Amphibia-Reptilia (2016) DOI:10.1163/15685381-00003037
A new small golden frog of the genus Pristimantis (Anura:
Craugastoridae) from an Andean cloud forest of Colombia
Mauricio Rivera-Correa1,2,∗, Faride Lamadrid-Feris3, Andrew J. Crawford4,5
Abstract. A new species of Pristimantis is described from an Andean cloud forest at 2650 m in the Cordillera Oriental
of Colombia. Pristimantis dorado sp. nov. is similar to and could be closely related to P. acutirostris, but can be readily
distinguished from this latter species by the absence of a tympanic annulus, vocal slits, vocal sac and reticulations on
concealed surfaces, and by having a metallic gold iris with a brown horizontal streak. The phylogenetic position of the
new species is recovered and we provide its advertisement call, which this species manages to emit despite lacking a vocal
sac and vocal slits. This discovery reminds us that despite the extensive research on the alpha-taxonomy of Pristimantis in
Colombia, fieldwork in high montane forests continues to yield previously unknown species.
Keywords: advertisement call, Amphibia, Brachycephaloidea, Eastern Cordillera, morphology, South America, Terrarana.
Introduction
The genus Pristimantis is a principal compo-
nent of anuran diversity in the Neotropics. Cur-
rently 465 species are recognized, 205 of which
are found in Colombia, making it the coun-
try with the highest diversity of Pristimantis
(AmphibiaWeb, 2015; Frost, 2015). Much of
our knowledge of these species comes from
the extensive taxonomic work exerted over the
last four decades by Dr. John D. Lynch (e.g.
Lynch, 1971; Lynch, 1980; Lynch and Rueda-
Almonacid, 1998). Despite these efforts, the
Andes of Colombia still continue to yield a
plethora of new species, especially from mon-
tane localities. Thus, the anuran diversity of
Colombia is still underestimated and many as-
pects of the biology of known species, such as
1 - Grupo Herpetológico de Antioquia, Instituto de Bi-
ología, Universidad de Antioquia, A.A. 1226, Medellín,
Colombia
2 - Seccional Oriente, Regionalización, Universidad de An-
tioquia, km 6 vía Rionegro – La Ceja, Carmen de Vibo-
ral, Colombia
3 - Programa de Biología, Facultad de Ciencias Básicas,
Universidad del Atlántico, Barranquilla, Colombia
4 - Department of Biological Sciences, Universidad de los
Andes, A.A. 4976, Bogotá, Colombia
5 - Smithsonian Tropical Research Institute, Apartado
0843-03092, Panama City, Republic of Panama
∗Corresponding author; e-mail:
mauriciorivera79@yahoo.com.ar
morphological variation, calls and reproduction
are still poorly understood. During field expe-
dition to cloud forest habitat in the buffer area
of the Chingaza National Park in the Eastern
Cordillera of Colombia, we discovered a small,
distinctive species of Pristimantis calling from
the bushes along a roadside. The aim of the
present paper is to describe this new species and
document its advertisement call.
Materials and methods
Morphology
Specimens were sacrificed in a solution of Lidocaine 2%
and fixed in 10% formaldehyde solution. Adult specimens
were transferred to and kept in 70% ethanol within five
days of fixation. Prior to fixation, tissue samples from some
specimens were collected and preserved in 96% ethanol.
Terminology used to describe the morphological charac-
ters of the frogs follows Lynch and Duellman (1997) and
Duellman and Lehr (2009). Abbreviations are SVL (snout-
vent length), HL (head length), HW (head width), ED (eye
diameter), END (eye-nostril distance), NSD (nostril to tip
of snout distance), IND (inter-nostril distance), AMD (dis-
tance between the anterior margins of eyes), FAL (fore-
arm length), FAB (forearm breadth); HAL (hand length),
TFD (third finger disc diameter), THL (thigh length), TL
(tibia length), TAL (tarsal length), FL (foot length) and FTD
(fourth toe disc diameter). All measurements were taken us-
ing dial callipers accurate to the nearest 0.1 mm. Observa-
tions on the colour of the frogs in life were based on field
notes and colour slides of specimens. Sex was determined
by examination of secondary sexual characters (i.e., nuptial
pads). Illustrations were made using a Zeiss stereomicro-
scope with a drawing tube attached.
