the previous steps (Table 1).
Despite the extensive mixing of the morphometric clusters within each
delimited lineage, the morphometric traits appear to be conserved at
many of the localities analysed (Fig. 3).
Results of Permutational Analysis of Variance (PERMANOVA) showing the
variation in the morphometric data from putative species validated in the pre­
sent study (=groups).
d.f. Sum of Squares Mean Squares F p (>F)
Groups 2 0.007601 0.003801 1.7029 0.1876
Residuals 96 0.21426 0.002232
The variation in the frequencies of the colour morphotypes among
the lineages in the P. hypochondrialis species complex and P. azureus
cannot be explained by chance (χ2 = 189.19; d.f. = 9; p < 0.001). All the
individuals of the species P. azureus (n = 17) are morphotype 4. In the
case of P. araguaius, morphotypes 1, 2, and 4 represent 10.0 %, 75.0 %,
and 15.0 % of the observed variation, respectively (n = 20). Pithecopus
hypochondrialis is the species with the greatest diversity of colouration
patterns, given the presence of individuals of all four morphotypes.
Morphotype 1 represents 17.4 % of the individuals in the species,
morphotype 2, 39.4 %, morphotype 3, 4.6 %, and morphotype 4, 38.5 %
(n = 109). All but one of the individuals of Pithecopus sp. presents
morphotype 3, with the other one being morphotype 1 (n = 33). Overall,
then, the colouration patterns are consistent with the species delimita­
tion outlined above, although they cannot be used as diagnostic
characters.
3.4. Dated species tree
The StarBEAST2 analysis recovered Pithecopus sp. as a sister of
P. araguaius plus the P. hypochondrialis clade (Fig. 4). However, the re­
lationships among these three lineages are weakly supported, as they are
in the concatenated tree (Fig. 2). The divergence time estimates indicate

Fig. 3. The Q-plot of the morphometric variation (left) of the specimens analysed in the present study for K = 3. The individuals are represented by the horizontal
bars and the colours (i.e., light grey, grey, and dark grey) represent the proportion of the individuals assigned to each cluster. The coloured vertical bars to the left of
the Q-plot represent the individuals that belong to the three candidate species validated by the species delimitation methods: Pithecopus sp. (red), P. araguaius
(orange), and P. hyponchondrialis (blue). The vertical lines or arrows with numbers to the right of the Q-plot indicate the collecting localities of the different in­
dividuals, which are shown on the map on the right, all within Brazil. The Brazilian states area as follow: AP = Amapá; BA = Bahia; GO = Goiás; MA = Maranhão;
MG = Minas Gerais; MT = Mato Grosso; PA = Pará; TO = Tocantins. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)

