Print
A new Terrarana frog of genus Pristimantis from an unexplored cloud forest from the eastern Andes, Colombia
expand article infoAndrés R. Acosta-Galvis, Ana M. Saldarriaga-Gómez§, Beatriz Ramírez|, Mario Vargas-Ramírez§
‡ Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, Boyacá, Colombia
§ Universidad Nacional de Colombia, Bogotá, Colombia
| Asociación de Becarios de Casanare, Yopal, Colombia
¶ Estación de Biología Tropical Roberto Franco, Villavicencio, Colombia
Open Access

Abstract

A new species of Pristimantis (Craugastoridae, subgenus Pristimantis) is described from a relict and unexplored cloud forest in the western slope from Cordillera Oriental of the Colombian Andes. The specific name was chosen by consensus expert scientists and local people. Pristimantis chamezensis sp. nov. is easily distinguished from congeneric species by having a gray iris with black reticulations in life, subconical tubercles on the upper eyelid, the chin edged with irregular, dark-brown blotches, and conical heel tubercles. The phylogenetic analyses suggest that the origin and radiation of its clade may have occurred in the highlands. With the description of P. chamezensis sp. nov., we identify 14 species distributed throughout the eastern slope of the Andes that are associated with the Orinoco Basin.

Keywords

Casanare, Cis-Andean, Cordillera Oriental, diversity, phylogeny, South America, taxonomy

Introduction

The amphibian fauna from Colombia is among the richest and most diverse in the world (Lynch 1999; Grant et al. 2008) and includes 843 species (Acosta-Galvis 2020). A significant number of these species is grouped in the so-called Terrarana; an unranked taxonomic grouping of at least four closely related families characterized by direct development, egg embryos, and terrestrial reproduction (Hedges et al. 2008; Heinicke et al. 2009, 2018). Terrarana richness in Colombia includes 268 species in 13 genera (Acosta-Galvis 2020), among which the frogs of the Pristimantis genus represent the greatest diversity with 83% of the described species.

Morphologically, frogs of the genus Pristimantis are easily recognizable among other features by terminal discs on expanded digits and T-shaped terminal phalanges, a dentigerous process of the vomers usually present, and toe IV much longer than toe III (Hedges et al. 2008; Duellman and Lehr 2009). However, this genus still has latent phylogenetic challenges (Navarrete et al. 2016), and recent proposals, based on molecular phylogenies (Hedges et al. 2008; Padial et al. 2014; Páez and Ron 2019; Reyes-Puig et al. 2020), reassigned or excluded members of the species groups from evolutionary arrangements, which were previously based solely on morphological evidence (Lynch and Duellman 1980, 1997).

The genus Pristimantis in Colombia is represented by 223 formally described species (Acosta-Galvis 2020). The geographic and ecological complexity of the Andes harbors the greatest richness and rate of endemism in contrast to the lowlands of the Amazon and Pacific basins (Lynch et al. 1997). Current geological evidence of the north-Andean region indicates that the northern formations in Colombia (Occidental, Central, and Oriental mountains ranges) have promoted speciation processes in the genus Pristimantis and, therefore, have high diversity and endemism (Lynch and Duellman 1997; Lynch 1999; García-R et al. 2012; Mendoza et al. 2015; Meza-Joya and Torres 2016; Acevedo et al. 2020). Among these geographical units, the Cordillera Oriental contains 44 species, with 13 species inhabiting the Andean and sub-Andean forests on the eastern slopes (Table 1), as part of the Orinoco basin (Acosta-Galvis et al. 2010; Rivera-Correa et al. 2016; Ospina-Sarria and Angarita-Sierra 2020; Acevedo et al. 2020).

Table 1.

Species of the genus Pristimantis from the eastern slope of Cordillera Oriental (Orinoco Basin) in Colombia.

Genus (Subgenus) Species Species group Ecoregional distribution Altitude(m a.s.l) Reference
Pristimantis (Pristimantis) vilarsi conspicillatus group sub-Andean, Amazonian and Orinoco. 200–600 Lynch 1975; 1980, 1994; Padial et al. 2014; Heyer and Barrio-Amorós 2009.
Pristimantis (Pristimantis) medemi conspicillatus group Andean and sub-Andean. 450–2400 Lynch 1994, 2006; Malambo and Marin 2006; Acosta-Galvis et al. 2010; Acosta-Galvis and Alfaro-Bejarano 2011.
Pristimantis (Pristimantis) carranguerorum conspicillatus group Andean. 1350–2060 Lynch 1994; Renjifo-Rey 2005; Acosta-Galvis and Alfaro-Bejarano 2011; Anganoy-Criollo and Ramírez 2017.
Pristimantis (Hypodictyon) w-nigrum ridens group Andean and sub-Andean. 800–3000 Cochran and Goin 1970; Lynch and Duellman 1980; Bernal and Lynch 2008.
Pristimantis (Pristimantis) savagei Unassigned Andean and sub-Andean. 600–3000 Pyburn and Lynch 1981; Lynch 1994; Ruiz-Carranza et al. 1996; Acosta-Galvis 2000; Lynch 2006; Bernal and Lynch 2008; Acosta-Galvis et al. 2010; Acosta-Galvis and Alfaro-Bejarano 2011.
Pristimantis (Pristimantis) frater Unassigned Andean and sub-Andean. 600–3000 Pyburn and Lynch 1981; Lynch 1994; Ruiz-Carranza et al. 1996; Acosta-Galvis 2000; Lynch 2006; Bernal and Lynch 2008; Acosta-Galvis et al. 2010; Acosta-Galvis and Alfaro-Bejarano 2011.
Pristimantis (Pristimantis) bogotensis Unassigned Andean, sub-paramos and paramos. 2410–3520 Cochran and Goin 1970; Ruiz-Carranza et al. 1996; Acosta-Galvis 2000; Bernal and Lynch 2008.
Pristimantis (Pristimantis) anolirex Unassigned Andean, sub-paramos and paramos. 1800–3550 Lynch 1983; Ardila-Robayo and Acosta-Galvis 2000; Bernal and Lynch 2008.
Pristimantis (Pristimantis) lynchi Unassigned Andean, sub-paramos and paramos. 1600–3590 Duellman and Simmons 1977; Bernal and Lynch 2008; Acosta-Galvis 2015.
Pristimantis (Pristimantis) dorado Unassigned Andean. 2650 Rivera-Correa et al. 2016
Pristimantis (Pristimantis) terrapacis Unassigned sub-Andean 713 Ospina-Sarria and Angarita-Sierra 2020
Pristimantis (Pristimantis) ardilae conspicillatus group sub-Andean 400–700 Acevedo et al. 2020
Pristimantis (Pristimantis) bowara Unassigned sub-Andean 500–665 Acevedo et al. 2020

During field studies along an unexplored cloud forest (2140 m a.s.l.) in the Cordillera Oriental, we collected several specimens of Pristimantis that, due to their morphological characters, are not assignable to any described species in this region. Based on the analysis of its molecular data and morphology, we describe a new species recognized by its molecular and morphological distinctiveness.

Methods

Study area

We collected by actively searching from September 2 to November 29, 2010, using intensive visual encounter surveys (Crump and Scott 1994) during evenings in the cloud forests in the municipality of Chámeza (05°15'24.4"N, 072°53'51.6"W), Department of Casanare, Colombia (Fig. 1). This locality is part of an elevated area between 1700–2200 m a.s.l. in an unexplored northern portion of the Cordillera Oriental. This mountainous area consists mainly of pristine natural forests of the Andes orobiome (Fig. 2) within the ecoregion of the Eastern Cordillera montane forests of Colombia (Dinerstein et al. 1995; Olson and Dinerstein 2002). We recorded geographical coordinates and elevations at collecting sites using a Garmin GPSMAP 60CSx (map datum WGS 84).

Figure 1. 

Geographic location in Colombia showing the type locality of Pristimantis chamezensis sp. nov. in the western slope of the Cordillera Oriental A red dot shows the type locality B the landscape of natural pristine forest on the eastern slopes of the Central Cordillera Oriental. Map produced using Arc Map, World Imagery.

Figure 2. 

A general landscape showing the mountains of the Vereda Centro Norte, Chámeza forest at Cerro Pan de Azúcar (black arrow); type locality of Pristimantis chamezensis sp. nov. B inside the cloud forests; microhabitat where individuals were found. Photographs by Andrés Acosta-Galvis.