© Koninklijke Brill NV, Leiden, 2016. DOI:10.1163/15685381-00003037
2 M. Rivera-Correa, F. Lamadrid-Feris, A.J. Crawford
Vocalization
Call recordings were made at the type locality on 26 Feb-
ruary 2009 at 22:08 h (ANDES-A 1028, temperature not
recorded), with an Astone mp3 player model Niva using the
incorporated omnidirectional microphone. All recordings
were made at a distance of approximately 0.6 m from the
signaller. Calls were analysed using the software RAVEN
version 1.3 (Charif et al., 2004) with a FFT of 512 points,
at a sampling rate of 44.1 kHz and 16-bit precision. The
following call variables were obtained as defined in Cro-
coft and Ryan (1995): call duration, interval between calls,
call type (tonal or pulsed), and peak (dominant) frequency.
Morphological information on additional species was taken
from preserved specimens (Appendix), photos in life, and
from the literature. Generic and familial allocations fol-
low Padial, Grant and Frost (2014). Institutional acronyms
are MHUA (Museo de Herpetología Universidad de Antio-
quia, Medellín, Colombia); ICN (Instituto de Ciencias Nat-
urales, Universidad Nacional de Colombia, Bogotá, Colom-
bia); ANDES (Museo de Historia Natural ANDES, Bogotá,
Colombia).
Molecular analysis
In order to provide a molecular genetic evaluation of the dis-
tinctiveness and phylogenetic position of the new species,
we sequenced DNA from two mitochondrial gene frag-
ments, a roughly 540 base pair (bp) fragment of the 16S
ribosomal RNA gene (16S) and 654 bp of the Folmer
fragment of cytochrome oxidase I (COI-5′), commonly re-
ferred to as the animal Barcode of Life (Hebert et al.,
2003), from two paratypes of the new species (see below)
and from one specimen each of P. lutitus from Colom-
bia and P. cf. fenestratus from Peru (see online supple-
mentary table S1). From liver tissue samples of these four
specimens we extracted genomic DNA following the stan-
dard instructions in a DNeasy Blood and Tissue Kit (QIA-
gen). We PCR-amplified the COI marker using the primers
dgHCO2198 (5′-TAA ACT TCA GGG TGA CCA AAR
AAY CA-3′) and dgLCO1490 (5′-GGT CAA CAA ATC
ATA AAG AYA TYG G-3′; Meyer et al., 2005) and in-
cluded 0.25 μg/μl of bovine serum albumin in the reac-
tion. The 16S fragment was amplified using 16SB-H (aka,
16Sbr-H) (5′-CCG GTC TGA ACT CAG ATC ACG T-
3′) and 16SA-L (aka, 16Sar-L) (5′-CGC CTG TTT ATC
AAA AAC AT-3′) (Kessing et al., 2004). All PCR con-
tained 2.0 mM Mg2+ and utilized an annealing temperature
of 49°C, with other reaction conditions standard (Kessing et
al., 2004). PCR products were cleaned using Exonuclease I
and Shrimp Alkaline Phosphatase enzymes (Werle et al.,
1994) and Sanger-sequenced on an ABI 3500 automated se-
quencer (Life Technologies). Chromatograms were assem-
bled and cleaned using Sequencher 5.0 (Gene Codes Corpo-
ration). Resulting DNA sequences were deposited in Gen-
Bank and chromatograms plus DNA sequences, collection
data and photographs were posted at the Barcode of Life
Data Systems (Ratnasingham and Hebert, 2007; table S1).
Phylogenetic inference
To compare genetic data from the new species with pub-
lished molecular data (supplementary table S1), we con-
ducted BLAST searches (Altschul et al., 1990) in GenBank
for sequences similar to the resulting COI and 16S gene
fragments. We found more published 16S data than COI
data for Pristimantis. Thus, we focused our phylogenetic
inference exclusively on 16S and report COI genetic dis-
tances below. The first 100 BLAST hits to the 16S fragment
obtained from the new species were visualized preliminarily
using neighbour-joining (NJ) trees (Saitou and Nei, 1987).
Redundant and genetically very distant sequences were re-
moved, leaving us with 13 published DNA sequences sim-
ilar to the new species, including the morphologically sim-
ilar P. sagittulus from central Peru. To this combined data
set of 13 published and four new DNA sequences, we added
as an out-group a sample from the sister genus Yunganastes
(Padial, Grant and Frost, 2014). For these 14 published 16S
sequences only six also had COI sequence available (Pinto-
Sánchez et al., 2012).
