Back from the deaf: integrative taxonomy revalidates an earless and mute species, Hylodes grandoculis van Lidth de Jeude, 1904, and confirms a new species of Pristimantis Jiménez de la Espada, 1870 (Anura: Strabomantidae) from the Eastern Guiana Shield

Abstract

Many anuran species remain to be formally named and described in Amazonia, notably in the Guiana Shield, and particularly in megadiverse groups such as Pristimantis. Several species in the Guiana Shield region have been confused with Pristi- mantis marmoratus and P. ockendeni. Hylodes grandoculis, a taxon previously placed in the synonymy of P. marmoratus, may be available for one of these species. To disentangle this confusing situation, we examined the external morphology and osteology (via μ-CT scans) of the holotype of H. grandoculis, the holotype of Pristimantis marmoratus, and of recently collected material for which we also analyzed molecular, acoustic, and morphological variation. We concluded that some populations from Suriname and northern Pará, Brazil, are distinct from P. marmoratus and correspond to Pristimantis gran- doculis. Other populations, from French Guiana, are closely related to P. grandoculis but their status remains ambiguous. Finally, some populations, from French Guiana and Amapá, Brazil, are conspicuously distinct from both P. marmoratus and P. grandoculis and are described herein as P. crepitaculus sp. nov. A third species, belonging to a “trans-amazon complex”, occurs in southern Suriname, Guyana, and Brazil and remains undescribed. Pristimantis grandoculis and related populations from French Guiana lack external tympanum, columella, pharyngeal ostia, vocal slits and do not vocalize. This represents a rare, perhaps unique, example of a deaf and mute species of frogs from the Amazonian lowlands

Full text

Vol.:(0123456789)
1 3
Organisms Diversity & Evolution
https://doi.org/10.1007/s13127-022-00564-w
ORIGINAL ARTICLE
Back fromthedeaf: integrative taxonomy revalidates anearless
andmute species, Hylodes grandoculis van Lidth de Jeude, 1904,
andconfirms anew species ofPristimantis Jiménez de la Espada, 1870
(Anura: Strabomantidae) fromtheEastern Guiana Shield
AntoineFouquet1 · PedroPeloso2,3· RawienJairam4· AlbertinaP.Lima5· AlexanderT.Mônico5· RaaelErnst6·
PhilippeJ.R.Kok7,8
Received: 30 April 2021 / Accepted: 4 May 2022
© Gesellschaft für Biologische Systematik 2022
Abstract
Many anuran species remain to be formally named and described in Amazonia, notably in the Guiana Shield, and particularly
in megadiverse groups such as Pristimantis. Several species in the Guiana Shield region have been confused with Pristi-
mantis marmoratus and P. ockendeni. Hylodes grandoculis, a taxon previously placed in the synonymy of P. marmoratus,
may be available for one of these species. To disentangle this confusing situation, we examined the external morphology
and osteology (via µ-CT scans) of the holotype of H. grandoculis, the holotype of Pristimantis marmoratus, and of recently
collected material for which we also analyzed molecular, acoustic, and morphological variation. We concluded that some
populations from Suriname and northern Pará, Brazil, are distinct from P. marmoratus and correspond to Pristimantis gran-
doculis. Other populations, from French Guiana, are closely related to P. grandoculis but their status remains ambiguous.
Finally, some populations, from French Guiana and Amapá, Brazil, are conspicuously distinct from both P. marmoratus and
P. grandoculis and are described herein as P. crepitaculus sp. nov. A third species, belonging to a “trans-amazon complex”,
occurs in southern Suriname, Guyana, and Brazil and remains undescribed. Pristimantis grandoculis and related populations
from French Guiana lack external tympanum, columella, pharyngeal ostia, vocal slits and do not vocalize. This represents a
rare, perhaps unique, example of a deaf and mute species of frogs from the Amazonian lowlands.
Keywords Amazonia· Amphibia· Morphology· Tomography
Introduction
Pristimantis Jiménez de la Espada, 1870 is the most species-
rich genus among all vertebrates, currently containing more
than 570 species mostly distributed throughout Amazonia,
the Andes, the Chocó, and Pantepui (Frost, 2021), and, albeit
more marginally, in Central America, the Caribbean, the
Cerrado, and the Atlantic Forest. Species in this genus are
being named at a fast pace, with over 133 species described
* Antoine Fouquet
fouquet.antoine@gmail.com
1 Laboratoire Evolution Et Diversité Biologique, UMR 5174,
CNRS, IRD, Université Paul Sabatier, Bâtiment 4R1, 118
Route de Narbonne, 31062cedex9Toulouse, France
2 Coordenação de Zoologia, Museu Paraense Emílio
Goeldi, Terra Firme, Avenida Perimetral, 1.901, Belém,
CEP66077-530, Brazil
3 Instituto Boitatá de Etnobiologia E Conservação da Fauna,
Goiânia, Goiás, Brazil
4 National Zoological Collection Suriname (NZCS), Anton de
Kom University of Suriname, Paramaribo, Suriname
5 Coordenação de Biodiversidade, Instituto Nacional de
Pesquisas da Amazônia, Avenida André Araújo 2936,
ManausAmazonas69080-971, Brazil
6 Museum ofZoology, Senckenberg Natural History
Collections Dresden, Dresden, Germany
7 Department ofEcology andVertebrate Zoology, Faculty
ofBiology andEnvironmental Protection, University
ofŁódź, 12/16 Banacha Str, 90-237Łódź, Poland
8 Department ofLife Sciences, The Natural History Museum,
Cromwell Road, LondonSW75BD, UK

A.Fouquet et al.
1 3
in the last decade, and over 220 species since 2000 (Frost,
2021). This, along with diversity estimates based on DNA
sequence data (e.g. Trevisan etal., 2020; Vacher etal.,
2020), suggests a vast number of unnamed species in this
genus.
Many Pristimantis species occur at very narrow alti-
tudinal ranges and have small distributions (Duellman &
Lehr, 2009). This pattern is most striking not only along
Andean slopes and in the Pantepui region, but also in
some lowland areas (e.g. Fouquet etal., 2013; Kok, 2013).
Direct mode of development (endotrophic larvae that hatch
as froglets) and narrow bioclimatic niches have been sug-
gested as possible causes of high micro-endemism in this
group (Gonzalez-Voyer etal., 2011; Vences & Wake,
2007). As a corollary, half of the 445 Pristimantis species
evaluated by the International Union for Conservation of
Nature (IUCN) are considered endangered (IUCN, 2020).
It is therefore important to improve our knowledge of the
actual diversity and distribution of Pristimantis species.
Pristimantis marmoratus (Boulenger, 1900) and P.
ockendeni (Boulenger, 1912) have for long been used to
designate multiple populations of superficially similar,
hard to identify, small brown Pristimantis species of the
“unistrigatus group” (sensu Hedges etal., 2008, see dis-
cussionin Kok etal., 2018). In the Guiana Shield lowlands
more specifically, P. marmoratus has been reported from
northern Brazil, in the state of Pará (Ávila-Pires etal.,
2010), Guyana (Ernst etal., 2005, 2006, 2007), French
Guiana (Lescure & Marty, 2000), and Suriname (Ouboter
& Jairam, 2012), whereas P. ockendeni has been reported
from the Reserva Florestal Adolpho Ducke (RFAD) in the
Amazonas state of Brazil (Lima etal., 2006). However,
Kok etal. (2018) clearly demonstrated that the range of
P. marmoratus (type locality “foot of Mount Roraima”,
Venezuela, ca. 1067m) is restricted to the eastern Pantepui
uplands and highlands between 600 and 1800m above sea
level (asl), and Elmer and Cannatella (2008) suggested
that the range of P. ockendeni (type locality “La Union,
Rio Huacamayo, Carabaya, [Departamento Puno,] Peru,
ca. 600m”) is restricted to the Andean foothills of south-
eastern Peru. Therefore, the Guiana Shield lowlands popu-
lations that were previously identified as P. marmoratus
and P. ockendeni likely remain unnamed, except for at least
some “P. marmoratus” populations from Suriname for
which a name may be available: Hylodes grandoculis van
Lidth de Jeude, 1904. The species was described based on a
single specimen from central Suriname (“the basin and the
sources of the Coppename”) and is considered a synonym
of P. marmoratus since Frost (1985). Several species co-
occur in the region, and the conspecificity of the holotype
of H. grandoculis with recently collected material needs,
therefore, to be clarified.
Vacher etal. (2020) delimited three major lineages cor-
responding to populations previously identified as P. mar-
moratus or P. ockendeni: (1) Pristimantis cf. marmoratus,
occurring in the easternmost part of the region (state of
Amapá in Brazil and French Guiana) and corresponding to
Pristimantis sp. 1 of Fouquet etal. (2013) and Kok etal.
(2018); (2) Pristimantis cf. ockendeni, occurring in southern
Suriname and central Guyana (Eleutherodactylus marmora-
tus of Ernst etal., 2005, 2006, 2007); and (3) Pristimantis sp.
“Guianas”, occurring in French Guiana, in southern Amapá
state (Brazil), Suriname, and at the border between Guyana
and the state of Pará, Brazil, corresponding to Pristimantis
sp. 4 of Fouquet etal. (2013) and Kok etal. (2018).
To (1) disentangle this confusing situation and determine
if one of these lineages would correspond to Hylodes gran-
doculis, and (2) facilitate the description of potential related
species, we examined the external morphology and osteol-
ogy (via µ-CT scans) of the holotype of H. grandoculis, of
the holotype of Pristimantis marmoratus, and of recently
collected material for which we analyzed genetic, acoustic,
and morphological variation.
Material andmethods
Field work anddeposition ofspecimens
We undertook extensive field work in French Guiana, Guy-
ana, the Brazilian states of Amapá and Amazonas, and in
Suriname (Bakhuis, Tafleberg, and Sipaliwini), including
areas nearby the type locality of Hylodes grandoculis, col-
lecting specimens and tissue samples, and recording adver-
tisement calls and ecological data of numerous Pristimantis
populations (Fig.1). Frogs were collected by hand and killed
with an intraperitoneal injection of Xylocaine® (lidocaine
hydrochloride). All specimens were individually tagged and
fixed in 10% formalin before being transferred to a solution
of 70% ethanol for permanent storage. Newly collected spec-
imens were deposited in the Museum National d’Histoire
Naturelle (Paris, France) (MNHN-RA-2020.0092–118),
Museu Paraense Emílio Goeldi (MPEG 41819–41825),
and the National Zoological collection of Suriname (NZCS
A1210–1212). Other specimens examined are deposited in
various zoological collections (Appendices 1 and 2).
Molecular analyses
We gathered 190 16S sequences of species of the “Pristi-
mantis unistrigatus group” from the eastern Guiana Shield
and the Pantepui region, as well as of populations south of
the Amazon River previously identified as P. cf. ockendeni
and closely related to Guiana Shield populations according to

Back fromthedeaf: integrative taxonomy revalidates anearless andmute species, Hylodes…
1 3
Vacher etal. (2020) (Appendix 1). We employed BLASTN to
verify that no other 16S sequence deposited on GenBank after
the work of Vacher etal. (2020) clustered with the species
used in this study. Twenty-eight sequences were newly gener-
ated while the other sequences were retrieved from GenBank
(Appendix 1). We completed these data with 13 sequences of
12S, eight retrieved from GenBank and five newly generated.
Genomic DNA was extracted from tissue samples (muscle or
liver) using either the Wizard® Genomic DNA Purification
Kit (Promega; Madison, WI, USA) or the DNeasy® Blood &
Tissue Kit (Qiagen; Hilden, Germany) following the manufac-
turer’s protocols. We amplified targeted loci by standard PCR
protocols and then used Sanger sequencing (primers 16RF:
TAT CCC TAG GGT AAC TTG; 16FR: TTA CCA AAA ACA
TCG CCT from Salducci etal., 2005). These sequences were
aligned with the mitogenome of P. thymelensis (JX564889,
which served as the outgroup), on the MAFFT7 online server
under default parameters except the E-INS-i strategy, which
is designed for sequences with multiple conserved domains
and long gaps (Katoh etal., 2017). We trimmed the alignment
to keep only regions where sequences overlap, resulting in
a final alignment of 2548 base pairs (bp). We investigated
the phylogenetic relationships among a total of 191 termi-
nals under maximum likelihood (ML), applying a GTR CAT
model with RAxML v.8.2.4 (Stamatakis, 2014). All RAxML
analyses were performed on the CIPRES Science Gateway
online server (Miller etal.,2010). Non-parametric bootstrap-
ping values (Felsenstein, 1985) were estimated using 1,000
pseudoreplicates.
Morphometric analysis
We examined 67 adult specimens (45 males, 22 females,
among which 27 were included in our molecular dataset) of
P. cf. marmoratus:[P. sp. 1 of Fouquet etal. (2013) and Kok
etal. (2018), 13 males, 3 females], P. cf. ockendeni (8 males,
6 females), and Pristimantis sp. “Guianas” from Suriname
(4 males, no females) and from French Guiana (13 males,
13 females) [P. sp. 4 of Fouquet etal. (2013) and Kok etal.
(2018)], and the holotype of Hylodes grandoculis (Appendix
2). The other sixspecimens were a priori assigned to one of these
species when they were collected from the same or nearby
populations and after morphological examination. Sex was
determined either in the field via calling activity, or in the
laboratory by examining the condition of vocal slits, nuptial
pads, and gonads. We measured 18 variables; abbreviations
for measurements are as follows: SVL—snout-vent length;
TiL—tibia length; FeL—femur length; TaL—tarsus length;
FL—foot length (from outer edge of metatarsal tubercle to
the tip of toe IV); HeL—head length; HW—head width;
Ind—internarial distance; IOD—interorbital distance; EN—
eye-nostril distance (straight line distance between anterior
corner of eye and nostrils); ED—horizontal eye diameter;
TD—horizontal tympanum length; FD—disc width of Fin-
ger III; 4TD—disc width of Toe IV; ETS—eye-tip of snout
distance (straight line distance between the anterior corner of
eye and tip of snout); 1FiL—length of Finger I (from proxi-
mal edge of palmar tubercle to the tip of Finger I); 2FiL—
length of Finger II (from proximal edge of palmar tubercle
to the tip of Finger II); HL—hand length (from distal edge
of palmar tubercle to the tip of Finger III).
Measurements were taken using a digital caliper to the
nearest 0.01mm. Mean and ranges were rounded to the
nearest 0.1mm (all measurements by AF, except for MPEG
specimens, measured by PP). We examined the variation of
morphometric data among adult males through a principal
component analysis (PCA) via the packageFactoMineR in R
v.3.2.4 (Lê etal., 2008; R Development Core Team,2016).
To control for variation in body size among individuals,
we performed additional analyses on a size-corrected data-
set (residuals) obtained by linear-regressing the original
morphometric measures of each variable on SVL (Strauss,
1985).
3D microtomography
We investigated the osteological variation across adult males
of P. cf. marmoratus, Pristimantis sp. “Guianas” and the hol-
otypes of Hylodes grandoculis (RMNH 4467) and P. mar-
moratus (BMNH 1947.2.16.92). Specimens were scanned
using an EasyTom 150 (at kV = 40–70, resolution < 20µm)
from the MRI platform of ISEM (Institute of Evolutionary
Sciences of Montpellier), except the holotype of H. gran-
doculis which was scanned using a CT/Zeiss Xradia at the
Naturalis Laboratories (Leiden Naturalis Biodiversity Center),
and the holotype of P. marmoratus that was scanned using
a Metris X-Tek HMX ST 225 System at the Natural His-
tory Museum, London. Segmentation of the full skeleton
and of the cranium was done using Avizo (FEI Visualiza-
tion Sciences Group, Burlington, MA, USA) and Biomedisa
(Lösel etal.,2020). µ-CT scans or surface renderings have
been deposited at www. morph osour ce. org (https:// www.
morph osour ce. org/ proje cts/ 00043 633).
Acoustic analysis
We gathered six call recordings of males of Pristimantis
cf. marmoratus (including one track file from Marty &
Gaucher, 1999). To our knowledge, Pristimantis sp. “Guia-
nas” has never been observed calling and no recording is
available. The external tympanum and pharyngeal ostia are
absent in that species, and males lack vocal slits. The calls
of P. cf. marmoratus consist in series of short notes, and
we measured seven call variables from waveforms using
Audacity v.2.1.1 following Köhler etal. (2017): call length,
silence between calls, number of notes, note length, silence