Data collection and laboratory procedures

Molecular distinctiveness and phylogenetic relationships of the new species were assessed by analyzing DNA sequences of mitochondrial DNA (mtDNA) which included a fragment of the 16S ribosomal RNA (16S) and a fragment of the cytochrome oxidase subunit 1 (COI) genes. We assembled a data set that included only the 16S gene fragment by aligning sequences from all known Pristimantis species from the eastern slopes of the Cordillera Oriental of Colombia together with the most similar sequences already published in Genbank (Table 2). For this, we conducted a search for sequences similar to the 16S gene fragment of the new species using the BLAST algorithm in GenBank. The most similar 127 BLAST hits to the sequences from the new species were downloaded, aligned, and assessed using Bayesian (BA) and maximum likelihood (ML) analyses. After removing distant and redundant sequences, the final dataset contained 58 sequences of 827 base pairs (bp) of the 16S, including the new species and Pristimantis medemi (Lynch, 1994) obtained in this study (Table 1). We assembled a complete data set comprising sequences of the 16S, concatenated with sequences of the COI gene for a subset, including the new species and its following six most-related species, selected based on the results of the analyses: Pristimantis carranguerorum (Lynch, 1994), P. bowara Acevedo et al., 2020, P. lutitus (Lynch, 1984), P. medemi (Lynch, 1994), P. nicefori (Cochran & Goin, 1970), and P. savagei (Pyburn & Lynch, 1981).

Table 2.

Species of Eleutherodactylus, Pristimantis, and GenBank accession numbers of the DNA sequences used in the phylogenetic analyses.

Species Accession numbers Voucher code Source
16S rRNA COI
E. johnstonei EF493561 USNM336018 Heinicke et al. 2007
P. acatallelus JN371032 UVC:15863 García-R et al. 2012
P. achatinus EF493660 KU217809 Heinicke et al. 2007
P. achatinus JN104676 UVC:15867 García-R et al. 2012
P. aniptopalmatus EF493390 KU291627 Heinicke et al. 2007
P. bipunctatus EF493702 KU291638 Heinicke et al. 2007
P. bogotensis JN991432 JN991362 NRPS003 Pinto-Sanchez et al. 2012
P. bowara MN215434 MCNUPH304 Acevedo, Armesto and Palma 2000
P. buccinator KY652650 MUSM:33269 von May et al. 2017
P. buckleyi EF493350 KU217836 Heinicke et al. 2007
P. caprifer EF493391 KU177680 Heinicke et al. 2007
P. carranguerorum KP149324 KP149128 LSB385 Guarnizo et al. 2015
P. chamezensis sp. nov. MK776946 MK789293 ARA5848 This study
P. chamezensis sp. nov. MK776947 MK789294 ARA5849 This study
P. citriogaster EF493700 KU212278 Heinicke et al. 2007
P. condor EF493701 KU217857 Heinicke et al. 2007
P. conspicillatus EF493529 QCAZ28448 Heinicke et al. 2007
p. curtipes EF493513 KU217871 Heinicke et al. 2007
P. devillei EF493688 KU217991 Heinicke et al. 2007
P. dorado KU496877 MRC636 Rivera-Correa et al. 2016
P. duellmani AY326003 WED 53050 Darst and Cannatella 2004
P. fenestratus EF493703 Heinicke et al. 2007
P. frater KP149461 AJC 4015 Guarnizo et al. 2015
P. gentryi EF493511 KU218109 Heinicke et al. 2007
P. koehleri EU192279 MNKA 6627 Padial and De la Riva 2009
P. lasalleorum KY494221 ICN55758 González-Durán et al. 2017
P. latro MK174413 LZA 1340 Cornelio et al. unpublished
P. leptolophus KY494226 JJS093 González-Durán et al. 2017
P. lutitus KP149401 KP149196 AJC3490 Guarnizo et al. 2015
P. lymani EF493392 KU218019 Heinicke et al. 2007
P. maculosus KY494240 ICN55760 González-Durán et al. 2017
P. malkini EU186663 QCAZ28296 Hedges et al. 2008
P. medemi MK776948 MK789295 ARA2655 This study
P. nicefori MN215436 MN218387 MCNUPH48 Acevedo, Armesto and Palma 2000
P. parectatus KY627810 MHUAA9977 Rivera-Correa et al. 2017
P. peraticus KY494224 WB1301 González-Durán et al. 2017
P. peruvianus EF493707 Heinicke et al. 2007
P. quinquagesimus EF493690 KU179374 Heinicke et al. 2007
P. rhabdolaemus EF493706 KU173492 Heinicke et al. 2007
P. sagittulus EF493705 KU291635 Heinicke et al. 2007
P. samaipatae EU192290 MNCN 42988 Padial and De la Riva 2009
P. savagei KP149382 KP149180 AJC3995 Guarnizo et al. 2015
P. scoloblepharus KY494236 ICN55768 González-Durán et al. 2017
P. skydmainos EF493393 Heinicke et al. 2007
P. simonbolivari EF493671 KU218254 Heinicke et al. 2007
P. simonsii EU186665 KU212350 Hedges et al. 2008
P. surdus EF493687 KU177847 Heinicke et al. 2007
P. sp.1 KY494239 JJS122 González-Durán et al. 2017
P. sp.2 KY494238 ICN55759 González-Durán et al. 2017
P. sp.3 KY494230 ICN55756 González-Durán et al. 2017
P. sp.4 KY494234 ICN55774 González-Durán et al. 2017
P. sp.5 KY494223 ICN55775 González-Durán et al. 2017
P. stictogaster EF493704 KU291659 Heinicke et al. 2007
P. thymalopsoides EF493514 KU177861 Heinicke et al. 2007
P. toftae EF493353 KU215493 Heinicke et al. 2007
P. unistrigatus EF493387 KU218057 Heinicke et al. 2007
P. uranobates KY494223 ICN55787 González-Durán et al. 2017
P. vertebralis EF493689 KU177972 Heinicke et al. 2007
P. vilarsi KP149391 KP149187 AJC2113 Guarnizo et al. 2015

From two tissue samples of the new species and a tissue sample of Pristimantis medemi we extracted total genomic DNA using a standard Phenol-Chloroform method (Sambrook et al. 1989). We amplified the gene fragments using the primers pairs 16Sbr-H/16SC-16L (Palumbi et al. 1991; Darst and Cannatella 2004, respectively) and LCO1490/HCO2198 (Folmer et al. 1994) for the 16S and COI, respectively. We carried out PCRs in a total volume of 30 μl containing one unit Taq polymerase (Bioline; Randolph, MA), 1× of a buffer (Bioline), a final concentration of 1.5 mM MgCl2 (Bioline), 0.5 μM of each primer, 0.2 mM of each dNTP (Bioline), 0.2 µg of bovine serum albumin (BSA), and approximately 50 ng of total DNA. We purified the PCR products using the ammonium acetate protocol (Bensch et al. 2000), and we sequenced them on an ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using the BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems). We stored the remaining DNA extractions at –80 °C in the tissue collection of the Instituto de Genética, Universidad Nacional de Colombia (for voucher numbers see Table 2). We performed the thermocycling conditions as indicated by the authors, who reported the primers for the obtained fragments. The GenBank accession numbers of the obtained sequences are MK776946MK776948 and MK789293MK789295. We edited and aligned the sequences using Chromas 1.51 (http://www.technelysium.com.au/chromas.html) and BioEdit v. 7.0.5.2 (Hall 1999). To exclude divergent regions and poorly aligned bases from the 16S dataset, we used the software Gblocks v. 0.91b (Castresana 2000; Talavera and Castresana 2007; available as a web server at http://molevol.cmima.csic.es/castresana/Gblocks server.html), which resulted in a final alignment of 528 base pairs (bp). The COI alignment consisted of 652 bp.

Phylogenetic and genetic divergence analyses

We analyzed the complete evidence dataset using the following partition scheme: (i) unpartitioned; (ii) partitioned by gene (i.e., each gene fragment treated as a distinct partition); and (iii) maximum partitioning (i.e., we treated each codon of the protein-coding gene COI and the ribosomal gene fragment as distinct partitions). We assessed the optimal partitioning scheme and best-fit evolutionary models using PartitionFinder v. 1.1.1 and the Bayesian Information Criterion (Lanfear et al. 2012), resulting in the selection of the maximum partitioning scheme. For the 16S dataset, the obtained model (SYM + G) was applied in a Bayesian analysis (BA) with MrBayes v. 3.2.1 (Ronquist et al. 2012). For the complete evidence dataset, we applied the 16S fragment model plus the following complementary COI fragment resulting models in a Bayesian analysis with MrBayes: COI 1st codon – TrNef + G, COI 2nd codon – HKY, COI 3rd codon – HKY. We incorporated these models into a single tree search (mixed model partition approach; Nylander et al. 2004). For both analyses, we carried out two parallel runs using four Markov chains, each starting from a random tree. We ran the Markov chains for 10 million generations. The burn-in was set to sample only the plateau of the most likely trees that were used for generating a 50% majority rule consensus. We used the software TRACER v. 1.5.4 (Rambaut and Drummond 2007) to assess an acceptable level of the MCMC chain mixing and to estimate effective sample sizes for all parameters. Additionally, maximum likelihood (ML) analyses were run using RAxML 7.2.8 (Stamatakis 2006) and the GTR+G model. We performed five independent maximum likelihood searches with different starting conditions and the rapid bootstrap algorithm to explore the robustness of the branching patterns by comparing the best trees. Afterward, 1000 non-parametric thorough bootstrap values were computed and plotted against the best tree. The Genbank sequence of Eleutherodactylus johnstonei Barbour, 1914, EF493561, was used as outgroup. To assess the genetic divergence between the new and the other Pristimantis species, we calculated uncorrected p genetic distances for the 16S and the COI fragments using MEGA v. 7.0.21 (Kumar et al. 2016).