Alignment of COI was trivial as no length variation was
inferred, whereas for 16S we aligned the sequences using
the ‘quick-set ML’ default parameter options in SATé-II ver-
sion 2.2.7, which iteratively optimizes both the tree and the
alignment (Liu et al., 2012). We then fixed the resulting 16S
alignment and conducted phylogenetic inference based on
maximum parsimony (MP; Fitch, 1971) and based on max-
imum likelihood (ML; Felsenstein, 1981). Because inferred
gaps clustered in the difficult-to-align loop regions of the
16S rRNA molecular, we conservatively removed all gapped
sites prior to phylogenetic inference, resulting in a 16S data
matrix of 474 total characters of which 40 were parsimony-
uninformative and 127 were parsimony-informative.
Heuristic tree searching for MP inference was conducted
using PAUP∗ (Swofford, 2000) version 4.0a136 for Unix
based on 5000 replicate searches each from random starting
trees (not just random addition sequence) with MaxTrees
set to 100 000 and the rearrangement limit on each tree set
also to 100 000 to indirectly constrain the search time. To
evaluate the potential completeness of the resulting search,
the same search conditions were run five more times and no
new trees were found after the first search, despite the use
of random starting trees.
Prior to ML inference we used jModeltest2 version 2.1.6
(Darriba et al., 2012) to select the best-fit model of molec-
ular evolution among seven possible substitution patterns
each with or without a gamma rate heterogeneity param-
eter (Yang, 1993). Akaike, Bayesian and Decision Theory
criteria agreed on recommending a general time-reversible
model with rate heterogeneity, GTR +  (Tavaré, 1986).
ML tree searches were performed using GARLI version
2.0.1019 for Unix (Zwickl, 2006) that uses a genetic algo-
rithm to more efficiently search tree space and optimize pa-
rameter values. Here we employed default search parameter
values and present the best tree obtained across ten indepen-
dent ML searches.
Statistical support for clades was assessed by non-
parametric bootstrapping (Felsenstein, 1985) under the MP
and ML criteria. For MP bootstrapping in PAUP∗, we
New species of Pristimantis from Colombia 3
used 2000 replicate samples (with replacement) of the
data matrix, with 100 tree searches performed on each re-
sampled character matrix starting from random trees and
with other search parameters as above. For ML bootstrap-
ping in GARLI we used 1000 re-sampling replicates with
two ML searches per re-sampled character matrix but with
the search parameter ‘number of generations without topol-
ogy improvement required for termination’ lowered from
the default of 20 000 to 5000. Results of ML bootstrap were
summarized using version 3.3.1 of SumTrees (Phylogenetic
Tree Split Support Summarization) for Unix (Sukumaran
and Holder, 2010).
As a heuristic measure of divergence between the new
species and other available samples, we calculated the raw,
uncorrected genetic distance, or p-distance, between each
of the eleven samples in the 16S phylogeny that also had
data available on the COI gene fragment. While we do not
support the concept of a widely applicable threshold of ge-
netic distance separating within- versus between-species di-
vergence levels, we find that mtDNA data may highlight
lineages of interest that may warrant further integrative tax-
onomic investigations (Padial et al., 2010). We chose the
p-distance measure because recent studies have shown that
uncorrected distances perform as well as other distance
measures in delimiting species (Collins et al., 2012; Sri-
vathsan and Meier, 2012), and by definition this measure
will provide a minimum estimate of divergence at our two
mtDNA markers.
Results
Pristimantis dorado sp. nov. (figs 1-3)
Pristimantis aff. acutirostris. Rivera-Correa
(2012:37; fig. 2, bottom left)
Holotype. MHUA-A 7313, adult male. Co-
lombia: Meta: Municipio San Juanito: Vereda
San Luis El Plan: Alto Buenavista, km 9
road San Juanito – Chingaza (4.487642,
−73.682584; 2650 m), collected on 16 April
2010 by Mauricio Rivera-Correa, Faride Lama-
drid-Feris, Marco Rada and Santiago Castro-
viejo-Fisher.
Paratypes. Six specimens. MHUA-A 7310,
7312, two adult males; MHUA-A 7311, sub-
adult female, collected with holotype. MHUA-
A 7308, ANDES-A 1028-29, three adult males.
Colombia: Meta: Municipio San Juanito: Ve-
reda San Luis El Plan: Alto Buenavista, km
9 road San Juanito – Chingaza (4.48947,
−73.68650; 2650 m), collected on 26 February
2009 by Faride Lamadrid-Feris, Diego Gonza-
lez, Olga Nieto and Ivan Sánchez.