9
R.F. Magalhães et al.
Fig. 4. Time-calibrated species tree produced by the StarBeast2 analysis. The values above the branches (in black) indicate the mean age of the node, while the blue
bars indicate the 95% credibility intervals of the divergence times. The values under the branch (in red) indicate the posterior probability of each node. Photograph of
a specimen of Pithecopus hypochondrialis from Oxiximiná (in Pará state, Brazil) by Samuel C. Gomides. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
that the most recent common ancestor of the P. hypochondrialis species
complex dates back to the Messinian stage of the Miocene (6.63 Ma, 95
%-HPD 8.78–3.60 Ma). Pithecopus araguaius and P. hypochondrialis
appear to have diverged at the end of the Zanclean stage of the Pliocene
(3.79 Ma, 95 %-HPD 5.18–0.90 Ma).
3.5. Integrative and heuristic validation
We ran two replicates of the iBPP for each of three possible phy­
logenies: (i) ((Pithecopus sp., P. araguaius), P. hypochondrialis), (ii)
((Pithecopus sp., P. hypochrodrialis), P. araguaius) and (iii) ((P. araguaius,
P. hypochondrialis), Pithecopus sp.). We tested four different scenarios for
each possible phylogeny, with four combinations of the parameters of
the species divergence times (τ) and population size (θ), which resulted
in a set of two replicates of 12 different analyses (Table S7). The three
putative species were validated in 20 of these 24 replicates, with PP >
70 % (Table S7). Only four scenarios did not recover all the putative
species with PP > 70 %, and these were all in one of the two replicates.
Three of these scenarios (Table S7) refer to the test of the phylogeny in
which P. hypochondrialis (lineage 3) is the sister species of the clade
containing Pithecopus sp. (lineage 1) and P. araguaius (lineage 2), with (i)
small ancestral population size and shallow divergence (replicate I.2a),
(ii) large ancestral population sizes and deep divergence (replicate I.4a)
and (iii) large ancestral population size and deep divergence (replicate
I.1b) (Table S7). The fourth scenario that did not recover all the three
species refers to the phylogeny in which P. araguaius is the sister species
of a Pithecopus sp. + P. hypochondrialis clade, with a small ancestral
population size and shallow divergence (replicate II.2b).
The results of the gdi vary significantly depending on the combina­
tion of guide trees, θ, and τ priors. In general, the evolutionary inde­
pendence of the validated species is uncertain (0.2 < gdi < 0.7;
Table S7). The divergence of P. hypochondrialis and P. araguaius, and of
Pithecopus sp. and P. hypochondrialis are well supported (gdi > 0.7) only
when each of these pairs are sister taxa in the guides trees and with
shallow divergence priors (Table S8). In the case of the genealogical
divergence between Pithecopus sp. and P. araguaius, there is only strong
support in the guide tree when they are sister taxa with the combination
of the priors for shallow divergence and small ancestral populations
(Table S8). However, there is no support (gdi < 0.2) for the divergence of
10
Molecular Phylogenetics and Evolution 190 (2024) 107959
Pithecopus sp. and P. araguaius in the guide tree where they are sister
taxa, with any other combination of θ and τ priors. There is no support
for any of the three species when deep divergences priors are applied, in
any of the other guide trees (Table S8).
3.6. Paleodistribution models and climatic stability over geological time
The SDM of the P. hypochondrialis species complex has a high AUC
value (0.805), with three bioclimatic variables contributing most to the
model – the seasonality of temperature (bio4, contribution = 32.4 %),
precipitation (bio15, contribution = 15.3 %), and the mean temperature
of the driest quarter (bio9, contribution = 11.1 %). In general, the po­
tential distribution of the species complex was ampler in the past than
that predicted for the present day (Fig. 5a–j). Under present-day climate
conditions, the predicted potential distribution of P. hypochondrialis
encompasses most of the transition zone between the Amazon Forest and
the Cerrado, with relatively high suitability (>0.6), in particular in the
northern Cerrado, in the Brazilian states of Tocantins and Maranhão,
and further north within the Amazon biome, including the Brazilian
state of Roraima and the open formations – the Llanos – of northern
Venezuela, and central and northwestern Colombia (Fig. 5J).
Projections of the current SDMs onto the paleoclimatic conditions of
Pliocene M2 (~3.3 Ma) indicate the largest and most continuous po­
tential area suitable for the P. hypochondrialis species complex, with high
