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A new rainfrog of the genus Pristimantis
(Anura, Brachycephaloidea) from
central and eastern Panama
Konrad Mebert1,2,5, Macario González-Pinzón3,6, Madian Miranda3,4,5,6,
Edgardo Grith7,8, Milan Vesely9, P. Lennart Schmid10, Abel Batista3,4,5,6,11
1Programa de Pós-graduação em Zoologia, Universidade Estadual de Santa Cruz, Rodovia Jorge Amado, Km
16, 45662-900, Ilhéus, Bahia, Brazil 2Global Biology, Waldmatt, Birr, 5242, Switzerland 3Universidad
Autónoma de Chiriquí (UNACHI), Vicerrectoría de investigación y Postgrado – Museo de Historia Natural,
Ciudad Universitaria El Cabrero David, Chiriquí, 427, Panama 4 Museo Herpetológico de Chiriquí
(MHCH), David, Chiriquí, 426–01459, Panama 5Los Naturalistas, David, Chiriquí, 426–01459, Panama
6Asociación ADOPTA el Bosque Panamá, 119x Gamboa, Panama 7El Valle Amphibian Conservation Center
Foundation, El Hato, El Valle de Antón, Coclé, Panama 8University of Panama, Vicerectoria de Investigación
y Posgrado, Regional University Center of Coclé. El Coco de Penonomé, Llano Marín, Vía Interamericana,
Coclé, Panama 9Department of Zoology, Faculty of Natural Sciences, Palacký University, 17. Listopadu 50,
77146 Olomouc, Czech Republic 10Institut für Organismische und Molekulare Evolutionsbiologie, Johannes
Gutenberg Universität, Mainz, Germany 11 Estación Cientíca COIBA AIP Edicio 205, Ocina 117
Ciudad del Saber, Clayton, Veraguas, Panama
Corresponding author: Abel Batista (abelbatista@hotmail.com)
Academic editor: J. Penner|Received 13 January 2021|Accepted 29 November 2021|Published 10 January 2022
http://zoobank.org/B9BC53C3-0A9B-44BB-9590-495AA68D4899
Citation: Mebert K, González-Pinzón M, Miranda M, Grith E, Vesely M, Schmid PL, Batista A (2022) A new
rainfrog of the genus Pristimantis (Anura, Brachycephaloidea) from central and eastern Panama. ZooKeys 1081: 1–34.
https://doi.org/10.3897/zookeys.1081.63009
Abstract
Substantial molecular and morphological character dierences lead us to the description of a new species
of the genus Pristimantis from the cloud forest of Cerro Chucantí, Maje Mountains, Darien Province, as
well as from several other mountain ranges in eastern and central Panama. Pristimantis gretathunbergae
sp.nov. is a sister species to the allopatric P. erythropleura-penelopus group from northern Colombia with
a mtDNA sequence divergence of > 4.4% at 16S and > 14.6% at COI. Its closest congener in sympatry is
P. cruentus that diers by a large sequence divergence of > 9.6% in 16S mtDNA and 19.0% at COI, and
ZooKeys 1081: 1–34 (2022)
doi: 10.3897/zookeys.1081.63009
https://zookeys.pensoft.net
Copyright Konrad Mebert et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Konrad Mebert et al. / ZooKeys 1081: 1–34 (2022)
2
from which it diers also by ventral and groin coloration, unusually prominent black eyes, a contrasting
light upper lip, commonly a single conical to spine-like tubercle on the upper eyelid, and a larger head.
While the habitat continuity at most sites in eastern Panama is moderate, habitats in central Panama are
severely fragmented. Cerro Chucantí and the surrounding Maje Mountains are highly threatened by rapid
deforestation and replaced by plantations and cattle pastures. us, investigations on the ecology of the
new species and its population status, especially at the type locality, are highly recommended. As a agship
species, this new frog can help to preserve the Chucantí cloud forest including several recently described
species known only from this isolated area in eastern Panama.
Keywords
Chucantí, Craugastoridae, Greta unberg’s Rainfrog, Maje, Pristimantis gretathunbergae sp. nov.,
Strabomantidae, Terraranae
Introduction
Tropical regions are extraordinarily rich in biodiversity which is caused by the combi-
nation of historical, climatic, and geographic characteristics that favor high speciation
rates, as for example in anurans (Myers et al. 2000). In particular, rainfrogs of the
genus Pristimantis (Family Strabomantidae, superfamily Brachycephaloidea, resp. Ter-
raranae, Padial et al. 2014, or family Craugastoridae, see Barrientos et al. 2021 for a
dierent arrangement due to paraphyletic issues), are a major component of anuran
diversity in the Neotropics (Rivera-Correa and Daza 2016). Although Pristimantis is
one of the most numerous genera of all vertebrates, containing at least 574 species
distributed primarily in Tropical Andes of Colombia, Ecuador, and Peru (Frost 2021),
it remains vastly understudied (Hedges et al. 2008; Meza‐Joya and Torres 2016; Am-
phibiaWeb 2021; Frost 2021). Species of the genus Pristimantis are highly variable in
coloration and morphology, often rendering it dicult to distinguish between species
based on external features alone (Batista et al. 2014a), while their phylogeny often
remains unclear. Indeed, more than 315 species of Pristimantis are not assigned to any
species group (Padial et al. 2014), and 124 species were described in the last 10 years
with a rate of 11.3 species/year (e.g., Rivera-Correa and Daza 2016; Reyes-Puig et al.
2020; Frost 2021). A relatively recent divergence and similar morphological character
variations among species indicate a remarkable cryptic diversity within Pristimantis
(Ortega-Andrade et al. 2015). is genus is the result of a mega-radiation event (Men-
doza et al. 2015; Heinicke et al. 2018; Waddell et al. 2018) and contains approxi-
mately 6.7% of all known amphibian and 7.4% of all anuran species (AmphibiaWeb
2021; Frost 2021). Its remarkable diversity is often explained by the evolution of di-
rect development. Hence, the lack of aquatic tadpole stages makes them independent
of water bodies for reproduction and provides greater habitat exibility (Duellman
and Lehr 2009). erefore, rainfrogs can ll niches unoccupied by other amphibians
(Teran-Valdez and Guayasamin 2010).
Currently, there are 13 species of Pristimantis frogs known to occur in Panama
(Batista et al. 2014a), or 14 species if P. educatoris Ryan, Lips & Giermakowski, 2010
A New Species of Pristimantis from Panama 3
is viewed as a separate species from P. caryophyllaceus Barbour, 1928 (Frost 2021).
Although this species richness is small compared to the richness of Pristimantis across
the much larger Choco bioregion of western Colombia and Ecuador (Cheza et al.
2020; Reyes-Puig et al. 2020), its variation in Panama still poses a major challenge
for taxonomic work (Crawford et al. 2010). Important revisions of the phylogeny
and distribution of this group in Central America have been conducted by Ibañez
and Crawford (2004) and Crawford et al. (2010) as well as by Pinto-Sánchez et al.
(2012) and Batista et al. (2014a) for Panama. In Panama there are currently three
endemic Pristimantis species: Pristimantis adnus Crawford, Ryan & Jaramillo, 2010;
Pristimantis pirrensis Ibáñez & Crawford, 2004 and Pristimantis museosus Ibáñez,
Jaramillo & Arosemena, 1994 (Frost et al. 2021).
Our study focuses on Darién Province with the principal material originating from
Cerro Chucantí. is mountain supports a remarkable diversity of organisms with 13
recently described new species, including plants (Ortiz et al. 2016; Flores et al. 2017),
insects (Miranda and Bemúdez 2010; Bezark et al. 2013; Martins and Galileo 2013),
amphibians (Batista et al. 2014b, 2016a), and reptiles (Batista et al. 2016b). ere is
also a relatively high diversity of herpetological species on Cerro Chucantí with a total
of 35 reptilian and 41 amphibian species recorded until 2020, including three endemic
ones and ten out of the 13 Pristimantis species from Panama (Batista et al. 2020).
Herein we describe a new species of Pristimantis based on molecular and mor-
phological characters of specimens from Cerro Chucantí, Maje Mountains, and other
mountain ranges in eastern and central Panama. Additionally, we present sequences
and photographic vouchers (photo panels in online Suppl. material 2) of closely related
Pristimantis taxa primarily from moist forests in Panama and Colombia, such as the
Magdalena-Urabá and Chocó-Darién regions (Fagua and Ramsey 2019), as well as
adjacent montane forests, providing valuable visual material for comparison.
Materials and methods
Sampling sites
e primary study site is Cerro Chucantí that includes the highest peak (1439 m
a.s.l. at 8.8046°N, 78.4595°W; Fig. 1) in the Maje Mountains, an isolated massif in
Darién, Panama. It is a sky island with a small cloud forest around its peak of < 5 km2
width. e nearest comparable cloud forests are at least 100 km away on Cerro Pirre
and Cerro Pechito Parao. e higher elevations of Cerro Chucantí are part of the
Eastern Panamanian Montane Forests ecoregion (Fund 2014). Annual precipitation
varies between 3,000 mm and 4,000 mm and occurs mainly from April to December
(Rio Maje Meteorological Station, 70 m a.s.l. http://www.hidromet.com.pa/, accessed
on 19 September 2015). e average temperatures on Cerro Chucantí, measured with
data loggers in 2018 and 2019, decreased with elevation from 23.5 °C at 770 m a.s.l.,
to 21.1 °C at 1025 m a.s.l., and 19.1 °C at 1269 m a.s.l., yet was with 22.1 °C again
Konrad Mebert et al. / ZooKeys 1081: 1–34 (2022)
4
higher on the top at > 1400 m a.s.l., possibly as a result of increased solar radiation
due to reduced canopy cover in the cloud forest. e following vegetation zones oc-
cur on Cerro Chucantí at dierent elevations: Lowland Moist Forest (0–500m a.s.l.),
Premontane Moist Forest (500–1000 m a.s.l.) and a small area of Premontane Wet
Forest (≈ cloud forest) higher than 1000 m a.s.l. (Holdridge 1966). All geographical
coordinates were recorded in the WGS 1984 datum and presented in decimal degrees.
e distributional map was created using QGIS (QGIS 2018) with an Open Street
Map (OSM) layer (OSM 2015).
Additional material for molecular and/or morphological analysis was obtained
from specimens collected in eastern Panama (location names in bold as used in article):
1) Maje Ambroya, Maje Mountains, Panama Province; 2) Cerro Chucantí, Province
Darién; 3) Río Tuquesa, Cerro Pechito Parao at Bajo Pequeño, Darien Mountains,
Embera-Wounaan Comarca (= indigenous autonomous region). ird party material
was collected in Central Panama from: 4) Cerro Brewster, Piedras-Pacora Mountains
Figure 1. Map showing locations of Pristimantis gretathunbergae sp. nov. and Pristimantis cruentus in
Panama. Numbers in the map correspond to localities mentioned in methods. Internal divisions in the
map correspond to provinces in black letters.
A New Species of Pristimantis from Panama 5
of Chagres National Park, Province Panama; 5) only photographic vouchers from
Cerro Bruja, Chagres National Park, Province Colon; 6) a single DNA sequence from
El Cope National Park at Rio Blanco, Penonomé Mountains, Province Cocle; and 7)
Altos del Maria, vicinity of Gaita Hills, Province Panama Oeste (Fig. 1).
Specimens and morphological characters
Molecular characters
For molecular analyses of Panamanian samples, DNA was extracted from fresh liver
tissue. e 16S mtDNA extraction and sequencing follow previously described rou-
tines (Batista et al. 2016a). e COI fragments were sequenced in the Southern China
DNA Barcoding Center. e mtDNA sequences obtained were compared and related
specimens from Colombia and Ecuador published in GenBank, with those retained
for the analysis that were informative per region (i.e., only one sequence/taxon/loca-
tion). e sequences were aligned with CLUSTAL W (Larkin et al. 2007) and edited
by eye using Geneious version 4.8.5 (Kearse et al. 2012). A list of specimens included
in the genetic analysis with corresponding GenBank accession numbers is appended
in the Suppl. material 1. GenBank sequences of Craugastor sagui and C. crassidigitus
were used as outgroups. e nal 16S alignment comprised 97 sequences obtained
from this study and from GenBank consisting of 500 bp, of which 339 sites were vari-
able, 187 parsimony-informative, and 50 singletons. For COI gene analyses Diasporus
diastema, C. longirostris and C. crassidigitus were used as outgroup. e nal alignment
comprised 8 sequences from our material and 30 ones from GenBank, consisting of
567 bp, of which 293 sites were variable, 282 parsimony-informative, and 11 single-
tons. e nal alignment for the COI and 16S genes together comprised 49 sequences
consisting of 1053 bp, of which 412 sites were variable, 359 parsimony-informative,
and 52 singletons.
A Maximum Likelihood analysis (MA) was conducted for both genetic markers
using IQ-TREE (Nguyen et al. 2015; Trinopoulos et al. 2016). To estimate support,
1000 replicates of ultrafast bootstrapping (Minh et al. 2013) were performed. A nodal
or branch support with SH-aLRT values ≥ 80% is considered reliable for a clade
(Guindon et al. 2010). A substitution model using JModeltest 0.1.1 (Posada 2008) was
selected for the Bayesian Inference (BI) analysis under the corrected Akaike Information
Criterion (AICc; Akaike 1974), for the 16S gene. However, the resulting TIM2+I+G
model was replaced by the GTR model (Lecocq et al. 2013). e 3-parameter model
with rate heterogeneity, TIM2+I+G (Kimura 1981) was implemented for the combined
gene data set. We ran a Bayesian phylogenetic analysis in MrBayes 3.1.2 (Huelsenbeck
and Ronquist 2001) for 10,000,000 generations with four default chains, sampling
every 1000 generations and for the nal consensus tree discarding the rst 25% as
burn-in. To test species delimitation among the Pristimantis species included in this
study, the Automatic Barcode Gap Discovery (ABGD) algorithm (Puillandre et al.
Konrad Mebert et al. / ZooKeys 1081: 1–34 (2022)
6
2012) was applied for the 16S gene under the following settings: steps = 20, distance
= Kimura 2-parameter model with transversion/transition ratio of 2.0. e setting for
the minimum relative gap width (X) was set to 0.5.
Collecting permits for 2009 (SC/A-8-09, SC/A-28-09), 2011 (SC/A-37-11), 2012
(SC/A-33-12), 2016 (SE/A-60-16) and 2018 (SE/A-33-18) as well as export permits
for 2012 (SC/A-33-12) and 2013 (SEX/A-7-13) were provided by UNARGEN-Minis-
terio de Ambiente, Panama. Finally, we applied the traditional Environmental Vulner-
ability Score (EVS) methodology by Wilson and McCranie (2004) to calculate the con-
servation status of this species. is method assigns increasing values to higher conser-
vation priorities based on geographic and habitat distribution, and reproductive mode;
in anurans from 1–17, or up to 20 in the revised version by Johnson et al. (2015).
