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Research Article
A new species of rain frog (Anura: Strabomantidae: Pristimantis) from the Guiana Shield and amended diagnosis of P. ockendeni (Boulenger, 1912)
expand article infoAlexander Tamanini Mônico, Miquéias Ferrão§, Juan Carlos Chaparro|, Antoine Fouquet#, Albertina Pimentel Lima
‡ Programa de Pós-Graduação em Biologia (Ecologia), Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil
§ Harvard University, Cambridge, United States of America
| Museo de Biodiversidad del Perú, Cusco, Peru
¶ Universidad Nacional de San Antonio Abad del Cusco, Cusco, Peru
# Université Paul Sabatier, Toulouse, France
Open Access

Abstract

Pristimantis is already the most speciose genus among vertebrates, yet the current number of species remains largely underestimated. A member of the P. unistrigatus species group from the Guiana Shield has been historically misidentified as P. ockendeni, a species described from southern Peru. We combined mitochondrial (16S and COI) and nuclear (RAG1) loci, external morphology, skull osteology (μ-CT scan), vocalization (advertisement and courtship calls), geographic distribution and natural history data to differentiate the Guiana Shield populations from P. ockendeni, and describe them as a new species. The new species is crepuscular and nocturnal and inhabits the understory of unflooded (terra firme) forests in Brazil, Guyana and Suriname. It is phylogenetically related to P. arda­lonychus, P. martiae and undescribed species from Brazilian Amazonia. The new species notably differs from P. ockendeni and its congeners in the P. unistrigatus species group occurring in the Guiana Shield by the combination of the following characters: absence of dentigerous processes of vomers, presence of vocal slits in males, body size (SVL 16.2–20.7 mm in males and 21.4–25.7 mm in females), advertisement call (call with 4–6 notes, call duration of 158–371 ms and dominant frequency of 3,466–4,521 Hz) and translucent groin coloration in life. To facilitate the recognition and description of cryptic species previously hidden under the name P. ockendeni, we provide an amended diagnosis of this taxon based on external morphology and advertisement call of specimens recently collected nearby the type locality and additional localities in southwestern Amazonia.

Keywords

Amazonia, Amphibia, Brachycephaloidea, integrative taxonomy, natural history, Terrarana

Introduction

With more than 590 currently valid nominal species (Frost 2022), Pristimantis Jiménez de la Espada, 1841 is the most speciose genus among vertebrates (Hedges et al. 2008; Padial and De la Riva 2009; Ortega-Andrade et al. 2015; Chavez and Catenazzi 2016). Moreover, this number is increasing at a fast pace suggesting that the number of extant species of Pristimantis remains largely underestimated (De Oliveira et al. 2019; Paéz and Ron 2019; Vacher et al. 2020; Zumel et al. 2022, Fouquet et al. 2022a).

Members of the Pristimantis unistrigatus species group occupy mostly the Andes and Amazonia and are generally cryptically colored and small-bodied. Although the monophyly of this speciose group remains highly contentious (Padial et al. 2014), our focal species belong to one of the well-suported clades supposedly forming the P. unistrigatus species group (Zabata-Brito et al. 2021; Frost 2022). Some populations of this species group from central Amazonia and the Guiana Shield have been historically identified as P. ockendeni (Boulenger, 1912)—a species described based on three syntypes from La Union, Huacamayo River, Carabaya, Departamento Puno, southeastern Peru, at about 800 m above sea level (a.s.l.). Pristimantis ockendeni has been subsequently reported from Brazil, Colombia, Ecuador, Guyana, Suriname (Zimmerman and Rodrigues 1990; Lima et al. 2006; Lima et al. 2012; Ocampo et al. 2016; Silva-e-Silva and Costa-Campos 2018; Azevedo et al. 2021; Torralvo et al. 2021; Fouquet et al. 2022b) and Bolivia (Padial et al. 2004; Elmer and Cannatella 2008; Ocampo et al. 2016; Frost 2022). However, the identification of these populations as P. ockendeni and, thus, the wide geographic distribution of the species, has been considered doubtful by Elmer et al. (2007a, 2007b), who suggested the existence of undescribed species erroneously associated with this taxon. Later, congruence between genetic and morphologic evidence supported the formal description of three new species historically hidden under the name P. ockendeni in Ecuador (Elmer and Cannatella 2008). Due to the unavailability of recently collected topotypical material, Elmer and Cannatella (2008) recognized the difficulties to clarify the identity of P. ockendeni sensu stricto. Similarly, populations from Brazil are unlikely conspecific to P. ockendeni (Fouquet et al. 2013; Ocampo et al. 2016). More recently, Fouquet et al. (2022b) highlighted specifically the case of these populations from central Amazonia and the Guiana Shield that have been identified as P. cf. ockendeni (also indicated by Vacher et al. 2020), showing that they form a species complex (“trans-Amazon complex”) which is distinct from all the other species of the Guiana Shield while possibly related, though also distinct, to western Amazonian species.