Morphology

We euthanized specimens using Clorethone, which were then fixed in 10% formalin, preserved in 70% ethanol, and deposited in the biological collections of the Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, Villa de Leyva, Boyacá, Colombia (IAvH-Am). Other specimens examined are listed in Suppl. material 1. The criteria for the definition of descriptions and diagnostic characters followed Duellman and Lehr (2009), Lynch and Duellman (1997), and Navarrete et al. (2016). To identify sex and sexual maturity, we made a small incision in the groin region for macroscopic observation of the gonads. Adult males have the granular testis, while females show enlarged, thickened, and convoluted oviducts. Morphometric measurements were made with digital calipers (nearest 0.01 mm) or a Nikon stereoscopic microscope SMZ-1B with high Intensity Illuminator NI-150 Nikon as follows: SVL (snout-vent length), HW (head width), HL (head length from the tip of the snout to the posterior border of the skull, posterior edge of prootic, noted through the skin), IOD (interorbital distance), ED (eye diameter), EN (eyes-nares distance), UEW (upper eyelid width), ETS (distance between the anterior edges of the eye to the tip of the snout), TD (horizontal tympanum diameter), RW (rostral width), InD (internarial distance), TL (tibial Length), FL (femur length), FtL (foot length), and HnL (hand length). Means are reported ± one standard error. We photographed habitats and specimens using Canon EOS 30D and EOS 5D Mark II digital cameras inside a Photo Safe-box using 5.500 kelvins LED lights.

Results

Phylogenetic and genetic divergence analyses

The resulting phylogenetic tree including all 58 sequence of the 16S fragment is shown in the Suppl. material 2: Fig. S1. A reduced phylogenetic tree including the 16S fragment sequences of the new species and its closest 29 sequences is shown in Figure 3. The following description is referring to the reduced tree. Based on the phylogenetic relationships, the new species could be assigned to the genus Pristimantis, subgenus Pristimantis. Both tree-building methods revealed Pristimantis chamezensis sp. nov. with maximum support within a supported monophyletic group comprising Pristimantis carranguerorum, P. bowara, P. lutitus, P. medemi, P. nicefori, and P. savagei (Fig. 3). Both analyses concurred in placing the new species as a sister taxon of P. nicefori with low support (ML: 40%; BA: 0.80). The other 23 Pristimantis species were revealed by both analyses within three separated, weakly supported clades, exhibiting low supported evolutionary relationships (Fig. 3). For the complete evidence dataset, both tree building methods revealed Pristimantis chamezensis sp. nov., as part of a monophyletic clade also comprising P. carranguerorum, P. bowara, P. lutitus, P. medemi, P. nicefori, and P. savagei with maximum support (Suppl. material 3: Fig. S2). Both analyses revealed that the new species is the sister taxon of a clade showing the following weakly supported phylogenetic relationships: (((P. lutitus + P. bowara) P. nicefori) P. carranguerorum). Finally, P. medemi and P. savagei were revealed as successive sister taxa of the that clade plus the new species, with low support (Suppl. material 3: Fig. S2). Genetic distances for the 16S gene between P. chamezensis sp. nov. and P. nicefori, P. carranguerorum, and P. savagei were 4.8%, 5.2%, and 5.9%, respectively. Distances between P. chamezensis sp. nov. and P. medemi, P. lutitus, and P. bowara were 6.2%, 6.2%, and 6.7%, respectively (Table 3). The sequence divergence range of the monophyletic group compared to the other analyzed taxa was 5.9–4.1% (Table 3). The uncorrected p distances for the COI gene revealed that sequence differentiation values between P. chamezensis sp. nov. and P. carranguerorum, P. nicefori, P. lutitus, P. savagei, and P. medemi were 6.2%, 6.4%, 6.7%, 6.7%, and 6.7%, in that order. For the same gene fragment, the distance between P. chamezensis sp. nov. and P. bowara was 7.8%.

Figure 3. 

Maximum likelihood inference tree showing the evolutionary relationships of Pristimantis chamezensis sp. nov. (bold) and its 28 more closely related Pristimantis species based on 528 bp of the 16S rRNA gene. Numbers before nodes: thorough maximum likelihood (ML) bootstrap percentages left and Bayesian analysis (BA) posterior probability values right. Bootstrap values below 50% and posterior probabilities below 0.5 not shown. Outgroup taxon removed.

Table 3.

Uncorrected p-distances for the fragment of 16S gene (528 bp) of the Pristimantis species, expressed as percentages (means).

n cha nic car sav med lut bow cit mal con buc lym ach con sti rha cap ach tof plu ani bip lat koe fen sam sag vil dor sky
chamezensis sp. nov. 2 0.0
nicefori MN215436 1 4.8
carranguerorum KP149324 1 5.2 4.8
savagei KP149382 1 5.9 5.2 5.5
medemi MK776948 1 6.2 5.9 6.2 5.0
lutitus KP149401 1 6.2 6.2 6.9 5.9 7.6
bowara MN215434 1 6.7 6.2 6.4 4.0 5.7 6.9
citriogaster EF493700 1 7.8 6.7 6.2 5.9 6.4 7.1 6.4
malkini EU186663 1 8.3 7.8 8.6 9.0 8.8 10.2 9.2 10.0
conspicillatus EF493529 1 8.3 8.3 9.3 9.5 8.8 10.0 10.0 10.2 3.1
buccinator KY652650 1 8.3 8.3 9.7 8.8 9.3 9.7 10.4 10.0 2.8 2.4
lymani EF493392 1 8.3 8.3 10.5 8.3 8.8 9.7 10.0 10.9 4.0 3.8 2.1
achatinus EF493660 1 8.8 8.3 9.0 7.8 8.8 9.5 9.0 9.7 3.8 4.7 3.8 5.0
condor EF493701 1 8.8 8.8 9.8 9.3 10.0 10.2 10.5 11.7 5.2 5.0 4.8 5.9 4.3
stictogaster EF493704 1 9.0 8.8 9.7 9.2 9.7 9.5 10.0 10.7 4.0 3.3 2.8 4.5 4.7 5.5
rhabdolaemus EF493706 1 9.0 9.0 9.8 9.7 8.8 10.9 10.5 9.8 9.7 9.5 9.3 10.2 9.5 9.8 9.7
caprifer EF493391 1 9.1 9.0 9.5 9.0 8.1 9.7 9.7 9.7 9.7 9.7 9.2 10.2 9.7 9.3 9.2 3.1
achatinus JN104676 1 9.2 9.1 9.1 9.3 9.1 10.3 9.8 9.1 9.3 8.6 8.4 9.5 8.4 7.9 9.5 9.3 8.6
toftae EF493353 1 9.2 9.2 10.2 10.9 11.9 11.1 11.1 13.1 7.1 7.3 6.9 7.6 7.8 7.6 5.9 10.9 10.7 11.2
aniptopalmatus EF493390 1 9.2 9.2 10.5 10.7 10.0 10.7 10.9 9.7 10.9 10.4 10.7 11.6 10.9 11.6 10.7 3.3 4.3 10.3 11.8 4.3
bipunctatus EF493702 1 9.5 9.2 9.5 9.5 9.0 11.1 10.2 9.5 9.7 10.0 9.2 10.2 9.2 10.5 10.2 2.4 3.1 9.5 11.1 3.6 2.8
latro MK174413 1 10.0 9.5 9.8 9.0 10.2 10.2 9.5 11.0 6.9 6.4 6.2 6.9 6.4 7.1 5.7 10.0 10.7 9.3 7.8 10.5 10.5 10.2
koehleri EU192279 1 10.0 10.0 11.5 10.5 9.4 12.2 10.8 11.3 7.9 7.7 8.6 9.8 9.1 8.6 8.4 9.6 9.3 9.4 11.2 9.8 11.0 10.0 9.4
fenestratus EF493703 1 10.0 10.0 11.4 11.4 10.2 11.1 11.6 11.6 7.8 8.1 8.8 9.2 7.6 8.6 7.6 10.9 10.4 10.0 8.5 10.4 11.8 11.1 8.6 6.9
samaipatae EU192290 1 10.4 10.0 11.9 11.4 10.0 10.7 11.6 11.6 8.3 7.6 8.8 9.2 7.8 8.8 7.6 11.2 10.2 10.0 8.8 10.2 12.1 10.9 9.3 6.7 1.2
sagittulus EF493705 1 11.1 10.4 10.5 10.2 9.7 10.2 10.9 10.2 8.1 7.6 7.6 8.8 7.6 8.6 7.8 10.5 10.2 10.0 10.2 10.9 11.1 9.7 9.3 7.2 3.3 3.6
vilarsi KP149391 1 11.4 11.1 11.4 10.2 10.2 12.1 11.1 10.5 11.6 11.4 11.1 11.6 11.1 11.4 11.6 3.6 4.0 10.7 13.0 4.3 4.5 3.1 11.6 11.0 12.1 11.8 11.1
dorado KU496877 1 11.4 11.4 10.0 9.5 10.0 12.1 11.2 11.4 8.1 7.6 7.8 8.6 7.6 7.6 7.8 11.2 10.9 10.8 10.9 12.8 13.1 11.4 9.0 9.1 10.2 10.5 9.3 12.6
skydmainos EF493393 1 11.6 11.4 13.1 11.4 14.1 13.6 12.8 14.1 9.6 9.6 8.9 9.4 11.4 11.9 9.6 12.1 13.3 12.7 12.6 13.8 13.8 12.6 10.4 11.2 11.9 11.9 11.6 14.1 12.8

Description of new species

Pristimantis chamezensis sp. nov.