Morphological characters
Specimens were collected by hand, photographed alive, euthanized with the Solution
Tanax T-61, xed with a preservative solution of 5 mL formalin (36%) in 1 L ethanol
(94%), and subsequently stored in ethanol (70%) following the protocol of Batista et
al. (2016a). Preserved specimens were later analyzed at the Zoological Laboratory of
the Universidad Autónoma de Chiriquí. All gures were assembled and some improved
using Adobe Photoshop CS6. Specimens are deposited at the Museo Herpetológico
de Chiriquí (MHCH, Universidad Autónoma de Chiriquí, David) in Panama, and at
the Senckenberg Forschungsinstitut Frankfurt (SMF) in Germany. e abbreviations
for museum collections follow Sabaj (2016), with eld numbers AB for initials of Abel
Batista and MG of Macario Gonzalez. Morphological nomenclature, measurements
and standardized diagnosis characters follow Duellman and Lehr (2009). Some com-
parative morphological data of similar Pristimantis species in Colombia were extracted
from the respective original descriptions, as well as a few online photo repositories (see
online Suppl. material 2). For color descriptions, we applied the code of Köhler (2012).
Sex of specimens was determined by morphometric characters and presence of
eggs in Panamanian samples. Measurements were taken to the nearest 0.1 mm, using
a stereomicroscope and precision digital calipers. Following variables were measured
according to Batista et al. (2016a) and Duellman and Lehr (2009):
SVL Snout-Vent Length
HW Head Width, measured between posterior end of jaws
HL Head Length, measured between posterior end of jaws and tip of snout
InD Internarial Distance as shortest line between inner edges of narial openings
IoD Interorbital Distance as shortest distance between visible eyes, reecting size of
braincase
EW Eyelid Width, perpendicular distance to the outer edge of the upper eyelid
ED Eye Diameter as length of exposed eye
EN Eye-Nostril distance as shortest distance between anterior corner of eye and
posterior margin of nostril
A New Species of Pristimantis from Panama 7
TY Tympanum Diameter
TL Tibial Length from knee to distal end of tibia
FL Foot Length between proximal edge of inner metatarsal tubercle to tip of fourth toe
FAL Forearm Length between elbow and hand
HAL Hand Length between proximal edge of palmar tubercle to tip of third nger
BW Body Width as largest width on trunk
AGD Axilla-Groin Distance as length between hind and front limbs along the trunk
TrL Trunk Length as SVL minus HL
3FW Width of 3rd Finger at penultimate phalanx just anterior to disc
3FD Disk Width of 3rd Finger
3TW Width of 3rd Toe at penultimate phalanx just anterior to disc
3TD Disk Width of 3rd Toe
4TW Width of 4th Toe at penultimate phalanx just anterior to the disc
4TD Disk Width of 4th Toe
Interspecic dierences among Pristimantis spp. and related species are known to be
relative lengths of heads, hind limbs, and feet (Duellman and Lehr 2009). Conse-
quently, multivariate analyses were conducted to investigate morphometric dierences
between sympatric P. cruentus and the new species. To reduce the impact of ontoge-
netic and gender dierences on measures of all body parts, 15 meaningful ratios of our
initially measured variables were applied. To account for potential head shape dier-
ences, the measured distances along the head were put into relation to head length,
i.e., InD/HL, IoD/HL, ED/HL, TY/HL, EN/HL, EW/HL, and proportionally larger
head size was reected by TrL/HL, whereas stockiness is investigated by BW/SVL.
Similar, sizes relating to limb length were put into relationship with the approximate
trunk length, i.e., FL/TrL and TL/TrL, whereas hand and foot length were measured
against forearm HAL/FAL, respectively shank FL/TL. Relative size of disk width to
digits of nger and toes were represented by 3FD/3FW, 3TD/3TW, and 4TD/4TW.
We applied a Principal Component Analysis (PCA) for variable selection and there-
fore removed redundant (highly correlated) ones. A Linear Discriminant Function
Analysis (LDFA), or simply Linear Discriminant Analysis (LDA), with the remaining
morphometric variables (ratios) followed to elucidate the potential dierences of body
proportions between the two sympatric Pristimantis species.
We conducted a Multiple Correspondence Analysis (MCA) in R (Version 4.0.3),
using the FactoMineR package, on categorical variables to compare the presence/ab-
sence of certain color pattern and tubercle characters between sympatric Pristimantis
gretathunbergae sp. nov. and P. cruentus. We assessed the character state of six variables
from photographic material of 26 P. gretathunbergae sp. nov. and 17 P. cruentus, where-
by several specimens were collected and inspected by us, and their taxonomic allocation
conrmed through molecular means. e value 0 was assigned to the morphological
state typical for P. gretathunbergae sp. nov., whereas the value 2 is typical for P. cruentus,
and the value 1 represents an intermediate expression. Follow do the descriptive states
for these six variables: A) iris coloration: 0 = blackish to very dark red; 2 = whitish,
Konrad Mebert et al. / ZooKeys 1081: 1–34 (2022)
8
golden, or light reddish, B) iris reticulation: 0 = no pattern, some with golden red
sparkling; 1 = some dark red, small patches; 2 = reticulation, C) upper eyelid tubercle:
0 = single conical to spine-like; 1 = short singular, but spine-like, higher than other
subtriangular humps; 2 = not singular or none at all, D) upper lip coloration: 0 = light
colored and sharply demarcated to darker snout coloration; 1 = light color with some
dark patches ingressing vertically from the snout, but upper border of light colored
parts on the lip still contrastingly sharp bordered; 2 = no light color or very diuse, no
upper dark border, E) groin coloration: 0 = colors are relatively uniform white, yellow,
light olive, or red, but some show a ecking pattern of these colors; 2 = dark brown to
black ecks or patches on a light ground, F) ventral coloration: 0 = unicolored or ne
spotting on white, yellow or orange: 2 = heavily dark mottled.
Results
Phylogenetic analyses
In the following, we present information on both genes (16S and COI) separately, as
well as their combined results. However, we focus more on 16S for the presentation
on closely related taxa, as 16S is more widely used and thus comparable with many
Neotropical anurans (Fouquet et al. 2007; Vacher et al. 2020). Furthermore, 16S tends
to be more appropriate when using a few phylogenetically and/or geographically close
taxa. Limitations with COI are the lack of a universal primer for the PCR amplica-
tions across numerous dierent species and high rates of intraspecic genetic variations
(Vences et al. 2005, 2012; Grosejan et al. 2015; Estupinan et al. 2016).
e ABGD analysis generated ten groups of species by initial partition with prior
maximal distance P = 1.45e-02 (Distance K80 Kimura MinSlope = 0.5) and a relative
width of barcoding gap of 0.05 X-value (Fig. 2). Genetic divergence values among
groups for 16S and COI genes combined are shown in Table 1, whereas the respective
values of each gene alone are shown in the Suppl. material 1: Tables S1, S2). Group 1
received a high SH-aLRT support of 96% (bootstrap support of 95%) and includes
all Panamanian specimens that have originally been labeled as P. a. latidiscus on
GenBank, but which show a large genetic divergence of > 11% at 16S to the original
P. latidiscus from Ecuador, South America. All other samples were grouped with high
bootstrap support in their corresponding known species, with the lowest, yet still
good support of 89.% for Group 3 (P. erythropleura) and 86.8% for Group 2 that
consisted of single specimens originally labeled as P. cruentus (SMF 97539), P.paisa
(AJC 1344) and P. viejas (EMM 247), see also Reyes-Puig et al. (2020). However,
these latter three specimens actually represent P. penelopus from northwestern Colom-
bia, errors that were already addressed/corrected by Restrepo et al. (2017). e third
specimen of Group 2 (P. cruentus SMF 97539) is a close relative collected by us from
a 200 km distant, montane site near the Pacic coast of Panama. Morphologically it
resembles P. cruentus but was provisionally labeled as P. a. sanguineus/penelopus due
A New Species of Pristimantis from Panama 9
to genetic results. Group 5 represents P. cruentus with a perfect SH-aLRT support
of 100% but a comparatively lower, yet still moderately well-supported bootstrap
value of 75%, that possibly is the result of a large genetic variation and indicates an
unresolved species complex (Crawford et al. 2012; Estupinan et al. 2016), which is
also shown by the large genetic distance of 6% at 16S between the distinct groups
cruentus and a. cruentus (see Suppl. material 1: Table S1). Furthermore, the cruentus
clade contains the specimen CH 6456 from Cana, Darien Province, Panama, origi-
nally labeled as P. a. latidiscus (Crawford et al. 2012). is specimen was relabeled
as P. cruentus, hence, P. latidiscus is removed from the list of Panamanian Pristimantis
species, because all other originally labeled P. latidiscus (members of Group 1) actually
Figure 2. Phylogenetic tree of Pristimantis spp. based on mtDNA genes 16S and COI performed by
a Shimodaira-Hasegawa approximate likelihood ratio test (SH-aLRT test). Numbers on nodes indicate
estimated SH-aLRT support/bootstrap support with SH-aLRT values ≥ 80% are considered reliable for a
clade (Guindon et al. 2010). e tree is drawn to scale, with branch lengths measured in the number of
substitutions per site. Colored bars and G-numbers represent the groups generated by ABGD analysis (see
results of phylogenetic analysis and Suppl. material 1: Table S1 for more details).
Konrad Mebert et al. / ZooKeys 1081: 1–34 (2022)
10
represent the new, undescribed species. e shortest genetic distance (16S and COI
combined) of this new rainfrog to any other Pristimantis species is 9.6% to P. penelo-
pus and 11% to P. erythropleura (Table 1).
On a wider perspective, the phylogenetic inference based on combined 16S and
COI sequences placed the Pristimantis erythropleura-penelopus clade, P. cruentus, P. cis-
nerosi (data of cisnerosi available only for 16S, see also Suppl. material 1: Table S1 and
Suppl. material 2: Figs S1, S3), and the new species into one larger clade with moderate
support, possibly reecting still unresolved species complexes. However, the new species
clearly formed a distinct lineage (Fig. 2). With the phylogeny analyzed by the approxi-
mative likelihood test (SH-aLRT test), the South American species were grouped in a
clade separated from P. gretathunbergae sp. nov. and P. cruentus, both from Panama. Ap-
plying only 16S, a ML analysis placed P. cisnerosi as sister species to the new species (not
shown). Pristimantis cisnerosi is a rainfrog of lower elevations, 70–600m a.s.l., from the
Choco forests of southwestern Colombia (see Reyes-Puig et al. 2020, and specimens
depicted in Suppl. material 2: Fig. S16), whereas P. gretathunbergae sp. nov. occupies el-
evations higher than 700 m a.s.l. at sites of more than 400 km further north in Panama.
In contrast to ML, results of 16S from a BI analysis placed P. cisnerosi as sister to the P.
erythropleura-penelopus clade, with P. erythropleura occurring at elevations primarily >
900 m and P. penelopus inhabiting lower to higher elevations around 150–200 km south
of the new species (Suppl. material 2: Fig. S3). Biologically and geographically, the BI
tree with 16S alone or combined with COI is in accordance with the current distribu-
tional patterns of the species included in our phylogenetic analyses (Fig. 2).
Pristimantis gretathunbergae sp. nov. reveals a genetic variation of < 3% for 16S
between our focus populations from Maje Mountains (Cerro Chucantí and Maje Am-
broya) to related populations from Rio Tuquesa (divergence of 1.5–2.8%) and Cer-
ro Brewster (1.9–2.9%), but also to a single sequence from farther west at El Cope,
Table 1. Estimates of net evolutionary divergence (mean %) between groups (G-numbers from the
ABGD analysis) of sequences of two mtDNA genes, 16S and COI. For every group, the estimated aver-
age evolutionary divergence over sequence pairs within groups is shown in parenthesis, with n: number
of samples included in each group, followed by origin of country: CO (Colombia), CR (Costa Rica), EC
(Ecuador), HO (Honduras), PA (Panama).
Species 16S and COI evolutionary divergence between groups
G1 G2 G3 G4 G5 G6 G7 G8 G9
G1 P. gretathunbergae sp. nov. (5%; n: 10, PA) – – – – – – – – –
G2 P. penelopus (3%; n: 3, CO) 9.6 – – – – – – – –
G3 P. erythropleura (1%; n: 2 CO) 11.0 8.1 – – – – – – –
G4 P. viejas (n.a.: n: 1, CO) 13.7 12.7 14.6 – – – – – –
G5 P. cruentus (12%; n: 22, PA) 14.9 13.4 13.1 17.0 – – – – –
G6 P. cerasinus (6%; n: 2, CR, PA) 14.6 12.8 14.3 13.2 17.5 – – – –
G7 P. calcaratus (0%; %; n: 2, EC) 15.1 12.2 13.2 13.2 16.5 15.9 – – –
G8 P. museosus (n.a; n:1, PA) 16.4 12.7 14.2 15.7 18.0 16.0 18.3 – –
G9 P. ridens (11%; n: 3, CR, HO) 18.6 15.4 15.7 17.3 19.2 17.9 16.7 21.1 –
G10 P. taeniatus (13%; n: 3, CO ) 22.0 20.6 20.5 22.4 22.7 22.0 20.6 22.3 21.7
A New Species of Pristimantis from Panama 11
Central Panama (2.3%). is corresponds to the suggested and applied minimum
sequence divergence of 3% between Neotropical frog species (Fouquet et al. 2007;
Vacher et al. 2020). Additional genetic variations by species for single genes 16S and
COI are displayed in Suppl. material 1: Tables S1, S2.
e shortest genetic distance for 16S mtDNA between the new Pristimantis spe-
cies and any other Pristimantis sample was 4.4% and pertains to two specimens of
allopatric Colombian relatives, one P. erythropleura (minimum of 14.6% at COI to
the new species) and one P. penelopus (min. of 15.5% at COI to the new species).
e mean dierence at 16S in these Colombian subsamples to P. gretathunbergae sp.
nov. is 4.8%, that increased with the addition of a few samples from other sites in
the same general region to 5.9% (P. erythropleura), respectively 6% (P. penelopus, see
also Suppl. material 1: Table S1). While a slightly shorter mean sequence divergence
to the new species is also reected at COI (16.0% in P. erythropleura vs. 16.3% in P.
penelopus; Suppl. material 1: Table S2), the combination of both genes reversed that
order, as P. penelopus exhibits a shorter distance to the new species (16S and COI
combined: 9.9% in P.penelopus vs. 11.0% in P. erythropleura, Table 1). Yet, their very
close relationship is displayed in the SH-aLRT phylogenetic tree (Fig. 2). Genetic di-
vergence of the new Pristimantis species is similarly low towards a single specimen of
P. viejas with a mean value of 5.5% at 16S, however, with considerable higher values
at COI (19.7%) and COI with 16S combined (13.7%). Furthermore, P. viejas was
placed in a clade with P.cerasinus (Fig.2), corroborating the results in Amezquita et
al. (2019). In contrast to allopatric Colombian species, sympatric Pristimantis spp.
in Panama are more distant, as P. cruentus is the closest relative with a divergence
at 16S of > 9.6% to the new species (19.0% at COI; and 14.9% at 16S and COI
combined; Table 1, Fig. 2).