In the present study, we evaluate the taxonomic status of the populations from the Guiana Shield (Brazil, Guyana and Suriname) previously identified as P. ockendeni and confirm that they belong to a new species that is described herein. We provide information regarding the ecology, phylogeny, call, and natural history of the new species. We also collected new morphological, bioacoustic and genetic data of P. ockendeni that permitted clarifying its phylogenetic position, and amend its diagnosis.

Material and methods

Sampling

Fifty-six adults of the new species were manually collected in five localities in Brazil between November 2019 and December 2021 [eighteen individuals at Reserva Floresta Adolpho Ducke (RFAD), municipality of Manaus (2°55′53.6″S; 59°58′25.7″W); six individuals from Km 144 of the BR-174 Highway (1°45′26.7″S; 60°08′24.2″W) and eight individuals from Cachoeira da Suçuarana in Balbina (1°54′45.5″S; 59°24′30.8″W), municipality of Presidente Figueiredo, state of Amazonas; ten individuals from vicinity of the municipality of São João da Baliza (0°57′08.9″N; 59°53′29.7″W), state of Roraima], one locality in Suriname in April 2014 [five individuals from Sipaliwini (2°01′40.0″N; 56°07′32.2″W)] and the last one in Guyana between November 2002 to June 2004 and August 2010 [nine individuals from Mabura Hill Forest Reserve (5°09′20.6″N; 58°42′00.4″W)]. Eight adults of Pristimantis ockendeni sensu stricto (Appendix 1) were collected, being six individuals from municipalities of Manoel Urbano (Floresta Estadual do Afluente; 8°42′16.6″S; 69°32′02.6″W, to 522 Km from Type Locality – TL) and two individuals from Feijó (8°38′46.5″S; 69°43′29.7″W, to 522 Km from TL), state of Acre, Brazil. Specimens were anaesthetized and killed with 5% lidocaine. Muscle or liver tissue was preserved in 100% ethanol for genetic analysis, whereas the specimens were fixed in 10% formalin and preserved in 70% ethanol. Specimens were deposited in the Herpetological Collection of the Instituto Nacional de Pesquisas da AmazôniaINPA-H (Manaus, Brazil), Museu Paraense Emílio GoeldiMPEG (Belém, Brazil), Museum National d’Histoire Naturelle – MNHN-RA (Paris, France), Staatliches Museum für NaturkundeSMNS (Stuttgart, Germany), and Museum für Tierkunde in DresdenMTD (Dresden, Germany).

Sequencing and phylogenetic analyses

Genomic DNA was extracted from tissues (muscle or liver) of 26 individuals of the new species, including three individuals from each locality previously mentioned, in addition to three (JJLR007, JJLR010 and JJLR016; Appendix 1) from Reserva Biológica do Uatumã, state of Amazonas, and one (MPEG 33750; Appendix 1) from Oriximiná municipality, Pará state, Brazil. We also extracted genomic DNA of five individuals of Pristimantis ockendeni from Brazil (previously mentioned) and three from Peru, deposited in Museo de Biodiversidad del Perú (MUBI): two (MUBI 10538 and MUBI 14568; Appendix 1) from Manu Province (Reserva Comunal Amarakaeri, to 154 Km from TL) and one (MUBI 13049; Appendix 1) from Paucartambo Province (Kosñipata District, to 196 Km from TL) (Appendix 1). We extract genomic DNA using PureLink™ Genomic DNA (Invitrogen by Thermo Fisher Scientific, Carlsbad, CA, USA). Fragments of two mitochondrial (16S and Cytochrome C Oxidase sub-unit 1 – COI) and a nuclear gene (Recombination Activating 1 – RAG1) were amplified through polymerase chain reaction (PCR).

The 16S was amplified using primers 16Saf (5’-CGCCTGTTTATCAAAAACAT-3’) and 16Sbr (5’-CCGGTCTGAACTCAGATCACGT-3’) (Palumbi 1996) under the following conditions: 5 m at 94°C followed by 35 cycles of 94°C (30 s), 50°C (50 s) and 72°C (120 s), and final extension of 7 minutes at 72°C. The final volume of the PCR reaction was 15 μL and contained 1.5 μL of 25 mM MgCl2, 1.5 μL of 10 mM dNTPs (2.5 mM each dNTP), 1.5 μL of tampon 10× (75 mM Tris HCl, 50 mM KCl, 20 mM (NH4)2SO4), 1,5 μL of each primer (2 μM), 6.4 μL of ddH2O and 0.1 μL of 1 U Taq DNA Polymerase and 1 μL of DNA (30–50 ng/μL). The COI was amplified using primers Chmf4f (5’-TYTCWACWAAYCAYAAAGAYATCGG-3’) and Chmr4r (5’-ACYTCRGGRTGRCCRAARAATCA-3’) (Che et al. 2012) under the following conditions: 60 s at 94°C followed by 35 cycles of 94°C (20 s), 50°C (50 s) and 72°C (90 s), and final extension of 10 minutes at 72°C. The final volume of the PCR reaction was 15 μL and contained 1.2 μL of 25 mM MgCl2, 1.2 μL of 10 mM dNTPs (2.5 mM each dNTP), 1.5 μL of tampon 10× (75 mM Tris HCl, 50 mM KCl, 20 mM (NH4)2SO4), 0,5 μL of each primer (10 μM), 8.95 μL of ddH2O and 0.15 μL of 1 U Taq DNA Polymerase and 1 μL of DNA (30–50 ng/μL). Finally, RAG1 was amplified using primers R182 (5’-GCCATAACTGCTGGAGCATYAT-3’) and R270 (5’-AGYAGATGTTGCCTGGGTCTTC-3’) (Heinicke et al. 2007) under the following conditions: 5 m at 94°C followed by 40 cycles of 94°C (30 s), 59°C (30 s) and 72°C (60 s), and final extension of 7 minutes at 72°C. The composition of the PCR reaction was the same used for 16S. PCR products were visualized in 1 agarose with SYBRSafe (Life Inc.) and purified using PEG 8000 protocol (Sambrook and Russell 2001) and submitted to sequencing using standard protocols of the Big DyeTM Terminator Kit (Applied Biosystems, Inc., Grand Island, NY, USA). Amplicons were sequenced in an ABI PRISMI 3130XL (Thermo Fisher) using the forward and reverse primers of each gene. Sequences were subjected to BLAST searches (Altschul et al. 1997) in GenBank to verify if the target had been amplified. Newly generated sequences were deposited in GenBank, and accession numbers are available in Appendix 2.