Figs 4, 5; Table 4

Holotype

IAvH-Am-10269 (field number ARA 5852. Figs 4, 5) an adult female collected on 3 September 2010 by Andrés R. Acosta-Galvis, Beatriz Ramirez, José Pérez, Luis Daniel Prada, and Natalia Novoa.

Figure 4. 

Pristimantis chamezensis sp. nov. preserved holotype, adult female, IAvH-Am-10269 (SVL = 23.8 mm) A dorsal view B ventral view C lateral view. White arrow = chin with irregular blotches of dark brown. Scale bar: 10 mm. Photographs by Andrés Acosta-Galvis.

Type locality

(Figs 1, 2). Colombia, Casanare Department, Chámeza Municipality, vereda Centro Norte, Chámeza forest, Cerro Pan de Azúcar, eastern flank of the Cordillera Oriental, Colombia. 05°15'24.4"N, 072°53'51.6"W, 2140 m a.s.l.

Paratypes

(11) (Fig. 5; Table 4). IAvH-Am-10267, IAvH-Am-10270–10274, adult males; IAvH-Am-10275–10277, IAvH-Am-10282, adult females, collected on 13 November 2010 by Andrés R. Acosta-Galvis, Beatriz Ramirez, José Pérez, Luis Daniel Prada, and Natalia Novoa; same locality as the holotype.

Figure 5. 

Pristimantis chamezensis sp. nov., live specimens. A Holotype, adult female, IAvH-Am-10269 (SVL= 23.8 mm) B juvenile, IAvH-Am-10283 (SVL = 17.5 mm) C paratype, adult female, IAvH-Am-10277 (SVL = 19.7 mm) D paratype, adult male, IAvH-Am-10267 (SVL = 22.6 mm). Photographs by Andrés Acosta-Galvis.

Referred specimens

IAvH-Am-10268, IAvH-Am-10278–10281, IAvH-Am-10283–10287, juveniles, same locality and date as paratypes.

Diagnosis

(Figs 47). A species of Pristimantis characterized by the following combination of morphological characters: (1) dorsal skin shagreen with scattered larger tubercles; dorsolateral folds absent; discoidal fold visible; skin on venter areolate. (2) Tympanic membrane and tympanic annulus present, its dorsoposterior border converges with supratympanic fold; its diameters are 35.6–56.0% of the eye diameter; small, barely visible, subconical postrictal tubercles. (3) Snout short, broadly rounded in dorsal view and rounded in lateral view; canthus rostralis sharp and concave. (4) Upper eyelid bearing one to three subconical tubercles, narrower than IOD. (5) Choanae small, subovoid; dentigerous processes of vomers prominent, oblique, and widely separated from each other, bearing 8 or 9 teeth. (6) Males with vocal slits; subgular vocal sac observable; nuptial pads not evident. (7) Finger I shorter than II, with discs expanded and rounded; bifid palmar tubercle. (8) Fingers bearing narrow lateral fringes. (9) Ulnar tubercles absent. (10) Tarsal tubercles present, subconical; heel tubercles present but nearly inconspicuous and conical. (11) Two metatarsal tubercles, with inner tubercle elongate, three times the length of the rounded and prominent outer tubercle; supernumerary plantar tubercles numerous, enlarged, and rounded. (12) Toes with lateral fringes and broad discs; toe V much longer than toe III (disc on toe III extends to the proximal edge of the medial subarticular tubercle on toe IV, disc on toe V extends beyond the distal edge of the penultimate subarticular tubercle on toe IV); webbing absent. (13) Dorsal surface pattern variable, with homogeneous color brown (with or without paravertebral line) or inverted V-shaped markings with dark brown blotches edged with pale cream; iris gray, medially reddish, with black reticulations; ventral surfaces cream-colored to light brown, finely peppered with irregular, diffuse, dark-brown reticulations or blotches; posterior surface of thighs brown; dark-brown labial bars present or absent; edge of the chin with irregular blotches of dark brown (Fig. 5). (14) apparently sexually dimorphic in size (Table 4), with an SVL in adult males 19.6–23.7 mm and 19.0–24.9 mm in adult females.

Table 4.

Morphometric (in mm) of the type series of Pristimantis chamezensis sp. nov. Abbreviations are given in Methods.

IAvH–Am Sex SVL HW HL IOD ED EN UEW ETS TD FL FtL InD RW TL HnL
10267 M 22.6 7.9 10.1 3.1 3.1 3.0 1.8 4.1 1.7 10.6 10.9 2.5 3.2 12.5 6.4
10271 M 23.7 9.2 9.1 3.6 2.8 3.0 2.0 4.6 1.6 12.6 10.8 2.5 3.8 12.6 6.5
10273 M 20.9 7.5 7.6 2.8 2.8 2.4 2.1 3.7 1.1 10.9 9.6 2.5 3.6 11.6 5.9
10270 M 19.6 7.9 8.9 2.6 2.6 2.8 1.7 3.5 1.1 10.6 9.9 2.3 2.7 11.7 6.1
10274 M 21.2 8.3 9.4 2.8 2.9 3.1 1.8 4.0 1.2 10.5 9.9 2.4 3.3 11.8 5.9
10272 M 20.3 8.0 9.3 3.0 2.7 2.4 1.8 3.7 1.0 10.0 10.1 2.5 2.8 12.2 6.3
Means 21.4 8.1 9.1 3.0 2.8 2.8 1.9 3.9 1.3 10.9 10.2 2.5 3.2 12.1 6.2
Standard error 1.4 0.5 0.8 0.3 0.2 0.3 0.1 0.4 0.3 0.8 0.5 0.1 0.4 0.4 0.2
10276 F 24.9 11.0 11.3 3.4 3.5 3.1 2.6 4.7 1.2 14.3 12.9 3.1 3.8 14.4 8.4
10277 F 19.7 8.1 9.0 2.9 2.8 2.4 1.6 4.1 1.0 11.0 8.8 2.5 2.7 12.1 6.0
10269 F 23.8 9.8 10.4 3.8 3.3 3.2 2.1 4.5 1.1 13.6 12.1 2.9 3.9 14.5 7.4
10275 F 19.0 7.8 9.0 2.9 2.4 2.8 1.7 4.1 1.0 9.7 9.4 2.1 3.1 11.5 5.6
Means 21.9 7.9 8.5 2.9 2.6 2.6 1.8 3.8 1.1 10.6 9.6 2.3 3.1 11.5 6.8
Standard error 2.9 1.4 1.1 0.4 0.4 0.3 0.4 0.2 0.1 2.1 1.9 0.4 0.5 1.5 1.3