A.Fouquet et al.
1 3
between the two first notes, silence between the two last
notes, dominant frequency (taken with a spectral slice over
the entire note). Whenever possible, up to four measure-
ments of each variable were taken per recorded male, and
the average was considered as a single measurement. Call
recordings have been deposited at www. sonot heque. mnhn.
fr (Appendix 3).
Results
Phylogenetic relationships
The relationships among species inferred from the ML anal-
ysis are poorly supported at several nodes (Fig.1). However,
sequences from individuals corresponding to the different
putative species form strongly supported clades. The popu-
lations previously identified as P. cf. ockendeni from the
Guiana Shield (Suriname and Guyana) form a clade related
to several populations south of the Amazon River, which
we hereafter refer to as the “trans-amazon complex”. The
Guiana Shield populations are more closely related to a sin-
gle specimen from São Sebastião, Rio Abacaxis, Amazonas
(p distance = 5.4%, Appendix 4). The “trans-amazon complex”
is highly divergent from other Guiana Shield species, and is
more closely related to western Amazonian species, although
probably not directly related to P. ockendeni (Vacher etal.,
2020). Pristimantis cf. marmoratus is circumscribed to the
easternmost part of the Guiana Shield and forms a poorly
supported clade with P. marmoratus sensu stricto. Pristi-
mantis sp. “Guianas” samples form a strongly supported
clade with uncertain relationships and are subdivided into
two main lineages (p distance = 4.5%, Appendix 4), one
occurring in the west (Suriname, the Amapá and Pará states in
Brazil) and the other one in the east (French Guiana). The
geographic range of the western lineage of Pristimantis sp.
“Guianas” encompasses the type locality of P. grandoculis.
Fig. 1 a Maximum Likelihood phylogenetic tree obtained from the
analysis of 191 sequences of 12S-16S (2548 bp) of Pristimantis.
Bootstraps > 50% are indicated on the left side of the nodes. For sake
of clarity, branches within species are collapsed. Only the species
occurring in the lowlands have their collapsed branches coloured in
the tree according to the map. b Distribution of the different species
included in the molecular dataset. The ranges of the species occur-
ring in the lowlands(except P. espedeus and P. inguinalis that are not
filled for clarity purpose) and P. marmoratus are depicted by filled
polygons. The population of P. cf. ockendeni from RFAD (Manaus
region) is only tentatively assigned to the same species as Guyana and
Suriname populations since it is only documented by pictures and a
call recording, and is indicated with a purplecircle with white con-
tour. The type locality of Pristimantis grandoculis is indicated by a
blue filled star as well as the type locality of P. marmoratus with a
green star

Back fromthedeaf: integrative taxonomy revalidates anearless andmute species, Hylodes…
1 3
Morphometric analyses
The PCA based on raw morphometric measurements pro-
vided two first components accounting for 63.4% of the total
variation (Fig.2a). The first component explains 46.7% of
the variation, and variable coefficients are all highly and
positively correlated. Overall, the variation along the first
principal component (PC) axis is related to body size; since
the three species largely overlap on this variable, they also
do along this axis. The variation along the second axis is
notably related to differences in TiL and FL (negatively cor-
related with PC2) and ED (positively correlated with PC2)
and segregates populations of Pristimantis sp. “Guianas”,
which largely overlap, from the other species that only partly
overlap with each other. The holotype of P. grandoculis is
positioned within the western populations of Pristimantis
sp. “Guianas”.
The PCA based on size-corrected morphometric measure-
ments provided two first components accounting for 48.1%
of the total variation (Fig.2b). The variation along the first
11
6
010203040
23
12
315
0510 15 20 25 30
a
TiL
FeL
TaL
FL
HeL
HW
Ind
IOD
EN
ED
FD
X4TD
ETS
X1FiL
X2FiL
HL
−1.0
−0.5
0.0
0.5
1.0
−1.0 −0.5 0.0 0.5 1.0
Dim 1 (46.71%)
Dim 2 (16.74%)
P. cf. ockendeni
P. grandoculis holotype
P. cf. ockendeni
P. grandoculis
holotype
FeL
TaL
FL
HeL
HW
Ind
IOD
EN
ED FD
X4TD
ETS
X1FiL
X2FiL
HL
−1.0
−0.5
0.0
0.5
1.0
−1.0 −0.5 0.0 0.5 1.0
Dim 1 (30.63%)
Dim 2 (17.47%)
0.48 0.50 0.52 0.54 0.56 0.58 0.60
TiL/SVL
0.22 0.23 0.24 0.25 0.26 0.27 0.28
HL/SVL
0.36 0.38 0.40 0.42 0.44 0.46
FL/SVL
P. sp. “Guianas“ East
P. crepitaculus sp. nov.
P.
cf. marmoratus
P. cf. ockendeni
P. grandoculis
P. sp. “Guianas“ West
P. grandoculis
holotype
b
c
P. sp. “Guianas“ East
P. grandoculis
P. sp. “Guianas“ West
P. crepitaculus sp. nov.
P. cf. marmoratus
P. grandoculis
P. sp. “Guianas“ West
P. crepitaculus sp. nov.
P. cf. marmoratus
P. sp. “Guianas“ East
Fig. 2 a PCA based on 17 external body measurements (without TD) across 43 Pristimantis males. b PCA based on 16 size-corrected (residu-
als of the linear regression of the measurements on SVL) external body measurements. c Boxplots of ratio between three measurements on SVL

A.Fouquet et al.
1 3
axis is notably related to differences in HL, FeL and FL
(positively correlated with PC1), and ED (negatively cor-
related with PC1) and segregates Pristimantis cf. ockendeni
from the other species that only partly overlap with each
other (eastern and western populations of Pristimantis sp.
“Guianas” completely overlap). The variation along the sec-
ond axis is notably related to differences in HW (positively
correlated with PC2) and TaL (negatively correlated with
PC2) and partly segregates P. sp. “Guianas” from P. cf.
marmoratus. The holotype of P. grandoculis is positioned
within populations of P. sp. “Guianas” but also within P.
cf. marmoratus.
The examination of the ratios between the aforemen-
tioned variables and SVL (Fig.2c) shows limited overlap
across species particularly considering HL/SVL and TiL/
SVL. Moreover, the ratios displayed by the holotype of P.
grandoculis are always within the range of French Guiana
and Suriname populations of P. sp. “Guianas” and only mar-
ginally overlaps with the range of the other species.
Taxonomic conclusion
External morphology and osteology (see below) strongly
suggest that the western populations that have been identi-
fied as Pristimantis sp. “Guianas” [P. sp. 4 of Fouquet etal.
(2013) and Kok etal. (2018)] correspond to Hylodes gran-
doculis. This species is geographically disjunct and phylo-
genetically distinct from P. marmoratus sensu stricto based
on molecular data (Fig.1; see also Kok etal., 2018). Our
morphological and osteological data also unambiguously
confirm the non-conspecificity of P. marmoratus and H.
grandoculis. Therefore, we formally rescue H. grandoculis
from the synonymy of P. marmoratus, and herein provide
an amended diagnosis of Pristimantis grandoculis n. comb.
(van Lidth de Jeude, 1904) based on the examination of the
holotype and four adult specimens from Suriname. It must
be mentioned that the specimens of Pristimantis sp. “Guia-
nas” from French Guiana, although morphologically very
similar to P. grandoculis, form a distinct, closely related
clade that upon the examination of a sufficient number of
adult specimens (not currently available) could be either
considered conspecific or a distinct species and will be
hereafter referred to as P. sp. “Guianas” East. The east-
ernmost species previously reported as Pristimantis sp. 1
(Kok etal., 2018) and Pristimantis cf. marmoratus (Vacher
etal., 2020) is also morphologically clearly distinct from
P. grandoculis and all known congeners in the area and is
formally described and named herein as P. crepitaculus sp.
nov. The “trans-amazon complex”, in which some popula-
tions have been erroneously identified as P. ockendeni, will
be treated elsewhere.
Species accounts
Pristimantis grandoculis (van Lidth de Jeude, 1904)
Hylodes grandoculis van Lidth de Jeude, 1904
Eleutherodactylus marmoratus Hoogmoed, in Frost, 1985
Eleutherodactylus marmoratus Avila-Pires etal.,2010
Pristimantis marmoratus Ouboter & Jairam, 2012
Pristimantis sp. 4 Jairam, 2019
Pristimantis sp. “Guianas” Vacher etal., 2020
Holotype. RMNH 4467. Type locality: “the basin and
the sources of the Coppename”, interior of Surinam. van
Lidth de Jeude stated that the specimen was collected “Sept.
10th 1901” during the Coppename expedition (Anonymous,
1903). The expedition took place between August and late
November 1901 “we went up the Coppename then the two
rivers that form it until they become unseaworthy even for
the korjales (boats) of the natives (3°57N)”. These indi-
cations suggest that the specimen was collected at the far-
thest point reached by the expedition and that 3.9500°N
56.7543°W are plausible coordinates for the type locality.
Referred specimens. MNHN-RA-2020.0118 (field no.
AF3435), an adult male collected by A. Fouquet, S. Cally
and R. Jairam on 30 April 2015 at Bakhuis Mountains, Suri-
name (4.7246°N 56.7638°W, ~ 200m asl; Fig.3). NZCS
A1212, an adult male collected by P. Ouboter and V. Kado-
soe on 20 August 2013 at Tafelberg, Suriname (3.8049°N
56.1539°W ~ 500m asl); NZCS A1210–11, two adult males
collected by R. Jairam and D. Baêta on 23 June 2016 at Lely
Mountain, Suriname (4.4158°N 54.6498°W, ~ 600m asl).
Definition. Pristimantis grandoculis is characterized by
the following unique combination of characters: (1) SVL
small, adult males 16.5 ± 1.1mm (range 14.7–17.9mm,
n = 5) (Table1), female unknown; (2) dorsal skin tubercu-
late, with two pairs of enlarged tubercles on the scapular
region embedded in a W-shaped scapular fold, ventral skin
granular particularly on the femoral region; (3) external tym-
panum absent, tympanic membrane not differentiated and
obscured by supratympanic fold on the posterodorsal edge
of tympanum, tympanic annulus indistinct; (4) pharyngeal
ostia absent; (5) columella absent; (6) tibia length 55–59% of
SVL; (7) snout broadly rounded in profile and dorsal view;
(8) each upper eyelid with two prominent tubercles; (9) cho-
anae round and small (0.3mm for MNHN-RA-2020.0118),
dentigerous processes of vomers oblique, narrowly sepa-
rated, each bearing 3–5 odontophores; (10) vocal slits
absent, vocal sac absent; (11) one unpigmented whitish nup-
tial pad located on the preaxial side of the thenar tubercle on
each thumb in male; (12) FI much shorter than FII, reaching

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subarticular tubercle of FII; (13) fingers with preaxial
fringes on FII and III; (14) finger discs broadly expanded,
elliptical, thenar tubercle oval, palmar often broken in three
distinct tubercles; (15) three enlarged ulnar tubercles often
flat and barely visible; (16) axillary tubercles (sensu Myers
& Donnelly, 2001) absent; (17) tarsal fold absent but tuber-
cles present; (18) toes basally webbed between TII–IV with
preaxial fringes on TII–V; (19) calcars absent, inner metatar-
sal tubercle oval, outer metatarsal tubercle round; (20) dorsal
colouration mostly chestnut brown, but highly variable in
colour and pattern, ventral colouration translucent brownish
grey with small cream spots, mostly on chest, throat suffused
with black melanophores and scattered with small irregular
cream blotches; (21) iris golden with a copper horizontal
band, absence of vertical streak; (22) posterior surface of
thighs and groin dark grey in life and brown in preservative,
absence of yellow spot on groin.
Fig. 3 a Dorsal, ventral, and lateral (head) views of the holotype
(RMNH 4467) of P. grandoculis from Coppename River, Suriname.
b Dorsal and ventral views and hand and foot of a recently collected
specimen of P. grandoculis (MNHN-RA-2020.0118) from Bakhuis,
Suriname. c A photograph in life of another recently collected speci-
men P. grandoculis (NZCS A1210)