levels of suitability within the current distribution of the Llanos, central-
northern Cerrado and eastern Amazonia (Fig. 5a). This supports the
conditions for an eastern Amazonian corridor during the Pliocene,
which would have persisted into the warm period of the mid-Pliocene
(~3.3 Ma), except for the fragmentation of suitable areas in the cen­
tral Cerrado (Fig. 5b). The models predicted gradual contractions over
time in the climatically suitable areas towards the transition zone be­
tween the Amazon and Cerrado domains (Fig. 5e–j). The climate of the
northern coast of South America was suitable for the species during
many periods in the past (Fig. 5c, f, h, i), which supports the hypothesis
of a coastal Atlantic corridor during the Pleistocene.
The map of climatic stability (Fig. 5k) reveals the existence of three
large climatically stable refugia for P. hypochondrialis over time, corre­
sponding to the Cerrado/eastern Amazonia in the southeast, the
Guianan (or Roraima) savannas in the centre, and the Llanos in the
R.F. Magalhães et al.
Fig. 5. (a)-(j) Distribution models for the Pithecopus hypochondrialis species complex that represent the areas of relative climatic suitability over time. (k) The map of
the areas of stability represents the sum of the maps of all the periods analysed for the P. hypochondrialis species complex. The colour scales represent the suitability
(a)-(j) or stability (k) of the areas.
northwest. Some areas between these predicted refugia, as well as others
to the west and south, were predicted to be relatively stable, but not
consistently by all the models (Fig. 5k).
3.7. Spatiotemporal phylogeographic dynamics
The most likely centre of diversification of the P. hypochondrialis
species complex would have been located in present-day south-eastern
Amazonia (Fig. 6a - R), at the end of the Messinian age of the Miocene
(~5.87 Ma). During the Zanclean age of the Pliocene, the complex
diversified and radiated northwards from this point through eastern
Amazonia and towards the southern limit between the Cerrado savanna
and the Amazon rainforest (Fig. 6b). During the Pleistocene, the lineages
spread to the Llanos and the savannas of Guyana, the central Cerrado,
and the interface between eastern Amazonia and the Cerrado
(Fig. 6c–d). Overall, then, our results support an eastern Amazonian
route during the Pliocene and rule out a possible Andean corridor during
the Pleistocene. The diffusion rate was rapid and constant, at a mean of
~ 316.74 km/Ma (=0.316 m/year; 95 %-HPD = 0.189–0.486 m/year),
with a subtle acceleration over the past two million years (Fig. 6e).
4. Discussion
4.1. Phylogenetic relationships
The phylogenetic relationships of the Pithecopus species in the
11
Molecular Phylogenetics and Evolution 190 (2024) 107959
concatenated phylogeny agree broadly with the previous hypotheses (e.
g., Faivovich et al., 2010; Bruschi et al., 2013; Duellman et al., 2016;
Haga et al., 2017; Andrade et al., 2020), that is, with the genus being
divided into two principal clades. The main differences are related to the
phylogenetic relationship of P. azureus and P. palliatus. The relationships
between P. palliatus and the other species of the genus have varied in
previous studies, depending on the dataset or the methods used in the
phylogenetic reconstruction (Faivovich et al., 2010; Bruschi et al., 2013;
Duellman et al., 2016; Haga et al., 2017; Andrade et al., 2020). In the
present phylogeny, P. palliatus was recovered as a sister of the campo
rupestre and Atlantic Forest leaf frogs (i.e., P. ayeaye, P. centralis,
P. megacephalus, P. oreades, P. rohdei, and P. rusticus). However, this
phylogenetic placement is only poorly supported (PP = 0.25), which
highlights the need for more independent markers to better elucidate its
position in the arrangement.
As in the present phylogeny, P. azureus has invariably been recovered
as the sister species of P. nordestinus in previous studies (Faivovich et al.,
2010; Bruschi et al., 2013; Haga et al., 2017; Andrade et al., 2023),
except for Duellman et al.’s (2016) analysis, in which it was shown as a
sister of P. hypochondrialis. In this phylogeny, P. hypochondrialis was
represented by a chimera of six DNA fragments (mtDNA: 12S, 16S, and
cytb; nDNA: CXCR4, H3A, and NCX1) from distinct sources. Three of
these fragments were sequenced from the same individual, whose origin
was given only as “South America” and was purchased from an animal
dealer [GenBank accession numbers: AY948748 (16S), AY948826
(NCX1), and FJ882741 (12S); Roelants et al., 2007; Van Bocxlaer et al.,
R.F. Magalhães et al. Molecular Phylogenetics and Evolution 190 (2024) 107959