Species comparisons

(Figs 57, Suppl. material 1). The new species is compared to other species of Pristimantis in the eastern slope of the Cordillera Oriental in the Orinoco basin of Colombia. The character states of the compared species are enclosed in parentheses. Pristimantis chamezensis is distinguished from P. carranguerorum by the absence of short dorsolateral folds in the scapular region (present); snout rounded in dorsal view (subacuminate; Fig. 6); the dorsum brown, with some lighter and diffuse reticulations (pale dorsolateral lines; Fig. 6); and subconical tubercles on the upper eyelid (absent). The new species differs from P. vilarsi (Melin, 1941) in having the posterior surfaces of the thighs brown in life (reddish); adult females smaller, SVL 19.0–24.9 mm (SVL 25.4–43.2 mm); and the snout broadly rounded in dorsal view (subacuminate). Pristimantis chamezensis can be easily confused with P. savagei by the presence of one to three subconical tubercles on the upper eyelid; however, it differs by the absence of ulnar tubercles (present); snout broadly rounded in dorsal view (subacuminate); and posterior surface of thighs brown in life (pale orange). Pristimantis chamezensis is distinguished from P. medemi by having subconical tubercles on the upper eyelids (absent); dorsal and ventral iris gray in life (Fig. 5), medially reddish, with black reticules (orange to yellow); and smaller size, SVL 19.6–26.4 mm (SVL 29.4–43.1 mm). Pristimantis chamezensis differs from P. anolirex (Lynch, 1983) (Fig. 6) in lacking dorsolateral folds (present on half of the body); ulnar tubercles absent (present and small; Fig. 7); and snout broadly rounded in dorsal view (subacuminate). Pristimantis chamezensis is distinguished from P. lynchi (Duellman & Simmons, 1977) in having the edge of the chin with irregular blotches (Fig. 4) of dark brown (uniformly brown); palmar tubercle bifid (elliptical); and snout broadly rounded in dorsal view (subacuminate). Compared to P. bogotensis (Peters, 1863) (Fig. 6), P. chamezensis has a prominent dentigerous process on the vomers, oblique and widely separated from each other (concealed in the palatine tissue); and broadly rounded snout in dorsal view (rounded). Pristimantis chamezensis differs from P. frater (Werner, 1899) (Fig. 6) in having a broadly rounded snout in dorsal view (acuminate); and toes IV and V with narrow discs (broader). Pristimantis chamezensis is distinguished from P. terrapacis Ospina-Sarria & Angarita-Sierra, 2020 by having subconical tubercles on upper eyelid and heel (absent) and webbing absent between the toes (basal webbing). Pristimantis chamezensis differ from P. ardilae Acevedo et al. 2020 by the absence of short dorsolateral folds in the scapular region (present); broadly rounded snout in dorsal view (subacuminate); and upper eyelid with subconical tubercles (without tubercles). Pristimantis chamezensis is distinguished from P. bowara in having the broadly rounded snout in dorsal view (subacuminate) and dorsal skin shagreen with scattered larger tubercles (smooth). Lastly, P. chamezensis that can be distinguished from P. nicefori (Fig. 6) in having the discs of the digits expanded (slightly expanded), snout broadly rounded in dorsal view (acuminate), and snout broadly rounded in lateral view (pointed).

Figure 6. 

Live specimens (lateral view) of Pristimantis currently known from the eastern Andean Cordillera associated with the Orinoco basin in Colombia. A Pristimantis carranguerorum, Medina Municipality, Cundinamarca Department, IAvH-Am-14954 (adult female, SVL = 22.6 mm) B Pristimantis medemi, Medina Municipality, Cundinamarca Department, IAvH-Am-15025 (adult male, SVL = 32.5 mm) C Pristimantis frater, Medina Municipality, Cundinamarca Department, IAvH-Am-14923 (adult female, SVL = 27.8 mm) D Pristimantis savagei, Medina Municipality, Cundinamarca Department, IAvH-Am-14933 (adult male, SVL = 23.2 mm) E Pristimantis vilarsi, La Macarena Municipality, Meta Department, IAvH-Am-15095(Adult female, SVL = 44.8 mm) F Pristimantis bogotensis, Cabrera Municipality, Cundinamarca Department, IAvH-Am-15345 (adult male, SVL = 21.9 mm) G Pristimantis anolirex, Santa Barbara Municipality, Santander Department, IAvH-Am-15654 (juvenile female, SVL = 22.3 mm) H Pristimantis lynchi, Tasco Municipality, Boyacá Department, IAvH-Am-15871 (adult male, SVL = 22.1 mm) I Pristimantis nicefori, Santa Barbara Municipality, Santander Department, IAvH-Am-15730 (SVL = 24.5 mm). Photographs by Andrés Acosta-Galvis.

Description of the holotype

An adult female (Figs 4, 5) with a snout-vent length (SVL) of 23.8 mm; the skin of cephalic region, dorsum, eyelids, lateral surfaces, and dorsal thighs shagreen with scattered larger tubercles; dorsolateral folds absent and discoidal folds visible; skin on venter areolate. Head length (HL), diagonally from the corner of mouth to tip of snout 10.4 mm; head width (HW) 9.8 mm, approximately equal to width of the body and 41.1% of the SVL. Snout broadly rounded in dorsal view (type F, sensu Duellman and Lehr 2009; Fig. 4) and rounded in lateral view (type A, sensu Duellman and Lehr 2009; Fig. 4); internarial distance (between center of naris) 2.9 mm; nostril moderately protuberant, directed dorsolaterally; canthus rostralis well defined; loreal region slightly concave; lips not prominent. Eye diameter (ED) from its posterior to anterior corner 3.3 mm; its length 73.3% of the ETS (distance between the anterior edge of the eye to the tip of snout); interorbital region wider than upper eyelid; the upper eyelid width (UEW) 55.2% of interorbital distance (IOD); upper eyelid bearing three smaller subconical tubercles (Figs 4, 5); no cranial crests. Supratympanic fold low and short. Tympanic membrane and tympanic annulus present, small, and rounded (Figs 4, 5), its dorsoposterior border converges with supratympanic fold; its diameter 1.1 mm and equivalent to 33% of eye diameter (ED). Choanae subovoid, not concealed by the palatal shelf of the maxillary arch; dentigerous processes of vomers prominent, nine teeth positioned posterior to level of choanae and widely separated from each other. Tongue rounded, its posterior border notched for half of its extension is adherent to the floor of mouth; teeth present on the maxillary arch.

Forelimbs of moderate size, forearm length 6.4 mm; ulnar tubercles absent. Hand length (HnL) 7.4 mm its length 31.0% of SVL. Palmar tubercle bifid, about two-thirds the length of oval thenar tubercle (Fig. 7). Supernumerary palmar tubercles present, rounded to elongated, and slightly elevated; subarticular tubercles large, round, and conical; fingers without lateral fringes; disks on all fingers rounded apically and extensively expanded (Fig. 7); disk of finger III equal in diameter to the tympanic annulus; disks bearing ventral pads; finger I shorter than II when appressed (Fig. 7). Relative lengths of appressed fingers III>IV>II>I. Subarticular tubercles 1–1–2–2.

Figure 7. 

Hand and toes of adult male paratype, IAvH-Am-10271 of Pristimantis chamezensis sp. nov. in ethanol 70%. A Ventral view of foot B ventral view of hand. Scale bar: 2 mm. Photographs by Andrés Acosta-Galvis.

Hindlimbs slender; foot length (FtL) 12.1 mm, 50.8% of SVL. Toe webbing and toe fringes absent. Relative lengths of appressed toes IV>V>III>II>I. Discs of the toes expanded; width of adjacent phalange 53.7% of disc of toe IV; disc of toe III does not reach penultimate subarticular tubercle of toe IV; toe V beyond that of the level of penultimate subarticular tubercle of toe IV. Femur length (FL) 13.6 mm, tibia length (TL) 14.5 mm, its length is equivalent to 60.9% of SVL. Subarticular tubercles 1–1–2–3–2; supernumerary plantar tubercles numerous, suboval, and low; inner metatarsal tubercle oval; outer metatarsal tubercle rounded, prominent, and smaller than inner metatarsal tubercle. Diameter outer metatarsal tubercle 52.8% of inner metatarsal tubercle; outer tarsal fold absent; inner tarsal fold short. Numerous supernumerary plantar tubercles rounded and barely visible; subarticular tubercles large, round, and conical; toes without lateral fringes; no webbing. Cloacal sheath absent; subcloacal tubercles absent.

Color of holotype in preservative (Fig. 4). Dorsum and flanks dark brown; hands in dorsal view, with fingers I and II cream-colored, while fingers III and IV brown with cream-colored bars; dorsal surfaces of the thigh with diffuse dark-brown transversal bars; hidden surfaces of thighs pale brown; venter light brown with a dark-brown suffusion and mottled brown; ventral surfaces of hindlimbs and forelimbs dark brown with a cream-colored suffusion; edge of chin with irregular blotches of dark brown; hands, in ventral view, with palmar tubercle cream-colored and palmar region dark brown.

Color of holotype in life (Fig. 5). Dorsal surfaces of body and limbs pink-orange; flanks salmon and sides of the head pink-orange; venter reddish cream-colored on chest and belly, cream-colored on throat; axillary region, groin, and anterior thigh pale orange; ventral surfaces of thighs light brown; iris gray, medially reddish, with black reticulations.