Morphology
Results of morphometric measurements of adult specimens of Pristimantis gretathun-
bergae sp. nov. are presented in Table 2. It generally resembles the sympatric P. cruentus,
yet diers from it, as well as all other known Pristimantis spp. occurring in Panama (see
Comparative diagnosis and Figs 3–6) by having poorly dened tympanic membrane,
absence of vocal slits, and absence of nuptial pads (illustrative examples in Figs 4, 5 and
Suppl. material 2: Figs S8–S11). Other qualitative variables (color pattern, tubercles)
and parametric variables (body proportions) have been analyzed statistically.
A PCA revealed the following relative variables to contribute mostly to the prin-
cipal components: TrL/HL, 3TD/3TW, 3FD/3FW, 4TD/4TW with strong load-
ings and IoD/HL, ED/HL, EW/HL with medium loadings (Suppl. material 2: Fig.
S4 and Suppl. material 1: Table S3 with loadings). In the rst PCA axis (67.64%),
P.gretathunbergae sp. nov. and P. cruentus display no dierences (Mann-Whitney-U-
Test, W =301, p = 0.834), whereas the second PCA axis (14.04%) reveals signicant
dierences between the two species (Welch Two Sample t-test, t = 6.74, df = 15.473,
p<0.001; Suppl. material 2: Fig. S5).
Konrad Mebert et al. / ZooKeys 1081: 1–34 (2022)
12
A subsequent Linear Discriminant Analysis LDA correctly separated P. gretathun-
bergae sp. nov. (n = 9) from P. cruentus from eastern Panama (n = 27) and western
Panama (n = 37). Pristimantis cruentus had to be split into two separate geographic
groups based on LDA-conditions (LDA graph in Suppl. material 2: Fig. S6). On av-
erage, 79.4% of the specimens from all three groups were classied correctly accord-
ing to our a priori groupings. Pristimantis gretathunbergae sp. nov. was classied cor-
rectly by 77.78%. e four morphometric variables that contributed the most to the
LDA groupings in order of relevance were: 1) IoD/HL, 2) EW/HL, 3) ED/HL, 4)
4TD/4TW, followed by TrL/HL and characters of nger disk proportions (coecients
of LDA in Suppl. material 1: Table S4). ese results indicate a principal dierence
between the three groups in head morphology, eye size (eyelid width EW and eye-
diameter ED likely relate similarly to eye size), and toe characters.
A nal univariate analysis corroborates that in four morphometric variables used
in the LDA P. gretathunbergae sp. nov. dier signicantly from P. cruentus, for which
Eastern and Western populations were combined (unlike in the LDA): P. cruentus ex-
hibits a relatively larger eye (mean ED/HL = 0.414; mean P. gretathunbergae sp.nov. =
Table 2. Morphometric characters of adult Pristimantis gretathunbergae sp. nov. with mean ± SD (range,
followed by n); all values are in mm and separated by sex. Abbreviations of raw variables: Snout-Vent
Length (SVL), Head Width (HW), Head Length (HL), Internarial Distance (InD), Interorbital Distance
(IoD), Eyelid Width (EW) Eye Diameter (ED), Eye-Nostril Distance (EN), Tympanum Diameter (TY),
Tibial Length (TL), Foot Length (FL), Forearm Length (FAL), Hand Length (HAL), Body Width (BW),
Axilla-Groin Distance (AGD), 3rd Finger Width (3FW), 3rd Finger Disk Width (3FD), 3rd Toe Width
(3TW), 3rd Toe Disk Width (3TD), 4th Toe Width (4TW), and 4th Toe Disk Width (4TD); see methods
for denitions.
Measurement Females Males
SVL 42.66±3.71(38.15–46.3; 4) 31.24±3.52(26.9–36.7; 8)
HW 19.17±0.99(17.84–20; 4) 12.39±1.55(10.7–15.9; 8)
HL 18.14±1.49(16.46–19.9; 4) 12.44±1.69(10–14.7; 7)
InD 3.05±0.21(2.9–3.2; 2) 2.37±0.55(1.67–3.3; 7)
IoD 5.1±0.71(4.6–5.6; 2) 3.11±0.45(2.6–3.9; 7)
EW 6.3±0.14(6.2–6.4; 2) 4.79±0.43(4.3–5.4; 7)
TL 23.15±2.05(21.7–24.6; 2) 14.61±5.07(3.5–18.8; 7)
FL 22.1±0.14(22–22.2; 2) 14.45±1.99(11.7–17.8; 7)
TY 2.85±0.92(2.2–3.5; 2) 1.34±0.49(0.65–2; 7)
ED 5.45±0.21(5.3–5.6; 2) 4.11±0.61(3.7–5.3; 7)
EN 5.65±0.49(5.3–6; 2) 3.55±0.67(3.02–5; 7)
FAL 11.2±1.13(10.4–12; 2) 7.73±0.85(6.5–8.86; 7)
HAL 9.15±6.72(4.4–13.9; 2) 9.22±1.27(7.5–11.4; 7)
3FW 1.45±0.21(1.3–1.6; 2) 0.72±0.28(0.41–1.1; 7)
3FD 3.15±0.07(3.1–3.2; 2) 1.66±0.26(1.2–2; 7)
3TW 1.25±0.07(1.2–1.3; 2) 0.59±0.28(0.19–0.94; 7)
3TD 2.35±0.07(2.3–2.4; 2) 1.24±0.35(0.66–1.7; 7)
4TW 1.4±0(1.4–1.4; 2) 0.62±0.16(0.39–0.83; 7)
4TD 2.5±0.14(2.4–2.6; 2) 1.39±0.11(1.3–1.6; 7)
BW 16.75±1.34(15.8–17.7; 2) 8.46±1.2(7.14–10.8; 7)
AGD 21.4±0.85(20.8–22; 2) 12.85±1.73(10.5–14.8; 5)
A New Species of Pristimantis from Panama 13
0.322; Welch Two Sample t-test, t = 6.297, df = 25.65, p < 0.001), and eyelid width
(mean sp. nov. EW/HL = 4.50, mean P. gretathunbergae sp. nov. = 0.376; Welch Two
Sample t-test, t = 4.667, df = 25.97, p < 0.001); a longer trunk (mean TrL/HL = 2.109;
mean P. gretathunbergae sp. nov. = 1.529; Mann-Whitney-U-Test, W = 548, p < 0.001),
Figure 3. Map of the Multiple Correspondence Analysis (MCA) of P. gretathunbergae sp. nov. (red dots)
and P. cruentus (black dots): Number labels for individual frog with lines pointing to specimen location
on the map. Following correlation ratio (Dimension 1/Dimension 2) resulted from the MCA: iris colora-
tion 0.937/0.001; iris reticulation 0.933/0.637; upper eyelid tubercle 0.751/0.331; upper lip coloration
0.735/0.326; groin coloration 0.852/0; ventral coloration 0.810/0.001. e qualitative scoring of the
variables and its species-specic expression is explained in the methods.
Konrad Mebert et al. / ZooKeys 1081: 1–34 (2022)
14
and a wider head (mean IoD/HL = 0.368; mean P. gretathunbergae sp. nov. = 0.259;
Welch Two Sample t-test, t = 7.591, df = 18.8, p < 0.001). ese results indicate that
the head morphology relates primarily to the separation of P. gretathunbergae sp. nov.
and all P. cruentus. Toe characters, important in the LDA, are only signicantly dif-
ferent between P. cruentus from eastern Panama and western Panama (4TD/4TW:
Mann-Whitney-U-Test, W = 686, p = 0.011), whereas they are marginal to not dierent
between P.gretathunbergae sp. nov. and P. cruentus from eastern Panama (4TD/4TW:
Mann-Whitney-U-Test, W = 73, p = 0.079) or from western Panama (4TD/4TW:
Mann-Whitney-U-Test, W =151, p = 0.683), respectively.
A Multiple Correspondence Analysis (MCA) of six categorical variables of color
pattern and tubercle properties results in a clear distinction between the new Pristim-
antis gretathunbergae sp. nov. and its closest relative in sympatry, P. cruentus. e most
frequent and/or typical expression of these variables in Pristimantis gretathunbergae sp.
nov. (with the comparative expression of P. cruentus in parenthesis) are: 1) blackish eyes
or iris (light colored iris in P. cruentus), 2) no iris reticulation (reticulated), 3) a single
conical tubercle on the upper eyelid (rarely so, generally more variable from subtrian-
gular to spine-like, and from none at all to several ones), 4) light upper lip contrast-
ingly bordered to dark coloration on snout above (none, diusely colored lips, or light,
but not demarcated), 5) coloration of groin, as well as 6) venter is unicolored whitish,
yellow or reddish, sometimes with ne spotting (heavily black and white to dark and
light mottled, see methods for a more detailed and expanded species-specic variable
denition and quality scoring). A photographic example of a Pristimantis gretathunber-
gae sp. nov. and a P. cruentus in a face-o position is depicted in Fig. 4C, while more
explicit photographic material for comparison between these two species can be viewed
in Suppl. material 2: Figs S10, S11.
e rst and second dimension of the MCA describe 73.89% of the total variance,
allowing a conclusive two-dimensional display of the scores (Fig. 3), with further graph-
ic variables representation and their weighing in Suppl. material 2: Fig. S7. All variables
correlate strongly with Dimension 1, with the iris coloration and reticulation having
the highest correlation ratio, 0.937 and 0.933, respectively. Dimension 2 is mainly cor-
related with the iris reticulation, with 0.637 producing the only correlation ratio > 0.5.
Two distinct clusters appear in the MCA that clearly represent the two sympatric spe-
cies P. gretathunbergae sp. nov. and P. cruentus (Fig. 3). e distinction of these species in the
rst and second dimension of the MCA is highly signicant (Dimension 1: Mann-Whitney
U-Test, W = 133, p < 0.001, and Dimension 2: Mann-Whitney U-Test, W = 321,
p = 0.026). ese results strongly separate the two Pristimantis species on qualitative
morphological characters, with the distinctive eye color and pattern being a particu-
lar easy and obviously useful character to separate P.gretathunbergae sp. nov. from P.
cruentus (see Suppl. material 2: Figs S10, S11). e distinctive black eyes without
reticulation of P. gretathunbergae sp. nov. also separates it from the even closer related,
but allopatric, P. erythropleura-penelopus clade from Colombia, and likewise from Co-
lombian and Ecuadorian P. cisnerosi, P. viejas, and P. paisa (consult respective species-
specic photographic panels in the Suppl. material 2: Figs S12–S16).
A New Species of Pristimantis from Panama 15
Based on molecular divergence and morphological consensus, we assign the unde-
scribed Pristimantis sp. with the type material from Cerro Chucantí, Maje Mountains
as a new species to science. It belongs to the Pristimantis ridens species group (sensu
Reyes-Puig et al. 2020), dened by having large digital disks, nger I shorter than
nger II, toe III shorter than toe V, tympanum concealed, vomerine odontophores
oblique, no toe webbing and vocal slits absent. It is most closely related to the allopat-
ric P. erythropleura-penelopus group, which inhabits similar montane forests along the
Andean slopes of western and central Colombia. Following is the formal description of
the new species of Pristimantis.
Taxonomic account
Pristimantis gretathunbergae sp. nov.
http://zoobank.org/F9121E09-EA7C-4B9A-9ABA-7F65A82CAC2A
Suggested English name: Greta unberg’s Rainfrog
Suggested Spanish name: Rana de Greta unberg
Holotype. MHCH 3082 (original eld number AB 1059), an adult male (Figs 4A, 5)
collected by Abel Batista & Konrad Mebert on the top of Cerro Chucantí (8.804621°N,
-78.45950°W; near 1439 m a.s.l.), Maje Mountains, Río Congo Arriba, Distrito de
Chepigana, Darién, Panama, on 03 December 2012 at 18:21 hrs.
Paratypes. Seven males, three females. Male and female SMF 97521–22 (AB
1056–7) respectively, male MHCH 3081 (AB 1058) same collecting attributes as
holotype (Fig. 4B, C, E-F); male MHCH 3111 (MG 28), male collected by Macario
Gonzalez on 27 June 2018 at 23:40 hrs; male MHCH 3112 (MG 31), male col-
lected by Macario Gonzalez on 07 August 2018 at 21:15 hrs, all from around the top
of Cerro Chucantí (8.80455°N, 78.45951°W; near 1439 m a.s.l.) Maje Mountains,
Río Congo Arriba, Distrito de Chepigana, Darién, Panama. Males MHCH 3113–4
(MG 48–9), males, collected by Macario Gonzalez on 27 June 2018 at 23:40 hrs
(8.80455°N, 78.45951°W; 1439 m a.s.l.); females MHCH 3115 and SMF 97517 (AB
654), from Ambroya (8.92111°N, -78.62786°W; 851 m a.s.l.), Cerro la Javillosa Torti,
Chepo, Panama, on 28 August 2012 at 19:40 hrs.
Diagnosis. Pristimantis gretathunbergae sp. nov., a member of the Pristimantis
ridens species group (sensu Reyes-Puig et al. 2020), is characterized by the following
combination of characters: (1) dorsal skin surfaces slightly areolate, with dispersed
tubercles; venter weakly areolate; discoidal fold present, dorsolateral folds absent;
(2)tympanum concealed, indistinguishable or poorly distinguished; annulus and tym-
panic membrane barely visible in males, not visible in females; tympanic fold from
the posterior edge of the eye to the arm insertion; (3) snout short, broadly rounded in
dorsal view, moderate in length, rounded and slightly protruding in prole; (4) upper
eyelid with a single conical to spine-like, some triangular tubercle, ED wider than IoD;
cranial crests absent; (5) dentigerous processes of vomers present, prominent, oblique,
Konrad Mebert et al. / ZooKeys 1081: 1–34 (2022)
16
Figure 4. Coloration in life of specimens of Pristimantis gretathunbergae sp. nov. and P. cruentus from
eastern Panama A holotype male (MHCH 3082), Cerro Chucantí B paratype female (SMF97520), Cerro
Chucantí C left, paratype female (MHCH 3081), right P. cruentus female (MHCH3034) D female from
Cerro Chucantí, not collected E female (MHCH3115) La Javillosa F female, Cerro La Javillosa, Ambroya,
Maje Mountain Range (SMF97517) G female (MHCH3079), Rio Tuquesa. Colored lines point to some
diagnostic characters as follow: red: blackish iris; yellow: single spine-like tubercle; turquoise: light-colored
upper lip; pink: cream, yellow to red groin (red groin also shown in Suppl. material 2: Fig. S10).