Fig. 6. (a)-(d) Snapshots of the spatiotemporal dynamics of the Pithecopus hypochondrialis species complex in central-northern South America (the complete
reconstruction is available as a KML file in Supplementary File 1). R in circle (a) represents the estimated root location of the complex. The cyan polygons represent
the 80% highest posterior density (HPD) interval of uncertainty in the location of ancestral branches, and the lighter the tone, the older the ancestral branch
associated with it. The polygons with a black border (d) represent the current distribution of the lineages revealed by the first iteration of the general mixed Yule-
coalescent (GMYC) analysis (see Fig. 2, S3). (e) Diffusion rates over time, showing the mean values (solid line) and 95% HPD interval (dashed line). The green shape
shows the distribution of the Brazilian Cerrado savanna, as defined by the Brazilian Institute for Geography and Statistics Cerrado (IBGE, 2019), and WWF’s savanna
ecoregions (Olson et al., 2001), but excluding the Uruguayan savanna, and the dry and humid Chaco.

2009]. In a BLAST nucleotide search (Altschul et al., 1990), we found
that sequence AY948748 was 98.87–99.72 % similar to the available
sequences of P. azureus, while sequence FJ882741 was 99.42–99.56 %
similar. In other words, the P. hypochondrialis sample analysed by
Duellman et al. (2016) is probably a chimera of P. azureus and
P. hypochondrialis, created by the misidentification of the purchased
frog, which would explain why the two species appear as sister taxa in
this phylogeny. Finally, the phylogenetic relationship of Pithecopus sp.,
P. araguaius, and P. hypochondrialis were poorly supported in both the
concatenated and the species trees, which emphasises the need for the
sequencing of additional independent loci to clarify the relationships
among these lineages, given that we were able to use only three inde­
pendent loci for the phylogenetic analyses. Despite this, we were able to
reassign specimens that had been mistakenly identified as
P. hypochondrialis to P. araguaius and Pithecopus sp.
4.2. Species delimitation and taxonomic implications
Our findings substantiate the conclusion that P. hypochondrialis
constitutes a species complex which encompasses at least three
morphologically cryptic species, including two that were previously
defined as P. araguaius and P. hypochondrialis. The third lineage may