Variation of type series

(Fig. 5, Table 4). In this section, coloration refers to specimens in life and is based on field notes and digital photographs, unless otherwise noted. Dorsal coloration reddish brown with mottled, dark-brown chevrons, usually surrounded by a thin band of lighter color; canthal stripe black; dorsal surfaces of thigh with dark-brown transversal bars; axillary region, groin, and anterior thigh bright orange (e.g., IAvH-Am-10283, IAvH-Am-10276; Fig. 5) or uniformly dark brown (e.g., IAvH-Am-10267–68, IAvH-Am-10272; Fig. 5). An adult female (IAvH-Am-10277) has a gold paravertebral line (Fig. 5). Labial bars dark brown, and postorbital and supratympanic stripe dark (e.g., IAvH-Am-10268, IAvH-Am-10270, IAvH-Am-10272, IAvH-Am-10276–7; Fig. 5). In IAvH-Am-10270 and IAvH-Am-10276, flanks with oblique, irregular, dark-brown bars (Fig. 5); IAvH-Am-10267 with a W-shaped, light-brown marking on scapula; some specimens with a dark-brown interorbital bar (e.g., IAvH-Am-10268, IAvH-Am-10273–4, IAvH-Am-10279–10280). Pristimantis chamezensis is metachromatic, being lighter in color at night. Teeth positioned posterior to level of choanae and widely separated from each other, which vary between eight to nine. The variation in the skin texture is noteworthy (Fig. 5), varying from smooth (e.g., IAvH-Am-10283) to shagreen with scattered tubercles (e.g., IAvH-Am-10267, IAvH-Am-10277). The SVL of adult males ranges from 19.6 to 23.7 mm (Table 4), and the SVL of adult females ranges from 19.0 to 24.9 mm (Table 4). The HW 35.9–40.3% of SVL in adult males and 41.2–44.1% in adult females. ED 61.6–75.0% of ETS in adult males and 59.1–74.9% in adult females. UEW 58.0–77.6% of interorbital distance (IOD) in adult males and 54.6–78.2% in adult females. TD 39.1–56.0% of ED in adult males and 33–41.8% in adult females. HnL in adult males 29.3% of SVL and 31.2% in adult females. FtL in adult males 45.6–50.4% of SVL and 44.9–51.8% in adult females.

Distribution and natural history

This species is only known from the type locality at an altitude between 2125–2160 m a.s.l. in an Andean and relictual cloud forest in the Casanare region on the eastern flank of the Cordillera Oriental of Colombia (Fig. 1). The locality is within the Cordillera Oriental montane forest ecoregion (sensu Dinerstein et al. 1995) in the Andean region (Middle Orobiome). The forest (Fig. 2) is unaffected by human activities and is typified by a canopt of medium-height (up to 20 m). The annual precipitation is between 4600 and 5600 mm with bimodal seasonality. Specimens were found active during the second annual rainy season (August to November) at a temperature of 14 °C resting on mosses and lower leaves of shrubs and ferns in the undergrowth. Pristimantis chamezensis is syntopic with an undescribed species of genus Pristimantis.

Etymology

The specific epithet is derived from the Municipality of Chámeza, a geopolitical area where the type locality is located. We decided on this name using a citizen science approach, where expert scientists and local people met and discussed a list of possible names and their corresponding meanings. There was consensus on P. chamezensis as the preferred name.

Conservation status

The direct evaluation of the landscape units (e.g., broad-leaved forest) at the type locality, as well as the map of land cover of Colombia (CORINE Land Cover, IDEAM 2010), allowed us to identify a rapid reduction and low connectivity of its habitat. Based on land cover maps of Chameza’s forest, the potential extent of occurrence is 301,624 km2. Consequently, we propose to categorize P. chamezensis as Vulnerable using the criteria B2a (IUCN Red List Categories and Criteria 2019).

Discussion

Colombian diversity of the genus Pristimantis in a biogeographical context

The genus Pristimantis, with 556 described species, comprises of a substantial number of identified taxa (Frost 2020). Colombia harbors 40% of this diversity. The Andean Cordilleras harbor 183 species (Acosta-Galvis 2020), evidencing the high rate of speciation and endemism of the genus in this ecoregion (Lynch 1999), while in the lowlands (Pacific, Middle Magdalena, and Amazon basins) there are just 52 species. The diversity of Pristimantis of the Andean-Cordillera and Sierra Nevada of Santa Marta reflects the geological history of these mountains (Lynch and Ruiz-Carranza 1985; Lynch et al. 1997). Consequently, the geological formations of the Cordillera occidental (25 Ma old, with the greater species richness), Cordillera Central, and the Central Massif exhibit a 30% similarity of species. While, the Cordillera Oriental (10 Ma old; Gregory-Wodzicki 2000) and Sierra Nevada de Santa Marta (2.6 Ma old; Idárraga et al. 2011) have allowed the evolution of an unparalleled diversity with a high degree of endemism (Lynch et al. 1997) (Fig. 8).

Figure 8. 

Geographic diversity of frogs of the genus Pristimantis in Colombia; the numerical values correspond to the number of species reported in each region.

Despite this rough correspondence between the geological history of the Colombian Andes and Pristimantis diversity, the inventory of species in each region is far from being completed. Socio-political factors affecting the various regions of Colombia have limited scientific access, leaving several crucial regions with pronounced gaps in our knowledge of amphibians. Among these regions, we highlight the northern lowland regions of the upper Amazon, including Putumayo, Caquetá, Guaviare, Guainía, and Vaupés departments, as well as neighboring areas such as the Darien region. Additionally, some other unsampled areas are the tropical rainforests in the Pacific basin and the Andean region, such as the Serranias of Perijá and San Lucas, southern Cordillera Oriental (including the Andean-Amazonian foothills) and mountainous areas associated with the Orinoco drainage (Fig. 8).

Over the past six years of scientific studies in unexplored mountainous areas within the Orinoco drainage, including cloud forests and foothills of the Cordillera Oriental, several species of Pristimantis have been described (e.g., Acosta-Galvis et al. 2010; Acosta-Galvis and Alfaro-Bejarano 2011; Pedroza-Banda et al. 2014; Rivera-Correa et al. 2016; Acevedo et al. 2020; Ospina-Sarria and Angarita-Sierra 2020). However, there is still a long way to go to characterize the amphibian fauna of this region.

Phylogenetic relationships of Pristimantis chamezensis

In our research, the integration of morphological and genetic data allowed us to establish that P. chamezensis is distinct from the other 13 Pristimantis species from Andean and sub-Andean forests on the eastern flank of the Cordillera Oriental. Taking into account the agreement between all phylogenetic analyses revealing a supported monophyletic group comprised of P. chamezensis, P. carranguerorum, P. bowara, P. lutitus, P. medemi, P. nicefori, and P. savagei, as well as the altitudinal (450–4170 m a.s.l.) and longitudinal distribution of those species along the Andean and sub-Andean forest on the eastern flank of the Cordillera Oriental (almost all are syntopic except by P. lutitus and P. nicefori from the western flank), it is probable that the origin of the new species and the radiation of the monophyletic group may have occurred at higher altitudes within this region. It might be possible that these Pristimantis lineages show the same pattern of recent diversification due to climatic changes, as seen in both, a high altitude dendrobatid frog (Hyloxalus felixcoperari Acosta-Galvis & Vargas-Ramírez, 2018) and a group of Andean anoles (Anolis heterodermus species group; Vargas-Ramírez and Moreno-Arias 2014) from the middle part of the eastern Cordillera.

Nevertheless, the generalized low support of the phylogenies emphasizes the need to increase the molecular dataset to reveal with confidence the evolutionary relationships within Pristimantis. This is clear from the recent changes in the phylogenetic position of several species (e.g., Hedges et al. 2008; Padial et al. 2014; Reyes-Puig et al. 2020). In addition, it is still required to incorporate a large number of unassigned Colombian taxa into evolutionary based species groups. There are about 117 species not yet analyzed using phylogenetic methods.

Our phylogenetic analyses unequivocally revealed that P. chamezensis is part of the subgenus Pristimantis. However, we do not force its allocation into one of the several species group (Hedges et al. 2008; Padial et al. 2014; Acevedo et al. 2020). Although our results validate some arrangements (e.g., conspicillatus or danae species groups; Fig. 3), some other individual assignments are weakly supported, and do not correspond to arrangements within the already proposed groups. Among the examples that we can identify, is the nesting of P. chamezensis with P. nicefori, which was formerly assigned within the unistrigatus group by Hedges et al. (2008) and later transferred to unassigned species group by Padial et al. (2014). Likewise, the close relationship of the chamezensis+ P. nicefori clade with the P. lutitus + P. medemi + P. carranguerorum clade (Fig. 3) is inconsistent with previous species groups assignments; P. medemi and P. carranguerorum were assigned to the conspicillatus species group by Hedges et al. (2008) and, later, validated by Padial et al. (2014). Additionally P. lutitus (Fig. 3), which was formerly assigned to the unistrigatus species group but subsequently transferred to an unassigned species group by Padial et al. (2014) and later inferred as sister to P. anolirex by Rivera et al. (2016).

Conclusion

Pristimantis chamezensis is described as an endemic species from Chámeza forest. This new species is closely related to P. carranguerorum, P. bowara, P. lutitus, P. medemi, P. nicefori, and P. savagei.

Acknowledgments

We extend our thanks to all inhabitants of the Municipality of Chámeza, who by referendum selected the name of the species described here. Many thanks go to our local guides, José Pérez and Antonio Montaña. Natalia Novoa and Luis Daniel Prada offered their support and helped with fieldwork during the monitoring of Chameza’s forest between 2010 and 2011. We also thank Dr José Rigoberto Ruiz Castillo and Dr Campo Elias Cardozo Tafur from the local government of Chámeza. Corporinoquia granted the collection permit (resolution 200.41–10.1409 on 8 October 2010). Fieldwork support was provided through several projects: “Discovering the biodiversity of Chámeza”, funded by Chameza’s municipality and executed by Asociación de Becarios de Casanare ABC; and “Detection of chytridiomycosis in Colombia”, funded by COLCIENCIAS-Javeriana University. The final development of this contribution was produced under the resolution 0041–2020 of the Ministerio de Ambiente y Desarrollo Sostenible de Colombia and Biological Collections of Research Institute of Biological Resources Alexander von Humboldt (IAvH). We thank Thomas Defler for his assistance with the English proofing of the manuscript. Finally, special thanks to Santiago Ron, Robert Forsyth, and Carolina Reyes-Puig provided comments, suggestions and corrections that greatly improved the manuscript.