A.Fouquet et al.
1 3
Table 1 Morphometric variables
SVL TiL FeL TaL FL HeL HW Ind IOD EN ED TD FD 4TD ETS 1FiL 2FiL HL
P. crepitaculus sp. nov M (n = 24) Mean 17.0 8.8 8.3 5.3 6.8 6.5 6.4 1.6 2.0 2.2 2.5 0.9 0.9 0.9 3.2 2.1 2.4 4.3
min 15.7 8.0 7.5 4.9 6.1 6.0 5.7 1.5 1.8 1.9 2.3 0.7 0.7 0.8 2.9 1.8 2.1 3.9
max 18.3 9.4 9.2 5.9 7.3 7.1 6.9 1.8 2.3 2.6 2.9 1.1 1.0 1.1 3.6 2.5 2.7 4.7
F (n = 4) Mean 23.4 11.4 10.8 6.7 9.1 8.5 8.4 2.0 2.4 2.9 3.1 1.1 1.2 1.3 4.3 3.0 3.5 5.5
min 22.9 10.9 10.3 6.4 8.9 8.3 7.7 1.8 2.4 2.9 2.7 0.9 1.1 1.2 4.0 2.8 3.1 5.1
max 23.9 11.8 11.0 7.0 9.5 8.6 9.0 2.2 2.5 3.0 3.3 1.3 1.2 1.3 4.5 3.2 4.0 5.8
P. cf. ockendeni M (n = 8) Mean 17.4 8.6 7.8 5.4 6.3 6.4 6.2 1.7 2.0 2.2 2.6 0.8 0.8 0.8 3.0 2.0 2.2 3.8
min 16.2 8.2 7.1 5.1 5.8 5.9 5.7 1.6 1.8 2.1 2.4 0.7 0.7 0.8 2.8 1.8 2.0 3.6
max 18.4 9.1 8.3 5.9 6.8 6.8 6.7 1.8 2.1 2.3 2.8 0.9 1.0 1.0 3.2 2.2 2.4 4.1
F (n = 6) Mean 24.1 11.4 10.4 6.9 8.2 8.4 8.6 2.1 2.7 3.0 3.2 1.0 1.1 1.2 4.1 2.6 3.0 5.1
min 21.4 10.7 10.0 6.5 7.9 7.6 7.5 1.9 2.6 2.8 3.1 0.8 1.0 1.0 3.9 2.2 2.6 4.7
max 25.7 12.2 11.5 7.3 8.6 9.6 9.6 2.3 3.1 3.2 3.4 1.2 1.2 1.3 4.5 2.9 3.6 5.4
P. sp “Guianas” East M (n = 13) Mean 16.0 9.1 8.4 5.2 7.1 5.8 5.5 1.7 2.0 2.0 2.3 NA 0.8 0.8 3.1 2.0 2.4 4.2
min 14.7 8.2 7.2 4.6 5.9 5.3 4.7 1.4 1.7 1.7 2.1 NA 0.7 0.6 2.8 1.7 1.9 3.7
max 17.5 10.4 9.4 5.9 8.0 6.5 6.5 1.9 2.2 2.5 2.5 NA 0.9 0.9 3.4 2.4 2.9 4.8
F (n = 13) Mean 22.5 12.0 11.1 6.6 9.6 8.1 8.2 2.1 2.5 2.9 3.2 NA 1.1 1.1 4.3 2.8 3.2 5.9
min 20.6 11.1 10.5 6.0 8.7 7.4 7.2 1.8 2.1 2.5 2.7 NA 0.9 0.9 4.1 2.5 2.7 5.3
max 25.0 13.1 12.7 7.3 10.6 9.3 9.0 2.3 2.8 3.4 3.5 NA 1.4 1.4 4.7 3.2 3.9 6.4
P. grandoculis M (n = 5) Mean 16.5 9.5 8.2 5.4 7.0 6.1 6.1 1.8 1.9 2.0 2.5 NA 0.8 0.8 3.1 2.0 2.5 4.4
min 14.7 8.7 7.3 4.9 6.1 5.5 5.6 1.7 1.9 1.8 2.3 NA 0.8 0.7 2.8 1.8 1.9 3.8
max 17.9 10.5 9.0 6.4 7.7 6.5 6.4 1.9 2.0 2.2 2.7 NA 0.9 0.9 3.4 2.4 3.0 4.9
F (n = 0)

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Morphological comparisons with other Guiana
Shield lowlands Pristimantis of the “unistrigatus group”.
Pristimantis grandoculis can mainly be distinguished
from P. espedeus by its smaller body size (SVL range in
males = 14.7–17.9mm vs. 20.7–24.8 in P. espedeus); exter-
nal tympanum absent (vs. visible in P. espedeus) and dark
grey groin colouration in life (vs. reddish in P. espedeus).
Pristimantis grandoculis can mainly be distinguished
from P. inguinalis by dark grey groin colouration in life (vs.
bright yellow inguinal mark in P. inguinalis); external tym-
panum absent (vs. visible in P. inguinalis) and translucent
brownish grey ventral colouration with small cream spots
(vs. entirely black in P. inguinalis).
All the other known Guiana Shield species of the “unistri-
gatus group” occur in the Pantepui region and are generally
associated with highlands (i.e. > 700m elevation; Fouquet
etal., 2013; Kok etal., 2018). Pristimantis grandoculis was
previously confused (and considered a synonym) with P.
marmoratus, one of these Pantepui species, from which it
can immediately be distinguished by iris colour in life (black
vertical streak running across the iris in P. marmoratus vs.
no such streak in P. grandoculis) and absence of columella
and vocal slits (vs. present in P. marmoratus). Among
the other Pristimantis species found at mid-elevation in
Pantepui, P. grandoculis can mainly be distinguished from P.
jester (Means & Savage, 2007) by having preaxial fringes on
FII and III (absent in P. jester), toes basally webbed between
TII–IV with preaxial fringes on TII–V (webbing and fringes
on toes absent in P. jester) and by iris colour in life (golden
to copper in P. grandoculis vs. upper 2/5 of iris blue-grey
in P. jester); from P. saltissimus (Means & Savage, 2007)
by tympanum condition (external tympanum absent, with
indistinct tympanic annulus in P. grandoculis vs. distinct
with distinct tympanic annulus in P. saltissimus), by having
preaxial fringes on FII and III (absent in P. saltissimus) and
toes basally webbed between TII–IV with preaxial fringes on
TII–V (webbing and fringes on toes absent in P. saltissimus);
from P. guaiquinimensis (Schlüter & Rödder, 2007; see also
Kok & Barrio-Amorós, 2013) mostly by tympanum condi-
tion (external tympanum absent, with indistinct tympanic
annulus in P. grandoculis vs. distinct with distinct tympanic
annulus in P. guaiquinimensis); from P. sarisarinama (Bar-
rio-Amorós & Brewer-Carías,2008) mostly by the absence
of vocal slits in males (present in P. sarisarinama); from P.
pulvinatus (Rivero, 1968) mostly by smaller body size in
males (14.7–17.9mm in P. grandoculis vs. 23.0–26.1mm
in P. pulvinatus) and distinct vomerine teeth (indistinct or
absent in P. pulvinatus); and from P. memorans (Myers &
Donnelly,1997) mostly by the absence of vocal slits in males
(present in P. memorans), and tympanum condition (external
tympanum absent, with indistinct tympanic annulus in P.
grandoculis vs. distinct with distinct tympanic annulus in
P. memorans).
Variation. The holotype is relatively well preserved.
However, it is highly discoloured, and overall tuberculation
is difficult to assess. Proportions are similar among the five
specimens examined; the amount of tuberculation on the
skin varies, especially on the dorsum and lateral surfaces
of the body. Colour pattern is variable across the recently
collected specimens. NZCS A1210 has a dark band run-
ning along the canthus rostralis which is absent in the other
specimens (Fig.2c). The W-shaped scapular mark is well-
developed anteriorly but less so posteriorly compared to
the other specimens. An interorbital band darker than the
dominant background is often present as well as dark bands
on the arms and legs but all these markings are variable in
sharpness across specimens.
Osteology of the holotype (RMNH 4467). Cranium
(Fig.4). Shape and proportions. The skull is well ossified,
widest posterior to the orbit at the level of the articulation of
the maxilla with the quadratojugal. The rostrum is moderate,
the braincase is broad.
Neurocranium and dorsal investing bones. The nasals
(separated medially from one another and covering all the
nasal capsules dorsally), frontoparietals, parasphenoid and
neopalatines are co-ossified with the sphenethmoid. The
frontoparietal and prootic are fused. Ventrally, the proot-
ics are fused with the parasphenoid alae. The exoccipitals
are fused. The dorsal surface of the otic capsule is ossified.
Frontoparietal crests are absent and the frontoparietal fon-
tanelle is exposed. The septomaxilla is roughly spiralled,
the medial ramus extending posterodorsal to the posterior
ramus; the anterior ramus is thick; the lateral ramus is
oblique with a long acuminate posterolateral extension; the
posterior ramus extends from the middle of the lateral ramus
ventromedially. The columellae (stapes) are absent.
Ventral investing bones. The parasphenoid cultriform
process extends anteriorly from the anterior edge of the otic
capsule and is co-ossified with the sphenethmoid. The par-
asphenoid alae are moderately long (about half of the length
of the cultriform process), perpendicular to the anteropos-
terior body axis, broadening slightly laterally. The vomers
are fused with the sphenethmoid; each vomer is composed
of an arcuate bone bordering the anteromedial, medial, and
posterior margins of the choana. The prechoanal ramus is
expanded medially and anteriorly and bears a ventral flange
along its medial edge. The postchoanal process is narrow
and acuminate, slightly posteriorly curved. Well-developed
dentigerous processes extend posteromedially from the
union of the pre- and postchoanal processes. Each dentiger-
ous process bears three teeth and is broadly separated from
its counterpart medially. The neopalatine is fused with the
maxilla distally. This complex is fused with the parasphe-
noid medially.
Maxillary arcade. The maxillary arcade bears many
small teeth on the premaxilla and maxilla. The arcade is

A.Fouquet et al.
1 3
complete and connected to the slender quadratojugal. The
premaxillae are separated medially, and their anterodorsal
alary process is weakly divergent from the midline. The
pars palatina is broad, with two well-defined processes:
the medial (palatine) process is relatively narrow and runs
roughly parallel toward its contralateral; the lateral process
is broader. The premaxilla and maxilla are in lateral contact
via a simple juxtaposition. The maxilla is long, with a broad
pars palatina along its lingual margin and a moderately
developed pars facialis.
Suspensory apparatus. The triradiate pterygoid bears a
slightly curved anterior ramus with a sculpted ventrolateral
face, oriented anterolaterally toward the maxilla, with which
it articulates at approximately the mid-length of the orbit.
Fig. 4 Volumetric renderings of µ-CT scans of the skeleton of the
holotype of Pristimantis grandoculis (RMNH 4467). a Full skeleton
in dorsal (left), ventral (middle), and lateral (right) views. b Skull in
dorsal (left), ventral (middle), and lateral (right) views. Abbreviations:
ang—angulosplenial, den—dentary, dp—dentigerous process, exo—
exoccipital, fro—frontal, max—maxillary, men—mentomeckelian,
nas—nasal, neo—neopalatine, par—parasphenoid, pre—premaxillary,
pro—prootic, pte—pterygoid, qua—quadratojugal, sep—septomaxilla,
sph—sphenethmoid, squ—squamosal, vom—vomer

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The pterygoid is fused to the maxilla. The medial and poste-
rior face of the medial rami of the pterygoid are about equal
in length. The medial ramus is broader than the posterior and
its posterior face is strongly sculpted. The lateral end of the
medial ramus overlaps the lateral edge of the prootic. The
quadratojugal is long, laterally curved, and slender, articulat-
ing anteriorly with the maxilla. It has a bulbous posteroven-
tral process and articulates dorsally with the ventral ramus
of the squamosal. The squamosal is dorsally bifurcated,
broad, and sculpted, extending anterodorsomedially from
the quadratojugal to the level of the otic capsule; the zygo-
matic ramus is short, whereas the otic ramus is long, almost
reaching the posterior end of the skull.
Mandible. The mandible is slim and edentate. The men-
tomeckelians are small and arcuate in ventral view, medially
and laterally broadened, and in narrow contact medially. The
dentary is short and thin, posteriorly acuminate, and overlaps
the angulosplenial for about a quarter of its length. Dentary
is in contact with the angulosplenial posteriorly. The angu-
losplenial is long and arcuate, laterally slightly grooved. The
coronoid process is weak.
Hyoid. The bony posteromedial processes of the hyoid
are expanded proximally and separated from one another.
No ossified parahyoid is present.
Axial skeleton (Fig.4).
Vertebral column. The vertebral column has eight pro-
coelous, presacral vertebrae. The vertebrae have complete
neural arches and low neural processes. Atlas (presacral I)
and presacral II are fused medially and laterally. The trans-
verse processes of presacrals II–III are slightly expanded
distally, thicker, and broader than those of other presacrals.
The transverse processes of presacrals II and III are oriented
ventrolaterally (II anteriorly, III posteriorly), whereas those
of presacrals IV–VIII extend dorsolaterally (IV–VII poste-
riorly, VIII anteriorly). The relative lengths of the transverse
processes and sacral diapophyses are: III > IV > Sacrum > V
≈ VI ≈ VII ≈ VIII > II. The sacral diapophyses are slightly
expanded distally. The urostyle has a well-developed dorsal
ridge that extends along half its length.
Pectoral girdle. The zonal portion has well-ossified cora-
coids, clavicles, scapulae, and cleithra. The clavicles are
long, slender, and oriented anteromedially; the medial tips
are narrowly separated from one another and located ante-
riorly from the level of anterolateral end of the clavicle that
articulates with the scapula; the coracoid is long and flared,
with its sternal end slightly broader than its glenoid end. The
scapula is long with a prominent pars acromialis that is not
separated from the pars glenoidalis. The scapula is about one
and a half the length of the clavicle. The cleithrum is ossi-
fied, well-developed, anteriorly thicker, thinning posteriorly.
The suprascapula is unossified.
Pelvis girdle. The long, slender iliac shafts bear dorsolat-
eral crests throughout their length. The overall length of the
girdle is more than two times the width between the anterior
ends of the iliac shafts. The iliac prominence is broad and
low, and pubes mineralized.
Manus and pes. The phalangeal formulae for the hand
and foot are standard, 2–2–3–3 and 2–2–3–4–3, respectively.
Terminal phalanges strongly T-shaped. Ossified prepollex
and prehallux visible.
Natural history. Our data suggest that Pristimantis gran-
doculis is terrestrial and scansorial. Males were often found
perched on low vegetation (0.5–1.5m above the ground),
at dusk, in a position suggesting calling activity. How-
ever, acoustic activity has never been observed neither in
Suriname nor in French Guiana for P. sp. “Guianas” East
(despite being locally abundant). It is noteworthy that both
groups of populations also lack a columella and that external
tympanum is absent in all specimens examined. Juveniles
have been found on the leaf litter during the day, and this
is also where juveniles, females and amplectant pairs of P.
sp. “Guianas” East have been found in French Guiana sug-
gesting that the reproduction takes place there. The species
inhabits pristine terra firme forests from 50 to 700m asl,
generally along the slopes of small massifs.
Distribution. Pristimantis grandoculis occurs throughout
the interior of Suriname (not along the coastal band), on the
southern border of Guyana with Brazil (Acari mountains),
and along the border between the Brazilian states of Pará and
Amapá. The species probably extends further in southern
Suriname and northern Pará state, Brazil. Phylogenetically
related populations occur in French Guiana (Pristimantis
sp. “Guianas” East), the Maroni River separating these two
groups of genetically divergent populations. The taxonomic
status of this group of populations needs to be clarified when
additional material becomes available. In French Guiana, P.
sp. “Guianas” East occurs throughout the territory except
the Approuague-Oyapock interfluvium and is probably
absent from northern Amapá state in Brazil. This pattern
of distribution strikingly mirrors that of Anomaloglossus
surinamensis (Vacher etal., 2017) and Pipa aspera (Vacher
etal., 2020), both species displaying similar geographical
boundaries and deep genetic structures across the Maroni
River. Interestingly, Pristimantis sp. “Guianas” East only
overlaps with P. cf. marmoratus (Pristimantis sp. 1) in the
southern half of French Guiana. Likewise, the distribution of
A. surinamensis only weakly overlaps with that of A. blanci
(Fouquet etal., 2018). Both are pairs of sister species with
similar ecology and body size.
Pristimantis crepitaculus sp. nov.
Pristimantis marmoratus Lescure & Marty, 2000
Pristimantis marmoratus Fouquet etal., 2007
Pristimantis marmoratus Dewynter etal., 2008
Pristimantis marmoratus Fouquet etal., 2012