12
R.F. Magalhães et al.
represent a new taxon, which requires further evaluation and descrip­
tion. Bruschi et al. (2013) obtained a tree with the same three-clade
layout. However, the samples in their subclade A and lineage 1 of the
present study are not from the same localities, which prevents us from
determining whether they correspond to the same entity. Given this, we
would recommend that any future studies assess whether the pop­
ulations from Belterra, in the Brazilian state of Pará, and Alta Floresta
(Mato Grosso) can belong to Pithecopus sp. If confirmed, the presence of
NORs in the short arm of the chromosome pair 8 (Bruschi et al., 2013)
emerges as a potential diagnosis for this candidate species.
The results of the present study align with the more conservative
hypothesis of the presence of three candidate species in the
P. hypochondrialis species complex. To reach this conclusion, we
implemented a two-iteration routine of the GMYC, given the evidence
that this increases accuracy when only a limited number of sequences is
available for each species (Talavera et al., 2013). This approach resulted
in a considerable decrease in the number of candidate species for vali­
dation, which prevented overestimating the number of species delimi­
ted, as found in some previous studies that have used single-locus
delimitation methods to resolve under-sampled genetic variation
(Lohse, 2009; Bergsten et al., 2012). It is important to note here that the
sample analysed in the present study has a number of geographical gaps,
in particular in the central portions of the Cerrado, Llanos and, Guiana
savannas, which may have led to the false identification of deep genetic
divergence between certain mitochondrial lineages. Given this, addi­
tional geographic sampling will be essential to ascertain whether the
substantial genetic distances observed between some lineages, such as
2.1, 2.2, and 2.3 (Fig. 2; Appendix A), are the result of the actual
divergence of the lineages or rather, of sampling artefacts. Multi-species
coalescent delimitation methods also tend to overestimate the number
of candidate species when isolation-by-distance is strong, taxa are
widespread, or sampling is not representative of the actual species
ranges (Chambers and Hillis, 2020; Mason et al., 2020). Given that all
three of these factors affect our dataset, the adoption of a three-species
layout, rather than a 12-species scheme, would appear to be justifiably
prudent, and avoid any gross overestimate of the number of valid
species.
Conversely, we might ask – why were these lineages considered to be
a single species, rather than three? The null hypothesis of a single species
was not rejected in the second iteration of the ML-GMYC, although
several pieces of evidence contradict this conclusion, including the
result of the bGMYC, the ample phylogenetic distances between the
clades (Fig. 2), and the clear gap between the intra- and inter-specific
16S rDNA distances (Appendix A). The Pithecopus sp. specimens also
diverged in their colouration pattern, given that this was the only line­
age in which morphotype 3 predominated. In fact, while morphotype 3
was present in 97 % of the Pithecopus sp. specimens, it was recorded in
only 4.6 % of the P. hypochondrialis specimens, and was completely
absent in P. araguaius, further supporting our model-based hypothesis of
the existence of three valid species. Haga et al. (2017) also distinguished
P. araguaius from P. hypochondrialis based on their longer, more pulsated
calls, which are emitted at higher frequencies. While there was an
overlap in the amplitude of 14 of the call traits analysed by these au­
thors, the differences could not explained by chance in four cases.
Ultimately, Solís-Lemus et al. (2015) concluded that the integrative
iBPP approach increases the accuracy of the species delimitation by
limiting the parameter of space in which the models may under- or over-
estimate the number of candidate species, as well as reducing the po­
tential impacts on the results of violations of the assumptions of the
coalescent model. In the present study, most of the tested parameter
combinations supported the existence of three species, even with the
lack of clear morphometric differentiation. This further corroborates the
three-species hypothesis. On the other hand, the results of the gdi were
influenced considerably by the combination of priors used for the dis­
tribution of θ and τ, and the a priori species tree used in the iBPP
delimitations. In most input combinations, the gdi values ranged from
13
Molecular Phylogenetics and Evolution 190 (2024) 107959
0.2 and 0.7, which is inconclusive for the confirmation of the evolu­
tionary independence of the lineages (Leaché et al., 2019). Overall, then,
the evolutionary independence among the three validated species can
only be unequivocally assessed when we determine the best-fitting
species tree and the θ and τ priors that best align with the observed
data. Given this, we would strongly recommend that any future phylo­
genetic investigations of this species complex incorporate more robust
data, such as genomic-scale markers or a more comprehensive matrix of
phenotypic traits.
Our results confirm the taxonomic scenario outlined by Bruschi et al.
(2013), which indicates that the P. hypochondrialis species complex is
distributed throughout the Cerrado, Amazon, Llanos, and Guiana do­
mains, whereas P. azureus is found in the Pantanal, Chaco, and Beni
savannas, without no clear diagnosis based on colouration patterns.
While this arrangement has been available for a decade, some studies
still adopt the classification of Caramaschi (2006), which considers the
Cerrado populations to be P. azureus (e.g., Santoro & Brandão, 2014;