Referred specimens. MHUA-A 7314-15 (two
juvenile), collected with the holotype. MHUA-
A 7309 (adult female) collected with the para-
types.
Generic allocation. We designate this new
species to the genus Pristimantis based on
its morphological similarity to other species
of Pristimantis, and corroborate this inference
using mitochondrial DNA (mtDNA) sequence
data (see below).
Diagnosis. This new species of Pristimantis
is characterized and defined by the following
combination of characters: (1) skin on dorsum
is shagreen, ventral skin areolate; dorsolateral
fold present, supratympanic fold barely evident;
(2) tympanic membrane present but inconspic-
uous and tympanic annulus absent; (3) snout
acuminate in dorsal view and rounded in profile,
canthus rostralis distinct, loreal region slightly
concave to flat; nostrils slightly protuberant, di-
rected laterally; area between nostrils convex;
(4) upper eyelid rugose and lacking an en-
larged supraocular tubercle; cranial crests ab-
sent; (5) choanae small, rounded and partly con-
cealed by palatal shelf of maxilla arch; dentiger-
ous process of vomers present; (6) vocal slits
and vocal sac absent; males with extensive nup-
tial pads on thumbs almost reaching the prox-
imal region of the disc of Finger I, without
thorny structures; (7) Finger I shorter than Fin-
ger II; disks on Fingers III and IV expanded;
(8) fingers and toes lacking lateral fringes;
(9) small ulnar tubercles present; (10) inner and
outer edges of tarsus with very small tubercles;
heel bearing single small and subconical tuber-
cle; (11) inner metatarsal tubercle ovoid and el-
evated; outer metatarsal tubercle small, subcon-
ical; a small and elongate inner tarsal; supernu-
merary plantar tubercles present; (12) rounded
and projecting subarticular tubercles on toes,
webbing absent, Toe III shorter than Toe V, toe
disks smaller than finger disks; (13) dorsum gol-
den with brown stripes and blotches; lip cream
4 M. Rivera-Correa, F. Lamadrid-Feris, A.J. Crawford
Figure 1. Pristimantis dorado sp. nov. in life (MHUA-A 7311, paratype, female, SVL 19.2 mm). (A, D) lateral view; (B)
ventral view; (C) frontal view. Photographs: S. Castroviejo-Fisher. This figure is published in colour in the online version.
New species of Pristimantis from Colombia 5
Figure 2. Pristimantis dorado sp. nov. (MHUA-A 7313, holotype). (A) Head in lateral view; (B) head in dorsal view; (C) left
hand in ventral view; (D) left foot in ventral view. Scale bar = 5 mm. Note the nuptial pad on Finger I. Drawings: MRC.
6 M. Rivera-Correa, F. Lamadrid-Feris, A.J. Crawford
Figure 3. Dorsal and ventral view of the holotype of Pristimantis dorado sp. nov. (MHUA-A 7313, SVL 17.9 mm). This
figure is published in colour in the online version.
with a brown line at lip margin; concealed sur-
faces of thigh immaculate light orange; gu-
lar translucent yellow, venter cream and dark
brown spots present; iris metallic golden with a
brown horizontal streak (14) SVL in adult males
14.2-17.9 mm (n = 6) and one female 25.6 mm.
Pristimantis acutirostris (Lynch, 1984) is su-
perficially similar to P. dorado, but differ in the
states of the following characters: vocal slits,
vocal sac and tympanic annulus present (absent
in P. dorado); concealed surfaces yellow with
brown reticulation (light orange without retic-
ulations in P. dorado); iris pale blue with red-
dish horizontal streak (metallic golden with a
brown horizontal streak in P. dorado). Accord-
ing to our phylogenetic evidence, the species
with closer genetic affinity is P. lutitus (Lynch,
1984), but this latter species has tympanic annu-
lus prominent, conical supraocular tubercle (ab-
sent in P. dorado), dorsum brown with darker
markings (dorsum golden with brown stripes
and blotches in P. dorado) and, limbs with bars
broad (absent in P. dorado). Pristimantis do-
rado is also superficially similar to P. sagit-
tulus (Lehr, Aguilar and Duellman, 2004), but
differs in the states of the following charac-
ters: nuptial pads in males absent (nuptial pads
present in P. dorado); fingers and toes with lat-
eral fringes (lateral fringes absent in P. dorado);
tympanic annulus present (absent in P. dorado);
and posterior surfaces of thighs brown with red
spots and broad red longitudinal stripe (light
orange without spots and stripe in P. dorado).