To infer the phylogenetic relationships of the new species, the above newly generated sequences were inserted into a data set containing homologous sequences retrieved from GenBank. Sequences retrieved from GenBank are detailed in Appendix 2. We selected sequences used in the most recent phylogenetic analyses that involved Pristimantis ockendeni and related Andean species (Zabata-Brito et al. 2021) and species historically assigned to the P. unistrigatus species group of the Guiana Shield (Fouquet et al. 2022b). In total, 252 sequences (Appendix 2) of the three genes were selected (135 for 16S, 66 for COI and 51 for RAG1), which corresponded to 136 terminals. We aligned sequences of each gene using MAFFT online server with default parameters for each gene, except by the use of E-INS-i strategy for 16S gene and G-INS-i for protein coding genes (Katoh and Standley 2013). The final matrix was concatenated in Mesquite (Maddison and Maddison 2021) and composed of 136 terminals with 1,843 bp (569 pb for 16S, 638 pb for COI, and 636 pb for RAG1).

Best-fit evolutionary models and partition schemes were determined through ModelFinder (Kalyaanamoorthy et al. 2017) using nine partitions: one for the 16S and one for each codon of protein-coding genes. The best evolutionary models for partitions in the concatenated matrix were: TIM2 + F + R5 for 16S, COI 2nd and RAG 3rd codons, TIM3 + F + R3 for COI 1st position, and HKY + F + G4 for COI 3rd, RAG 1st and 2nd codons. Phylogenetic relationships were reconstructed under Maximum Likelihood inference (ML). The ML tree was inferred using IQTREE (Nguyen et al. 2015) as implemented in the webserver http://iqtree.cibiv.univie.ac.at (Trifinopoulos et al. 2016). Clade support was estimated with 10,000 ultrafast bootstrap replications (Hoang et al. 2018), 1,000 maximum iterations, and a minimum correlation coefficient of 0.99. We calculated pairwise genetic distances (p and Kimura-two-parameter distances; Kimura 1980) among populations of new species and P. ockendeni sensu stricto using MEGA 11 (Tamura et al. 2021). Interspecific distances were also calculated among the new species and other species of the P. unistrigatus species group from the Guiana and the Pantepui regions. Genetic distances were calculated using pairwise deletion.

Morphology

We measured 25 morphometric measurements from adults of the new species (males = 43 and females = 13) and Pristimantis ockendeni sensu stricto (males = 9 and females = 2, including three syntypes). Measurements follow Duellman and Lehr (2009) ( eye diameter [ED], eye-nostril distance [EN], foot length [FL], interorbital distance [IOD], internarial distance [IND], head length [HL], head width [HW], snout-vent length [SVL], tibia length [TL] and tympanum diameter [TD]), Caldwell et al. (2002) ( forearm length [FAL], hand length [HAND], snout length [SL], disc width of Finger III [WFD]), Heyer et al. (1990) ( tarsus length [TAL], thigh length [THL], upper arm length [UAL]) and Lima et al. (2007) ( hand length from proximal edge of palmar tubercle to tip of Finger I [HANDI], Finger II [HANDII] and Finger IV [HANDIV]). Additionally, we measured foot length from proximal edge of outer metatarsal tubercle to tip of Toe I (FLI), Toe II (FLII), Toe III (FLIII), Toe V (FLV), and disc width of Toe IV (WTD).

We obtained μ-CT scans of the skull to confirm the presence/absence of dentigerous processes of vomers in one male (MNHN-RA-2020.0115) and one female (MNHN-RA-2020.0114). Specimens were scanned (kV = 40–70, resolution < 20 µm) using an EasyTom 150 from the MRI platform of ISEM (Institute of Evolutionary Sciences of Montpellier, France). Segmentation of the full skeleton and of the cranium was done using Avizo (FEI Visualization Sciences Group, Burlington, MA, USA) and Biomedisa (Lösel et al. 2000).