A.Fouquet et al.
1 3
Pristimantis sp. 1 Fouquet etal., 2013
Pristimantis sp. 1 Kok etal., 2018
Pristimantis sp. 1 Fouquet etal., 2019
Pristimantis cf. marmoratus Vacher etal., 2020
Holotype. MNHN-RA-2020.0102 (field no. AF2786), an
adult male, collected by Antoine Fouquet, Maël Dewynter
& Nicolas Vidal on 26 February 2015 at Mitaraka, French
Guiana (2.2358N 54.4493W, ~ 200m asl; Fig.5).
Paratypes. Twenty-seven specimens (23 males and four
females) from French Guiana and the state of Amapá, Bra-
zil: MNHN-RA-2020.0092–0112; MPEG 41819–41825
(Appendix 2).
Etymology. The specific epithet is a latinised adjective
referring to the call structure of the new species, which
sounds like a rattle (crepitaculum).
Definition. The new species is characterized by the follow-
ing unique combination of characters: (1) SVL small, adult
males 17.0 ± 0.8mm (range 15.7–18.3mm, n = 24) and adult
females 23.4 ± 0.5mm (range 22.9–23.9mm, n = 4) (Table1);
(2) dorsal skin tuberculate, two pairs of enlarged tubercles on
the scapular region embedded in a W-shaped scapular fold,
ventral skin granular particularly on the femoral region; (3)
tympanum visible, tympanic membrane not or only poorly
differentiated, tympanic annulus partially visible externally
obscured by supratympanic fold on the posterodorsal edge of
tympanum, tympanum c.a. 1/3 of eye length; (4) pharyngeal
ostia present; (5) columella present; (6) tibia length 48–54% of
SVL; (7) snout broadly rounded in profile and slightly acumi-
nate in dorsal view; (8) each upper eyelid with two prominent
tubercles; (9) choanae round and small (0.5mm for the holo-
type), dentigerous processes of vomers oblique, narrowly sepa-
rated, each bearing 2–4 small odontophores; (10) vocal slits
present, vocal sac median, subgular; (11) one unpigmented
whitish nuptial pad located on the preaxial side of the thenar
tubercle on each thumb in male; (12) FI slightly shorter than
FII, reaching disc of FII; (13) fingers without lateral fringes;
(14) finger discs broadly expanded, elliptical, thenar tubercle
ovoid, palmar tubercle ill-defined; (15) three enlarged ulnar
tubercles often flat and barely visible; (16) axillary tubercles
(sensu Myers & Donnelly, 2001) absent; (17) small tarsal
tubercles present; (18) toes without lateral fringes but with
weak lateral keels, webbing rudimentary basal between TII–V;
(19) calcars absent, inner metatarsal tubercle oval, much larger
than the round outer tubercle; (20) dorsal colouration highly
variable from greenish to brownish with various patterns, ven-
tral colouration light grey with small black spots denser on
throat and legs; (21) iris reddish to copper with a darker hori-
zontal band and a black vertical streak; (22) posterior surface
of thighs and groin dark grey in life and brown in preservative,
lack of yellow circumscribed spot on groin; (23) anterior sur-
face of arms, posterior surface of flanks and dorsal surface of
thighs often yellow in reproductive males; (24) advertisement
call characterized by series (0.50–0.75s long) of 6–12 very
short notes (0.02–0.05s long) with a dominant frequency rang-
ing between 3.2–3.6kHz and emitted every 5.4–8.6s; (25)
male calling activity exclusively crepuscular, usually upside
down on tree trunks of low diameter.
Morphological comparisons with other Guiana Shield
lowlands Pristimantis of the “unistrigatus group”. Pristi-
mantis crepitaculus sp. nov. can mainly be distinguished
from P. espedeus by its smaller body size (SVL range in
males = 15.7–18.3mm vs. 20.7–24.8 in P. espedeus); dark
grey groin colouration in life (vs. reddish in P. espedeus);
and advertisement call composed of more notes and higher
dominant frequency (> 6 notes and > 3.1kHz).
Pristimantis crepitaculus sp. nov. can mainly be distin-
guished from P. inguinalis by dark grey groin colouration in
life (vs. bright yellow inguinal mark in P. inguinalis); light
grey ventral colouration with small black spots (vs. entirely
black in P. inguinalis); and advertisement call composed of
series of notes (vs a single note in P. inguinalis).
Pristimantis crepitaculus sp. nov. can mainly be distin-
guished from P. grandoculis by visible tympanum (external
tympanum absent in P. grandoculis); more tuberculate dor-
sal skin; enlarged tubercles on eyelids being equally distant
from the eye (vs. posterior tubercles closer to the eye in P.
grandoculis); fingers and toes without fringes (fringed in
P. grandoculis), smaller tibia length (48–54% of SVL vs.
58–60%); and presence of vocal slits in males (absent in P.
grandoculis).
All the other species of the unistrigatus group known
from the Guiana Shield occur in the Pantepui region and
are generally associated with highlands (i.e. > 700m eleva-
tion; Fouquet etal., 2013; Kok etal., 2018). One of these
Pantepui species, P. marmoratus, is possibly its closest
relative. Pristimantis crepitaculus sp. nov. can mainly be
distinguished from P. marmoratus by its fingers and toes
without fringes (fringes present in P. marmoratus) and its
advertisement call composed of a series of notes (vs. a single
note in P. marmoratus). Among the other Pristimantis spe-
cies found at mid-elevation in Pantepui, P. crepitaculus sp.
nov. can mainly be distinguished from P. jester (Means &
Savage, 2007) by the presence of a tympanum (absent in P.
jester), from P. saltissimus (Means & Savage, 2007) by iris
colour in life (black vertical streak running across the iris in
P. crepitaculus vs. no such streak in P. saltissimus), from P.
guaiquinimensis (Schlüter & Rödder, 2007) by its smaller
body size (females 32.4–33.6mm in P. guaiquinimensis
[see Kok & Barrio-Amorós, 2013] vs. 22.9–23.9mm in P.
crepitaculus sp. nov.), a distinct tympanum (weakly distinct
in Pristimantis crepitaculus sp. nov.), from P. sarisarinama
(Barrio-Amorós & Brewer-Carías,2008) by iris colour in
life (black vertical streak running across the iris in P. crepi-
taculus sp. nov. vs. no such streak in P. sarisarinama), and
distinct vocalization (1–2, rarely 3 notes in P. sarisarinama

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Fig. 5 a Holotype of Pristimantis crepitaculus sp. nov. in dorsal, ventral views. b Hand, foot and lateral view of the head. c Photographs of the
holotype in life. d Several photographs of additional specimens in life

A.Fouquet et al.
1 3
vs. 6–12 notes in P. crepitaculus sp. nov.), from P. pulvinatus
(Rivero, 1968) by its smaller body size (males 23.0–26.1mm
in P. pulvinatus vs. 15.7–18.3mm in P. crepitaculus sp. nov.)
and distinct vomerine teeth (indistinct or absent in P. pulvi-
natus), from P. memorans (Myers & Donnelly,1997) by iris
colour in life (black vertical streak running across the iris in
P. crepitaculus sp. nov. vs. no such streak in P. memorans), a
shorter inter-note interval (0.05–0.12s in P. crepitaculus sp.
nov. vs. 0.20–0.29s in P. memorans), and by the posterior
thigh surface being dark grey in life (vs. blackish with yel-
low flecking in P. memorans).
Description of the holotype. An adult male, SVL
15.8mm. Head only slightly longer than wide, widest at
corner of the mouth; snout slightly acuminate in dorsal and
round in lateral views; canthus rostralis distinctly concave;
nostrils nearly elliptical, directed almost completely later-
ally; interorbital region flat, loreal area slightly concave.
Eyes large and protuberant, directed laterally, diameter of
eyes much larger than tympanum diameter (ED/TD = 3.0),
pupil elliptical. Supratympanic fold slightly distinct; tympa-
num small, ovoid, tympanic membrane completely covered
by skin, but tympanic annulus distinct anteriorly and ven-
trally, covered by the M. depressor mandibulae posteriorly
and dorsally. Vocal sac present, single, subgular, extending
towards pectoral region between forearms. Choanae small
(0.5mm), round, not concealed by palatal shelf, larger than
vomerine odontophores; a pair of small vomerine odonto-
phores present; tongue ovoid, posteriorly free; vocal slits
present, extending diagonally from lateral base of tongue to
almost to the angle of the jaw.
Arms slender, not hypertrophied; lateral margins of arm
and forearm free of fringes, folds, but with three tubercles;
finger discs elliptical, expanded in all fingers, disc on FI
the smallest, on FIII the largest; relative lengths of fingers
I < II < IV < III; subarticular tubercles round, narrower than
finger width; fringes absent on all fingers. Subarticular
tubercles present on all fingers, one on FI and FI, two on
FIII and FIV; all subarticular tubercles round and about the
same size; inner metacarpal (thenar) tubercle present, oval;
outer metacarpal tubercle large, W-shaped, ill-defined. Web-
bing between fingers absent.
Legs relatively long and slender, with rows of large and
flat tubercles on tarsus and postaxially on foot (but lacking
fringes or folds). All toes well developed, with expanded,
elliptical, medium sized discs; discs on TIV and TV the
largest, those on TI and TII the smallest; relative lengths of
toes I < II < III < V < IV. Toes lacking fringes. Subarticular
tubercles present on all toes; one tubercle present on TI, TII,
and TV, two tubercles on TIII, three on TIV; all subarticular
tubercles round, similar in size. Inner metatarsal tubercle
oval; outer metatarsal tubercle small, round.
Skin on dorsum, head, dorsal surfaces of limbs, flanks,
and groin tuberculate with enlarged tubercles on the scapular
region and on eyelids, posterior surface of the dorsum,
and dorsal surface of hindlimbs. Skin on the gular region
smooth; chest, belly and undersurfaces of limbs granular.
Cloacal opening directed posteriorly; cloacal region lack-
ing tubercles.
Colour of holotype in life. Dorsal colour dark brown
with V-shaped black band on the scapular region, a large
middorsal black blotch and an ill-defined black blotch near
groin (Fig.5). Black bands and blotches located in the inter-
orbital region, on the loreal region, the nasal region, below
the eyes and the tympanic region. Interorbital region and
V-shaped scapular mark delimited anteriorly by reddish
bands. Lower flank as dorsum with a black blotch. Axillary
region and upper arm yellow. Lower arm as dorsum with two
black transversal bands. Dorsal colouration of legs as dor-
sum with four black transversal bars on thigh, four on tibia
and three on tarsus. Throat background grey covered with
minute black melanophores; belly skin grey covered with
small cream spots and by black melanophores that become
sparse near groin; ventral surfaces of thighs and arms as
throat. Iris with copper metallic pigmentation and pupil ring
interrupted dorsally and ventrally by transversal pigmenta-
tion (Fig.5).
Colour of holotype in preservative. After four years in
70% ethanol, colours of the specimen faded, notably glan-
dular supracarpal pad and throat pigmentation (Fig.5). The
snout is dorsally dark brown and well-delimited by a trans-
versal light grey interorbital band extending on half of the
eyelids. Posteriorly, the interorbital region is covered by a
dark brown band. A V-shaped dark brown mark is well-
defined on the scapular region posteriorly followed by two
oblique traversal dark brown bands. Similar, transversal dark
brown bands are present on the arms, hands and legs and feet
(for more details see Fig.5a).
Variation in the type series. Proportions vary little
among the 21 paratypes. Males are smaller than females
(15.7–18.3mm vs. 22.9–23.9). The amount of tubercula-
tion on the skin varies, especially on the dorsum and flanks.
Outer metatarsal tubercle is always present, but its size/vis-
ibility vary among specimens.
Colour pattern is highly variable among specimens
and several patterns are observed. A pattern similar to
the holotype (middorsal and interobital bands) is present
in female MNHN-RA-2020.0104 and in males MNHN-
RA-2020.0110 and MPEG 41821. A white band between
the anterior corner of the eyes is present in most individu-
als but some specimens have less conspicuous markings
(e.g. MNHN-RA-2020.0102, MNHN-RA-2020.0107). The
scapular V-shaped mark is light grey in a few specimens
(MNHN-RA-2020.0101, MNHN-RA-2020.094) or reddish
(MNHN-RA-2020.0102, MNHN-RA-2020.0109) and dis-
play brightly coloured upper arms and posterior portions of
flanks in life. Two specimens (MNHN-RA-2020.0098 and

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MPEG 41825) have a large well-defined cream spot on top
of the snout and another one (MNHN-RA-2020.0100) has a
light brown scapular mark posteriorly prolonged along the
back forming a Y-shaped pattern delimited by darker flanks.
Two specimens, one male (MPEG 41823) and one female
(MPEG 41821) have large cream blotches on the dorsum.
Osteology of the holotype (MNHN-RA-2020.0102).
Cranium (Fig.6). Shape and proportions. The skull is wid-
est posterior to the orbit at the level of the articulation of the
maxilla with the quadratojugal. The rostrum is moderate, the
braincase is broad.
Fig. 6 Volumetric renderings of µ-CT scans of the skeleton of the
holotype of Pristimantis crepitaculus (MNHN-RA-2020.0102). a Full
skeleton in dorsal (left), ventral (middle), and lateral (right) views. b
Skull in dorsal (left), ventral (middle), and lateral (right) views. Colu-
mellae are highlighted in red. Abbreviations: ang—angulosplenial,
col—columella, den—dentary, exo—exoccipital, fro—frontal, max—
maxillary, men—mentomeckelian, nas—nasal, neo—neopalatine,
par—parasphenoid, pre—premaxillary, pro—prootic, pte—pterygoid,
qua—quadratojugal, sep—septomaxilla, sph—sphenethmoid, squ—
squamosal, vom—vomer