Pristimantis dorado differs from P. merostictus
(Lynch, 1984) because the males of latter has
vocal slits and tympanic annulus present (ab-
sent in P. dorado), posterior surfaces of thighs
black with small yellow spots (light orange with
dark brown spots in P. dorado). In addition, P.
atratus (Lynch, 1979) is similar in dorsal pat-
tern to P. dorado, but the former has more abun-
dant ridges, hidden areas with black spots, less
acuminate snout, dentigerous process of vomers
conspicuous and a conic heel tubercle more
prominent.
Description of the holotype. A small frog,
17.9 mm SVL; head wider than body, head
width 35% of SVL; head barely wider than
long; head length 34% of SVL. Canthus ros-
tralis sharp, essentially straight, loreal region
slightly concave to flat; nostrils slightly protu-
berant, directed laterally; area between nostrils
convex; eyes directed laterally; eye-to-nostril
distance equal to eye diameter; lips not flared;
upper eyelid rugose but lacking an enlarged
supraocular tubercle; eyes extend beyond jaw
in dorsal view; snout acuminate in dorsal view
and rounded in profile; tympanic membrane
present although depressed and inconspicuous,
tympanic annulus absent; supratympanic fold
New species of Pristimantis from Colombia 7
barely evident; postrictal tubercle indistinct, but
post-tympanic fold evident; interorbital distance
greater than width of upper eyelid; choanae
small, rounded and partly concealed by palatal
shelf of maxilla arch; dentigerous processes
of vomers present but inconspicuous; tongue
longer that wide, posterior one-half free from
floor of mouth; vocal sac and vocal slits absent.
Skin of dorsum is finely shagreen; dorsolat-
eral folds low; skin of throat smooth; ventral
and lateral skin areolate; discoidal folds absent;
small ulnar tubercles present, without forming
a distinct fold; thenar tubercle oval, smaller
than divided palmar tubercles; supernumerary
palmar tubercles present; subarticular tubercles
prominent, rounded, including the most distal
tubercle; lateral fringes absent; Finger I shorter
than Finger II; tips of digits round to subtrun-
cated; inner digits of hand bearing much nar-
rower discs than outer digits; all fingers bearing
pads on digital tips; tip of Finger IV reaches be-
yond distal subarticular tubercle on Finger III;
tip of Finger II barely reaches distal subarticular
tubercle of Finger III; nuptial pads on thumbs
almost reaching the proximal region of the disc
of Finger I, without thorny structures.
Hind limbs moderate; tibia long, 47% of
SVL; foot long, 44% of SVL; toe length for-
mula I < II < III < V < IV; tip of Toe V
reaches distal subarticular tubercle on Toe IV;
tip of Toe III does not reach the distal subartic-
ular tubercle on Toe IV; discs of toes slightly
smaller to those on outer fingers, round to sub-
truncated; webbing absent; subarticular tuber-
cles on toes distinct, projecting and ovoid; few
supernumerary plantar tubercles at bases of toes
present; inner metatarsal tubercle ovoid and el-
evated, almost half the length of Toe I; lateral
fringes absent; outer metatarsal tubercle small,
subconical; tarsus without fold, but small sub-
conical tubercles along inner and outer border; a
small subconical heel calcar; cloacal sheath and
tubercles present, cloacal opening directed pos-
teroventrally at level of thighs.
Coloration in life. Dorsum with many fine
dark brown blotches forming middorsal, par-
avertebral and dorsolateral stripes (interrupted
partially); canthal stripe dark brown, reaches the
tip of the snout; post-ocular stripe dark brown;
lip pale cream, unmarked except for thin brown
line at lip margin; limbs, flanks and thighs
cream with scattered brown blotches; axillae,
undersides and posterior surfaces of thighs im-
maculate with light orange colouration, venter
and throat white with darker brown spots; pal-
mar and plantar side light orange; ventral side of
limbs and thighs light orange with dark brown
spots; iris metallic golden with a brown hori-
zontal streak.
Colouration in preservative. Dorsum and
flanks dirty brown to grey with numerous darker
brown blotched, partially forming a middorsal
and dorsolateral stripes; canthal and postocular
stripes darker brown; arms and legs cream with
dark brown spots and blotches, cream belly with
dark brown spots.