Bioacoustics

We recorded advertisement calls of 34 males of the new species and seven males Pristimantis ockendeni sensu stricto with a digital recorder “Marantz PMD660” (Marantz, Japan) coupled to a Sennheiser K6/ME66 unidirectional microphone (Sennheiser, Germany). Air temperatures during calls ranged between 22.4 and 25.6°C. We recorded three minutes of calls per individual using frequency rate of 44 kHz and 16 bits of resolution in the mono pattern. Call recordings were deposited at Fonoteca Neotropical Jacques Vielliard (FNJV) of the Universidade de Campinas (UNICAMP, Campinas, SP – Brazil) and Sonothèque du Museum National d’Histoire Naturelle – MNHN-SO, Paris, France.

Bioacoustic variables were analyzed with Raven Pro 1.6 software, 64-bit version (Bioacoustics Research Program 2014) set as follow: window = Blackman, Discrete Fourier Transform = 2,048 samples and 3dB filter bandwidth = 80.0 Hz. Dominant frequency was measured using the Peak frequency function; maximum and minimum frequencies were measured 20dB below the peak frequency to avoid background noise interference. Call centered terminology and categorization follows Köhler et al. (2017): call duration (CD), number of notes (NN), note duration (ND), inter-note interval (SBN), minimum (LF), maximum (HF), and dominant frequency (DF). Inter-call interval was not measured because it is affected by microclimatic conditions at the time of recording (i.e., on rainy days males call more often in a short period of time than on days without rain). Spectrogram and oscillogram were generated in R environment (R Core Team 2019) through the ‘seewave’ package 2.0.5 (Sueur et al. 2008) using a Hanning window, 256 points of resolution (Fast Fourier Transform) and an overlap of 85.

Statistical Analyses

We used a principal component analysis (PCA) via FactoMineR package in R v.3.2.4 (Lê et al. 2008; R Core Team 2019) associated with analysis of variance (ANOVA) to investigate the variation of morphometric and bioacoustic data between the new species and P. ockendeni sensu stricto.

We conducted the analysis with morphometric ratios (morphometric measurements divided by SVL) using only males since the P. ockendeni sensu stricto has insufficient data for females. We normalized data with the function ‘data.Normalization’ of the package ‘clusterSin’ (Walesiak and Dudek 2020) and tested for homoscedasticity with the Levene Test using the function ‘leveneTest’ of the package ‘car’ (Fox and Weisberg 2019). PCAs were run using the function ‘prcomp’ of the package stat with parameters ‘scale’ and ‘center’ set as ‘True’. We obtained the number of retained principal components (PCs) with the broken stick model using the function ‘screeplot’ of the package ‘vegan’ (Oksanen et al. 2022), which retained the first two PCs for both PCAs. Finally, we prepared the graphs using the function ‘autoplot’ of the package ‘ggfortify’ (Horikoshi and Tang 2016; Tang et al. 2016).

We conducted an ANOVA for PC1 and PC2 for morphometric and bioacoustic analyses to test the existence of statistical difference in the space occupied by males of the new specie and P. ockendeni. Morphological and bioacoustic analyses were conducted on the R platform (R Core Team 2021).

Results

Phylogenetic relationships and genetic distances

The two focal species are not closely related, even though they are both nested in a large strongly supported clade formed by species of the Pristimantis unistrigatus species group. Relationships inferred from the Maximum Likelihood analysis within that clade are well-supported (Bootstraps > 70%; Fig. 1). The individuals of the new species from the Guiana Shield form a monophyletic group, and display low intraspecific genetic distances on 16S (p-distance = mean 0.4%, maximum 1.6%). The new species is nested within a strongly supported clade (100%) formed with four additional major lineages (mean p-distance between 5.8 and 7.3% to the new species) located south of the Amazon River that were historically confused with P. ockendeni. This clade corresponds to the “trans-Amazon complex” of Fouquet et al. (2022b) and is distantly related to two species from Peru: P. ardalonychus (Duellman and Pramuk, 1999), p-distance = 13.3%, and P. martiae (Lynch, 1974), p-distance = 10.4% (Table 1).

Table 1.

Interspecific and intraspecific genetic distances between Pristimantis guianensis sp. nov. and closely related taxa. Uncorrected p-distances (%; lower diagonal) and Kimura-2-parameter (%; upper diagonal) for sequences in a matrix with 562 bp from 16S mtDNA gene and expressed as percentages. Bold numbers in the diagonal represent intraspecific p-distance.

Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
1 P. guianensis sp. nov. 0.4 6.1 6.2 7.3 7.7 17.9 13.4 10.2 16.1 14.2 15.8 8.3 16.5 10.4 14.1 15.9 13.2 13.0 10.4
2 P. sp._South PA 5.8 1.4 5.3 4.5 4.9 16.8 13.7 11.0 16.8 15.1 16.6 5.9 16.4 10.5 12.4 16.7 11.6 13.0 10.2
3 P. sp. Abacaxis1 5.9 5.11 7.0 6.7 16.1 13.7 8.9 15.6 12.8 15.8 5.9 14.0 8.6 11.7 16.2 12.6 12.2 8.3
4 P. sp. South AM, RO 6.9 4.3 6.6 1.6 5.6 17.1 15.0 11.3 17.2 15.3 17.9 8.7 16.1 11.2 12.5 16.3 13.6 14.0 10.6
5 P. sp. Abacaxis2 7.3 4.7 6.4 5.3 17.6 14.4 11.4 18.2 14.9 17.7 6.5 15.8 12.5 14.1 16.7 14.2 14.4 10.9
6 P. ockendeni SS 15.7 14.8 14.1 15.1 15.6 0.2 14.8 8.1 14.1 12.4 12.9 5.4 13.6 8.0 7.0 14.7 10.5 10.6 8.0
7 P. ardalonychus 12.1 12.4 12.5 13.4 13.1 13.2 7.2 12.2 9.7 9.5 5.7 13.3 7.1 9.5 11.6 10.1 8.4 6.9
8 P. bogotensis 9.4 10.1 8.3 10.3 10.4 7.6 6.8 8.8 6.5 5.9 5.0 11.8 7.3 9.1 4.8 6.7 5.1 5.7
9 P. buenaventura 14.2 14.7 13.9 15.1 15.9 12.6 11.1 8.1 9.7 10.1 4.8 15.1 8.9 10.6 14.9 10.4 11.5 7.4
10 P. cajamarcensis 12.8 13.5 11.7 13.7 13.4 11.3 9.0 6.1 9.0 8.5 4.8 13.9 6.5 9.4 12.0 10.7 8.5 5.6
11 P. ceuthospilus 14.1 14.7 14.1 15.6 15.7 11.7 8.9 5.6 9.3 8.0 7.0 11.6 6.6 8.2 11.6 8.8 8.0 6.0
12 P. daquilemai 7.7 5.6 5.6 8.0 6.1 5.1 5.4 4.8 4.6 4.6 6.6 0.0 7.6 8.0 5.9 6.9 6.7 5.6 5.1
13 P. delius 14.6 14.6 12.7 14.3 14.2 12.3 12.0 10.7 13.4 12.6 10.6 7.4 12.4 13.5 13.9 15.8 8.9 10.7
14 P. martiae 9.6 9.6 8.1 10.3 11.4 7.5 6.7 6.8 8.2 6.1 6.3 7.5 11.3 9.6 7.1 10.0 5.4 6.2
15 P. matidiktyo 12.7 11.3 10.7 11.4 12.7 6.6 8.9 8.5 9.8 8.8 7.7 5.6 12.3 9.0 0.0 8.5 10.5 7.2 8.4
16 P. miyatai 14.2 14.8 14.3 14.4 14.9 13.1 10.6 4.6 13.1 10.9 10.6 6.6 12.5 6.7 8.0 8.0 10.2 6.6
17 P. taeniatus 11.9 10.6 11.5 12.2 12.8 9.7 9.3 6.4 9.5 9.8 8.1 6.4 14.0 9.2 9.7 7.5 3.8 8.1 8.7
18 P. unistrigatus 11.9 11.8 11.3 12.7 13.0 9.8 7.9 4.9 10.5 8.0 7.6 5.3 8.3 5.2 6.8 9.4 7.6 4.4
19 P. zophus 9.6 9.4 7.8 9.7 10.1 7.5 6.5 5.4 7.0 5.3 5.7 4.9 9.9 5.9 7.9 6.3 8.1 4.2 4.9
Figure 1. 

Part of the phylogenetic reconstruction showing the relationships of Pristimantis guianensis sp. nov. and P. ockendeni. Maximum likelihood tree inferred based on 16S, COI and RAG1. Non-parametric bootstrap support is shown above branches. The species name is preceded by the specimen voucher number (continuation of the tree in Appendix 3).

Pristimantis ockendeni from Peru (near to the type locality) clusters with specimens from Acre state (Brazil; p-distance = 0.2%) and Peru and this clade is phylogenetically related to another undescribed species from Peru (voucher MUBI 17035[Appendix 1], p-distance 10.9%) which altogether form a well-supported clade with P. matidiktyo Ortega-Andrade and Valencia, 2012, p-distance 7.5 (Fig. 1; Table 1).

Morphometric and acoustic analyses

The first two Principal Components (PCs) of morphometric and bioacoustic PCAs explained together ~47 and ~82% of data variance, respectively. Neither morphometric (Fig. 2A) nor bioacoustic (Fig. 2B) spaces occupied by the new species and Pristimantis ockendeni overlap in graphic representations. ANOVAs showed that these species are significantly different along the first (S2 = 259.6, F = 142.3, DF = 47, p < 0.0001) and second morphometric (ANOVA: S2 = 20.69, F = 5.5, DF = 47, p = .023) and first bioacoustic PCs (first: S2 = 153.8, F = 207.6, DF = 35, p < 0.0001). However, they do not significantly differ along bioacoustic PC2 (ANOVA: S2 = 1.0, F = 1.3, DF = 35, p = .258). The three main variables contributing to the variation captured in morphometric PC1 are FLIII, HANDII and HAND, and in bioacoustics PC1 are DC, LF and DF. Contribution of other variables in PC1 and PC2 of morphometric and bioacoustic analyses are shown in Table 2 and Table 3, respectively.