A New Species of Pristimantis from Panama 17
each bearing from 5 to 10 teeth; (6) vocal slits and nuptial pads absent; (7) Finger I
shorter than Finger II; discs on outer ngers truncate, more than twice width of digit
proximal to disc; (8) ngers bearing narrow lateral fringes; (9) three to four low ulnar
tubercles, barely visible in preservative; (10) heel bearing a conical tubercles, outer edge
of tarsus with three to four low and small conical tubercles, inner edge of tarsus lacking
tubercles; (11) inner metatarsal tubercle large and elliptical, 4–5× size of outer, ovoid
metatarsal tubercle; supernumerary plantar tubercles low; subarticular tubercles coni-
cal; (12) toes bearing narrow lateral fringes; webbing absent; Toe V much longer than
Toe III; discs as large as those on outer ngers; (13) dorsal ground coloration usually
shades of brown with individual tones of red or yellow with or without scattered or-
ange ecks, and/or larger reddish or distinct brown blotches, or light dorsolateral band;
(14) venter uniform dirty white (some specimens exhibit dark spotting) or patternless
yellow to orange; (15) groin and inner thighs white, yellow or orange-red, some with
ecks matching the dorsal ground color or red; (16) blackish iris, some individuals
show very dark red iris and/or red-golden speckling; (17) prominent light upper lip
in all females and in some males, while other males exhibit some blotches extending
from the nose vertically across the lip, however, the upper border of the light-colored
lip patches is still demarcated by the darker nose coloration, except in generally light-
colored specimens; (18) SVL up to 36.7 mm in males, up to 46.3 mm in females.
Comparative diagnosis to sympatric rainfrogs. Pristimantis gretathunbergae
sp.nov. diers markedly from all other Pristimantis species in central and eastern Panama
by its very dark to black, non-reticulated iris, respectively entire eyes (iris pale and/or
with heavy pale ecking in other species). Some ne golden to dark red speckling or
ecking might be visible in some P. gretathunbergae sp. nov. In sympatry, the new species
is most similar to the equally large and bulky P. cruentus (Fig. 4C) from which it can
be distinguished as follows (characters of P. gretathunbergae sp. nov. in parentheses): P.
cruentus has venter heavily mottled with dark pigment to almost uniform black (white,
dirty white or yellow, see Suppl. material 2: Fig. S10 G–I, M), upper surfaces gray,
brown, brownish black (reddish brown, light gray to yellow-brown); lips mottled or
with patches, whereas specimens with light upper lip usually show an irregular border
with the dark snout coloration (upper lip uniformly colored white or yellow, but some
males have upper lips with dark patches, yet the light parts are still sharply and straight-
bordered by the dark snout coloration above, whereas the colored demarcation in
specimens of P. cruentus with a light upper lip is normally diuse or irregularly shaped,
see Suppl. material 2: Fig. S11 for a multi-specimen comparison); tympanic annulus
partially evident in females (not visible); P.cruentus exhibits a variable number and shape
of tubercles on the eyelid (usually only one single conical to spine-like tubercle over the
eyelid (see Fig. 4 and Suppl. material 2: Fig. S10A, B). Pristimantis gretathunbergae sp.
nov. diers from other coexisting species of the P. ridens species group in Panama by
being larger in size, and by having white, cream, yellow, or orange-reddish coloration on
inguinal area, often suused with red pigment (Suppl. material 2: Fig. S10C–F, K, L). A
more detailed comparison by sympatric species from Panama follows: P. caryophyllaceus,
dorsum smooth (slightly areolate, scattered with tubercles), sharp and projecting snout
Konrad Mebert et al. / ZooKeys 1081: 1–34 (2022)
18
(short, broadly rounded in dorsal view); P. cerasinus, P. ridens, and P. taeniatus have
general dorsal color brown (reddish brown or yellow) and tympanic membrane distinct
(tympanic membrane indistinct); P. gaigei is black with orange dorsolateral stripes
(reddish brown or yellow); P. museosus and P. moro general dorsal color is green (reddish
brown or yellow); P. pardalis has silvery white spots on side and anterior portion of
thighs (anterior portion of thighs yellow, suused with reddish color).
Comparative diagnosis to related, allopatric rainfrogs. is comparison includes
only members of the Pristimantis ridens species group sensu Reyes-Puig et al. (2020).
Pristimantis gretathunbergae sp. nov. is genetically most closely related to the allopatric
rainfrog Pristimantis erythropleura. Like P. gretathunbergae sp. nov., P. erythropleura
inhabits cloud forests higher than 980 m in the western and partly central Cordilleras
in the Department Antioquia, Caldas, Cauca, Chocó, Quindío, Risaralda, Tolima,
and Valle del Cauca (e.g., Lynch 1992; Atehortua-Vallejo et al. 2020). It is also highly
polymorphic and sexually dimorph (see 41 examples in Suppl. material 2: Fig. S12).
According to data from Lynch (1992) some P. erythropleura share a few characters with
P. gretathunbergae sp. nov. by exhibiting: a dirty white venter, frequently also yellow to
red ash colors on the concealed inner, some also outer, surface of the upper thigh and
groin (however extended color variation is depicted in Suppl. material 2: Fig. S12),
vocal slit absent and other characters shared within the P. ridens species group. But
P. erythropleura dier in a few characters from P. gretathunbergae sp. nov. (character
expression of P. gretathunbergae sp. nov. in parenthesis): body size regionally variable
but always smaller, even in the population with the largest individuals from Calarca,
Colombia, with SVL for males 21.2–25.4 mm, females 28.2–34.8 mm (substantially
larger: SVL 26.9–36.7 mm males, 38.2–45.0 mm females), golden to red eyes, resp.
iris, with some heavy reticulation (fully black eyes with golden or dark-red speckling/
ecking in some individuals), subconical tubercle on upper eyelid (conical to spine-
like single tubercle), glandular nuptial pad on thumb of males (lacking nuptial pads).
Two additional rainfrog taxa inhabit northwestern Colombia that are closely re-
lated to P. gretathunbergae sp. nov. First, Pristimantis penelopus, sister species to P. eryth-
ropleura, was originally known to inhabit montane areas higher than 1000 m a.s.l.
in northwestern Colombia (Lynch and Rueda-Almonacid 1999), but has also been
found as low as 94 m a.s.l. (Restrepo et al. 2017). e two conrmed samples of P.
penelopus from the Cordillera Central exhibit a short 16S mtDNA genetic divergence
of 4.8% to P. gretathunbergae (Table 1). Second, one sample in our analysis (SMF
97539), originally labeled as “P. cruentus”, clustered with the two penelopus-samples
(16S mtDNA divergence < 1%) but showed a large dierence to P. gretathunbergae
of 8.2%. It was collected in the Jingurudó (Pacic coastal) Mountain range, Comarca
Emberá-Wounaan, Panama, and its external appearance resembles P. sanguineus from
the Pacic versant of the Cordillera Occidental, Antioquia, and the coastal mountains
of Choco (Lynch 1998). Although, no sequence of P. sanguineus was available to verify
its taxonomic allocation to specimen SMF 97539 from this little studied region (Pa-
cic coastal border Panama-Colombia), morphological resemblance to former species
A New Species of Pristimantis from Panama 19
and molecular proximity to P. penelopus are sucient to provisionally label it as P. a.
sanguineus/penelopus pending further investigation.
Both, P. penelopus and P. sanguineus (examples in Suppl. material 2: Fig. S13), dier
similarly from P. gretathunbergae sp. nov. (with the character expression of P. gretat-
hunbergae sp. nov. in parenthesis), tympanum in P. penelopus and P. sanguineus more
prominent (tympanum mostly concealed), upper eyelid with a subconical tubercle,
with several non-pungent tubercles only in P. penelopus (triangular, conical to spine-like
single tubercle); venter color cream to dull orange with brown spotting and/or more or
less prominent dark reticulation in P. penelopus, brown stippling in P. sanguineus (uni-
formly dirty white to orange), groin and concealed surfaces of limbs black with light-
colored spots (groin and inner thighs white, yellow or orange-red, some mixed with
speckling of brown or yellow), iris copper or red with black reticulum (iris blackish,
some golden or dark red speckling visible in some specimens), upper lips with marked
labial bars (prominent light–uniformly colored upper lip in females and some males),
smaller body size in P. penelopus/P. sanguineus with SVL in mm: 16.3/16.9–22.2/24.0
males, 31.2/29.1–37.835.2 females (SVL 26.9–36.7 in males, 38.2–46.3 females).
Further detailed comparisons to similar rainfrog species, e.g., P. viejas, P. latidiscus,
P. laticlavius, P. cisnerosi, and P. paisa is provided in the Suppl. material 2. In addition,
photo panels in the Suppl. material 2: Figs S12–S16 show color pattern variations of
these related rainfrog taxa, as well as the two closest relatives of P. gretathunbergae sp.
nov., P. erythropleura and P. penelopus. With regards to the blackish eyes, which is the
most conspicuous character of P. gretathunbergae sp. nov., few other Pristimantis spp.
from north-western South America exhibit very dark eyes (resp. iris), but none are
related to the P. ridens group treated herein. Examples are P. farisorum, P. orcesi, P. parec-
tatus, P. acerus, and P. piceus, which are primarily species of higher (> 2000 m a.s.l.)
elevations of the Andean Mts., in which blackish iris coloration is only one morph and
that tends to be more of a very dark grey, brown, or red, whereas other specimens of
these species have lighter colored iris. One notable exception appears to be P. chalceus
from the Chocoan lowlands and adjacent western Andean slopes up to 1970 m a.s.l. in
western Colombia and Ecuador (e.g., Padial et al. 2014; Frenkel et al. 2021).
Description of the holotype (Figs 4A, 5). Adult male (SVL 34.6 mm; head
approximately as wide as long (HL/HW = 1.11); snout short, broadly rounded in dorsal
view, moderate in length, rounded and slightly protruding in prole, eye to nostril
distance 10% of SVL. Canthus rostralis and loreal region slightly concave, nares situated
near tip of snout and slightly dorso-laterally directed, clearly visible in frontal and dorsal
view, but not ventrally; interorbital area smooth, the upper eyelid is 1.4 of the IoD; a low
and conical upper eyelid tubercle, rest of the head with scattered tubercles, but visible
only in live specimens, without crests; tympanic annulus slightly visible, tympanum
indistinguishable, tympanic annulus concealed by skin, tympanum of moderate size,
ratio TY/EW 0.39, supratympanic fold present, from the outer edge of the eye to
posterior the insertion point of the jaw, skin around the tympanum with scattered small
tubercles; clearly visible choanae rounded and moderate in size, dentigerous processes
Konrad Mebert et al. / ZooKeys 1081: 1–34 (2022)
20
of vomer in transverse row between choanae, separated by half of a vomer size, with ve
teeth on right side and seven on left side; vocal slits absent; tongue slightly longer than
wide, 2/3 attached to mouth oor, shagreen in texture, with an evident papillae at the
anterior 1/4 of the tongue; dorsal skin surface shagreen with scattered tubercles, ventral
Figure 5. Preserved holotype of Pristimantis gretathunbergae sp. nov. (MHCH 3082) A left hand in
ventral view B left foot in ventral view C dorsal view D ventral view E ventral view of head F lateral view
of head. Scale bars: 10 mm.
A New Species of Pristimantis from Panama 21
surface weakly areolate, without dorsolateral folds, discoidal fold present, extended from
level of arm pit to the groin; cloaca partially smooth, granular in the lower part; hands
moderate in size, 30% of SVL, four or ve low and small ulnar tubercles; nger II longer
than nger I, expanded disks on ngers II, III, & IV; relative lengths of adpressed ngers
I < II < IV < III; nger II subequal in size to nger VI, nger II reaching the disc on
nger IV when adpressed; nger III disc 2.4× wider than distal end of adjacent phalanx;
subarticular tubercles rounded, and elevated on lateral view, thenar tubercle long, oval
and low; palmar and supernumerary present, slightly visible, no nuptial pads, narrow
lateral fringes on ngers; hindlimbs of moderate length, TL 51% of SVL; relative lengths
of adpressed toes I < II < III < V <IV; when adpressed, tip of toe I reach tubercle of
toe II; disc of toe IV expanded, 1.9× wider than distal end of adjacent phalanx; narrow
lateral fringes on toes; between one and three non-protuberant subarticular tubercles
present (one each on toes I and II, two on toes III and V, and three on toe IV); inner
metatarsal tubercle elongated; outer metatarsal tubercles slightly pointed and smaller
than inner; tarsal ridge absent, outer tarsal tubercles absent; hands and feet without
webbing; nger and toe discs broadly expanded.
Measurements of the holotype. SVL 34.6, HW 12.8, HL 14.2, InD 2.4, IoD
4.1, EW 5.4, ED 4.6, EN 3.6, TY 1.9, TL 17.7, FL 16.2, FAL 8.8, HAL 10.2, BW
8.7, 3FW 0.8, 3FD 1.5, 3TW 0.9, 3TD 1.4, 4TW 0.7, 4TD 1.3.
Coloration of holotype in life (MHCH 3082; Fig. 4A): Color codes of Köhler
2012 in parenthesis: In life, the dorsum is cream color (Light Yellow Ocher 13), with
reddish (Chrome Orange 74) irregular big blotches, except in the anks; inferior part
of supratympanic fold suused with brown color (Russet 44); thighs and anterior por-
tion of tibia and foot with transverse bars. Groin is reddish (Scarlet 69) above and
yellow (Orange Yellow 8) below. e margin of the upper lip is yellow (Sulphur Yellow
80). e iris is almost black (Black Carmine 61) with paler (Geranium 66) spots. e
area between anks and venter is suused with cream color (Cream Yellow 82), the
venter is dirty white.
Coloration in preservative (Fig. 5): Dorsal ground color cream (Pale Pinkish Bu
3), suused with minute dark pigments (Hair Brown 277), pale (Light Orange Yellow
7) groin, forelimbs, and hind limbs and with diuse dark (Hair Brown 277) transverse
bands; ventral areas cream (Cream Color 12); underparts of nger and toe disks dif-
fused with dark (Hair Brown 277) pigments.
Variation (Fig. 4, Suppl. material 2: Figs S8–S11): Most specimens correspond
with the general description of the holotype, but some specimens show variation, in-
cluding pale brown (Clay Color 18, 20) dorsum, with or without reddish (Chrome
Orange 74) irregularly distributed and sized large blotches on dorsum; a specimen from
Cerro Chucantí photographed in 2016 had anks with reddish color (Scarlet 69), in
between the dorsal (Clay Color 20) and ventral (dirty white) color. Other specimens
from Ambroya presented spots (GE) or bands on dorsum. One female had uniform
yellow color (Orange Yellow 8) on venter. Morphometric variation is shown on Table 2.
Etymology. e specic name is a noun in the genitive case and is a patronym
in honor for Greta unberg, a Swedish student, and her global climate activism.
Konrad Mebert et al. / ZooKeys 1081: 1–34 (2022)
22
Greta initiated a “School Strike for Climate Action” outside the Swedish parliament
to demand a radical response to the threat by the ongoing climate change. en six-
teen-year-old unberg’s example has inspired students worldwide to carry out similar
strikes called Fridays For Future that started in August 2018. In December 2018 she
addressed world leaders at the COP24 climate talks in Katowice, Poland, with sharp
and unmasked words, and equally impressed a global audience in January 2020 with
her unpolitical, direct speech down to the point on “Averting a Climate Apocalypse” at
the WEF (World Economic Forum) in Davos, Switzerland. Just recently, she publicly
slammed the world leaders at the 26th UN Climate Change Conference of the Parties
(COP26) in Glasgow, November 2021, for not doing enough to meet the demands
of the climate emergency. Greta unberg represents the authentic voice that exposes
the motivations behind the diplomatic curtain of politicians and business stakeholders.