Measurements of holotype (in mm). SVL 17.9;
HL 6.1; HW 6.3; ED 2.0; END 2.1; NSD 1.2;
IND 1.9; AMD 4.1; FAL 3.5; FAB 1.3; HAL
4.9; THL 7.2; TL 8.5; TAL 4.5; FL 7.8; TFD
1.0; FFD 1.0.
Variation. Morphometric variation in adult
males is described in table 1. The only known
adult female is noticeably larger than males
[SVL 25.6 vs. 14.2-19.2 mm (mean = 17.0), re-
spectively]. The forearm of males is swollen and
nuptial pads are evident covering dorsal surface
of Finger I. In the female, the dentigerous pro-
cesses of the vomers are conspicuous, oblique,
each bearing three teeth and separated by a dis-
tance approximately the size of the choanae.
Female is more pigmented ventrally relative to
males and its dorsum is darker than that of the
males. Dorsal colour is reddish-gold in the fe-
male and golden in males.
Distribution and natural history. The new
species is known only from the type local-
ity, the buffer zone of Chingaza National Park
and Farallones de Gachalá, Municipio de Med-
ina, Departamento de Cundinamarca, Colombia
8 M. Rivera-Correa, F. Lamadrid-Feris, A.J. Crawford
Table 1. Morphological variation (in mm) of the type series
of Pristimantis dorado sp. nov. See text for abbreviations.
Min = minimum value; Max = maximum value; Mean =
arithmetic mean value; S.D. = standard deviation.
Measurements Males (n = 6)
Min Max Mean S.D.
SVL 14.2 19.2 17.0 1.5
HL 5.4 7.0 6.1 0.5
HW 5.7 7.1 6.3 0.5
ED 1.9 2.2 2.0 0.1
END 1.7 2.3 2.0 0.2
NSD 1.2 1.8 1.4 0.2
IND 1.0 2.2 1.8 0.4
AMD 3.4 4.5 4.1 0.4
FAL 3.0 4.6 3.6 0.6
FAB 1.2 1.5 1.3 0.1
HAL 4.4 5.5 5.0 0.4
THL 7.2 9.5 8.0 0.8
TL 8.0 9.9 8.7 0.6
TAL 4.3 5.1 4.6 0.3
FL 5.2 8.7 7.3 1.1
TFD 0.8 1.1 1.0 0.1
FFD 0.7 1.0 0.9 0.1
(fig. 4). Specimens were found in an Andean
cloud forest between 19:00 h and 22:30 h during
a light drizzle. Pristimantis dorado was found
along the roadside in association with plants
such as Miconia summa, Weinmannia spp. and
Hedyosmum bomplandianum and near palms of
the species Ceroxylon quindiuense. Pristiman-
tis dorado is vocally active at night; all adult
males captured were calling from vegetation at
a height of 50 to 150 cm. We observed two or
more males vocalizing from the same plant at
different heights above the ground. By placing
males together to be photographed, we observed
that the frogs produced a typical advertisement
call but increased the call rate and decreased the
intercall interval, which may represent an ag-
onistic behaviour. However, no orientation be-
haviour or physical contact was observed. Pris-
timantis dorado was found in sympatry with P.
frater.
Vocalization. The advertisement call is char-
acterized by a series of short, indistinctly pulsed
clicks with notes not modulated in amplitude
(fig. 5). The peak frequency ranged between
2930.0 and 3320.5 Hz; the mean value of the
maximum energy among calls was 60.4 dB ±
3.51, n = 22 calls (fig. 5b). The mean note
length was 0.011 s with an internote interval
ranging between 0.0010 and 0.041 s (mean =
0.026 ± 0.03 s; n = 20 calls). The mean inter-
val between calls was 1.223 s, and call duration
ranged from 0.001 to 0.049 s.
Molecular phylogenetics and similarity. MP
tree searching found 28 shortest trees of 488
steps length. MP trees varied widely in topol-
ogy among each other and in comparison to the
ML tree (fig. 6). Bootstrap analyses under MP
and ML criteria, however, supported roughly
the same nodes and with quite similar bootstrap
scores (fig. 6). The ML tree and all 28 MP trees
placed P. dorado as sister to P. lutitus (reported
here for the first time) + P. cf. anolirex. Because
these latter two samples are so genetically sim-
ilar, and the latter voucher has not been recov-
ered, we hypothesize that both of these samples
may, in fact, be P. lutitus. Little agreement ex-
isted among MP trees in the placement of the P.