Thomassen & Ziade, 2020). This misconception may be especially
problematic in some contexts, such as morphological diagnoses and
population comparisons (Santos et al., 2018), and in particular in
ecological niche modelling (see Fig. 1 in Bandeira et al., 2021). Santos
et al. (2018), for example, reported variation in the formula of the tooth
row between individuals from the Argentinean Chaco [2(2)/2(1)] and
the Brazilian Cerrado [2(2)/3(1)], and attributed this difference to
insufficient sampling in Argentina. However, the results of the present
study indicate that these populations are distinct species and that the
variation observed by Santos et al. (2018) would be diagnostic of these
species.
While colouration patterns have been used historically for the
diagnosis of phylomedusine taxa (e.g., Brandão, 2002; Caramaschi,
2006; Giaretta et al., 2007), some authors have highlighted potential
problems in the use of this type of trait, such as the pronounced intra­
specific variation and interspecific overlap (Baêta et al., 2009; Bruschi
et al., 2013; Brunes et al., 2014; Magalhães et al., 2018). The results of
the present study are consistent with this, and reinforce the need for a
taxonomic review of Pithecopus using other types of characters, such as
geometric morphometry, internal anatomy, larval morphology,
bioacoustics, cytogenetics, and behaviour.
4.3. Dynamics of range limits over time
The paleo-distribution and spatiotemporal reconstructions presented
here reject the hypothesis of a Pleistocene corridor through the foothills
of the Andes (Webb, 1991; Silva and Bates, 2002). On the other hand, an
eastern Amazonian Pliocene route (Dowsett et al., 2016; Azevedo et al.,
2020) is supported by both approaches. The diversification of the
P. hypochondrialis species complex was estimated to have occurred be­
tween the Miocene and Pliocene, in south-eastern Amazonia. During the
Pliocene, this area was predicted to have had a highly suitable climate
for the distribution of the species complex. The dispersal of the common
ancestor of the three lineages appears to have been restricted to this
region, which is consistent with the presence of savannas during this
period, as also predicted by Downset et al. (2016). To our knowledge,
only Leite et al. (2015) have provided any biological evidence for a
Pliocene connection between the Cerrado and the Guiana Shield, spe­
cifically for rodents, although they did not investigate or discuss the
location or characteristics of this route.
Our evidence of a central Amazonian or coastal Atlantic corridor for
the P. hypochondrialis species complex is more uncertain, however. The
paleo-distribution models projected highly suitable climates in eastern
Amazonia and on the northern coast of South America throughout the
Pleistocene, which may have favoured the establishment of a corridor in
these regions. The continuous spatial reconstruction also indicates that
the Amazonian corridor was the most likely route connecting pop­
ulations during this period. Despite proposals of a central Amazonian
corridor (Silva and Bates, 2002), the sum of the ecological modelling
R.F. Magalhães et al.
evidence – including that from the present study – indicates that the
most likely location of this route would have been in eastern Amazonia
(e.g., Bueno et al., 2017; Buzatti et al., 2017; Buzatti et al., 2018). Ox­
ygen isotope data from speleothems also demonstrate that eastern
Amazonia has been drier since at least the last two glacial-interglacial
cycles (Cheng et al., 2013), although pollen fossils indicate that the
tropical evergreen forests were replaced by seasonal forests rather than
being replaced by savanna or grassland (Kern et al., 2023). However, the
extent to which these changes affected the connectivity of savanna-
associated species in South America remains unclear, and the general
paucity of fossils continues to be the principal challenge for the defini­
tive determination of if, when, and how this corridor may have been
formed (Azevedo et al., 2020).
In the present study, sampling is especially deficient across the
northern portion of the species’ distribution, with very poor represen­
tation from Colombia, French Guiana, Guyana, and Suriname, and no
sequences from Venezuelan specimens. As continuous phylogeographic
methods are known to be affected strongly by sampling gaps (Kalkaus­
kas et al., 2021), we suspect that the apparent long-distance jump in
dispersal from the Cerrado to the Llanos through central Amazonia is
almost certainly a sampling artefact. Given this, a more comprehensive
sample of the lineages delimited in the present study, in particular from
the areas with greatest data deficiencies, should provide a more realistic
overview of the history of the geographical distribution of the species
complex. At this point, the only Pleistocene route that can be discarded
with confidence is the Andean corridor. A more comprehensive sample,
especially from the northern extreme of its range, will be crucial to
determine whether the coastal, eastern Amazonian, or a combination of
both routes may account for the connectivity of the P. hypochondrialis
species complex in the Pleistocene savannas of South America.
The mean diffusion rate of the P. hypochondrialis species complex was
considerably higher than those estimated previously for other
Neotropical anurans. Bokermannohyla saxicola has a mean diffusion rate
approximately 4.5 times lower than the rate we estimated here (Oswald
et al., 2022), for example, while the mean diffusion rates of P. ayeaye
and P. megacephalus were around 2.3 times lower, with a comparable
rate only being observed during a period of expansion followed by an
introgression event that occurred between the two species at approxi­