A.Fouquet et al.
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Neurocranium and dorsal investing bones. The nasals
are broadly separated from one another and from the
sphenethmoid; they cover most of the nasal capsules dor-
sally. The frontoparietals are well developed, co-ossified
with the sphenethmoid. Frontoparietal crests are absent
and the frontoparietal fontanelle (T-shaped) is exposed.
The parasphenoid and neopalatines are co-ossified with the
sphenethmoid. The frontoparietal and prootic are fused.
Ventrally, the prootics are fused with the parasphenoid alae.
The exoccipitals are fused. The dorsal surface of the otic
capsule is mostly ossified. The septomaxilla is roughly spi-
ralled, the medial ramus extending posterodorsal to the pos-
terior ramus; the anterior ramus is thick; the lateral ramus
is oblique with a long acuminate posterolateral extension;
the posterior ramus extends from the middle of the lateral
ramus ventromedially. The columellae (stapes) are well ossi-
fied, formed by the synostotic fusion of the long, thin pars
media plectri (stylus) and the pars interna plectri (baseplate),
which is curved.
Ventral investing bones. The parasphenoid cultriform
process extends anteriorly from the anterior edge of the otic
capsule and is co-ossified with the sphenethmoid. The par-
asphenoid alae are moderately long (about half of the length
of the cultriform process), perpendicular to the anteropos-
terior body axis, broadening slightly laterally. The vomers
are in narrow contact with the sphenethmoid; each vomer
is composed of an arcuate bone bordering the anteromedial,
medial, and posterior margins of the choana. The prechoanal
ramus is expanded medially and anteriorly and bears a ven-
tral flange along its medial edge. The postchoanal process
is narrow and acuminate, slightly anteriorly curved. Well-
developed dentigerous processes extend posteromedially
from the union of the pre- and postchoanal processes, left
process bears two small odontophores. Each dentigerous
process is broadly separated from its counterpart medially.
The neopalatine is narrowly in contact with the maxilla dis-
tally. This complex is fused with the parasphenoid medially.
Maxillary arcade. The maxillary arcade bears many small
teeth on the premaxilla and maxilla. The arcade is complete
and connected to the slender quadratojugal. The premaxillae
are separated medially, and their anterodorsal alary process
is weakly divergent from the midline. The pars palatina is
broad, with two well-defined processes: the medial (palatine)
process is relatively narrow and runs roughly parallel toward
its contralateral; the lateral process is broader. The premax-
illa and maxilla are in lateral contact via a simple juxtaposi-
tion. The maxilla is long, with a broad pars palatina along
its lingual margin and a moderately developed pars facialis.
Suspensory apparatus. The triradiate pterygoid bears a
slightly curved anterior ramus with a sculpted ventrolat-
eral face, oriented anterolaterally toward the maxilla, with
which it articulates at approximately the mid-length of the
orbit. This pterygoid is fused to the maxilla. The medial
and posterior face of the medial rami of the pterygoid are
about equal in length. The medial ramus is broader than
the posterior and its posterior face is strongly sculpted. The
lateral end of the medial ramus overlaps the lateral edge of
the prootic. The quadratojugal is long, laterally curved, and
slender, articulating anteriorly with the maxilla. It has a bul-
bous posteroventral process and articulates dorsally with the
ventral ramus of the squamosal. The squamosal is dorsally
bifurcated, broad, and sculpted, extending anterodorsomedi-
ally from the quadratojugal to the level of the otic capsule;
the zygomatic ramus is very short, whereas the otic ramus is
long, almost reaching the posterior end of the skull.
Mandible. The mandible is slim and edentate. The men-
tomeckelians are small and arcuate in ventral view, medially
and laterally broadened, and medially separated. The dentary
is short and thin, posteriorly acuminate, and overlaps the
angulosplenial for about a quarter of its length. Dentary is
not in contact with the angulosplenial posteriorly. The angu-
losplenial is long and arcuate, laterally slightly grooved. The
coronoid process is weak.
Hyoid. The bony posteromedial processes of the hyoid
are expanded proximally and separated from one another.
No ossified parahyoid is present.
Axial skeleton (Fig.6).
Vertebral column. The vertebral column has eight pro-
coelous, presacral vertebrae. The vertebrae have complete
neural arches and low neural processes. Atlas (presacral I)
and presacral II are fused medially and laterally. The trans-
verse processes of presacrals II–III are slightly expanded
distally, thicker, and broader than those of other presacrals.
The transverse processes of presacrals II and III are ori-
ented ventrolaterally (II anteriorly, III slightly posteriorly),
whereas those of presacrals IV–VIII extend dorsolaterally
(IV–V posteriorly, VI–VIII anteriorly). The relative lengths
of the transverse processes and sacral diapophyses are:
Sacrum > III > IV > V ≈ VI ≈ VII ≈ VIII > II. The sacral
diapophyses are slightly expanded distally. The urostyle has
a well-developed dorsal ridge that extends along most of
its length.
Pectoral girdle. The zonal portion has well-ossified cora-
coids, clavicles, scapulae, and cleithra. The clavicles are
long, slender, and oriented anteromedially; the medial tips
are in contact and located anteriorly from the level of ante-
rolateral end of the clavicle that articulates with the scapula;
the coracoid is long and flared, with its sternal end slightly
broader than its glenoid end. The coracoids overlap medially.
The scapula is long with a prominent pars acromialis that is
not separated from the pars glenoidalis. The scapula is about
one and a half the length of the clavicle. The cleithrum is
ossified, well-developed, anteriorly thicker, thinning poste-
riorly. The suprascapula is unossified.
Pelvis girdle. The long, slender iliac shafts bear dorsolat-
eral crests throughout their length. The overall length of the

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girdle is more than two times the width between the anterior
ends of the iliac shafts. The iliac prominence is broad and
low, and pubes mineralized.
Manus and pes. The phalangeal formulae for the hand
and foot are standard, 2–2–3–3 and 2–2–3–4–3, respectively.
Terminal phalanges strongly T-shaped. Ossified prepollex
and prehallux visible.
Advertisement call. Six specimens calling from the
underwood vegetation were recorded from about 2m away
at air temperatures between 23‒25°C and 90‒100% rela-
tive humidity. Descriptive statistics of call parameters are
presented in Table2. Pristimantis crepitaculus sp. nov. emits
every 7.04s on average (range 5.44–8.62) series (call length
mean = 0.59, range 0.50–0.75s) of 6–12 short notes (note
length mean = 0.004s; range 0.003–0.006s). These notes are
emitted with increasing inter-note intervals within the call
(mean 1st interval = 0.068s; range 0.054–0.085s; mean 2nd
interval = 0.102s; range 0.078–0.125s). The notes have a
clear harmonic structure (i.e. with extensive amount of energy
in the harmonics). The dominant frequency is 3.50kHz on
average (range 3.16–3.91kHz) with a slight upward modula-
tion within the call (ca. 0.2kHz) (Fig.7, Table2).
Natural history. Pristimantis crepitaculus sp. nov. is a
scansorial species. Males were found calling perched on low
vegetation (0.5–2.0m above the ground). Calling activity
was limited to a short period (about 1h) at dawn and early
night. Calling males often adopt a position head down along
the trunks, small twigs, or leaves. They form groups of 2–10
males usually spaced by at least 2m between individuals.
Juveniles and females are very rarely found, but at least one
female (MPEG 41825) was collected very close to a calling
male (MPEG 41824, calling from a leaf at about 2m above
ground). The species inhabits terra firme forests from 50 to
700m asl and seems to particularly thrive near ecotones,
such as tree falls, clear-cuts, roads, rivers, maybe because
the penetrating light allows denser understory cover and thus
more abundant calling sites.
Distribution. The species is apparently endemic to the
easternmost part of the Guiana Shield lowlands, i.e. through-
out the Amapá state of Brazil and French Guiana (at the
exception of the northwest part of the territory). It occurs in
the upper Maroni region, which corresponds to the border
with Suriname, and probably extends at least in southeastern
Suriname, like other species that are also endemic to the
easternmost part of the Guiana Shield (e.g. Amazophrynella
teko, Boana dentei, Pristimantis gutturalis). However, it is
unlikely that the range of P. crepitaculus sp. nov. extends to
the Coppename river.
Discussion
Species diversity, endemism, andbiogeography
oftheEastern Guiana Shield
As a further step from Kok etal. (2018), the present study
clarifies the understanding of the megadiverse genus Pristi-
mantis in the Guiana Shield, and of overall anuran diver-
sity pattern in the region. Many anuran species remain to
be described and named in Amazonia and notably in the
Guiana Shield region, as suggested by almost all studies
exploring particular groups (e.g. Gehara etal., 2014; Fou-
quet etal.,2021a; Kok etal., 2017; Jaramillo etal., 2020) and
molecular diversity (Vacher etal., 2020). The fact that many
Amazonian frog species are circumscribed to particular and
often relatively small subregions within Amazonia is pro-
gressively unveiling as taxonomic progresses are being made.
Vacher etal. (2020) notably identified three bioregions within
the Guiana Shield, one in the easternmost part, one in the
southern part, and one in the western part. The distributions
of P. espedeus, P. crepitaculus, P. sp. “Guianas” East, and P.
grandoculis in the eastern portion, of P. cf. ockendeni in the
southern, and of P. marmoratus, P. pulvinatus, and P. saltissi-
mus in the western portions strongly corroborate this pattern.
The determining factors of this pattern remain ambiguous,
but probably originate from both historical climatic and land-
scape changes and current bioclimatic heterogeneity, notably
the fact that the easternmost part of the Guiana shieldnotably
receives more precipitation seasonally than other parts of the
region (Silva etal., 2019; Vacher etal., 2020).
A few lineages remain to be investigated for a better
understanding of their taxonomic status, such as Pristim-
antis sp. “Guianas” East and the five lineages forming what
we call a “trans-amazon complex” (P. cf. ockendeni). These
candidate species require a thorough and integrative system-
atic review. Although P. grandoculis and P. sp. “Guianas”
East are morphologically very similar (the latter also lacking
a columella and external tympanum, see Fig.8), the degree
of divergence on the 16S fragment that we used is > 4% sug-
gesting that these groups of populations may correspond to
Table 2 Acoustic variables
Call length Inter-call Note length Inter-note 1 Inter-note 2 Dom. Freq Nb notes
P. crepitaculus sp. nov Mean (n = 6) 0.586 7.038 0.004 0.068 0.109 3497 7.9
Min 0.504 5.440 0.003 0.054 0.078 3163 6
Max 0.752 8.623 0.006 0.085 0.125 3908 12

A.Fouquet et al.
1 3
distinct species (Fouquet etal., 2007; Vieites etal., 2009).
However, the lack of acoustic data and the low number of
available specimens from Suriname so far hamper any taxo-
nomic decision about P. sp. “Guianas” East. Pristimantis
diversity in the Pantepui region also requires further inves-
tigation, as notably illustrated by candidate species from
Neblina, Mount Ayanganna, Mount Wokomung (B. Means,
pers. comm.). Probably many more yet undocumented iso-
lated lineages occur in poorly explored areas of Pantepui,
including isolated tepui summits. A larger-scaled phyloge-
netic investigation is needed to test whether a Guiana Shield
clade of Pristimantis might have diversified insitu. A few
groups have been documented to have extensively diversified
throughout the Guiana Shield lowlands and highlands such
as Anomaloglossus (Vacher etal., 2017), and Otophryne
(Fouquet etal., 2021a). These cases are nonetheless rela-
tively rare since most lowland amphibians have diversified
throughout Amazonia. The Pristimantis species belonging
to the “unistrigatus group” could represent an additional
case of Guiana Shield restricted clade, whose monophyly
and timing of diversification would be worth investigating.
The case of the “trans-amazon complex” (P. cf. ockend-
eni) is no less complex. It belongs to a lineage apart from
the other species of the Guiana Shield that most likely origi-
nated from western Amazonian ancestors and secondarily
dispersed to the Guiana Shield across the Amazon River.
Similar and possibly concomitant trans-Amazon dispersals
are documented in Allobates (A. aff. tapajos Réjaud etal.,
2020) and Boana (Boana gr. albopunctata Fouquet etal.,
2021b), Synapturanus (Fouquet etal., 2021a), Scinax (Ferrão
etal., 2016). From 9 million years ago (Ma) onwards, the
Amazon basin has been draining eastward into the Atlan-
tic Ocean and the Amazon River was forming a barrier for
South-North dispersal around 5Ma. The processes that
Fig. 7 Sonograms and oscil-
lograms on a 0.7-s window of a
characteristic call of Pristi-
mantis crepitaculus sp. nov.
from Oiapoque, Amapá, Brazil
(paratype, MPEG 41823)

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may have fostered multiple trans-Amazon dispersals around
5Ma remain highly speculative. Considerable uncertainty
remains about the timing and amplitude of historical topo-
graphic, hydrological, and vegetational changes in Amazo-
nia (Albert etal., 2018; Bicudo etal., 2019; Hoorn etal.,
2017; Latrubesse etal., 2010). The sediment discharge in the
Amazon fan may have been relatively modest until 5Ma and
vastly increased in the Pliocene–Pleistocene (Hoorn etal.,
2017; Albert etal.,2018). The lower course of the Amazon
River may have become an impassable barrier for these taxa
only from the Miocene-Pliocene boundary onward. Moreo-
ver, this period also coincides with vegetational and climatic
changes, notably the expansion of grasslands not only in the
Andes and the Cerrado but also within Amazonia (Kirschner
& Hoorn, 2019).
Osteological comparisons
At this stage, comprehensive osteological comparisons
among species in the “unistrigatus group” are impossible
due to the paucity of data, as the osteology of most of these
species has not been described or illustrated. However, our
osteological investigation of the holotypes of P. marmoratus
and P. grandoculis unambiguously confirms the non-con-
specificity of these taxa. The main diagnostic osteological
characters are the lack of a columella and a more ossi-
fied skull in P. grandoculis (Figs.4, 6and8). The skull of
MNHN-RA-2020.0118 is, however, poorly ossified, which
could be an artefact of preservation as suggested by the lack
of ossification of the brain case for instance. The skull of
the holotype of P. grandoculis, although that specimen was
collected in 1904, is still strongly ossified. Unfortunately,
further comparisons with the holotype of P. marmoratus are
made difficult by the poor quality of the µ-CT scans, and
we refrain to describe its osteology until more µ-CT scans
of additional specimens of P. marmoratus sensu stricto are
available. Spatial distribution, external morphology, and
osteological data converge in suggesting the conspecificity
of the populations previously identified as P. sp. “Guianas”
from Suriname and P. grandoculis. The absence of a colu-
mella in P. grandoculis is in line with the absence of vocal
slit, pharyngeal ostia, and calling activity in that species (see
below). The sister species (population from French Guiana)
also lacks a columella and external tympanum. A complete
investigation of the osteology of the “unistrigatus group”
would certainly be of taxonomic/evolutionary interest.
Vertical niche partitioning amongspecies
andrelated traits onlimbs
Juveniles, females, and amplectant pairs of Pristimantis sp.
“Guianas” East (sister to P. grandoculis) are often found on
the leaf litter, suggesting that the reproduction takes place
directly either on the ground or at low height in the vegeta-
tion. In contrast, females of P. crepitaculus, P. espedeus,
P. inguinalis and P. cf. ockendeni are rarely seen. Males
of Pristimantis sp. “Guianas” East are also found on the
ground but more frequently perched at low height in the
underwood at night. However, juveniles of P. espedeus are
also frequently seen on the ground as are juveniles of Pristi-
mantis sp. “Guianas” East, but during the day. Small-bodied,
terrestrial, and nocturnal anurans are very rare (Pough &
Magnusson, 1992), possibly because predators such as spi-
ders are very abundant in the leaf litter at night (Magnusson,
Fig. 8 Volumetric renderings
of µ-CT scans of the skull (in
lateral view) of the holotype
of Pristimantis grandoculis (a;
RMNH 4467), a specimen of
P. grandoculis from Suriname
(b; MNHN-RA-2020.0118),
the holotype of P. marmora-
tus (c; BMNH 1947.2.16.92),
and the holotype of P. crepi-
taculus sp. nov. (d; MNHN-
RA-2020.0102). Absence/pres-
ence of columellae (highlighted
in red) is pointed by a red arrow