Table 2.

Loadings of 23 morphometric ratios on the first two principal components. Values generated by a principal component analysis based on 43 males of Pristimantis guianensis sp. nov. and nine males of Pristimantis ockendeni sensu stricto.

Variables PC1 PC2
HW 0.038 –0.332
HL –0.017 –0.382
SL –0.013 –0.223
IND –0.024 –0.139
EN –0.071 –0.078
IOD 0.068 –0.131
ED –0.171 –0.141
TD 0.197 0.013
TL 0.064 –0.362
TAL –0.214 –0.184
FL 0.311 –0.133
FLI 0.241 –0.197
FLII 0.293 –0.078
FLIII 0.339 –0.101
FLV –0.167 –0.268
WTD –0.079 –0.161
UAL –0.302 –0.184
FAL –0.136 –0.274
HAND 0.332 –0.137
HANDI 0.265 –0.031
HANDII 0.334 0.129
HANDIV 0.279 –0.117
WFD –0.009 –0.014
Table 3.

Loadings of seven bioacoustic measurements on the first two principal components. Values were generated by a principal component analysis based on the advertisement call of 34 males of Pristimantis guianensis sp. nov. and seven males of Pristimantis ockendeni sensu stricto.

Variables PC1 PC2
CD 0.430 0.235
ND 0.345 –0.479
NN 0.320 0.675
INI 0.365 0.272
LF –0.405 0.191
HF –0.366 0.054
DF –0.403 0.382
Figure 2. 

Principal Component Analysis (PCA) comparing Pristimantis guianensis sp. nov. (red circles) and Pristimantis ockendeni sensu stricto (yellow triangles). A morphometric data, B bioacoustic data. Red star indicates holotype of P. guianensis sp. nov. and yellow star indicates syntype of Pristimantis ockendeni.

Taxonomic account

Pristimantis ockendeni (Boulenger, 1912)

Figs 3, 4, 5

Hylodes ockendeniBoulenger (1912)

Eleutherodactylus ockendeniLynch (1996)

Pristimantis ockendeniHeinicke et al. (2007), Hedges et al. (2008), Padial et al. (2014), Ocampo et al. (2016), von May et al. (2017), Villacampa et al. (2017)

Syntypes

BMNH 1947.2.16.88, BMNH 1947.2.16.89 and BMNH 1947.2.16.90 (Fig. 3), collected on 07 May 1907 from La Union, Rio Huacamayo, Carabaya, southeastern Peru (13°31′58.3″S, 69°45′06.7″W; 783 m elevation).

Figure 3. 

Dorsal, ventral and lateral views of syntypes of Pristimantis ockendeni. A, B and C female, NHM 1947.2.16.88; D, E and F female, NHM 1947.2.16.89; and G, H and I male, NHM 1947.2.16.90. Photographs: Natural History Museum of London.

The specimens are in a good state of preservation, allowing for an easy view, for example, of the dark bar between the eyes, of oblique black streak in front of and behind the eye, oblique brown bars on the tibia, supratympanic stripe and melanophores in ventral surface.

We compared the newly collected individuals with the morphological diagnosis described by Boulenger (1912) and the syntypes of species (through high quality images made available by the Natural History Museum – London) to ensure that we are dealing with the same taxon. Moreover, the molecular analyses confirmed that our material is conspecific to a specimen (voucher RvM5_12) collected near the type locality (112 km linear distance) and made available by von May et al. (2017). Our samples are distributed at altitudes ranging from 223 m (in Brazil) to 924 m (in Peru), range that includes the altitude of the type locality (783 m).

The characters available in the species description are relatively vague. Boulenger (1912) defined the size of the species as 34 mm from snout to vent, based on the two female syntypes (NHM 1947.2.16.88 and NHM 1947.2.16.89). Boulenger might not have included the third syntype (NHM 1947.2.16.90) because he believed it was a juvenile individual, but our examination showed this specimen to be an adult male.

We acknowledge that >100 km separates the type locality from the closest newly collected population that could corresponds to Pristimantis ockendeni. Therefore, on the sole basis of geographical distance and considering the megadiversity and the cryptic morphology of this clade we cannot completely reject the hypothesis that our populations in fact belong to another species than P. ockendeni.

Nevertheless, four other nominal species of the Pristimantis unistrigatus species group are known to occur in the area [i.e. P. altamazonicus (Barbour & Dunn, 1921), P. carvalhoi (Lutz, 1952), P. divnae Lehr & von May, 2009, and P. ventrimarmoratus (Boulenger, 1912); Duellman 2005; Villacampa et al. 2017] and one undescribed (“Pristimantis sp. 3” sensu Villacampa et al. 2017). However, all of them have distinctive features on groin (yellow, orange or red marks) or belly (black marble) that readily distinguishes them from P. ockendeni. Also, the main lineages of the P. unistrigatus species group related to P. ockendeni [i.e., P. matidiktyo, P. daquilemai Brito-Zabata, Reyes-Puig, Cisneros-Heredia, Zumel & Ron, 2021, and P. delius (Duellman & Mendelson, 1995)], and possible any undescribed species related to them, can be easily distinguished from P. ockendeni. For example, P. matidiktyo differs by the dorsum without any marks and the presence of a small tubercle on the upper eyelid, P. daquilemai differs by its flanks densely tuberculated and the presence of a prominent rostral papilla at tip of snout, and P. delius is distinguished by the snout acutely rounded in dorsal view and the absence of dentigerous processes of vomers. Finally, the characters described by Boulenger (1912), although vague, but completed by the examination of the syntypes (see below), strongly suggests that the newly collected specimens are conspecific to P. ockendeni.