mately 300 ka (Magalhães et al., 2021). However, B. saxicola, P. ayeaye,
and P. megacephalus all have considerably more restricted geographic
ranges than the P. hypochondrialis species complex, given that they are
endemic to highland campo rupestre and are specialised in the use of
rocky environments for reproduction (Magalhães et al., 2021; Oswald
et al., 2022).
Even so, the mean diffusion rate predicted here for P. hypochondrialis
is only approximately a third of that estimated for Pseudopaludicola
falcipes by Langone et al. (2016). This leptodactylid frog has an extensive
geographical distribution and thrives in the Pampa grasslands of
southern South America, where it is relatively abundant. A high diffu­
sion rate was projected for this species because of its presumed mech­
anisms of passive dispersal, such as basin captures and vegetation rafts
(Langone et al., 2016). These assumptions are derived from the obser­
vation that these frogs vocalise in waterlogged areas adjacent to streams,
environments that are conductive to dispersal events of this type. The
passive dispersal hypothesis is further supported by the diminutive size
of Ps. falcipes, which would limit considerably its potential for active
long-distance dispersal. By contrast, the members of the
P. hypochondrialis species complex are neither miniaturised nor do they
breed in riparian environments, which contradicts any hypothesis of
passive dispersal. In this case, the high diffusion rate observed in this
group is likely explained by its generalist habits (Barrio-Amorós, 2009)
and its environmental tolerance, which would underpin a much greater
potential for active dispersal and the efficient colonisation of new
regions.
14
Molecular Phylogenetics and Evolution 190 (2024) 107959
4.4. Conclusions and perspectives
We have presented an extensive and integrated analysis of the mo­
lecular, morphological, and ecological data, which offers valuable in­
sights into the diversification of the P. hypochondrialis species complex.
It is nevertheless important to acknowledge the limitations of the sam­
ple, in particular in the northern extreme of the clade’s geographic
distribution and narrow genomic sampling. The prevalence of cryptic
diversity in the genus Pithecopus has been revealed in several previous
studies of molecular markers (Faivovich et al., 2010; Ramos et al., 2019;
Andrade et al., 2020). In line with these studies, our findings also
indicate the existence of at least one undescribed species in the
P. hypochondrialis species complex. It is interesting to note that only
Haga et al. (2017) and Andrade et al. (2020) described formally the
cryptic species detected in their molecular studies. Haga et al. (2017)
described P. araguaius, which is indistinguishable from P. hypochon
drialis, based on its morphology and bioacoustics, while Andrade et al.
(2020) described P. gonzagai, which was similarly indistinguishable
from P. nordestinus. Even so, we do not rule out altogether the possibility
that these supposedly cryptic species and Pithecopus sp. may become
distinguishable when analysed through other character systems. Given
the ongoing global biodiversity crisis and widespread Linnaean short­
falls (Hortal et al., 2015), it will be essential to review and describe the
cryptic species identified within the genus Pithecopus. This will permit
their inclusion in future assessments of threat risks and the development
of conservation policies. In particular, future research efforts on Pith­
ecopus should focus on the existing geographic gaps, the expansion of
genomic sampling, and the integration of phenotypic data from multiple
sources. These additional perspectives will contribute to a more
comprehensive understanding of the characteristics and geographic
distribution of these species.
Although our results offer mixed support for the eastern Amazonian
and coastal Atlantic Pleistocene corridors, our evidence clearly supports
the occurrence of pulses of connectivity between the northern and
southern Neotropical savannas throughout both the Pliocene and the
Pleistocene. Previous studies have already presented biological evidence
of the connectivity of organisms that inhabited the open areas of the
Brazilian and Guyana shields during the Pliocene (Leite et al., 2015).
However, our findings represent the first phylogeographic evidence of
the existence of a corridor in eastern Amazonia during the late Pliocene.
Although we have assessed the spatiotemporal dynamics of the
P. hypochondrialis species complex, a number of other phylogeographic
questions remain unanswered, such as how Plio-Pleistocene climate
changes affected the demographic history and dynamics of the gene flow
within the group. Given its ample distribution across multiple
Neotropical domains and its occurrence within the interface of open and
forested habitats, the P. hypochondrialis species complex appears to be a
promising model for the investigation of the environmental dynamics of
the Neotropics.
CRediT authorship contribution statement
Rafael F. Magalhães: Conceptualization, Investigation, Formal
analysis, Resources, Writing – original draft. Elisa K. S. Ramos: Inves­
tigation, Formal analysis, Writing – review & editing. Lucas N. Ban­
deira: Investigation, Formal analysis, Writing – review & editing.
Johnny S. Ferreira: Data curation, Writing – review & editing. Fer­
nanda P. Werneck: Conceptualization, Supervision, Writing – review &
editing. Marina Anciães: Conceptualization, Supervision, Writing –
review & editing. Daniel P. Bruschi: Conceptualization, Data curation,
Resources, Supervision, Funding acquisition, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
R.F. Magalhães et al. Molecular Phylogenetics and Evolution 190 (2024) 107959