A.Fouquet et al.
1 3
2016). Among the species of the “unistrigatus group” occur-
ring in the Guiana Shield lowlands, Pristimantis grandoculis
and P. sp. “Guianas” East can be considered as the lowest
in terms of vertical partitioning. Vertical niche partition-
ing in Pristimantis has already been described notably in
western Ecuador (Lynch & Duellman, 1997; Guyasamin &
Funk,2009) and in Pristimantis, Craugastor, and Diasporus
in Costa Rica (Miyamoto, 1982). These converging obser-
vations strengthen the idea that vertical partitioning among
Pristimantis species is frequent, even at low elevations such
as in the Amazonian lowlands, and may even play a direct
role in speciation.
It is noteworthy that Pristimantis grandoculis and P. sp.
“Guianas” East have longer limbs (TL, TiL, FL) and more
terrestrial habits than scansorial P. crepitaculus, P. cf. ock-
endeni and arboreal P. inguinalis and P. espedeus, species.
These differences in limb proportions may be related to
jumping ability (Citadini etal., 2018; Emerson, 1985; Zug,
1972). We hypothesize that long distance jumping perfor-
mance may be selected in a leaf litter and scansorial environ-
ment which is relatively free of obstacles and that may be
risky because predators are abundant in the leaf litter (Pough
& Magnusson, 1992), whereas scansorial and arboreal spe-
cies require less power but more agility in dense vegetation.
Lack ofear andofcalling activity
Males of P. grandoculis and P. sp. “Guianas” East were
often found perched on the vegetation in a position sug-
gesting calling activity. However, acoustic activity has never
been heard in that clade, neither in Suriname nor in French
Guiana (despite being locally abundant), and these are the
only species in the Guiana Shield lowlands with unknown
call. Moreover, tympanum, columella, pharyngeal ostia,
and vocal slits are all absent in that clade. These observa-
tions suggest that P. grandoculis may be both deaf and mute.
The loss of hearing structures has occurred multiple times
throughout the evolutionary history of anurans (Jaslow
etal.,1988; Boistel etal., 2013; Pereyra etal.,2016). It is
particularly common in Bufonidae with 30% of the species
being earless (Pereyra etal.,2016). It is also common in Ter-
rarana (Hedges etal., 2008; Padial etal.,2014, Goutte etal.,
2017; Von May etal., 2018) and is particularly prevalent
in several clades distributed at high elevations (e.g. Bry-
ophryne, Phrynopus; Duellman & Lehr, 2009). Only a few
Pristimantis of the “unistrigatus group” have been reported
to lack tympanum and vocal slits (e.g. P. imthurni and P.
jamescameroni, see Kok, 2013; P. yaviensis, see Myers &
Donnelly,1996). However, these Pristimantis do call. As
a matter of fact, most earless frogs, although anatomically
deaf, still call and can perceive acoustic signals via other
organs (e.g. Boistel etal., 2013; Goutte etal., 2017). But
only a few frog species have been reported to be mute in
addition to being deaf. Most of these frogs are living near
noisy environments such as streams and advertise via other
cues such as visual or chemical signalling (Toledo etal.,
2015). The cases of a dull-coloured Amazonian lowland spe-
cies such as P. grandoculis being deaf and mute are remark-
able. Hypotheses regarding the loss of ear and of acoustic
activity in this species remain open.

Back fromthedeaf: integrative taxonomy revalidates anearless andmute species, Hylodes…
1 3
Appendix1 Molecular data
12S access 16S access Genus Species Voucher Voucher2 Locality Country lat lon
NA JQ742162 Pristimantis abakapa VUB3749 Abakapa-tepui Venezuela 5.1892 − 62.2944
NA JQ742163 Pristimantis abakapa VUB3750 Angasima-tepui Venezuela 5.0431 − 62.0808
NA JQ742151 Pristimantis aureoventris VUB3741 Roraima-tepui Guyana 5.2500 − 60.7167
NA JQ742152 Pristimantis aureoventris VUB3748 Wei-Assipu-tepui Guyana 5.2175 − 60.7058
NA JQ742153 Pristimantis aureoventris VUB3742 Wei-Assipu-tepui Guyana 5.2175 − 60.7058
NA JQ742154 Pristimantis aureoventris VUB3747 Wei-Assipu-tepui Guyana 5.2175 − 60.7058
NA JQ742155 Pristimantis aureoventris VUB3744 Wei-Assipu-tepui Guyana 5.2175 − 60.7058
NA JQ742156 Pristimantis aureoventris VUB3745 Wei-Assipu-tepui Guyana 5.2175 − 60.7058
NA JQ742157 Pristimantis aureoventris VUB3746 Wei-Assipu-tepui Guyana 5.2175 − 60.7058
NA JQ742158 Pristimantis aureoventris VUB3743 Wei-Assipu-tepui Guyana 5.2175 − 60.7058
NA JQ742159 Pristimantis aureoventris VUB3499 Wei-Assipu-tepui Guyana 5.2175 − 60.7058
NA KDQF01000101 Pristimantis espedeus AF0265 Nouragues French Guiana 4.0917 − 52.7000
NA KDQF01000107 Pristimantis espedeus AF0277 R116 Nouragues French Guiana 4.0917 − 52.7000
NA KDQF01000108 Pristimantis espedeus AF0278 R117 Nouragues French Guiana 4.0917 − 52.7000
NA KDQF01000150 Pristimantis espedeus AF0576 Itoupe French Guiana 3.0250 − 53.0800
NA KDQF01000422 Pristimantis espedeus AF1156 R118 Trinité French Guiana 4.6025 − 53.4143
NA KDQF01000423 Pristimantis espedeus AF1157 Trinité French Guiana 4.6025 − 53.4143
NA KDQF01000439 Pristimantis espedeus AF1190 R119 Trinité French Guiana 4.6025 − 53.4143
NA KDQF01000446 Pristimantis espedeus AF1203 R120 Trinité French Guiana 4.6025 − 53.4143
NA KDQF01000452 Pristimantis espedeus AF1219 Trinité French Guiana 4.6025 − 53.4143
NA KDQF01000570 Pristimantis espedeus AF1520 R121 Grande Montagne
Tortue
French Guiana 4.2927 − 52.3495
NA KDQF01000571 Pristimantis espedeus AF1521 R122 Grande Montagne
Tortue
French Guiana 4.2927 − 52.3495
NA KDQF01000572 Pristimantis espedeus AF1522 R123 Grande Montagne
Tortue
French Guiana 4.29273 − 52.3495
NA KDQF01000952 Pristimantis espedeus AF2430 Nassau Suriname 4.8041 − 54.5555
NA KDQF01000953 Pristimantis espedeus AF2431 Nassau Suriname 4.8041 − 54.5555
NA KDQF01000955 Pristimantis espedeus AF2433 Nassau Suriname 4.8041 − 54.5555
NA KDQF01001020 Pristimantis espedeus AF2616 Atachi Bakka French Guiana 3.5444 − 53.9128
NA KDQF01001025 Pristimantis espedeus AF2629 Atachi Bakka French Guiana 3. 5444 − 53.9128
NA KDQF01001028 Pristimantis espedeus AF2632 Atachi Bakka French Guiana 3. 5444 − 53.9128
NA KDQF01001048 Pristimantis espedeus AF2670 Alikéné French Guiana 3.2156 − 52.3980
NA KDQF01001057 Pristimantis espedeus AF2684 Alikéné French Guiana 3.2156 − 52.3980
NA KDQF01001058 Pristimantis espedeus AF2685 Alikéné French Guiana 3.2156 − 52.3980

A.Fouquet et al.
1 3
12S access 16S access Genus Species Voucher Voucher2 Locality Country lat lon
NA KDQF01001061 Pristimantis espedeus AF2690 Alikéné French Guiana 3.2157 − 52.3973
NA KDQF01001062 Pristimantis espedeus AF2694 Alikéné French Guiana 3.2157 − 52.3973
NA KDQF01001063 Pristimantis espedeus AF2695 Alikéné French Guiana 3.2157 − 52.3973
NA KDQF01001529 Pristimantis espedeus AG268 Lucifer French Guiana 4.7774 − 53.9475
NA KDQF01001653 Pristimantis espedeus AG507 Saul French Guiana 3.7193 − 53.4128
NA KDQF01001654 Pristimantis espedeus AG508 R124 Saul French Guiana 3.7193 − 53.4128
NA KDQF01002024 Pristimantis espedeus BPN2908 RAP Basecamp 1 Suriname 2.5269 − 55.7700
JN690706 JN691314 Pristimantis espedeus CM395 Lucifer French Guiana 4.7774 − 53.9475
NA KDQF01003878 Pristimantis espedeus PG477 R131 Kotika French Guiana 3.9333 − 54.1972
NA ON113943 Pristimantis espedeus PG524 R132 Pic Coudreau de l'Est French Guiana 3.3005 − 52.9493
NA KDQF01004301 Pristimantis espedeus ST300 Parna Tumucumaque AP 0.9091 − 53.2285
NA KDQF01004302 Pristimantis espedeus ST301 Parna Tumucumaque AP 0.9091 − 53.2285
NA ON113944 Pristimantis espedeus ST315 Parna Tumucumaque AP 0.9091 − 53.2285
NA ON113939 Pristimantis imthurni PK3671 Ptari-tepui Venezuela 5.7639 − 61.8112
NA KDQF01000063 Pristimantis inguinalis AF0157 Brownsberg Suriname 4.9365 − 55.1948
NA KDQF01000248 Pristimantis inguinalis AF0803 Kaw French Guiana 4.5507 − 52.1610
NA KDQF01000253 Pristimantis inguinalis AF0813 Savane Virginie French Guiana 4.1959 − 52.1490
NA KDQF01000256 Pristimantis inguinalis AF0820 Chutes Voltaire French Guiana 5.0312 − 54.0878
NA KDQF01000295 Pristimantis inguinalis AF0883 Nouragues French Guiana 4.0850 − 52.6810
NA KDQF01000514 Pristimantis inguinalis AF1386 Aratai French Guiana 3.9909 − 52.5902
NA KDQF01000523 Pristimantis inguinalis AF1415 Savane Virginie French Guiana 4.1959 − 52.1490
NA KDQF01000556 Pristimantis inguinalis AF1498 Nouragues French Guiana 4.0848 − 52.6806
NA ON113918 Pristimantis inguinalis AF1503 Grande Montagne
Tortue
French Guiana 4.2927 − 52.3496
NA KDQF01000609 Pristimantis inguinalis AF1626 Saul French Guiana 3.5732 − 53.1985
NA KDQF01000814 Pristimantis inguinalis AF2054 Sipaliwini Suriname 2.1751 − 56.0832
NA KDQF01000937 Pristimantis inguinalis AF2398 Nassau Suriname 4.8170 − 54.6037
NA KDQF01000954 Pristimantis inguinalis AF2432 Nassau Suriname 4.8041 − 54.5555
NA KDQF01001019 Pristimantis inguinalis AF2613 Atachi Bakka French Guiana 3.5520 − 53.9509
NA KDQF01001043 Pristimantis inguinalis AF2659 Alikéné French Guiana 3.2091 − 52.4020
NA KDQF01001121 Pristimantis inguinalis AF2858 Mitaraka French Guiana 2.2358 − 54.4493
NA KDQF01001229 Pristimantis inguinalis AF3083 Mitan G French Guiana 2.6284 − 52.5540
NA KDQF01001402 Pristimantis inguinalis AF3404 Bakhuis Suriname 4.6837 − 56.7721
NA KDQF01001416 Pristimantis inguinalis AF3432 Bakhuis Suriname 4.6837 − 56.7721
NA ON113921 Pristimantis inguinalis AF3531 Itoupe French Guiana 3.0230 − 53.0955
NA ON113922 Pristimantis inguinalis AF3772 Voltzberg Suriname 4.6817 − 56.1857
NA ON113923 Pristimantis inguinalis AF4777 Galbao French Guiana 4.4880 − 52.0445

Back fromthedeaf: integrative taxonomy revalidates anearless andmute species, Hylodes…
1 3
12S access 16S access Genus Species Voucher Voucher2 Locality Country lat lon
NA ON113925 Pristimantis inguinalis AF5402 Camp Savane Roche
Dachine
French Guiana 3.4696 − 53.2300
NA ON113928 Pristimantis inguinalis AF5448 Trois-Saut French Guiana 2.1969 − 52.9017
NA ON113929 Pristimantis inguinalis AF5524 Haute Courcibo French Guiana 4.4723 − 53.2459
NA KDQF01001509 Pristimantis inguinalis AG102 Kaw French Guiana 4.5710 − 52.2206
NA KDQF01001519 Pristimantis inguinalis AG225 Trinité French Guiana 4.6709 − 53.2843
JN690710 JN691317 Pristimantis inguinalis AG229 Trinité French Guiana 4.6709 − 53.2843
NA KDQF01001538 Pristimantis inguinalis AG291 Trinité French Guiana 4.6025 − 53.4143
NA KDQF01001595 Pristimantis inguinalis AG424 Atachi Bakka French Guiana 3.6553 − 53.8440
NA KDQF01001643 Pristimantis inguinalis AG491 Saul French Guiana 3.7193 − 53.4128
NA KDQF01001691 Pristimantis inguinalis AM015 Inini Tolenga French Guiana 3.6632 − 53.9283
NA KDQF01002875 Pristimantis inguinalis MPEG30059 Serra do Acari. N
ESEC Grão Pará
PA 1.2854 − 58.6959
NA KDQF01002876 Pristimantis inguinalis MPEG30060 Serra do Acari. N
ESEC Grão Pará
PA 1.2854 − 58.6959
NA KDQF01003609 Pristimantis inguinalis MTR24299 Oiapoque AP 3.8794 − 51.7710
NA KDQF01003778 Pristimantis inguinalis NZCS51 Brownsberg Suriname 4.9365 − 55.194826
NA KDQF01004105 Pristimantis inguinalis QM0406 Montagne de fer French Guiana 5.4074 − 53.5548
NA KDQF01004107 Pristimantis inguinalis QM0426 Montagne de fer French Guiana 5.4074 − 53.5548
NA ON113942 Pristimantis inguinalis QM1074 Gaa_Kaba French Guiana 4.4509 − 54.4117
NA JQ742170 Pristimantis inguinalis VUB3626 Cacao Mountain French Guiana 4.5667 − 52.4667
EU186739 EU186721 Pristimantis jamescameroni SBH268110 Bolivar, Aprada-tepui Venezuela 5.4243 − 62.4208
NA ON113932 Pristimantis jamescameroni IRSNB4163 Bolivar, Aprada-tepui Venezuela 5.4243 − 62.4208
NA JQ742169 Pristimantis jester VUB3493 Maringma-tepui Guyana 5.21524 − 60.5834
NA MF037223 Pristimantis marmoratus CPI10703 Mt. Wokomung Guyana 5.1175 − 59.8225
EU186734 EU186716 Pristimantis marmoratus ROM43302 Mt. Wokomung Guyana 5.1175 − 59.8225
EU186741 EU186723 Pristimantis marmoratus KU181015 km 127, El Dorado-
Santa Elena de Uairen
Rd
Venezuela 5.8983 − 61.4428
NA JQ742166 Pristimantis marmoratus VUB3485 Kaieteur NP Guyana 5.2686 − 59.7686
NA JQ742167 Pristimantis marmoratus VUB3491 Maringma-tepui Guyana 5.2152 − 60.5834
NA KDQF01002108 Pristimantis marmoratus BPN3814 Bay Camp Guyana 5.0112 − 59.63930
NA KDQF01002109 Pristimantis marmoratus BPN3815 Bay Camp Guyana 5.0112 − 59.6393
NA KDQF01002110 Pristimantis marmoratus BPN3816 Bay Camp Guyana 5.0112 − 59.6393
NA JQ742164 Pristimantis pulvinatus VUB3751 Iwokrama Guyana 4.6714 − 58.6850
NA JQ742165 Pristimantis pulvinatus VUB3674 La Escalera Venezuela 5.9158 − 61.4347
NA KDQF01004007 Pristimantis pulvinatus PK3509 Iwokrama Guyana 4.6720 − 58.6840
EU186692 EU186692 Pristimantis saltissimus ROM43913 Mt. Ayanganna Guyana 5.3996 − 59.9505
EU186693 EU186693 Pristimantis saltissimus ROM43310 Mt. Wokomung Guyana 5.1175 − 59.8225