References

  • Acevedo A, Armesto O, Palma RE (2020) Two new species of Pristimantis (Anura: Craugastoridae) with notes on the distribution of the genus in northeastern Colombia. Zootaxa 4750(4): 499–523. https://doi.org/10.11646/zootaxa.4750.4.3
  • Acosta-Galvis AR, Alfaro-Bejarano JP (2011) Anfibios del Casanare. In: Usma JS, Trujillo F, Ayala LT (Eds) Biodiversidad del Departamento del Casanare, identificación de ecosistemas estratégicos. Gobernación de Casanare-WWF, Bogotá, 134–147.
  • Acosta-Galvis AR, Señaris JC, Rojas-Runjaic F, Riaño-Pinzón DR (2010) Anfibios y reptiles. In: Lasso CA, Usma JS, Trujillo F (Eds) Biodiversidad de la cuenca del Orinoco: bases científicas para la identificación de áreas prioritarias para la conservación y uso sostenible de la biodiversidad. Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, WWF Colombia, Fundación Omacha, Fundación La Salle, and Instituto de Estudios de la Orinoquia (Universidad Nacional de Colombia). Bogotá, 258–289.
  • Acosta-Galvis AR (2020) Lista de los Anfibios de Colombia: Referencia en línea. V. 10.2020.0. http://www.batrachia.com [Accessed on: 2020-02-01]
  • Anganoy-Criollo M, Ramírez JP (2017) New records of Pristimantis carranguerorum (Anura: Craugastoridae) from the Cordillera Oriental of Colombia. Check List 13(3): 1–2138. https://doi.org/10.15560/13.3.2138
  • Ardila-Robayo MC, Acosta-Galvis AR (2000) Anfibios. In: Rangel C (Ed.) Colombia diversidad biotica III la region de vida paramuna de Colombia. Universidad Nacional de Colombia, Bogota, 617–628.
  • Barbour T (1914) A contribution to the zoogeography of the West Indies, with special reference to amphibians and reptiles. Memoirs of the Museum of Comparative Zoology 44: 205–359. https://doi.org/10.5962/bhl.title.49187
  • Bensch S, Stjernman M, Hasselquist D, Örjan Ö, Hannson B, Westerdahl H, Torres-Pinheiro R (2000) Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds. Proceedings of the Royal Society of London, Series B 267: 1583–1589. https://doi.org/10.1098/rspb.2000.1181
  • Crump ML, Scott NJ (1994) Visual Encounter Surveys. In: Heyer WR, Donnelly MA, McDiarmid RW, Hayek LC, Foster MS (Eds) Measuring and Monitoring Biological Diversity Standard Methods for Amphibians, Smithsonian Press, Washington, 84–92.
  • Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9(8): 1–772. https://doi.org/10.1038/nmeth.2109
  • Darst CR, Cannatella DC (2004) Novel relationships among hyloid frogs inferred from 12S and 16S mitochondrial DNA sequences. Molecular Phylogenetics and Evolution 31(2): 462–475. https://doi.org/10.1016/j.ympev.2003.09.003
  • Dinerstein E, Olson DM, Graham DJ, Webster AL, Primm SA, Bookbinder MP, Ledec G, Young KR (1995) A Conservation Assessment of the Terrestrial Ecoregions of Latin America and the Caribbean. World Bank Washington, 176 pp. https://doi.org/10.1596/0-8213-3295-3
  • Duellman WE, Simmons JE (1977) A new species of Eleutherodactylus (Anura: Leptodactylidae) from the Cordillera Oriental of Colombia. Proceedings of the Biological Society of Washington 90(1): 60–65. https://www.biodiversitylibrary.org/page/39057808
  • Duellman WE, Lehr E (2009) Terrestrial-Breeding Frogs (Strabomantidae) in Peru. Naturund Tier-Verlag, Naturwissenschaft, Münster, 382 pp.
  • Faivovich J, Haddad CFB, Garcia PCA, Frost DR, Campbell JA, Wheeler WC (2005) Systematic review of the frog family Hylidae, with special reference to the Hylinae: phylogenetic analysis and taxonomic revision. Bulletin of the American Museum of Natural History 294: 1–240. https://doi.org/10.1206/0003-0090(2005)294[0001:SROTFF]2.0.CO;2
  • Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates Molecular Marine Biology and Biotechnology 3(5): 294–299.
  • Fouquet A, Noonan BP, Rodrigues MT, Pech N, Gilles A, Gemmell NJ (2012) Multiple quaternary refugia in the eastern Guiana Shield revealed by comparative phylogeography of 12 frog species. Systematic Biology 61(3): 461–489. https://doi.org/10.1093/sysbio/syr130
  • García-R JC, Crawford AJ, Mendoza ÁM, Ospina O, Cardenas H, Castro F (2012) Comparative phylogeography of direct-developing frogs (Anura: Craugastoridae: Pristimantis) in the southern Andes of Colombia. PloS ONE 7(9): e46077. https://doi.org/10.1371/journal.pone.0046077
  • Grant T, Acosta-Galvis AR, Lynch JD (2008) Brief overview of the amphibians of Colombia. In: Stuart SN, Hoffmann M, Chanson JS, Cox NA, Berridge RJ, Ramani P, Young BE (Eds) Threatened Amphibians of the World. Lynx Edicions, Barcelona, 103–104.
  • González-Durán GA, Targino M, Rada M, Grant T (2017) Phylogenetic relationships and morphology of the Pristimantis leptolophus species group (Amphibia: Anura: Brachycephaloidea), with the recognition of a new species group in Pristimantis Jimenez de la Espada, 1870. Zootaxa 4243: 42–74. https://doi.org/10.11646/zootaxa.4243.1.2
  • Guarnizo CE, Paz A, Muñoz-Ortiz A, Flechas SV, Mendez-Narvaez J, Crawford AJ (2015) DNA Barcoding Survey of Anurans across the Eastern Cordillera of Colombia and the Impact of the Andes on Cryptic Diversity. Plos ONE 10(5): e0127312. https://doi.org/10.1371/journal.pone.0127312
  • Hedges SB, Duellman WE, Heinicke MP (2008) New World direct-developing frogs (Anura: Terrarana): molecular phylogeny, classification, biogeography, and conservation. Zootaxa 1737: 1–182. https://doi.org/10.11646/zootaxa.1737.1.1
  • Heinicke MP, Duellman WE, Trueb L, Means DB, MacCulloch RD, Hedges SB (2009) A new frog family (Anura: Terrarana) from South America and an expanded direct-developing clade revealed by molecular phylogeny. Zootaxa 2211: 1–35. https://doi.org/10.11646/zootaxa.2211.1.1
  • Heinicke MP, Duellman WE, Hedges SB (2007) Major Caribbean and Central American frog faunas originated by ancient oceanic dispersal. Proceedings of the National Academy of Sciences 104(24): 10092–10097. https://doi.org/10.1073/pnas.0611051104
  • Heyer WR, Barrio-Amorós CL (2009) The advertisement calls of two sympatric frogs, Leptodactylus lithonaetes (Amphibia: Anura: Leptodactylidae) and Pristimantis vilarsi (Amphibia: Anura: Strabomantidae). Proceedings of the Biological Society of Washington 122(3): 282–291. https://doi.org/10.2988/09-02.1
  • Heinicke MP, Lemmon AR, Lemmon EM, McGrath K, Hedges SB (2018) Phylogenomic support for evolutionary relationships of New World direct-developing frogs (Anura: Terraranae). Molecular Phylogenetics and Evolution 118: 145–155. https://doi.org/10.1016/j.ympev.2017.09.021
  • Idárraga-García J, Posada BO, Guzmán G (2011) Geomorfología de la zona costera adyacente al piedemonte occidental de la Sierra Nevada de Santa Marta entre los sectores de Pozos Colorados y el río Córdoba, Caribe colombiano. Boletín de Investigaciones Marinas y Costeras 40(1): 41–58. http://ref.scielo.org/rd7xzr
  • IDEAM (2010) Leyenda Nacional de Coberturas de la Tierra. Metodología CORINE Land Cover adaptada para Colombia Escala 1:100.000. Instituto de Hidrología, Meteorología y Estudios Ambientales. Bogotá, 72 pp.
  • Kumar S, Stecher G, Amura KT (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular biology and Evolution 33(7): 1870–1874. https://doi.org/10.1093/molbev/msw054
  • Lanfear R, Calcott B, Ho SY, Guindon S (2012) PartitionFinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Molecular Biology and Evolution 29(6): 1695–1701. https://doi.org/10.1093/molbev/mss020
  • Lynch JD (1975) The identity of the frog Eleutherodactylus conspicillatus (Günther) with descriptions of two related species from Northwestern South America (Amphibia, Leptodactylidae). Contributions in Science, Natural History Museum,Los Angeles County 272: 1–21. https://www.biodiversitylibrary.org/part/214211