the work reported in this paper. Funding

Acknowledgements
We thank all the museum and collection curators, in particular, Célio
F. B. Haddad, Luís F. Toledo and Natan M. Maciel, for making the ma­
terial under their care available to us for analysis. We also thank Fausto
Nomura for helping with the verification of the material. We would
especially like to thank Shirlei Maria Recco-Pimentel for her guidance
during the initial study, and her help with its design and the technical
and financial infrastructure, and Luciana Bolsoni Lourenço for her
valuable comments during the research and on a previous version
of the manuscript. We are also very grateful to Melisa Olave and three
anonymous reviewers for their valuable contributions that helped to
improve this paper greatly.
This study was supported by Minas Gerais State Research Founda­
tion, FAPEMIG (grant APQ00325-21), the São Paulo State Research
Foundation, FAPESP (grant 2012/12169-7), the Amazonas State
Research Foundation, FAPEAM (grant 01.02.016301.03263/2021-47),
the Brazilian National Council for Scientific and Technological Devel­
opment, CNPq (grants 425571/2018-1, 303646/2021-7), the Rufford
Foundation (grants 23763-1, 27629-2); DPB was funded by a FAPESP
scholarship (2010/17464-1) and CNPq (grant 303646/2021-7). LNB
was supported by a CAPES fellowship (grant 001), EKSR was funded by a
FAPESP doctoral scholarship (grant 2018/01236-1), and; FPW was
supported by a CNPq productivity fellowship (grant 311504/2020-5).

Appendix A

Mean uncorrected p-distances recorded between the 16S sequences. Intra-lineage distances are shown in the diagonal (in bold type) and the inter-
lineage distances are given in the lower half of the table. The individuals of the lineages resulting from the first and second iterations of the GMYC are
shown in Fig. 2. NA = not applicable.

Lineage 1st iteration of GMYC

1.1 1.2 1.3 2.1 2.2 2.3 3.1 3.2 3.3 3.4 3.5 3.6

1.1 0.001
1.2 0.025 NA
1.3 0.023 0.012 0.003
2.1 0.038 0.024 0.030 0.002
2.2 0.051 0.044 0.045 0.031 NA
2.3 0.052 0.044 0.046 0.025 0.037 0.009
3.1 0.049 0.042 0.040 0.024 0.049 0.044 0.004
3.2 0.049 0.046 0.046 0.035 0.056 0.052 0.023 0.006
3.3 0.048 0.043 0.042 0.031 0.051 0.047 0.017 0.026 0.009
3.4 0.046 0.043 0.041 0.031 0.052 0.047 0.019 0.024 0.009 0.002
3.5 0.051 0.049 0.048 0.036 0.055 0.051 0.022 0.029 0.015 0.014 0.007
3.6 0.044 0.041 0.040 0.027 0.051 0.047 0.020 0.023 0.013 0.011 0.016 0.006

Lineage 2nd iteration of GMYC

1 2 3
1 0.015
2 0.043 0.023
3 0.045 0.042 0.017

Appendix B. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ympev.2023.107959.

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