A.Fouquet et al.
1 3
12S access 16S access Genus Species Voucher Voucher2 Locality Country lat lon
NA JQ742168 Pristimantis saltissimus VUB3490 Maringma-tepui Guyana 5.2152 − 60.5834
JN690708 JN691315 Pristimantis crepitaculus AF0021 Saul French Guiana 3.6256 − 53.2072
NA KR811102 Pristimantis crepitaculus AF0563 Savane Virginie French Guiana 4.1959 − 52.1490
NA KR811105 Pristimantis crepitaculus AF0757 St Georges French Guiana 3.8112 − 51.8896
NA KDQF01000316 Pristimantis crepitaculus AF0910 MNHN-
RA-2020.0093
Pic Coudreau du Sud French Guiana 2.2534 − 54.3534
NA KDQF01000366 Pristimantis crepitaculus AF1014 Saul French Guiana 3.6359 − 53.2726
NA KDQF01000382 Pristimantis crepitaculus AF1046 Saul French Guiana 3.6112 − 53.2886
NA ON113933 Pristimantis crepitaculus AF1387 MNHN-
RA-2020.0094
Aratai French Guiana 3.9909 − 52.5902
NA ON113934 Pristimantis crepitaculus AF1388 MNHN-
RA-2020.0095
Aratai French Guiana 3.9909 − 52.5902
NA ON113935 Pristimantis crepitaculus AF1598 MNHN-
RA-2020.0097
Nouragues French Guiana 4.0848 − 52.6806
NA KDQF01000601 Pristimantis crepitaculus AF1607 MNHN-
RA-2020.0100
Saul French Guiana 3.5732 − 53.1985
NA KDQF01000749 Pristimantis crepitaculus AF1921 MNHN-
RA-2020.0101
Flat de la Waki French Guiana 3.0840 − 53.4107
NA KDQF01000754 Pristimantis crepitaculus AF1941 Flat de la Waki French Guiana 3.0836 − 53.4121
NA KDQF01001103 Pristimantis crepitaculus AF2786 MNHN-
RA-2020.0102
Mitaraka French Guiana 2.2358 − 54.4493
NA ON113936 Pristimantis crepitaculus AF2799 MNHN-
RA-2020.0103
Mitaraka French Guiana 2.2358 − 54.4493
NA KDQF01001186 Pristimantis crepitaculus AF3002 Mémora D AP 3.3129 − 52.1804
NA KDQF01001205 Pristimantis crepitaculus AF3047 Mitan G French Guiana 2.6284 − 52.5540
NA KDQF01001276 Pristimantis crepitaculus AF3161 MNHN-
RA-2020.0108
Mitan D AP 2.6276 − 52.5419
NA KDQF01001437 Pristimantis crepitaculus AF3539 MNHN-
RA-2020.0092 Itoupe French Guiana 3.0230 − 53.0955
NA ON113924 Pristimantis crepitaculus AF5399 Camp Saut Richard French Guiana 3.47363 − 53.2086
NA ON113927 Pristimantis crepitaculus AF5440 Trois-Saut French Guiana 2.1969 − 52.9017
JN690707 EU201063 Pristimantis crepitaculus AG_BM110 Kaw3 French Guiana 4.5481 − 52.1519
NA KDQF01001542 Pristimantis crepitaculus AG321 Kaw French Guiana 4.5161 − 52.1005
NA KDQF01001568 Pristimantis crepitaculus AG378 Montagne Maripa French Guiana 4.4088 − 52.34301
NA KDQF01001645 Pristimantis crepitaculus AG494 Saul French Guiana 3.7193 − 53.4128
NA KDQF01001886 Pristimantis crepitaculus BOAM048 Borne 4 French Guiana 2.3709 − 53.7728
NA KDQF01002129 Pristimantis crepitaculus CAAM15 Montagne Cacao French Guiana 2.3491 − 53.2160
NA EU201062 Pristimantis crepitaculus CM077 Trois-Sauts French Guiana 2.2480 − 52.8746
NA KDQF01002243 Pristimantis crepitaculus CM171 Trijonction French Guiana 2.3333 − 54.6000
NA KDQF01002504 Pristimantis crepitaculus FL04 Plote PPBIO AP 0.9793 − 51.6149

Back fromthedeaf: integrative taxonomy revalidates anearless andmute species, Hylodes…
1 3
12S access 16S access Genus Species Voucher Voucher2 Locality Country lat lon
NA KDQF01002521 Pristimantis crepitaculus FTA128 Rio Vila Nova AP 0.4493 − 52.0221
NA KDQF01002528 Pristimantis crepitaculus FTA165 Rio Amapá Grande AP 2.1271 − 51.1913
NA KDQF01002543 Pristimantis crepitaculus FTA239 Rio Amapá Grande AP 2.1271 − 51.1913
NA ON113931 Pristimantis crepitaculus FTA96 Rio Vila Nova AP 0.4493 − 52.0221
NA KDQF01003282 Pristimantis crepitaculus MTR13884 Lourenço AP 2.32361 − 51.6453
NA KDQF01003293 Pristimantis crepitaculus MTR13930 Lourenço AP 2.32361 − 51.6453
NA KR811104 Pristimantis crepitaculus MTR24285 Lourenço AP 2.3216 − 51.6109
NA KR811103 Pristimantis crepitaculus MTR24300 Oiapoque AP 3.8794 − 51.7710
NA KDQF01003952 Pristimantis crepitaculus PG748 St Georges French Guiana 3.8061 − 51.8934
NA KDQF01004462 Pristimantis crepitaculus WAAM011 Flat de la Waki French Guiana 3.18341 − 53.4775
NA KDQF01003322 Pristimantis sp. Neblina 1 MTR15532 São Gabriel da Cach-
oeira
AM 0.7195 − 66.0878
NA KDQF01003323 Pristimantis sp. Neblina 1 MTR15534 São Gabriel da Cach-
oeira
AM 0.7195 − 66.0878
NA KDQF01003325 Pristimantis sp. Neblina 2 MTR15536 São Gabriel da Cach-
oeira
AM 0.7195 − 66.0878
NA KDQF01000102 Pristimantis sp. “Guianas”
East
AF0266 Nouragues French Guiana 4.0917 − 52.7000
NA KDQF01000103 Pristimantis sp. “Guianas”
East
AF0268 Nouragues French Guiana 4.0917 − 52.7000
NA KDQF01000104 Pristimantis sp. “Guianas”
East
AF0272 Nouragues French Guiana 4.0917 − 52.700
NA KDQF01000354 Pristimantis sp. “Guianas”
East
AF0978 Saul French Guiana 3.6359 − 53.2726
NA ON113917 Pristimantis sp. “Guianas”
East
AF1360 Grande Montagne
Tortue
French Guiana 4.2927 − 52.3496
NA KDQF01000558 Pristimantis sp. “Guianas”
East
AF1505 Grande Montagne
Tortue
French Guiana 4.2927 − 52.3496
NA KDQF01001024 Pristimantis sp. “Guianas”
East
AF2625 Atachi Bakka French Guiana 3.5444 − 53.9128
NA ON113919 Pristimantis sp. “Guianas”
East
AF2626 Atachi Bakka French Guiana 3.5444 − 53.9128
NA ON113920 Pristimantis sp. “Guianas”
East
AF2818 Mitaraka French Guiana 2.2358 − 54.4493
NA ON113926 Pristimantis sp. “Guianas”
East
AF5414 Savane Roche Dachine French Guiana 3.4696 − 53.2298
NA ON113930 Pristimantis sp. “Guianas”
East
AF5525 Haute Courcibo French Guiana 4.4723 − 53.2459
NA KDQF01001850 Pristimantis sp. “Guianas”
East
BOAM001 Borne 4 French Guiana 2.3709 − 53.7728

A.Fouquet et al.
1 3
12S access 16S access Genus Species Voucher Voucher2 Locality Country lat lon
NA KDQF01001860 Pristimantis sp. “Guianas”
East
BOAM012 Borne 4 French Guiana 2.3709 − 53.7728
NA KDQF01002249 Pristimantis sp. “Guianas”
East
CM184 Trijonction French Guiana 2.3333 − 54.6000
JN690709 JN691316 Pristimantis sp. “Guianas”
East
CM317 CisameG French Guiana 4.1833 − 52.3667
NA ON113940 Pristimantis sp. “Guianas”
East
QM0423 Montagne de fer French Guiana 5.4074 − 53.5548
NA ON113941 Pristimantis sp. “Guianas”
East
QM1073 Gaa Kaba French Guiana 4.4520 − 54.4193
ON117803 KDQF01004305 Pristimantis sp. “Guianas”
East
ST310 Parna Tumucumaque AP 0.9091 − 53.2285
NA KDQF01004306 Pristimantis sp. “Guianas”
East
ST311 Parna Tumucumaque AP 0.9091 − 53.2285
NA ON113937 Pristimantis grandoculis UFRJA024 NZCS A1211 Lelygebergte Suriname 4.4158 − 54.6498
NA ON113938 Pristimantis grandoculis UFRJA023 NZCS A1210 Lelygebergte Suriname 4.4158 − 54.6498
NA KDQF01002880 Pristimantis grandoculis MPEG30085 Serra do Acari PA 1.2854 − 58.6959
NA KDQF01002881 Pristimantis grandoculis MPEG30088 Serra do Acari PA 1.2854 − 58.6959
NA KDQF01003777 Pristimantis grandoculis NZCS50 Brownsberg Suriname 4.9366 − 55.1948
NA KDQF01003795 Pristimantis grandoculis NZCS A1019 Tafelberg Suriname 3.8049 − 56.1539
NA KDQF01003796 Pristimantis grandoculis NZCS A1212 Tafelberg Suriname 3.8049 − 56.1539
NA KDQF01001388 Pristimantis grandoculis AF3365 Bakhuis Suriname 4.6562 − 56.7865
NA KDQF01001400 Pristimantis grandoculis AF3402 Bakhuis Suriname 4.6837 − 56.7721
NA KDQF01001403 Pristimantis grandoculis AF3408 Bakhuis Suriname 4.6837 − 56.7721
NA KDQF01001417 Pristimantis grandoculis AF3435 MNHN-
RA-2020.0118
Bakhuis Suriname 4.6837 − 56.7721
EU186676 EU186676 Pristimantis sp. Ayangana ROM40164 Mt. Ayanganna Guyana 5.3995 − 59.9505
ON117804 KDQF01004221 Pristimantis cf. ockendeni SMNS11989 Mabura Hill forest
reserve
Guyana 5.1553 − 58.6997
ON117805 KDQF01004222 Pristimantis cf. ockendeni SMNS11990 Mabura Hill forest
reserve
Guyana 5.1553 − 58.6997
ON117806 KDQF01004223 Pristimantis cf. ockendeni SMNS11994 Mabura Hill forest
reserve
Guyana 5.1553 − 58.6997
ON117807 KDQF01000865 Pristimantis cf. ockendeni AF2155 Sipaliwini Suriname 2.02682 − 56.1256
NA KDQF01003210 Pristimantis cf. ockendeni MTR12690 Igarapé Açu, Rio
Abacaxis
AM − 4.3442 − 58.6350
NA KDQF01001829 Pristimantis cf. ockendeni BM153 UHE Belo Monte,
Vitória do Xingu, Rio
Xingu
PA − 2.9506 − 51.9366
NA KDQF01004256 Pristimantis cf. ockendeni SMS155 Campo Catuquira AM − 4.9136 − 61.1092

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1 3
12S access 16S access Genus Species Voucher Voucher2 Locality Country lat lon
NA KDQF01004257 Pristimantis cf. ockendeni SMS156 Campo Catuquira AM − 4.9136 − 61.1092
NA KDQF01002705 Pristimantis cf. ockendeni JOG847 MINA PALITO PA − 6.3173 − 55.7986
NA KDQF01003224 Pristimantis cf. ockendeni MTR12855 São Sebastião, Rio
Abacaxis
AM − 4.3089 − 58.6364
NA KDQF01000889 Pristimantis cf. ockendeni AF2257 Sipaliwini Suriname 2.0244 − 56.1251
NA KDQF01002605 Pristimantis cf. ockendeni H2773 UHE Jirau RO − 9.4459 − 64.8247
NA JQ742160 Pristimantis yuruaniensis VUB3717 Yuruani-tepui Venezuela 5.3139 − 60.8639
NA JQ742161 Pristimantis yuruaniensis VUB3720 Yuruani-tepui Venezuela 5.3139 − 60.8639
Appendix2 Specimens examined andtypes
Genus Species Museum Field N Sex Type Locality Country Lat Lon Collector Date Acoustic
Pristimantis crepitaculus MNHN-RA-2020.0093 AF0910 F Paratype Pic Coudreau
du Sud
French
Guiana
2.2534 − 54.3534 M. Blanc 01_02_2013
Pristimantis crepitaculus MNHN-RA-2020.0104 AF2815 F Paratype Mitaraka French
Guiana
2.2358 − 54.4493 A. Fouquet − M.
Dewynter − N. Vidal
28_02_2015
Pristimantis crepitaculus MNHN-RA-2020.0105 AF2816 F Paratype Mitaraka French
Guiana
2.2358 − 54.4493 A. Fouquet − M.
Dewynter − N. Vidal
28_02_2015
Pristimantis crepitaculus MNHN-RA-2020.0094 AF1387 M Paratype Aratai French
Guiana
3.9909 − 52.5902 A. Fouquet − J.
Devillechabrolle
16_12_2013
Pristimantis crepitaculus MNHN-RA-2020.0095 AF1388 M Paratype Aratai French
Guiana
3.9909 − 52.5902 A. Fouquet − J.
Devillechabrolle
16_12_2013
Pristimantis crepitaculus MNHN-RA-2020.0096 AF1597 M Paratype Nouragues French
Guiana
4.0848 − 52.6806 E. Courtois 09_02_2014
Pristimantis crepitaculus MNHN-RA-2020.0097 AF1598 M Paratype Nouragues French
Guiana
4.08489 − 52.6806 E. Courtois 09_02_2014
Pristimantis crepitaculus MNHN-RA-2020.0098 AF1599 M Paratype Nouragues French
Guiana
4.08489 − 52.6806 E. Courtois 09_02_2014