  • Lynch JD (1983) A new leptodactylid frog from the Cordillera Oriental de Colombia. In Rhodin AGJ and Miyata K (eds). Advances in Herpetology and Evolutionary Biology: Essays in Honor of Ernest E. Williams. Museum of Comparative Zoology, Cambridge, 52–57.
  • Lynch JD (1984) New frogs (Leptodactylidae: Eleutherodactylus) from cloud forest of the northern Cordillera Oriental, Colombia. Contributions in Biology and Geology, Milwaukee Public Museum 60: 1–19.
  • Lynch JD (1994) Two new species of the Eleutherodactylus conspicillatus group (Amphibia: Leptodactylidae) from the Cordillera Oriental of Colombia. Revista de la Academia Colombiana de Ciencias Exactas, Físicas y Naturales 19(72): 187–193. http://www.accefyn.com/revista/Vol_19/72/187-193.pdf
  • Lynch JD, Duellman WE (1997) Frogs of the genus Eleutherodactylus in western Ecuador. Special Publication 23, University of Kansas, Natural History Museum: 1–236.
  • Lynch JD, Ruiz-Carranza PM (1985) A synopsis of the frogs of the genus Eleutherodactylus from the Sierra Nevada de Santa Marta. Occasional Papers of the Museum of Zoology, University of Michigan 711: 1–59. http://hdl.handle.net/2027.42/57147
  • Lynch JD, Ruiz-Carranza PM, Ardila-Robayo MC (1997) Biogeographic Patterns of Colombian Frogs and Toads. Patrones biogeográficos de las ranas y los sapos de Colombia. Revista de la Academia Colombiana de Ciencias Exactas, Físicas y Naturales 21(80): 237–248. http://www.accefyn.com/ranas/frogs/Rev237.html
  • Malambo C, Marin A (2006) Geographic distribution: Eleutherodactylus medemi. Herpetological Review 37(4): 1–487.
  • Melin D (1941) Contributions to the knowledge of the Amphibia of South America. Göteborgs Kungl. Vetenskaps-och Vitterhets-samhälles, Handlingar, Serien B, Matematiska och Naturvetenskapliga Skrifter 1: 1–71.
  • Mendoza ÁM, Ospina OE, Cárdenas-Henao H, García-R JC (2015) A likelihood inference of historical biogeography in the world’s most diverse terrestrial vertebrate genus: diversification of direct-developing frogs (Craugastoridae: Pristimantis) across the Neotropics. Molecular Phylogenetics and Evolution 85: 50–58. https://doi.org/10.1016/j.ympev.2015.02.001
  • Meza‐Joya FL, Torres M (2016) Spatial diversity patterns of Pristimantis frogs in the Tropical Andes. Ecology and Evolution 6(7): 1901–1913. https://doi.org/10.1002/ece3.1968
  • Navarrete MJ, Venegas PJ, Ron SR (2016) Two new species of frogs of the genus Pristimantis from Llanganates National Park in Ecuador with comments on the regional diversity of Ecuadorian Pristimantis (Anura, Craugastoridae). ZooKeys 593: 139–162. https://doi.org/10.3897/zookeys.593.8063
  • Olson DM, Dinerstein E (2002) The Global 200: priority ecoregions for global conservation. Annals of the Missouri Botanical Garden 89(2): 199–224. https://doi.org/10.2307/3298564
  • Ospina-Sarria JJ, Angarita-Sierra T (2020) A new species of Pristimantis (Anura: Strabomantidae) from the eastern slope of the Cordillera Oriental, Arauca, Colombia. Herpetologica 76: 93–92. https://doi.org/10.1655/Herpetologica-D-19-00048
  • Padial JM, Grant T, Frost DR (2014) Molecular systematics of terraranas (Anura: Brachycephaloidea) with an assessment of the effects of alignment and optimality criteria. Zootaxa 3825: 1–132. https://doi.org/10.11646/zootaxa.3825.1.1
  • Páez NB, Ron SR (2019) Systematics of Huicundomantis, a new subgenus of Pristimantis (Anura, Strabomantidae) with extraordinary cryptic diversity and eleven new species. ZooKeys 868: 1–112. https://doi.org/10.3897/zookeys.868.26766
  • Palumbi SR, Martin AP, Romano SL, McMillan WO, Stice L, Grabowski G (1991) The Simple Fool’s Guide to PCR. Department of Zoology, University of Hawaii, Honolulu, 45 pp.
  • Pinto-Sanchez NR, Ibanez R, Madrinan S, Sanjur OI, Bermingham E, Crawford AJ (2012) The great American biotic interchange in frogs: multiple and early colonization of Central America by the South American genus Pristimantis (Anura: Craugastoridae). Molecular Phylogenetics and Evolution 62(3): 954–972. https://doi.org/10.1016/j.ympev.2011.11.022
  • Pedroza-Banda R, Ospina-Sarria JJ, Angarita-Sierra T, Anganoy-Criollo M, Lynch JD (2014) Estado del conocimiento de la fauna de anfibios y reptiles del departamento de Casanare, Colombia. Revista de la Academia Colombiana de Ciencias Exactas, Físicas y Naturales 38(146): 17–34. https://doi.org/10.18257/raccefyn.37
  • Peters WCH (1863) Über eine neue Schlangen-Gattung, Styporhynchus, und verschiedene andere Amphibien des zoologischen Museum. Monatsberichte der Königlichen Preussische Akademie des Wissenschaften zu Berlin 1863: 399–413.
  • Pyburn W, Lynch JD (1981) Two little-known species of Eleutherodactylus (Amphibia: Leptodactylidae) from the Sierra de La Macarena, Colombia. Proceedings of the Biological Society of Washington 94(2): 404–412. https://www.biodiversitylibrary.org/page/34608261
  • Renjifo-Rey JM (2005) Rana de lluvia carranguera, Eleutherodactylus carranguerorum. In: Rueda-Almonacid JV, Lynch JD, and Amézquita A (Eds) Libro Rojo de Anfibios de Colombia. Serie Libros Rojos de Especies Amenazadas de Colombia. Conservación Internacional Colombia, Instituto de Ciencias Naturales-Universidad Nacional de Colombia, Ministerio del Medio Ambiente, Bogotá, 384 pp.
  • Reyes-Puig C, Yánez-Muñoz MH, Ortega JA, Ron SR (2020) Relaciones filogenéticas del subgénero Hypodictyon (Anura: Strabomantidae: Pristimantis) con la descripción de tres especies nuevas de la región del Chocó. Revista Mexicana de Biodiversidad 91: 1–38. https://doi.org/10.22201/ib.20078706e.2020.91.3013
  • Rivera-Correa M, Daza JM (2016) Molecular phylogenetics of the Pristimanstis lacrimosus species group (Anura: Craugastoridae) with the description of a new species from Colombia. Acta Herpetologica 11(1): 31–45. http://www.fupress.net/index.php/ah/article/view/16434
  • Rivera-Correa M, Lamadrid-Feris F, Crawford AJ (2016) A new small golden frog of the genus Pristimantis (Anura: Craugastoridae) from an Andean cloud forest of Colombia. Amphibia-Reptilia 37(2): 153–166. https://doi.org/10.1163/15685381-00003037
  • Rivera-Correa M, Jimenez-Rivillas C, Daza JM (2017) Phylogenetic analysis of the Neotropical Pristimantis leptolophus species group (Anura: Craugastoridae): molecular approach and description of a new polymorphic species. Zootaxa 4242: 313–343. https://doi.org/10.11646/zootaxa.4242.2.6
  • Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic biology 61(3): 539–542. https://doi.org/10.1093/sysbio/sys029
  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2028 pp.
  • Stebbins RC, Hendrickson JR (1959) Field studies of Amphibians in Colombia, South America. University of California Publications in Zoology 56(5): 497–540.
  • Talavera G, Castresana J (2007) Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Systematic Biology 56(4): 564–577. https://doi.org/10.1080/10635150701472164
  • Vargas-Ramírez M, Moreno-Arias R (2014) Unknown evolutionary lineages and population differentiation in Anolis heterodermus (Squamata: Dactyloidae) from the eastern and central Cordilleras of Colombia revealed by DNA sequence data. South American Journal of Herpetology 9(2): 131–141. https://doi.org/10.2994/SAJH-D-13-00013.1
  • von May R, Catenazzi A, Corl A, Santa-Cruz R, Carnaval AC, Moritz C (2017) Divergence of thermal physiological traits in terrestrial breeding frogs along a tropical elevational gradient. Ecology and Evolution 7(9): 3257–3267. https://doi.org/10.1002/ece3.2929