Her voice is essential if we want to revert to and maintain a healthy environment on
the planet we all share, and not least, learn to respect its magnicent mega-diversity of
life that took millions of years to evolve.
Distribution. Pristimantis gretathunbergae sp. nov. is endemic to Panama, but it
could occur on near mountains along the border in Colombia. Its currently known
distribution covers eastern Panama with records from the Darien Mountains within
Embera Comarca and the Maje Mountains within Darien and Panama Provinces, in-
cluding the type locality at Cerro Chucantí. e distribution continues west into Cen-
tral Panama, including records from Piedras-Pacora Mountains, Panama Province, and
Cerro Bruja, Colon Province, both within Chagres National Park. Farther west across
the Panama Canal, P. gretathunbergae sp. nov. is present at Altos del Maria, region of
Gaita Hills, Panama Oeste Province, and in the region of El Cope, Omar Torrijos Na-
tional Park, Coclé Province.
Color pattern of specimens from Cerro Brewster, not included in the LDA (DFA)
analysis, are consistent with the specimens from Maje Mountains in having a cream
dorsum coloration, the margin of the upper lip in females yellow, an iris nearly black
with pale dots or speckles, venter dirty white, and general stocky body and head. Due
to the unique combination of characters of P. gretathunbergae sp. nov., in particular
the blackish non-reticulated iris and light, unpatterned upper lip, that diers from any
other related rainfrog in Panama and Colombia, we condently allocate specimens
available only as photo vouchers from Cerro Bruja, Colon Province, and Altos del
Maria, Gaita Hills, Panama Oeste Province to the same species. e latter two locali-
ties substantially reduce the gap to El Cope, Cocle Province, the origin of the most
western specimen of our Group 1. So far, we have not received photographic vouchers
for the specimen from El Cope, but the low 16S-divergence of 2.3% clearly links it to
the undescribed species from the Maje Mountains (see above).
Natural history. Pristimantis gretathunbergae sp. nov. has been recorded at altitudes
between 718–1439 m a.s.l. and occupies most frequently montane forest, a cloud forest
consisting predominantly of trees covered with moss and a large variety of understory
and midstory bromeliads (Flores et al. 2018). At night, this species was observed
A New Species of Pristimantis from Panama 23
Figure 6. Habitat, mating, and parental care in females of Pristimantis gretathunbergae sp. nov. from
Cerro Chucantí A Habitat on Cerro Chucantí at ca. 1300 m a.s.l. B understory bromeliad with a P.
gretathunbergae sp. nov. in situ (blue line) and zoomed in on inset (MHCH 3115) C amplectant pair on
axillary part of bromeliad leaf (not collected) D same female after amplexus guarding eggs E female of
P. gretathunbergae taking care of its eggs with a male P. cruentus species holding on the female in reverse
position (not collected) F female with eggs about to hatch, note the transparency of the egg membrane
(not collected).
Konrad Mebert et al. / ZooKeys 1081: 1–34 (2022)
24
between 0.5–3 m above the ground on tree bark and in the bromeliad foliage (Fig.
6). During daytime, individuals were found hiding between bromeliad leaves. At the
top of Cerro Chucantí, males were calling (a sporadic “chack”) during the rainy season
in December. Reproductive activities beginning with the rain period have also been
observed at Altos del Maria, near Gaita Hills. ree females have been seen guarding
clutch of eggs for at least four nights in bromeliads and moss-covered tree branches
(Fig. 6, Suppl. material 2: Fig. S8E). Diet is not known, but as in other Pristimantis, it
likely consists of a variety of arthropods, mostly ants, orthopterans, and spiders (Lynch
and Duellman 1997; Garcia et al. 2015).
Conservation. Habitats occupied by P. gretathunbergae sp. nov. are under latent
threat. For example, anthropogenic pressure around Cerro Chucantí and the Maje
Mountains most likely will lead to declines of populations through habitat destruction
(Batista et al. 2020). Similar scenarios are known and can be expected from the other
known sites of P. gretathunbergae sp. nov., as they mostly represent restricted montane
areas surrounded by agriculture and pastures, and only a few sites are within protected
areas (Chucantí Private Reserve, Chagres National Park, General de División Omar
Torrijos Herera). Greta unberg’s Rainfrog is, thus far, known only from patches of
primary forest and slightly disturbed areas. Unfortunately, in the areas surrounding
P.gretathunbergae sp. nov. localities, population declines are related to the chytrid fun-
gus (Batrachochytrium dendrobatidis) and pose an additional serious threat (Rebollar et
al. 2014; Voyles et al. 2018). Consequently, P. gretathunbergae sp. nov. should be listed
as “Vulnerable (VU)” in the global Red List of the IUCN (2018) according to criteria
B2ab(iii), because: i) its reduced area of occupancy is less than 2000 km2, ii) it is known
from fewer than ten localities, iii) its range is severely fragmented with continuing de-
cline in extent and/or quality of habitat. e Environmental Vulnerability Score (EVS)
of this species is 18, placing it in the upper segment of the high vulnerability categories.
is score is based on a contributory score of 6 for distribution limited to Central Amer-
ica in the vicinity of the type locality; 8 for ecological distribution, because it is known
only from one forest type, and 4 for reproductive mode, because eggs are laid in moist
arboreal situations, and tadpoles undergo direct development (Johnson et al. 2015).
Dichotomous key for the species of the genus Pristimantis occurring in Panama
1 Dorsal ground color uniform blackish or grayish, with white or orange
blotches on groin, if not, the color is gray to pink, some species have an or-
ange or yellow dorsolateral stripe, continuous or interrupted ......................2
– Dorsal ground color, cream, reddish, brown tones, green or olive, uniform or
darker blotches or reticulations, without white or orange blotches on groin ....5
2 White or orange blotches on groin ..............................................................3
– Groin uniform ............................................................................................ 4
3 Well dened white blotches on groin ..........................Pristimantis pardalis
– Well dened orange blotches on groin .............................Pristimantis altae
A New Species of Pristimantis from Panama 25
4 Dorsal ground color uniform gray to pink without dorsolateral stripes .........
.................................................................................. Pristimantis pirrensis
– Orange or yellow dorsolateral stripes, continuous or interrupted, some speci-
mens lack stripes, but dorsal color is blackish, never gray or pink ..................
...................................................................................... Pristimantis gaigei
5 Dorsal ground color green or olive green, uniform or with darker blotches or
reticulations ............................................................................................... 6
– Dorsal ground color, cream, reddish, brown, dark brown, or olive, uniform or
with darker blotches or reticulations ...........................................................7
6 Dorsal ground color uniform green, with or without a reddish brown trans-
verse interorbital band, dorsal skin smooth ..................... Pristimantis moro
– Dorsal color green or olive, with irregular blotches or reticulations, brown,
olive or reddish color, dorsal skin tuberculated ..........Pristimantis museosus
7 Heel smooth or with one to several similar small sized tubercles scattered over
upper surface of hind limb; enlarged tubercle on upper eyelid present or not ... 8
– Well-developed pointed calcars, usually enlarged tubercle on upper eyelid ...10
8 Presence of a dorsolateral granular folds, dorsal pattern with chevrons ..........
................................................................................ Pristimantis achatinus
– Dorsolateral region smooth, dorsal pattern uniform, never with chevron pat-
tern ............................................................................................................. 9
9 Anterior and posterior surfaces of thighs, calves, and feet red .......................
..................................................................................... Pristimantis ridens
– Uniform posterior surface of thigh ............................ Pristimantis taeniatus
10 Posterior thighs uniform ..........................................................................11
– Posterior thighs dark brown with red-orange dots .........Prisitimantis adnus
11 Dorsal skin granulate or tuberculate, rarely smooth, head about as broad as
long; snout rounded..................................................................................12
– Dorsal skin smooth, long and pointed snout ...Pristimantis caryophyllaceus
12 No W-shape on dorsum, iris variable in color, usually highly reticulated or
blackish .................................................................................................... 13
– W-shape ridge that extent from the back of the head to the shoulder region,
groin, anterior, and posterior thigh red, iris usually pale golden without re-
ticulation, eyes usually with an orange perimeter ......Pristimantis cerasinus
13 Iris variably light colored, cream, yellow or reddish and strongly reticulated,
venter heavily mottled with dark pigment to almost uniform black, upper
surfaces gray, brown, brownish black; tympanic annulus partially evident in
females, upper lips with dark patches, with light colored lips or parts of it lit-
tle or not dark-bordered above ................................... Pristimantis cruentus
– Iris black, some very dark red, without reticulation, venter white, dirty white,
yellow or red, upper surfaces reddish brown or yellow, white to yellow upper
lips, contrastingly dark-bordered above, some with dark patches, tympanic
annulus not visible in females .......... Pristimantis gretathunbergae sp. nov.
Konrad Mebert et al. / ZooKeys 1081: 1–34 (2022)
26
Discussion
e genus Pristimantis is one of the most species rich genera of amphibians in the Neo-
tropics (Lehr and Duellman 2009). It is primarily distributed in South America with
a few species reaching Central America. Based on a combination of molecular data
(small sequence divergence of mtDNA 16S and COI) and a consensus of conspicuous
morphological characters (e.g., unusually dark to black eyes, spine-like single tuber-
cle on upper eyelid, sharply dark-bordered upper light lip, large body size), we could
identify seven locations in Darien and Central Panama that relate to a new species,
Greta unberg’s Rainfrog Pristimantis gretathunbergae sp. nov. is is the 14th or 15th
known Pristimantis species in Panama, depending on the author (see Introduction).
Initially, P. gretathunbergae sp. nov. thought to be related to P. latidiscus (Crawford et
al. 2012). However, it is signicantly dierent from P. latidiscus, both genetically and
morphologically (Table 2, Fig. 2, Suppl. material 2). One record labeled P. a. latidis-
cus reported from Cana, Darien Province, Panama (Crawford et al. 2012) is conspecic
with, P. cruentus (data not shown). Consequently, P. latidiscus is restricted to the Choco
Bioregion of South America.
Within Panama, Pristimantis gretathunbergae sp. nov. is most closely related to
P.cruentus, a rainfrog species with a large variation in morphology and genetics (Savage
2002; Crawford et al. 2012) that will require more work to discern potentially dier-
ent lineages. Including taxa from northern South America, the phylogenetic inferences
match well with previous phylogenetic hypotheses, that place P. cruentus with P. erythro-
pleura, P. penelopus (mis-labeled as P. paisa in Pinto-Sanchez et al. 2012 according to Re-
strepo et al. 2017), and P. cisnerosi into the same clade (Reyes-Puig et al. 2020). Among
all these rainfrogs, P. gretathunbergae sp. nov. is more closely related to the allopatric (in
decreasing relatedness) P. erythropleura and P. penelopus, P. cisnerosi, and possibly also
P. viejas, from western Colombia than to P. cruentus (Table 1, Fig. 2). e increase of
genetic divergence at 16S of P. erythropleura to P. gretathunbergae sp. nov. from 4.8% to
5.9% when comparing the two initially analyzed samples (P. erythropleura nrps_0055
and -57) and after inclusion of additional two samples (UVC:15886 and UVC:15933),
probably reects unresolved taxonomic relationships or misidentication in such a
highly variable species across an insuciently explored region, northwestern Colombia
(Suppl. material 1: Table S1 and Suppl. material 2: Figs S1, S3). Nonetheless, these
Colombian rainfrogs occupy approximately the same elevation and cloud forests as
P.gretathunbergae sp. nov., but south of the Darien Mountains, where P. penelopus and
P. viejas include also lowland areas in their large vertical distribution (Restrepo et al.
2017, IUCN SSC Amphibian Specialist Group 2019a). ese neighboring distribu-
tions suggests, that their shared ancestors expanded from Colombia into Panama and
evolved into a separate species, P. gretathunbergae sp. nov., probably between 3–15 mil-
lion years ago, when they diverged from the same ancestor of P. cruentus prior to the
closure of the Panamanian Isthmus (Pinto-Sanchez et al. 2012; Ramirez et al. 2020).
Whereas all interpopulation divergence between the type series with any of the
other sites remains below 3%, other population comparisons can vary and increase up
A New Species of Pristimantis from Panama 27
to 5.4% between single individuals from Cerro Brewster and El Cope, both central
Panama. Similar minimum genetic dierentiation of > 3–4% of the 16S rRNA gene
have been found to associate to CCS and UCS (not yet described Conrmed and
Unconrmed Candidate Species) of frogs in Madagascar, Africa, and Amazon Basin,
South America (Vieites et al. 2009; Vacher et al. 2020). In this context, some genetic
dierences might reect observed regional morphological variation in P. gretathun-
bergae sp. nov. For example, the specimens from Rio Tuquesa, Darien Mountains are
more light-colored and have the inguinal region mostly white with only little pinkish
pigments, instead of yellowish suused with reddish blotches as in other populations
of P. gretathunbergae sp. nov. But because of the low sample size it remains unclear
whether the currently perceived local morphological variation and some distant ge-
netic grouping between P. gretathunbergae sp. nov. populations are part of a wider
intraspecic geographic variation, potentially reecting isolation by distance and in-
creased regional selection by separating mountain blocks. Consequently, with only a
few data points per population available, we consider it inappropriate to separate such
population dierences into distinct CCS or UCS, in as much as geographic variation
is more prominent in its better-studied closest relatives, P. erythropleura (Lynch 1992;
Suppl. material 2: Fig. S12) and P. penelopus (Lynch and Rueda-Almonacid 1999;
Suppl. material 2: Fig. S13). Moreover, the consensus of a few conspicuous morpho-
logical characters among all investigated populations of P. gretathunbergae sp. nov. and
the generally low genetic dierence (< 3% from the type locality to all other popula-
tions) is sucient that they be considered as conspecic, at least until more material
(morphological, advertisement calls, and molecular) becomes available in the future.
Cloud forests in general and isolated mountain tops in particular are highly vulner-
able to climate change due they low range of mobility and high habitat specialization of
its denizen (Davies et al. 2004; Paaijmans et al. 2013). Consequently, species or popula-
tions restricted to such sky islands as Cerro Chucantí, the type locality of P. gretathunber-
gae sp. nov., are tremendously susceptible to ne changes in the environment and face a
constant risk of extinction (Batista et al. 2020). An urgent conservation plan is required
to protect the cloud forests and the distribution of this new, unique and endemic species.
Acknowledgements
We thank Luis DeLeon, Jesús Pérez, Juan Zarzavilla, Yorlis Cáceres, Hugo Martínez,
and Gilberto Torres for eld assistance, and Guido Berguido for his support during our
stay at the Chucantí private reserve. We also thank to Sarah Farinelli, Juan Daza, Mau-
ricio Rivera-Correa, and an anonymous reviewer for their valuable comments during
the review process, which greatly improved the manuscript. To Marcos Ponce and
Angel Sosa a thank you for providing photos of a specimen from Cerro Brewster. is
work was nancially supported by Asociación ADOPTA el Bosque Panama, Sistema
Nacional de Investigación (SNI) of the Secretaría Nacional de Ciencia, Tecnología e
Innovación (SENACYT, Panamá), and the Rainforest Trust (US). We thank Sebastian
Konrad Mebert et al. / ZooKeys 1081: 1–34 (2022)
28
Lotzkat, for his assistance in the eld and on the manuscript. Many colleagues have
provided photographs for the species panels in the Suppl. material 2, for which would
like to thank and give them credits by naming them in the respective legends.