Referred material

Available in Appendix 1.

Amended diagnosis

The species is characterized by the combination of the following characters, based on the description of Boulenger (1912) with additions to this study, obtained through examination of the syntypes (Fig. 3) and to the material recently collected (Fig. 4): (1) dorsal skin smooth to shagreen, frequently with distinctly enlarged tubercles, with or without W-shaped on scapular region, a narrow light vertebral line may be present; (2) tympanum very indistinct, its length 30 to 44% of eye length, tympanic membrane and tympanic annulus visible but sometimes inconspicuous; with or without supratympanic stripe; (3) snout short and subacuminate in dorsal view, with moderately strong, curved canthus and very oblique, concave loreal region; (4) upper eyelid usually bearing tubercles; dark bar between the eyes, and oblique black streak in front of and behind the eye; cranial crests absent; (5) nostril near the tip of the snout; interorbital space hardly as broad as the upper eyelid; (6) tongue oval, entire or indistinctly nicked behind; (7) dentigerous processes of vomers in two oblique oval groups just behind the level of the choanae, clearly visible; (8) vocal slits present, vocal sac median to subgular; nuptial pads absent; (9) Finger I shorter than Finger II; fingers moderate, discs ovoid to expanded (widest on fingers III and IV); (10) fingers without lateral fringes; (11) three to four ulnar tubercles, ill defined; (12) tibia length 49–56% of SVL; (13) heel bearing one rounded tubercle; small tarsal tubercles, ovoid, poorly visible in fixed specimens; inner edge of tarsus with a short fold; (14) two feebly prominent metatarsal tubercles, being thenar tubercle ovoid to elliptical and small palmar tubercle ovoid; subarticular tubercles well developed but small; (15) toes without lateral fringes; scarcely a rudiment of web between them; (16) Toe I clearly smaller than Toe II, toes moderate, discs ovoid to expanded (widest on toes III to V); (17) dorsal coloration very variable; two or three oblique brown bars on the tibia; ventral surface smooth to slightly areolate, variable concentration of melanophores; and ventral region of the femur externally slightly areolate; (18) absence of spot on groin; (19) dichromatic iris in life, the lower part being coppery and the upper part cream; (20) SVL in adult males of 18.3–22.8 mm (n = 9; Table 4) and females of 30.4–30.6 mm (n = 2; Table 4); and (21) advertisement call with average call duration of 541 ± 151 ms, inter-note interval of 44 ± 5 ms (68 ± 11 ms), minimum frequency of 2,046–2,610 Hz, maximum frequency of 2,964–3,641 Hz and dominant frequency of 2,519–3,143 Hz.

Table 4.

Morphometric measurements in millimeters of adults of Pristimantis guianensis sp. nov. and P. ockendeni. The measurement of the two P. ockendeni females correspond to the syntypes. Values express mean ± standard deviation (range). Measurement abbreviations are listed in the Material and Methods.