A.Fouquet et al.
1 3
Genus Species Museum Field N Sex Type Locality Country Lat Lon Collector Date Acoustic
Pristimantis crepitaculus MNHN-RA-2020.0099 AF1606 M Paratype Saul French
Guiana
3.5732 − 53.1985 A. Fouquet − E.
Courtois − J.P.
Vacher
20_02_2014 MNHN-
SO-2022-559
Pristimantis crepitaculus MNHN-RA-2020.0100 AF1607 M Paratype Saul French
Guiana
3.5732 − 53.1985 A. Fouquet − E.
Courtois − J.P.
Vacher
20_02_2014
Pristimantis crepitaculus MNHN-RA-2020.0101 AF1921 M Paratype Flat de la
Waki
French
Guiana
3.0840 − 53.4107 J.P. Vacher 07_04_2014
Pristimantis crepitaculus MNHN-RA-2020.0102 AF2786 M Holotype Mitaraka French
Guiana
2.2358 − 54.4493 A. Fouquet − M.
Dewynter − N. Vidal
26_02_2015 MNHN-
SO-2019–23
Pristimantis crepitaculus MNHN-RA-2020.0103 AF2799 M Paratype Mitaraka French
Guiana
2.2358 − 54.4493 A. Fouquet − M.
Dewynter − N. Vidal
27_02_2015
Pristimantis crepitaculus MNHN-RA-2020.0106 AF2860 M Paratype Mitaraka French
Guiana
2.2358 − 54.4493 A. Fouquet − M.
Dewynter − N. Vidal
04_03_2015
Pristimantis crepitaculus MNHN-RA-2020.0107 AF3054 M Paratype Mitan G French
Guiana
2.6284 − 52.5540 A. Fouquet − P. Nunes 20_03_2015
Pristimantis crepitaculus MNHN-RA-2020.0108 AF3161 M Paratype Mitan D AP 2.6276 − 52.5419 A. Fouquet − P. Nunes 23_03_2015
Pristimantis crepitaculus MNHN-RA-2020.0109 AF3530 M Paratype Itoupe French
Guiana
3.0230 − 53.0955 A Fouquet − E
Courtois − B
Villette − M
Dewynter
06_01_2016 MNHN-
SO-2022-561
Pristimantis crepitaculus MNHN-RA-2020.0092 AF3539 M Paratype Itoupe French
Guiana
3.0230 − 53.0955 A Fouquet − E
Courtois − B
Villette − M
Dewynter
06_01_2016 MNHN-
SO-2022-560
Pristimantis crepitaculus MNHN-RA-2020.0110 AF3548 M Paratype Itoupe French
Guiana
3.02218 − 53.1105 A Fouquet − E
Courtois − B
Villette − M
Dewynter
06_01_2016
Pristimantis crepitaculus MNHN-RA-2020.0111 AF3664 M Paratype Itoupe French
Guiana
3.0256 − 53.0525 A Fouquet − E
Courtois − B
Villette − M
Dewynter
11_01_2016
Pristimantis crepitaculus MNHN-RA-2020.0112 AF3687 M Paratype Itoupe French
Guiana
3.0230 − 53.0955 A Fouquet − E
Courtois − B
Villette − M
Dewynter
14_01_2016
Pristimantis grandoculis MNHN-RA-2020.0118 AF3435 M Bakhuis Suriname 4.7246 − 56.7638 A. Fouquet − S.
Cally − R. Jairam
30_04_2015
Pristimantis grandoculis RMNH 4467 M Holotype Coppenname Suriname
Pristimantis grandoculis NZCSA1212 M Tafleberg Suriname 3.8049 − 56.1539 P. Ouboter − V.
Kadosoe
Pristimantis crepitaculus MPEG 41,819 PLVP 289 M Paratype Oiapoque Brazil 3.79889 − 51.87993 P. Peloso − L. Brandão 04_12_2015

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1 3
Genus Species Museum Field N Sex Type Locality Country Lat Lon Collector Date Acoustic
Pristimantis crepitaculus MPEG 41,820 PLVP 290 M Paratype Oiapoque Brazil 3.79889 − 51.87993 P. Peloso − L. Brandão 05_12_2015
Pristimantis crepitaculus MPEG 41,821 PLVP 301 M Paratype Oiapoque Brazil 3.79889 − 51.87993 P. Peloso − L. Brandão 05_12_2015
Pristimantis crepitaculus MPEG 41,822 PLVP 352 M Paratype Oiapoque Brazil 3.79889 − 51.87993 P. Peloso − L. Brandão 06_12_2015 MNHN-
SO-2022-562
Pristimantis crepitaculus MPEG 41,823 PLVP 353 M Paratype Oiapoque Brazil 3.79889 − 51.87993 P. Peloso − L. Brandão 06_12_2015 MNHN-
SO-2022-563
Pristimantis crepitaculus MPEG 41,824 PLVP 354 M Paratype Oiapoque Brazil 3.79889 − 51.87993 P. Peloso − L. Brandão 06_12_2015
Pristimantis crepitaculus MPEG 41,825 PLVP 355 F Paratype Oiapoque Brazil 3.79889 − 51.87993 P. Peloso − L. Brandão 06_12_2015
Pristimantis grandoculis NZCS A1210 UFRJA023 M Lely Mountain Suriname 4.2667 − 54.7333 R. Jairam – D. Baeta
Pristimantis grandoculis NZCS A1211 UFRJA024 M Lely Mountain Suriname 4.2667 − 54.7333 R. Jairam – D. Baeta
Pristimantis cf. ockendeni MNHN-RA-2020.0113 AF2155 F Sipaliwini Suriname 2.0268 − 56.1256 A. Fouquet − J.P.
Vacher
23_04_2014
Pristimantis cf. ockendeni MNHN-RA-2020.0114 AF2258 F Sipaliwini Suriname 2.0244 − 56.1251 A. Fouquet − J.P.
Vacher
27_04_2014
Pristimantis cf. ockendeni MTD47769 F Mabura
Hill forest
reserve
Guyana 5.1553 − 58.6997 R Ernst 18 08 2010
Pristimantis cf. ockendeni SMNS11990 F Mabura
Hill forest
reserve
Guyana 5.1553 − 58.6997 R Ernst 25 04 2003
Pristimantis cf. ockendeni SMNS11991 F Mabura
Hill forest
reserve
Guyana 5.1553 − 58.6997 R Ernst 25 04 2003
Pristimantis cf. ockendeni SMNS11995 F Mabura
Hill forest
reserve
Guyana 5.15538 − 58.6997 R Ernst 01 06 2004
Pristimantis cf. ockendeni MNHN-RA-2020.0115 AF2255 M Sipaliwini Suriname 2.0244 − 56.12518 A. Fouquet − J.P.
Vacher
27_04_2014 MNHN-SO-
2021-XXX
Pristimantis cf. ockendeni MNHN-RA-2020.0116 AF2256 M Sipaliwini Suriname 2.02449 − 56.1251 A. Fouquet − J.P.
Vacher
27_04_2014
Pristimantis cf. ockendeni MNHN-RA-2020.0117 AF2289 M Sipaliwini Suriname 2.0334 − 56.1318 A. Fouquet − J.P.
Vacher
30_04_2014 MNHN-SO-
2021-XXX
Pristimantis cf. ockendeni MTD47770 M Mabura
Hill forest
reserve
Guyana 5.1553 − 58.6997 R Ernst 18 08 2010
Pristimantis cf. ockendeni SMNS11987 M Mabura
Hill forest
reserve
Guyana 5.1553 − 58.6997 R Ernst 18 11 2002
Pristimantis cf. ockendeni SMNS11989 M Mabura
Hill forest
reserve
Guyana 5.1553 − 58.6997 R Ernst 17 04 2003

A.Fouquet et al.
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Genus Species Museum Field N Sex Type Locality Country Lat Lon Collector Date Acoustic
Pristimantis cf. ockendeni SMNS11993 M Mabura
Hill forest
reserve
Guyana 5.1553 − 58.6997 R Ernst 26 03 2004
Pristimantis cf. ockendeni SMNS11994 M Mabura
Hill forest
reserve
Guyana 5.1553 − 58.6997 R Ernst 01 06 2004

Back fromthedeaf: integrative taxonomy revalidates anearless andmute species, Hylodes…
1 3
Appendix3 Call recordings list
Pristimantis crepitaculus
PLPDR 126 (MPEG 41822), Paratype, Oiapoque –
Amapá, BrazilMNHN-SO-2022-562
PLPDR 128 (MPEG 41823), Paratype, Oiapoque –
Amapá, BrazilMNHN-SO-2022-563
track59 Marty and Gaucher (NA), Kaw montain − French
Guiana
160106_0692 (AF3530), Itoupé − French GuianaMNHN-
SO-2022-561
160106_0693(AF3539), Holotype, Itoupé − French Gui-
anaMNHN-SO-2022-560
LS110010 (AF1606), Saul Limonade − French Gui-
anaMNHN-SO-2022-559
Appendix4
Mean pairwise p distance
Acknowledgements We thank Ate Alma-Cohen and Esther Dondorp
from the Leiden Naturalis Biodiversity Center (National Research Insti-
tute for Biodiversity, The Netherlands), and Mark Wilkinson from the
Natural History Museum (UK) who provided the µ-CT scans of the
holotype of Hylodes grandoculis and the holotype of Pristimantis mar-
moratus, respectively. Renaud Lebrun and Quentin Martinez (ISEM)
helped with data acquisition with the µ-CT facilities of the MRI platform
member of the National Infrastructure France-BioImaging (supported
by the French National Research Agency (ANR-10-INBS-04, «Invest-
ments for the future»), the Labex CEMEB (ANR-10-LABX-0004),
and NUMEV (ANR-10-LABX-0020)). We also thank E. Courtois, L.
Brandão, M. Dewynter, N. Vidal, J. Devillechabrole, J.-P. Vacher, P.
Nunes, B. Villette, S. Cally, D. Baêta, P. Ouboter, V. Kadosoe, M. Blanc,
J. Dias Lima, P. Gaucher, M.T. Rodrigues, and V. Rufray who helped
to collect some of the specimens and tissue samples used in this work.
Permission to conduct biodiversity research in Guyana was provided by
the Environmental Protection Agency of Guyana under research permit
number 180609 BR 112, and fieldwork was made possible through the
Forestry Department of the Guyana Forestry Commission (GFC-PRDD),
particularly R. Thomas. Collection permits for Brazil were provided by
Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio).
Funding This study benefited from an “Investissement d’Avenir” grant
managed by the Agence Nationale de la Recherche (CEBA, rANR-
10-LABX-25–01). PP and ATM were supported by fellowships from
Conselho Nacional de Desenvolvimento Científico e Tecnológico
(302501/2019–3 and 300822/2021–9 to PP; 142153/2019–2 to ATM).
PP was also supported by an award from the Maxwell/Hanrahan
Foundation. The work of RE was funded by the German Academic
Exchange Service (DAAD) and Deutsche Forschungsgemeinschaft
(DFG; ER 589/2–1). The work of PJRK was funded by the Fonds voor
Wetenschappelijk Onderzoek (FWO12A7614N and FWO12A7617N).
Availability of data and material All new DNA sequences, call record-
ings, and µ-CT scans have been deposited in public repositories.
Declarations
Ethics approval Experiments have been conducted in accordance with
relevant national legislation on the use of animals for research.
Competing interests The authors declare no competing interests.
P. crepitaculus sp. nov.
P. marmoratus 0.116
P. grandoculis 0.1260.118
P. sp “Guianas”East0.123 0.1200.045
P. cf._ockendeni0.2070.202 0.1840.184
P. cf._ockendeni_Abacaxis_20.183 0.1840.168 0.1680.054
P. cf._ockendeni_PA0.187 0.1930.167 0.1710.0610.061
P. cf._ockendeni_RO, AM 0.2010.193 0.1770.173 0.0730.0740.045
P. cf._ockendeni_Abacaxis_10.185 0.1800.164 0.1650.0730.0710.0470.054
P. abakapa0.1060.0800.0960.0880.1840.167 0.1720.168 0.152
P. imthurni 0.1120.0870.0970.0880.1840.167 0.1760.176 0.1580.036
P. jamescameroni0.1070.0890.0980.0870.1860.169 0.1710.165 0.1470.0260.023
P. pulvinatus0.1120.0990.1020.0990.1800.165 0.1670.174 0.1610.0630.0680.060
P. aureoventris0.1080.0820.1020.0860.1890.167 0.1750.181 0.1650.0530.0580.0600.063
P. yuruaniensis0.1170.0820.1000.0830.1900.168 0.1790.181 0.1650.0530.0550.0580.0590.015
P. inguinalis0.1380.121 0.1370.131 0.1870.178 0.1790.180 0.1510.0940.1100.101 0.1160.107 0.115
P. saltissimus 0.1230.115 0.1260.108 0.2070.179 0.1980.198 0.1860.0750.0820.0770.0770.0590.0530.133
P. jester 0.1300.0990.1010.0930.1860.169 0.1750.179 0.1630.0700.0670.0720.0740.0420.0430.1200.077
P. sp. “Ayanganna”0.120 0.0790.109 0.0990.1950.173 0.1860.191 0.1750.0650.0700.0720.0670.0380.0410.1150.067 0.060
P. espedeus 0.1380.121 0.1390.142 0.1990.179 0.1870.197 0.1780.110 0.1310.121 0.1110.111 0.1160.137 0.129 0.1330.114
P. sp. “Neblina 1” 0.1290.095 0.1160.100 0.1850.164 0.1760.182 0.1640.0770.0790.0790.0690.0590.0510.1190.083 0.0770.0640.114
P
. sp. “Neblina 2” 0.1310.116 0.1190.115 0.1840.170 0.1680.185 0.177
0.0770.0770.0830.0890.0720.072
0.120
0.097 0.0890.089
0.140
0.085

A.Fouquet et al.
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