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34
Supplementary material 1
Tables S1–S5
Authors: Konrad Mebert, Macario González-Pinzón, Madian Miranda, Edgardo Grif-
th, Milan Vesely, P. Lennart Schmid, Abel Batista
Data type: docx le
Explanation note: A new rainfrog of the genus Pristimantis (Anura, Brachycephaloidea)
from central and eastern Panama
Copyright notice: is dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). e Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.
Link: https://doi.org/10.3897/zookeys.1081.63009.suppl1
Supplementary material 2
Figures S1–S16
Authors: Konrad Mebert, Macario González-Pinzón, Madian Miranda, Edgardo Grif-
th, Milan Vesely, P. Lennart Schmid, Abel Batista
Data type: docx le
Explanation note: A new rainfrog of the genus Pristimantis (Anura, Brachycephaloidea)
from central and eastern Panama
Copyright notice: is dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). e Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.
Link: https://doi.org/10.3897/zookeys.1081.63009.suppl2
Supplementary Material - Figures
A new rainfrog of the genus Pristimantis (Anura,
Brachycephaloidea) from central and eastern Panama
Konrad Mebert, Macario González-Pinzón, Madian Miranda, Edgardo Grifith, Milan
Vesely, P. Lennart Schmid, Abel Batista
Pristimantis gretathunbergae sp. nov. - holotype
Remarks
•the list of specimens included in the genetic analysis with corresponding
GenBank accession numbers is presented in Table S5 of the Supplementary
Material – Tables.
•literature references in this supplementary are listed in the main article.
Supplementary Figure S1. Phylogenetic tree of Pristimantis spp. based on mtDNA
16S performed by a Shimodaira–Hasegawa approximate likelihood ratio test (SH-
aLRT test). Numbers on nodes indicate estimated SH-aLRT support/bootstrap. The
tree is drawn to scale, with branch lengths measured in the number of substitutions
per site.
Supplementary Figure S2. Phylogenetic tree of Pristimantis spp. based on mtDNA
COI performed by a Shimodaira–Hasegawa approximate likelihood ratio test (SH-
aLRT test). Numbers on nodes indicate estimated SH-aLRT support/bootstrap. The
tree is drawn to scale, with branch lengths measured in the number of substitutions
per site.
Supplementary Figure S3. Bayesian consensus phylogenetic tree of Pristimantis spp. based on mtDNA 16S gene. Numbers on nodes indicate
estimated posterior probabilities. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.
Supplementary Figure S4. Weighted presentation of 15 ratios of morphometric
variables (e.g., TrL.HL = TrL/HL) by a Principal Component Analysis PCA of P.
gretathunbergae sp. nov. and P. cruentus. Ctr = Contribution of the variables.
Abbreviations of raw variables used (before applying a ratio): Snout–Vent Length
(SVL), Head Width (HW), Head Length (HL), Internarial Distance (InD), Interorbital
Distance (IoD), Eyelid Width (EW) Eye Diameter (ED), Eye–Nostril Distance (EN),
Tympanum Diameter (TY), Tibial Length (TL), Foot Length (FL), Forearm Length
(FAL), Hand Length (HAL), Body Width (BW), Axilla-Groin Distance (AGD), Trunk
Length (TrL), 3rd Finger Width (3FW), 3rd Finger Disk Width (3FD), 3rd Toe Width
(3TW), 3rd Toe Disk Width (3TD), 4th Toe Width (4TW), and 4th Toe Disk Width (4TD).
Supplementary Figure S5. Individual map of the PCA of P. gretathunbergae sp.
nov. (red dots) and P. cruentus (black dots) based on 15 ratios of morphometric
variables, depicted and defined in Fig. S4 above.
Supplementary Figure S6. Linear Discriminant Analysis LDA of P. gretathunbergae
(green), P. cruentus from Eastern Panama (red) and P. cruentus from Western
Panama (blue). The LDA resulted from morphometric data that have been
standardized by conversion into ratios, as described in the methods and selected
after a PCA reduced the numbers of relevant variables to six (see article text).
Variables used here: TrL/HL, IoD/HL, ED/HL, 3TD/3TW, 3FD/3FW, 4TD/4TW.
Abbreviations of raw variables see Fig. S4 above.
Supplementary Figure S8: In situ specimens of female Pristimantis
gretathunbergae sp. nov. from central Panama. A, B a female photographed by
Marcos Ponce in 2010 from Cerro Brewster, Piedras-Pacora Mountains, Chagres
National Park, female (MHCH3115); C female from Cerro Brewster, Chagres
National Park, Panama, photograph by Angel Sosa; D female from Cerro Bruja,
Chagres National Park, Panama, (MHCH 9191), photograph by Angel Sosa; E
female with eggs from Altos del María, near Gaita Hills, Panama Oeste; F in situ
female from Cerro Chucantí, Panama. Colored lines point to diagnostic characters as
follow: red: blackish iris; yellow: single spin-like tubercle; turquoise: white upper lip;
pink: cream, yellow to red groin; photographs E and F by authors.
Supplementary Figure S9: Male Pristimantis gretathunbergae sp. nov. from eastern
Panama. A) Cerro Chucanti (SMF 97521); B, C, both males from Cerro La Jabillosa,
ca. 2–3 km west from Ambroya, Maje Mountain Range, (MHCH3091) and
(SMF97533) respectively; D, E right and left side of (SMF97519), Ambroya; F Rio
Tuquesa, Pechito Parao (MHCH 3080). Colored lines point to diagnostic characters
as follow: red: blackish iris; yellow: single spin-like tubercle; turquoise: light-colored
upper lip demarcated by dark color, even in males C, D, and E that exhibit some dark
blotches stretching vertically over the lip; pink: cream, yellow to red groin. All
photographs taken by authors.
Supplementary Figure S10: Pristimantis gretathunbergae sp. nov. from Panama
with morphological key characters exemplified in subfigure B, primarily black eyes
(red line), single spine-like tubercle on upper eyelid (yellow line), a light, contrastingly
demarcated upper lip (light blue line). Specimens are: A female, Chucantí (MHCH
3082); B female, Chucantí MHCH3081; further key characters of the new species are
unicolored white, yellow, to red groin/venter (pink line), e.g. C-G female SMF97517,
Ambroya; D, I male, Ambroya (SMF 97519); E female Rio Tuquesa (MHCH 3079); F-
H female, Ambroya (MHCH 3082). In contrast P. cruentus in all photographs right of
blue line J-M, with a very variable arrangement of upper eyelid tubercle, light colored
eyes, no light dark-bordered upper lip, a bicolored light and dark groin, and mottled
venter. Photograph on the bottom with P. gretathunbergae sp. nov. facing syntopic P.
cruentus. All photographs taken by authors.
Supplementary Figure S11: Comparison of morphological key characters between
sympatric Pristimantis gretathunbergae sp. nov. (two left columns) and P. cruentus
(two right columns) from Eastern Panama. Lines in uppermost left portrait show
characters typical for P. gretathunbergae sp. nov., such as primarily black eyes (red
line), single spine-like tubercle on upper eyelid (yellow line), and light, contrastingly
demarcated upper lip (light blue). The larger photos at the bottom corners show a P.
gretathunbergae sp. nov. with a syntopic P. cruentus for direct comparison. All
photographs taken by authors.
Extended comparative diagnosis between Greta Thunberg’s Rainfrog, Panama, to
adjacent related allopatric rainfrogs from Colombia:
Following figure panels show those rainfrogs from the ridens-cerasinus-group from north-
western South America (primarily Colombia) that represent the closest relatives (the
shortest genetic distances) to Greta Thunberg’s Rainfrog P. gretathunbergae sp. nov.
from Panama, and one example of species complex from the externally equally variable
taeniatus-group (Supplementary Figs S12–S16). The designation of “© by Name”
includes also a personal permission by the photographer. Images available under the
Attribution-NonCommercial-NoDeriv or Share-Alike International license are indicated by
CC BY-NC-ND or -SA. We would like to thank all authors by providing photographic
vouchers or making them available through Creative Commons. Full versions of these
panels can be requested from the lead author, Konrad Mebert.
The following two Supplementary Figures show examples color pattern variation in the
two closest relatives of Greta Thunberg’s Rainfrog, that are Pristimantis erythropleura
(Fig. S12) and P. penelopus (Fig. S13). Morphological differentiation from P.
gretathunbergae sp. nov. is further discussed in the article text.
Supplementary Figure S12: Examples of Pristimantis erythropleura complex from
northwestern Colombia. Correct identification and exact locations remain with the
sources, whereas some can also be retraced in iNaturalist. Location,
copyrights/permissions and photo credits are: 1) Bremen, Quindío_© by E. Caroline
Guevara-Molina; 2) Dapa, Yumbo, Valle del Cauca _© by Peter Dexter Hoell; 3) Dagua,
Valle del Cauca_© by Juan Manuel de Roux; 4)–5) Urrao, AN_© by Daniel
Bocanumenth E.; 6) Vereda El Chuscal, Urrao_© by Victor Fabio Luna; 7) Colombia_©
by Cristian Gonzalez-Acosta; 8)–9) Jardin, AN_© by Esteban Alzate Basto; 10) Urrao,
AN_© by Yojan Seawolf; 11) San Rafael, AN_© by Khristian Venegas Valencia; 12)
Colombia_Diego Gomez (CC BY-NC 2.0); 13) Frontino, AN_© by Daniel Bocanumenth
E.; 14) Urrao, AN_© by Daniel Bocanumenth E.; 15) Yarumal, AN_© by Diego A.
Botero-Álvarez; 16) Valdivia, AN_© by Khristian Venegas Valencia; 17) Natural
Reserva Patasola, Salento, Quindio_© by Sebastián Duarte-Marín; 18-19) Urrao, AN_©
by Daniel Bocanumenth E.; 20) Dagua, Valle del Cauca_© by Juan Manuel de Roux;
21-22) Darien, Valle del Cauca_© by Esteban Alzate Basto; 23) Jardin, AN_© by Laura
Rubio-Rocha; 24) Urrao, AN_© by Daniel Bocanumenth E.; 25) Filandia, Quindio_© by
Luis Felipe Estrada; 26-27) Colombia_© by Jhonattan Vanegas; 28) Urrao, AN_© by
Yojan Seawolf; 29) Natural Reserve Rincon Santo, Pijao, Quindío_© by Sebastián
Duarte-Marín; 30) Natural Reserva Patasola, Salento, Quindio_© by Sebastián Duarte-
Marín; 31) Filandia, Quindio_© by Luis Felipe Estrada; 32) Natural Reserve Rincon
Santo, Pijao, Quindío_© by Sebastián Duarte-Marín; 33) National Natural Park Selva de
Florencia, Pensilvania, Caldas_© by Sebastián Duarte-Marín; 34) Natural Reserve
Rincon Santo, Pijao, Quindío_© by Sebastián Duarte-Marín; 35) Farallones de Cali,
Valle del Cauca_© by Marco Rada; 36) Cerro Munchique, Tambo, Valle del Cauca_©
by Marco Rada; 37) Jardin, AN_© by Esteban Alzate Basto; 38)–39) Jardin, Vereda la
Herrera, AN_© by Mauricio Rivera Correa; 40) Natural Reserve Rincon Santo, Pijao,
Quindío_© by Sebastián Duarte-Marín; 41) Mistrató, Risaralda_© by Giovanni Chaves-
Portilla.
Supplementary Figure S13: Examples of Pristimantis penelopus from northwestern
Colombia (more examples in Fig. 3 of Restrepo et al. 2017). Correct identification and
exact locations remain with the sources, whereas some can be retraced in iNaturalist.
Locations, copyrights/permissions and photo credits are: 1) San Carlos, AN_© by
Esteban Alzate Basto; 2) Maceo, AN_© by Esteban Alzate Basto; 3) Frontino, AN_© by
Esteban Alzate Basto; 4) Yolombó, AN_© by Yeison Tolosa; 5) Gómez Plata, AN_© by
Esteban Alzate Basto; 6) Granada, AN_© by Claudia Molina-Zuluaga; 7) San Rafael,
AN_© by Laura Rocha-Rubin; 8) San Rafael, AN_© by Juan D. Vásquez-Restrepo; 9)
Granada, AN_© by Juan D. Vásquez-Restrepo; 10) National Natural Park Selva de
Florencia, Corregimiento de Florencia, Samaná, Caldas_© by Sebastian Duarte Marin;
11-12) dark and light state of same specimen from Campamento, AN_© by Juan D.
Vásquez-Restrepo; 13) San Rafael, AN_© by Juan D. Vásquez-Restrepo; 14) Maceo,
AN_© by Khristian Venegas Valencia; 15) Antioquia Dept._MHUAA7139 Creative
Commons - CC BY-NC-SA 2013; 16) Yarumal, AN_© by Mauricio Rivera Correa; 17)
Yarumal, AN_© by Mauricio Rivera Correa; 18) National Natural Park Selva de
Florencia, Corregimiento de Florencia, Samaná, Caldas_© by Sebastian Duarte Marin.
The following three Supplementary Figures (Figs S14-S16) show examples of color
pattern variation in other rainfrogs of the P. ridens-cerasinus groups sensu lato from
northwestern Colombia (Pristimantis viejas, P. latidiscus, P. laticlavius; P. cisnerosi and
P. paisa) that are related to Greta Thunberg’s Rainfrog. Morphological differentiation from
P. gretathunbergae sp. nov. is further discussed below. Comparison of Pristimantis
gretathunbergae sp. nov.
Pristimantis viejas inhabits a variety of vegetation zones from cloud forests to lowland
forest of Antioquia, Caldas, Tolima, Cundinamarca, Santander, and Cordoba
Departments (Lynch and Rueda-Almonacid 1999; Lynch and Ardilla-Robayo 1999; IUCN
SSC Amphibian Specialist Group 2019a). Pristimantis viejas differs from P.
gretathunbergae sp. nov. by (in parenthesis): smaller body size, with SVL for males 15.3–
19.1 mm, females 24.0–29 mm (much larger: SVL 26.9–36.7 in males, 38.2–45.0
females), pale-copper to reddish-copper iris with thick black reticulation (fully black eyes
with faintly dark-red speckling in some individuals), non-conical tubercle on upper eyelid
(conical to spine-like single tubercle), tympanum round and prominent (tympanum
concealed), vocal slit short (vocal slit absent), posterior surface of thighs dark brown with
prominent and circular pale, yellow to orange spots (frequently red, but also mixed with
speckling of browns or yellow). Some color pattern variations of this species are displayed
in Figure S14.