dorado + P. lutitus clade, nor was there any sup-
port in either MP or ML bootstrap analyses. The
morphologically similar P. sagittulus is placed
with high statistical support in a clade with an-
other Peruvian and a Bolivian sample (fig. 6),
and this clade was not sister to the P. dorado +
P. lutitus clade in any of the 28 MP trees.
Based on the COI data set, the species most
genetically similar to P. dorado was P. lutitus,
which showed a p-distance of 17.4%, while at
the 16S markers, the most similar species to P.
dorado were P. affinis and P. gaigei, with ge-
netic distances of 11.1% and 11.8%, respec-
tively (table 2). These distances are far beyond
any thresholds used previously to identify cryp-
tic species (Crawford et al., 2013), adding fur-
ther support for the distinctiveness of P. dorado,
based on available DNA sequence data.
Etymology
The specific name is an adjective in Spanish
meaning “golden”, in allusion to the gold colour
of the dorsum in this species. This name is also a
New species of Pristimantis from Colombia 9
Figure 4. Map showing the known localities of Pristimantis dorado sp. nov.: Municipio de San Juanito, Departamento de
Meta, type locality (white star), and Farallones de Gachalá, Municipio de Medina, Departamento de Cundinamarca (white
circle).
Figure 5. Advertisement call of Pristimantis dorado sp. nov. (A) Oscillogram showing the amplitude and call duration;
(B) spectrogram showing the frequency range of calls; (C) power spectrum showing the peak frequency of the call.
10 M. Rivera-Correa, F. Lamadrid-Feris, A.J. Crawford
Figure 6. Maximum likelihood (ML) phylogenetic inference based on 474 aligned base pairs of the 16S gene, including
127 parsimony-informative sites, for a Ln-likelihood value of −2866.67. Numbers on branches represent support values
for likelihood bootstrap percentages above the branch and maximum parsimony bootstrap percentages below the branch.
Bootstrap values below 50% are not shown. Scale bar shows ML branch length estimated under a General Time Reversible
model of substitution with four gamma-distributed rate categories described by an α = 0.21305 shape parameter.
tribute to “El Dorado”, a legendary but mythical
city of gold sought by Spaniard conquistadores
throughout South America.
Discussion
In the Neotropics, the terraranan frogs of the
genus Pristimantis are extraordinary diverse,
particularly in the northern region of the Andean
Mountains of South America (Frost, 2015). The
heterogeneous Andean topography accompa-
nied by the life histories of Pristimantis (such
as direct development, high endemism and the
ability to colonize a large variety of habitat
types including high altitudes) could be partly
responsible for this notorious biodiversity. Pris-
timantis is characterized by remarkable mor-
phological variability (Hedges, Duellman and
Heinicke, 2008). For example, body sizes range
from tiny species such as P. andinognomus with
an adult male SVL of 10.0-14.5 mm (Lehr
and Coloma, 2008) to relatively large species
such as P. labiosus, with males reaching up
to 50.8 mm (Lynch et al., 1994). Pristimantis
dorado belongs to the group of small species
(sensu Lynch and Duellman, 1997). In our type
series, for example, one male (MHUA-A 7308)
with SVL = 14.2 mm has well-developed nup-
tial pads, a secondary sexual character found
exclusively in reproductively active individuals
New species of Pristimantis from Colombia 11
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19
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P.
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is
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99
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11
11
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∗
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0.
16
79
P.
n
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sJ
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99
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0.
11
27
0.
10
66
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11
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0.
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10
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0.
12
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0.
12
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0.
04
08
∗
0.
20
04
0.
17
90
P.
sa
va
ge
iJ
N
99
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01
0.
11
63
0.
11
84
0.
12
35
0.
12
42
0.
10
70
0.
13
68
0.
13
74
0.
06
71
0.
05
09
∗
0.
20
85
P.
sa
va
ge
iJ
N
99
14
02
0.
11
45
0.
10
22
0.
13
10
0.
10
41
0.
10
31
0.
14
42
0.
14
49
0.
06
72
0.
05
71
0.
07
32
∗
(Lynch, 1971; Luna et al., 2012), indicating that
this small animal was a reproductively mature
adult.
Nuptial excrescences are secondary sexual
traits consisting of modified epidermal and der-
mal tissues typically located on the first finger
(Noble, 1931), and occasionally on the remain-
ing fingers (Lynch, 1971; Cisneros-Heredia and
McDiarmid, 2007). Their function is to facili-
tate the male’s grip on the female during am-
plexus (Lataste, 1876; Boulenger, 1897; Noble,
1931; Liu, 1936). Well-developed nuptial pads
may be associated with breeding in water, with
less developed structures being found in ter-
restrial breeders (Duellman and Trueb, 1986),
presumably because females would be more
difficult to clasp in water (Wells, 2007), al-
though this has not been experimentally tested.