Pristimantis paisa occurs adjacent-west of P. viejas above 1800 m elevation in
Antioquia and Caldas Department, Colombia, and is partly overlapping northeast of P.
erythropleura’s range (Lynch and Ardilla-Robayo 1999; IUCN SSC Amphibian Specialist
Group 2019b). Pristimantis paisa differs from P. gretathunbergae sp. nov. (in
parenthesis) by: smaller body size, with SVL for males 19.9–24.2 mm, females 27.5–
30.7 mm (significantly larger: SVL 26.9–36.7 in males, 38.2–45.0 females), pale-copper
iris with thick black reticulation and a reddish horizontal band crossing the pupil (fully
black eyes with faintly dark-red speckling in some individuals), low tubercle on upper
eyelid (conical to spine-like single tubercle), tympanum round, not prominent
(tympanum concealed), vocal slit present (vocal slit absent), posterior surface of thighs
dark brown (frequently red, but also mixed with speckling of browns or yellow), nuptial
pad present (lacking nuptial pads). Some color pattern variations of this species is
displayed in Figure S15.
Other similar and related species from northern South America are the recently
described P. cisnerosi (Reyes-Puig et al., 2020; see Figure S16), as well as P.
latidiscus and P. laticlavius. Pristimantis cisnerosi has dark brown dorsum with blackish
blotches and two ocher-colored postparietal patches (dark reddish-brown, light brown or
yellow), venter dark brown mottled (dirty white, yellow, or orangish), brown upper lips
with darker blotches (light-colored, some darker males with blotches), supratympanic
fold absent (present); P. laticlavius and P. latidiscus have tympanic membrane and
tympanic annulus prominent (tympanum concealed, indistinguishable or poorly
distinguished); venter white with some brown stippling or mottling (uniform dirty white or
yellow), bicolored or reddish eyes (black), no or short conical tubercle above the upper
eyelid (short conical, but mainly thin and prominently pointed, elongated tubercle),
variably colored upper lips with darker blotches, some with light colored upper lips with
suffused edges (uniformly light-colored with more defined edges, some darker males
with blotches); posterior surfaces of thighs brown with small cream spots (yellow
suffused with reddish color or white).
Supplementary Figure S14: Examples of Pristimantis viejas and P. aff. viejas from
northwestern Colombia, members of the P. cerasinus-taeniatus group acc. to this
analysis, as well as Amezquita et al. (2019) and Reyes-Puig et al. (2020). Correct
identification and exact locations remain with the sources, whereas some can be
retraced in iNaturalist. Locations, copyrights/permissions and photo credits are: 1)
Granada, AN_© by Juan D. Vásquez-Restrepo; 2) Granada, AN_© by Juan D.
Vásquez-Restrepo; 3) Compamento_© by Juan D. Vásquez-Restrepo; 4-5)
Colombia_© by Juan Mauricio Rivera-Correa; 6) Cocorná, AN_© by Claudia Molina-
Zuluaga; 7) San Rafael, AN_© by Laura Rubio-Rocha; 8) Colombia_© by Esteban
Alzate Basto; 9) Pailania, San Francisco, AN_© by Esteban Alzate Basto; 10) San
Carlos, AN_© by Eduardo Posada S.; 11) Otanche, Boyacá _© Andrés Leonardo
Ovalle; 12) Cocorná, AN_© by Wilmar Agudelo Sánchez; 13) Vereda El Porvenir,
Carmen de Viboral, AN_© Andres Rymel Acosta Galvis_IAvH-Am-14607; 14) Vereda El
Porvenir, Carmen de Viboral_© Andres Rymel Acosta Galvis_IAvH-Am-14462; 15)
Vereda El Porvenir, Carmen de Viboral_© Andres Rymel Acosta Galvis_IAvH-Am-
14459-60; 16) San Rafael, AN_© by Daniel Bocanumenth E.; 17) Vereda El Porvenir,
Carmen de Viboral_© Andres Rymel Acosta Galvis_IAvH-Am- 14463); 18) San Carlos,
AN_Verónica VH, (CC BY-NC-SA).
Supplementary Figure S15: Examples of Pristimantis paisa (sensu lato) from north-
western Colombia. This species is a complex and not part of the cruentus-subgroup
(incl. P. greathunbergae-erythropleura-penelopus-cisnerosi), but rather belongs to the
cerasinus-taeniatus group. This plate provides an example to show the similar variation
in color pattern as observed in other taxa of the ridens-group in Figures S12–S14 and
S16. Correct identification and exact locations remain with the sources, some can be
retraced in iNaturalist. An effort was taken to exclude specimens for which external
features are not consistent with those known for the species. Locations,
copyrights/permissions and photo credits are: 1) Medellin, AN_ © by Jorge J Restrepo
A.; 2) Sonsón, AN_© by Khristian Venegas Valencia A.; 3) Medellin, AN_© by Khristian
Venegas Valencia A.; 4) Rionegro, AN_© by Khristian Venegas Valencia A. 5)
Colombia_Sebastian Duarte Marin; 6) Hoyorrico, Santa Rosa de Osos, AN_© Andrés
Mauricio Forero Cano; 7) Hacienda Corrales, Cerro Quitasol, Bello, AN_© Jhon Steven
Murillo Serna; 8) Guarne, AN_© by Sebastian Serna Muñoz, some rights reserved (CC
BY-NC); 9) Alto de San Miguel, Caldas_© Mauricio Rivera Correa; 10) Vereda Gaviria,
Municipio de Marinilla, AN_© Juan M Daza; 11) Caldas, AN_© Alto de San Miguel,
some rights reserved (CC BY-NC); 12) Caldas, AN_© Alto de San Miguel, some rights
reserved (CC BY-NC); 13) Envigado, AN_© by Mauricio Rivera Correa; 14) Caldas,
AN_Alto de San Miguel, some rights reserved (CC BY-NC); 15) Alto de San Miguel,
AN_© by Juan M Daza;16) La Ceja, AN_© by Nicolas Steven; 17) La Union, AN_© by
Khristian Venegas Valencia; 18) Caldas, AN_© by Alto de San Miguel, some rights
reserved (CC BY-NC); 19) Caldas, AN_© by Alto de San Miguel, some rights reserved
(CC BY-NC); 20) La Unión-El Carmen De Viboral La Unión, AN_© by Diego A. Botero-
Álvarez; 21) La Union, AN_© by Diego A. Botero-Álvarez; 22) Valdivia, AN_© by
Khristian Venegas Valencia; 23) La Ceja, AN_© by Nicolas Steven; 24) Medellin, AN_©
Nicolas Betancourt, some rights reserved (CC BY-NC); 25) Jardin, AN_© by Juan
Farias aka luissierra, some rights reserved (CC BY-NC); 26) La Unión, AN_© by Diego
A. Botero-Álvarez; 27) La Unión, AN_© by Diego A. Botero-Álvarez; 28) Medellín,
AN_© by Marisol Zapata Caro, some rights reserved (CC BY-NC); 29) Anorí, AN_© by
Daniel Velez; 30) El Carmen de Viboral, AN_© by Ángela María Gómez ; 31) La Clara,
Caldas, AN_© by Esteban Alzate Basto; 32) Granada, AN_© by Daniel Bocanumenth
E.; 33) Vereda Gaviria, Municipio de Marinilla_© by Juan M Daza ; 34) Copacabana,
AN_© by Sebastian Serna Muñoz, some rights reserved (CC BY-NC); 35) Medellin,
AN_© by Efray Alzate; 36) Copacabana, AN_© by C. Julio Montoya; 37) Envigado,
AN_© Pablo Cifuentes; 38) Medellin, AN_© by Efray Alzate.
Supplementary Figure S16: Pristimantis cisnerosi from northwestern Ecuador.
Sources and copyrights/permissions under the Attribution-NonCommercial-NoDeriv 4.0
International license (CC BY-NC-ND 4.0) from
bioweb.bio/faunaweb/amphibiaweb/UsoDatos/: specimen in 1a, b, c) QCAZ65528 from
Reserva Tesoro Escondido. Río Gualpí, Esmeraldas; 2a, b) QCAZ65533 from Reserva
Tesoro Escondido. Río Gualpí, Esmeraldas; 3a, b) QCAZA65554 from Reserva Tesoro
Escondido. Río Gualpí, Esmeraldas; 4a, b) QCAZA32120 from San Francisco,
Esmeraldas.
Supplementary Material - Tables
A new rainfrog of the genus Pristimantis (Anura, Brachycephaloidea) from central and
eastern Panama
Konrad Mebert, Macario González-Pinzón, Madian Miranda, Edgardo Grifith, Milan Vesely, P. Lennart Schmid, Abel Batista
Pristimantis cruentus (left) and P. gretathunbergae sp. nov. (right)
Table S1. Estimates of net evolutionary divergence (mean %) between groups (G-numbers) of sequences based on 16S mtDNA are
shown. Groups correspond to those resulted from a previous ABGD analysis; for every group, the estimates of average evolutionary
divergence over sequence pairs within groups is shown in parenthesis, with n: number of samples included in each group, followed by
origin of country: CO (Colombia), CR (Costa Rica), EC (Ecuador), HO (Honduras), PA (Panama); * in G4 represents specimens labeled
as P. cruentus from El Cope, Panama (see article text). Shaded values relate to group comparisons with P. gretathunbergae. # Genetic
divergence P. gretathunbergae with P. penelopus shown for the two confirmed samples, in parenthesis the mean value including the P.
aff. sanguineus/penelopus sample (see article text). # Genetic divergence P. gretathunbergae with P. erythropleura shown for the two
samples nrps_0055 and nrps_0057, in Colombia parenthesis the mean value including two additional samples from the same general
region (see article).
Species
16S evolutionary divergence between groups
G1
G2
G3
G4
G5
G6
G7
G8
G9
G10
G11
G12
G13
G14
G15
G1 P. cruentus (3%; n: 9, PA)
G2 P. penelopus (0.6%; n: 3#, CO)
10.3
G3 P. cisnerosi (0.4%; n: 2, EC)
12.1
8.5
G4 P. aff. cruentus (0%; n: 2, PA*)
6.0
10.6
13.1
G5 P. erythropleura (2%; n: 4 CO)
8.9
4.5
8.3
9.1
G6 P. gretathunbergae (2%; n: 14, PA)
10.2
4.8(6.0*)
10.4
11.2
4.8(5.9#)
G7 P. latidiscus (1%; n: 2, EC)
11.7
7.6
13.0
13.1
8.6
10.1
G8 P. ridens (1%; n: 2, CR, HO)
14.1
10.9
13.2
14.3
11.0
12.6
12.7
G9 P. taeniatus (3%; n: 2, CO)
16.4
17.0
17.2
17.5
16.1
17.7
17.9
17.2
G10 P. viejas group (2%; n: 2, CO)
8.0
7.2
6.4
7.1
6.4
5.5
5.3
10.4
16.4
G11 P. museosus (n: 1, PA)
13.7
9.0
15.8
15.5
10.0
11.1
9.1
13.1
17.5
7.4
G12 P. caryophyllaceus (n: 1, PA)
15.8
11.7
17.2
16.6
12.6
14.4
15.3
12.8
18.5
8.3
15.1
G13 P. caryophyllaceus (n: 1, PA)
15.4
11.3
15.9
15.9
12.6
14.6
14.5
13.8
17.8
9.2
14.9
7.8
G14 P. cerasinus (2%; n: 2, CR, PA)
11.2
7.2
8.3
9.9
7.2
7.2
8.1
10.4
17.8
5.1
9.4
10.6
9.2
G15 C. crassidigitus (n: 1, CR)
23.0
17.1
20.7
24.0
17.9
20.8
21.8
19.9
22.3
15.4
22.6
24.8
25.2
16.4
G16 C. sagui (n: 1, PA)
26.9
21.6
23.1
27.5
23.1
23.8
23.7
27.0
29.3
22.6
24.2
25.8
26.4
22.7
22.9
Supplementary Table S2. Estimates of net evolutionary divergence (mean %) between groups (G-numbers from the ABGD analysis) )
of sequences on mtDNA gene COI. For every group, the estimates of average evolutionary divergence over sequence pairs within
groups is shown in parenthesis, with n: number of samples included in each group, followed by origin of country: CO (Colombia), CR
(Costa Rica), EC (Ecuador), HO (Honduras), PA (Panama).
Species
COI evolutionary divergence between groups
G1
G2
G3
G4
G5
G6
G7
G8
G9
G1 P. gretathunbergae (8%; n: 9, PA)
G2 P. penelopus (4%; n: 3, CO)
16.3
G3 P. erythropleura (2%; n: 2 CO)
16.0
15.2
G4 P. cruentus ( 16%; n: 23, PA)
19.0
18.5
17.3
G5 P. calcaratus (0%; %; n: 2, EC)
18.2
19.9
17.9
19.0
G6 P. viejas (n.a.; n: 1, CO)
19.7
19.8
21.0
23.3
17.5
G7 P. museosus (2%: n:5, PA)
20.7
19.1
19.0
20.7
21.5
21.0
G8 P. cerasinus (10%; n: 3, CR, PA)
20.7
22.8
21.3
23.3
22.0
20.3
21.4
G9 P. ridens (15%; n: 3, CR, HO)
24.7
23.4
21.4
23.4
21.7
23.3
27.0
25.1
G10 P. taeniatus (18%; n: 3, CO )
25.9
27.2
24.6
27.4
23.4
26.4
26.2
25.7
25.8
Supplementary Table S3. Loadings of the PCA of morphometric characters (ratios) of
P. gretathunbergae sp. nov. and P. cruentus. Abbreviations of raw variables see Fig. S4
above.
Supplementary Table S4. Coefficients of linear discriminants of the LDA of
morphometric characters of P. gretathunbergae sp. nov. and P. cruentus. Abbreviations
of raw variables see Fig. S4 above.
Variable
Dim.1
Dim.2
Dim.3
Dim.4
Dim.5
TrL/HL
-0.179
0.730
0.375
0.431
-0.111
InD/HL
-0.015
0.034
0.039
0.031
0.016
IoD/HL
-0.035
0.108
0.055
0.043
-0.006
ED/HL
-0.033
0.110
0.027
0.023
0.053
TY/HL
-0.011
0.019
0.017
0.027
-0.005
EN/HL
-0.014
0.072
0.043
0.050
-0.018
EW/HL
-0.034
0.095
0.025
0.048
-0.025
TL/TrL
0.009
-0.050
0.117
-0.294
0.032
FL/TL
0.006
-0.181
-0.566
0.746
0.073
FAL/TrL
0.017
-0.063
-0.040
-0.019
-0.095
HAL/FAL
-0.029
0.115
0.076
0.003
0.969
BW/SVL
-0.007
-0.005
0.013
0.008
0.024
4TD/4TW
-0.100
0.053
0.083
0.053
-0.168
3FD/3FW
0.769
0.487
-0.364
-0.169
-0.019
3TD/3TW
0.601
-0.368
0.609
0.361
0.014
Variable
LD1
LD2
TrL/HL
0.607
1.294
IoD/HL
19.30
-8.618
ED/HL
5.415
2.217
EW/HL
-5.8404
7.058
4TD/4TW
-5.293
-5.386
3FD/3FW
0.228
0.439
3TD/3TW
0.279
0.398
Supplementary Table S5: List of specimens included in the genetic analysis with corresponding GenBank accession numbers. A “not
epithet” in parenthesis refers to an older species name for that particular sample that is preceded by the new, corrected species name.
Museum
ID
Field ID Species Locality Lat Lon Elev BIN
16S
Accession
SMF97520 AB1055
Pristimantis
gretathunbergae
Chucanti 8.805 -78.46 1460
SMF97521 AB1056
Pristimantis
gretathunbergae
Chucanti 8.805 -78.46 1460
SMF97522 AB1057
Pristimantis
gretathunbergae
Chucanti 8.805 -78.46 1460
MHCH3081 AB1058
Pristimantis
gretathunbergae
Chucanti 8.805 -78.46 1460
MHCH3082 AB1059
Pristimantis
gretathunbergae
Chucanti 8.805 -78.46 1460
SMF97517 AB654
Pristimantis
gretathunbergae
Ambroya 8.923 -78.625 852
SMF97518 AB661
Pristimantis
gretathunbergae
Ambroya 8.923 -78.625 852
SMF97519 AB662
Pristimantis
gretathunbergae
Ambroya 8.923 -78.625 852 BOLD:ACJ9377
MHCH3078 AB842
Pristimantis
gretathunbergae
Bajo pequeño,
Rio Tuquesa
8.48 -77.519 859 BOLD:ACJ9376
MHCH3079 AB868
Pristimantis
gretathunbergae
Bajo pequeño,
Rio Tuquesa
8.479 -77.528 718 BOLD:ACJ9376
MHCH3080 AB869
Pristimantis
gretathunbergae
Bajo pequeño,
Rio Tuquesa
8.479 -77.528 718
SMF97529 AB 48
Pristimantis aff.
cruentus
Darien 8.896 -78.566 518 BOLD:AAA5626
SMF97530 AB055
Pristimantis aff.
cruentus
Darien 8.896 -78.566 518
SMF97531 AB068
Pristimantis aff.
cruentus
Darien 8.892 -78.561 911 BOLD:AAA5626
SMF97532 AB089
Pristimantis aff.
cruentus
Darien 8.892 -78.56 911 BOLD:ACJ9377
SMF97486 AB1006 Pristimantis cruentus Darien 8.795 -78.449 824
MHCH3088 AB102
Pristimantis aff.
cruentus
Darien 8.892 -78.56 928
MHCH3033 AB1020 Pristimantis cruentus Darien 8.798 -78.462 1295
MHCH3034 AB1047 Pristimantis cruentus Darien 8.797 -78.463 1342
MHCH3089 AB106
Pristimantis aff.
cruentus
Darien 8.892 -78.561 904
MHCH3090 AB108
Pristimantis aff.
cruentus
Darien 8.892 -78.562 886
SMF97487 AB1129 Pristimantis cruentus Darien 8.002 -78.347 290 BOLD:AAA5626
SMF97488 AB1139 Pristimantis cruentus Darien 7.961 -77.704 1303 BOLD:ACA9764
MHCH3035 AB1140 Pristimantis cruentus Darien 7.961 -77.704 1303
MHCH3036 AB1169 Pristimantis cruentus Darien 7.959 -77.704 1230
SMF97489 AB1191 Pristimantis cruentus Darien 7.942 -77.703 1463 BOLD:ACA9764
SMF97528 AB1275 Pristimantis cruentus Darien 7.988 -77.708 1135
SMF97545 AB324 Pristimantis cruentus Darien 7.683 -78.038 943
SMF97546 AB331 Pristimantis cruentus Darien 7.683 -78.039 955
SMF97539 AB370
Pristimantis
penelopus/sanguineus
(not cruentus)
Darien 0
MHCH3016 AB484 Pristimantis cruentus Darien 8.029 -77.413 155
SMF97476 AB611 Pristimantis cruentus Darien 8.917 -78.618 485 BOLD:AAA5626
SMF97477 AB612 Pristimantis cruentus Darien 8.917 -78.618 485
MHCH3024 AB625 Pristimantis cruentus Darien 8.916 -78.629 906
MHCH3025 AB626 Pristimantis cruentus Darien 8.916 -78.629 906
SMF97478 AB676 Pristimantis cruentus Darien 9.035 -78.026 289 BOLD:ACK0001
SMF97479 AB694 Pristimantis cruentus Darien 9.049 -77.998 433
MHCH3026 AB695 Pristimantis cruentus Darien 9.049 -77.998 433 BOLD:ACJ9510
MHCH3027 AB718 Pristimantis cruentus Darien 9.061 -77.98 340
SMF97480 AB742 Pristimantis cruentus Darien 9.059 -77.984 553
SMF97481 AB745 Pristimantis cruentus Darien 9.059 -77.984 553
MHCH3028 AB759 Pristimantis cruentus Darien 9.061 -77.98 344
MHCH3029 AB790 Pristimantis cruentus Darien 9.034 -78.022 227 BOLD:ACK0001
SMF97482 AB791 Pristimantis cruentus Darien 9.034 -78.022 227
SMF97483 AB870 Pristimantis cruentus Darien 8.479 -77.528 718 BOLD:ACJ9403
SMF97484 AB871 Pristimantis cruentus Darien 8.479 -77.528 718
MHCH3030 AB872 Pristimantis cruentus Darien 8.479 -77.528 718
MHCH3031 AB873 Pristimantis cruentus Darien 8.479 -77.528 718
MHCH3032 AB898 Pristimantis cruentus Darien 8.479 -77.528 718
SMF97485 AB941 Pristimantis cruentus Darien 7.764 -78.101 655 BOLD:ACJ9785
CH 9651 AJC 2042
Pristimantis aff.
cruentus
Nurra, Darién, 140
m
9.0489 -77.997 KC129345
USNM
572403
KRL 0683
Pristimantis aff.
cruentus
El Copé, Coclé,
700 m
8.668 -80.602 FJ784334
MVUP
1796
KRL 0739
Pristimantis aff.
cruentus
El Copé, Coclé,
700 m
8.668 -80.602 FJ784354
USNM
572404
KRL 0861
Pristimantis aff.
cruentus
El Copé, Coclé,
700 m
8.668 -80.602 FJ784409
CH 9653 AJC 1670
Pristimantis
gretathunbergae
Cerro Chucantí,
Darién, 1365 m
8.805 -78.46 KC129350
CH 6046 CH 6046
Pristimantis
gretathunbergae
Brewster,Panamá,
900 m
9.35 -79.25 KC129352
CH 6265 CH 6265
Pristimantis
gretathunbergae
Cerro Chucantí,
Darién, 1240 m
8.805 -78.46 KC129349
CH 6266 CH 6266
Pristimantis
gretathunbergae
Cerro Chucantí,
Darién, 1240 m
8.805 -78.46 KC129347
CH 6271 CH 6271
Pristimantis
gretathunbergae
Cerro Chucantí,
Darién, 1240 m
8.805 -78.46 KC129348
swab EVACC 096
Pristimantis
gretathunbergae
Brewster,
Panamá, 810 m
9.35 -79.25 KC014937
swab EVACC 097
Pristimantis
gretathunbergae
Brewster,
Panamá, 810 m
9.35 -79.25 N/A
swab EVACC 098
Pristimantis
gretathunbergae
Brewster,
Panamá, 810 m
9.35 -79.25 KC014936
swab EVACC 217
Pristimantis
gretathunbergae
Brewster,
Panamá, 810 m
9.35 -79.25 KC014939
swab EVACC 218
Pristimantis
gretathunbergae
Brewster,
Panamá, 810 m
9.35 -79.25 KC014938
USNM
572470
KRL 1489
Pristimantis
gretathunbergae
Río Blanco,
Coclé, 1100 m
8.668 -80.602 KC129351
CH 6456 CH 6456
Pristimantis cruentus
(not latidiscus)
Cana, Darién,
1320 m
7.945 -77.685
KC129363
CH 9618 AJC 1133 Pristimantis cruentus
Altos del María,
930 m
8.664 -80.061 KC129367
CH 9621 AJC 1139 Pristimantis cruentus
Altos del María,
950 m
8.664 -80.061 KC129365
CH 9622 AJC 1140 Pristimantis cruentus
Altos del María,
940 m
8.664 -80.061 KC129372
CH 9624 AJC 1145 Pristimantis cruentus
Altos del María,
940 m
8.664 -80.061 KC129373
CH 9625 AJC 1147 Pristimantis cruentus
Altos del María,
940 m
8.664 -80.061 KC129364
CH 9626 AJC 1150 Pristimantis cruentus
Altos del María,
940 m
8.664 -80.061 KC129371
MVUP
2029
AJC 1204 Pristimantis cruentus
Altos del María,
900 m
8.664 -80.061 KC129370
MVUP
2038
AJC 1213 Pristimantis cruentus
Altos del María,
895 m
8.664 -80.061 KC129369
CH 9641 AJC 1917 Pristimantis cruentus
Brewster,
Panamá, 810 m
9.35 -79.25 KC129366
CH 9643 AJC 1930 Pristimantis cruentus
Brewster,
Panamá, 810 m
9.35 -79.25 KC129368
CH 6721 CH 6721 Pristimantis cruentus
Brewster,
Panamá, 810 m
9.35 -79.25 JN991442
USNM
572788
KRL 0811 Pristimantis cruentus
El Copé, Coclé,
700 m
8.668 -80.602 FJ784380
USNM
572361
KRL 1333 Pristimantis cruentus
El Copé, Coclé,
700 m
8.668 -80.602 FJ784502
USNM
572375
KRL 1407 Pristimantis cruentus
El Copé, Coclé,
700 m
8.668 -80.602 FJ784520
USNM
572362
KRL 1420 Pristimantis cruentus
El Copé, Coclé,
700 m
8.668 -80.602 FJ784525
USNM
572364
KRL 1455 Pristimantis cruentus
El Copé, Coclé,
700 m
8.668 -80.602 FJ784531
USNM
572365
KRL 1462 Pristimantis cruentus
El Copé, Coclé,
700 m
8.668 -80.602 FJ784535
USNM
572366
KRL 1474 Pristimantis cruentus
El Copé, Coclé,
700 m
8.668 -80.602
FJ784538
USNM
572367
KRL 1528 Pristimantis cruentus
El Copé, Coclé,
700 m
8.668 -80.602 FJ784548
USNM
572369
KRL 1550 Pristimantis cruentus
El Copé, Coclé,
700 m
8.668 -80.602 FJ784557
CH 9616 AJC 1128
Pristimantis aff.
cruentus
Altos del María,
930 m
8.664 -80.061 JN991444
CH 9617 AJC 1129
Pristimantis aff.
cruentus
Altos del María,
930 m
8.664 -80.061 KC129344
CH 9650 AJC 1998
Pristimantis aff.
cruentus
Rio Chico,
Panamá, 135 m
KC129346
AJC 1957 Pristimantis cruentus Panamá 9.230999947
-
79.40299988
KR863312
CH 6867 Pristimantis cruentus Panamá 9.265000343
-
79.50800323
KR863315
AJC 1983 Pristimantis cruentus Panamá 9.312999725
-79
KR863314
AJC 1913 Pristimantis cruentus Panamá 9.3167 -78.9833
KR863308
CH 6679 Pristimantis cruentus Panamá 9.319999695
-
79.28900146
KR863313
AJC 1943 Pristimantis cruentus Panamá 9.32 79.289
KR863309
AJC 1954 Pristimantis cruentus Panamá 9.32 79.289
KR863311
AJC 1951 Pristimantis cruentus Panamá 9.32 79.289
KR863310
AJC 1767 Pristimantis cruentus Panamá 9.231 79.403
KR863316
AJC 1948 Pristimantis cruentus Panamá 9.32 79.289
KR863307
MVZ 203826
Eleutherodactylus
cruentus
Costa Rica AY948758
AJC 0475 Pristimantis cruentus
Costa Rica,
Tapantí
9.65 83.85 JN991441
AJC 0524 Pristimantis cruentus Costa Rica 10.2 83.75
JN991440
AJC 0581 Pristimantis cruentus
Panamá, Darien,
Cana
7.76 77.72 JN991443
EMM 250 Pristimantis viejas
Colombia,
Antioquia
6.371 74.019 JN991476
CH 6747 Pristimantis museosus Panamá, Chepo 9.32 79.289
KC129374
KU218016 Pristimantis latidiscus
Ecuador,
Pichincha
EF493698
MZUTI 2992 Pristimantis latidiscus
Ecuador,
Pichincha, Mindo
KU999197
nrps 0055
Priatimantis
erythropleura
Colombia, Valle
del Cauca
4.802 76.196 JN991445
nrps 0057
Pristimantis
erythropleura
Colombia, Valle
del Cauca
4.802 76.193 JN991446
QCAZ45432 Pristimantis cisnerosi
Ecuador,
Pichincha
S 01.04186 78.62405 243 MT372697
QCAZ32120 Pristimantis cisnerosi
Ecuador, San
Francisco
1.05151 78.41145 77 MT372698
QCAZ:28430 Pristimantis variabilis Ecuador -1.095 -77.925
MH516198
EMM 247
Pristimantis
penelopus (not viejas)
Colombia,
Antioquia
6.371 74.019 JN991477
NRPS 0011
Pristimantis viejas
Colombia
JN991475
AJC 1344
Pristimantis
penelopus (not paisa)
Colombia,
Antioquia
6.54 74.64 JN991459
KU177252
Pristimantis
cremnobates
Ecuador, Napo,
Río Salado
EF493528
UVC:15881 Pristimantis calcaratus Colombia 3.4289 76.6592
JN104658
UVC:15889 Pristimantis calcaratus Colombia 3.4289 76.6592
JN104659
AJC 0126 Pristimantis ridens
Costa Rica,
Puntarenas, Río
Claro
8.74 82.96 JN991466
ENS 10722 Pristimantis ridens
Honduras,
Olancho, Sierra
de Agalta
14.96 86.14 JN991464
CH 6776 Pristimantis ridens Panamá, Chilibre 9.231 79.403
KR863319
AJC 0527
Pristimantis
cerasiunus
Costa Rica, Limón 10.04 83.55 JN991437
AJC 1142
Pristimantis
cerasiunus
Panamá, Altos del
María
8.61 80.09 JN991438
AB434
Pristimantis
caryophyllaceus
AB679
Pristimantis
caryophyllaceus
Panamá, Darién,
Wargandí, Nurra
9.035 78.036 KJ201962
AB253 Pristimantis
caryophyllaceus
Panamá, Darién,
Pinogana, Río
Cana
7.756 77.686 KJ201961
AJC 1126 Pristimantis taeniatus
Panamá, Colón,
Barro Colorado
9.15 79.85 JN991472
CJD 069 Pristimantis taeniatus
Colombia,
Santander, Mesa
de los Santos
6.766 73.083 JN991473
nrps 0016 Pristimantis taeniatus
Colombia,
Cundinamarca,
Yacopi
5.459 74.337 JN991474
MVZ:207248
Craugastor
crassidigitus
Costa Rica,
Puntarenas
EU186715
EVACC_024 Craugastor tabasarae
Panamá, Cerro
Brewster
9.31985 79.2889 KC014806
UCR 22737
Craugastor
aenigmaticus
Costa Rica 9.322 83.203 MK211617
SMF 104017 Craugastor sagui Panamá 8.5 81.772 1700
MK279370