In Pristimantis, a genus without aquatic breed-
ing, nuptial pads are taxonomically widely dis-
tributed (Lynch and Duellman, 1997; Duellman
and Lehr, 2009). Despite the lack of direct ob-
servations of the reproductive behaviour of P.
dorado sp. nov., we assume that, as with the rest
of the genus, these species are terrestrial breed-
ers. Clearly, the presence of nuptial pads is not
strictly related to breeding in water. The func-
tional implications of variation in nuptial pad
morphology are presently unknown for Pristi-
mantis. More research is needed to understand
the diversity and evolution of these characters.
A distinctive feature of P. dorado is the abil-
ity of males to call in the absence of both a
vocal sac and vocal slits, morphological struc-
tures widely associated with the production of
calls (Wells, 2007; Duellman and Lehr, 2009).
Among Terrarana, several species of the Crau-
gastor gollmeri species group lack these struc-
tures yet they may emit calls (Ibañez et al.,
2012; Salazar-Zuñiga and García-Rodríguez,
2014), conditions not previously reported for
Pristimantis (J.D. Lynch, pers. comm.). We sus-
pect that in more species of Pristimantis, and
perhaps of other genera, males without these
structures may also be able to vocalize, but that
this has been overlooked.
12 M. Rivera-Correa, F. Lamadrid-Feris, A.J. Crawford
The discovery of a morphologically distinc-
tive species not far from a major metropolis
such as Bogotá emphasizes the fact that, despite
recent advances in the alpha taxonomy of Pris-
timantis (or Eleutherodactylus sensu lato), our
knowledge of the true diversity of this genus re-
mains quite incomplete. The large genetic dis-
tances among species observed here based on
the conservative p-distance measure of diver-
gence among the COI and 16S genes (table 2)
support the idea that many species of Pristiman-
tis are missing from our tree. The unsatisfactory
resolution especially of the basal relationships
in our tree highlights the fact that much larger
molecular datasets are needed in order to con-
fidently designate new species to well-defined
species groups within the genus or infer the bio-
geographic history of these lineages.
Acknowledgements. We thank M. Rada, S. Castroviejo,
D. Gonzalez, O. Nieto and I. Sánchez for field assistance
and A. Muñoz-Ortiz for conducting all molecular laboratory
work. We are grateful to J. M. Daza (MHUA), J. D. Lynch
(ICN) and S. Ron (QCAZ) for allowing access to collections
under their care. We are thankful to S. Castroviejo-Fisher for
allowing us to use his photographs. Edgar Lehr and Karl-
Heinz Jungfer kindly provided suggestions and comments
on the manuscript. This biological collection was made with
financial support from the technology cooperation agree-
ment No. 7-24100-925-2007 between Bogota’s Aqueduct
water company and Conservation International – Colom-
bia. Additional samples were obtained and DNA-sequenced
under research and collecting permit no. 15 and access to
genetic resources permit no. 44 to A.J.C. by the Ministe-
rio de Ambiente, Vivienda y Desarrollo Territorial. Labora-
tory work was financed by grant no. 156-09 from Ecopetrol.
Special thanks to J.V. Rodríguez-Mahecha for supporting
the study of the diversity of amphibians in Colombia and
to S. Guerra and P. Bejarano for their interest and support
to make this article a product of the conservation corridor.
Postdoctoral Fellow support for M.R.C. was provided by
Consejo Nacional de Investigaciones Científicas y Técnicas
de Argentina (CONICET).
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14 M. Rivera-Correa, F. Lamadrid-Feris, A.J. Crawford
Appendix. Specimens examined
Pristimantis acutirostris: COLOMBIA: Santander, Charalá,
Virolín, 1780, ICN 12374 (holotype); Santander, Gám-
bita, ICN 11063; Santander, Charalá, ICN 11281.
Pristimantis atratus: ECUADOR: Morona Santiago, Ran-
cho Suro, USNM 199675 (holotype).
Pristimantis lutitus: COLOMBIA: Santander, Charalá, Rio
Luisito, ICN 5192 (holotype).
Submitted: July 3, 2015. Final revision received: January
19, 2016. Accepted: February 4, 2016.
Associate Editor: Julian Glos.