Variables Pristimantis guianensis sp. nov. Pristimantis ockendeni
Holotype Males (n = 43) Females (n = 13) Males (n = 9) Females (n = 2)
Syntype Newly collected (n = 8)
SVL 17.7 17.9 ± 0.8 (16.2–20.7) 23.8 ± 1.3 (21.4–25.7) 18.6 19.6 ± 1.3 (18.4–22.8) 30.4–30.6
HW 6.4 6.5 ± 0.4 (5.7–7.6) 8.6 ± 0.5 (7.5–9.6) 6.8 7.0 ± 0.5 (6.7–8.2) 10.4–10.7
HL 6.7 6.8 ± 0.4 (5.9–7.9) 8.7 ± 0.7 (7.6–10.0) 6.9 7.2 ± 0.4 (6.7–8.3) 10.8–10.8
SL 2.9 3.0 ± 0.2 (2.6–3.5) 4.1 ± 0.2 (3.8–4.5) 2.9 3.2 ± 0.3 (2.7–3.6) 4.4–4.6
IND 1.9 1.8 ± 0.2 (1.5–2.2) 2.3 ± 0.2 (1.9–2.5) 1.8 2.0 ± 0.1 (1.8–2.2) 2.6–2.7
EN 2.1 2.1 ± 0.1 (1.7–2.3) 2.9 ± 0.1 (2.8–3.2) 1.8 2.2 ± 0.3 (1.8–2.6) 3.3
IOD 3.6 2.1 ± 0.1 (1.8–2.5) 2.8 ± 0.2 (2.4–3.1) 2.3 2.4 ± 0.2 (2.0–2.7) 3.8–3.9
ED 2.7 2.7 ± 0.2 (2.3–3.0) 3.2 ± 0.1 (3.1–3.4) 2.7 2.7 ± 0.1 (2.6–3.0) 4.1–4.3
TD 0.8 0.8 ± 0.1 (0.6–1.0) 1.0 ± 0.1 (0.8–1.2) 0.9 1.0 ± 0.1 (0.9–1.2) 1.9
UAL 4.7 4.7 ± 0.3 (4.0–5.2) 6.4 ± 0.3 (5.8–6.7) 4.9 5.2 ± 0.4 (4.8–6.2) 6.5–7.7
FAL 4.6 4.4 ± 0.2 (3.8–4.9) 5.5 ± 0.8 (3.8–6.3) 4.2 4.4 ± 0.4 (3.8–5.1) 5.2–5.3
HAND 4.5 4.3 ± 0.3 (3.6–5.1) 5.4 ± 0.4 (4.7–6.1) 5.0 5.3 ± 0.5 (4.9–6.6) 6.3–4.5
HANDI 2.2 2.2 ± 0.2 (1.8–2.9) 2.8 ± 0.3 (2.2–3.2) 2.6 2.7 ± 0.3 (2.4–3.4) 3.9–4.0
HANDII 3.0 2.8 ± 0.3 (2.0–3.4) 3.5 ± 0.5 (2.6–4.1) 3.2 3.5 ± 0.3 (3.2–4.2) 5.6–5.8
HANDIV 3.5 3.7 ± 0.2 (3.2–4.3) 4.7 ± 0.3 (4.3–5.1) 4.1 4.3 ± 0.4 (3.9–5.3) 7.0–7.3
WFD 0.8 0.8 ± 0.1 (0.5–1.0) 1.0 ± 0.1 (1.0–1.2) 0.8 0.8 ± 0.1 (0.8–0.9) 1.9
THL 9.2 8.7 ± 0.6 (7.1–10.4) 10.7 ± 0.7 (10.0–12.0) 9.2 9.3 ± 0.5 (8.7–10.5) 14.9–15.6
TL 9.6 9.3 ± 0.5 (8.2–10.7) 11.5 ± 0.6 (10.7–12.5) 10.1 10.2 ± 0.6 (9.5–11.4) 16.1–16.2
TAL 5.6 5.2 ± 0.3 (4.4–5.9) 6.6 ± 0.5 (5.7–7.3) 5.0 5.2 ± 0.4 (4.9–5.7) 6.8–7.8
FL 7.2 6.9 ± 0.4 (5.8–7.8) 8.6 ± 0.5 (7.9–9.5) 7.9 8.3 ± 0.8 (7.5–10.1) 12.3–12.5
FLI 2.4 2.3 ± 0.2 (2.0–2.6) 3.0 ± 0.2 (2.7–3.3) 2.4 2.6 ± 0.3 (2.4–3.2) 4.1–4.5
FLII 2.9 3.0 ± 0.2 (2.7–3.5) 3.8 ± 0.2 (3.6–4.2) 3.4 3.5 ± 0.2 (3.3–4.0) 5.5–8.4
FLIII 4.5 4.5 ± 0.2 (4.1–5.1) 5.7 ± 0.3 (5.4–6.3) 5.1 5.4 ± 0.5 (4.9–6.6) 8.4–8.9
FLV 5.7 5.6 ± 0.4 (4.3–6.4) 7.4 ± 0.3 (6.9–7.8) 6.3 6.6 ± 0.7 (5.8–8.2) 9.9–10.8
WTD 0.8 0.8 ± 0.1 (0.5–1.0) 1.1 ± 0.1 (0.9–1.3) 0.8 0.9 ± 0.1 (0.7–1.0) 1.7–1.9
Figure 4. 

Newly collected specimens of Pristimantis ockendeni preserved, in dorsal and ventral view, respectively. A and B INPA-H 43946; C and D INPA-H 43949; and E and F INPA-H 43948. Photographs: A.T. Mônico.

Additional comments about P. ockendeni morphology

Dorsal pattern of males is highly variable, showing three main patterns (n = 8 specimens): (i) coloration strongly delimited and different from sideview, with or without dark transverse stripes on dorsum (n = 4 specimens; Fig. 4A; in life, Fig. 5A, 5B, 5D and 5F); (ii) irregular dark markings present on dorsum (Fig. 4C); and (iii) similar to the previous pattern, but lighter (Fig. E); mostly, interorbital region is covered by a dark band (n = 5 specimens). In addition, in some individual, W-shaped mark on scapular region present, even if not very evident (Fig. 4C; Fig. 5E and 5G). Ventral surface can have different shades, varying according to the concentration of melanophores: darker (Fig. 4B), intermediary (Fig. 4D) or lighter (Fig. 4F). Ventral skin texture is smooth (arms and chest region) to areolate (especially in belly and on femur region).

Figure 5. 

Pristimantis ockendeni. A INPA-H 43946 (SVL 20.1 mm); B INPA-H 43952 (SVL 18.9 mm); C INPA-H 43948 (SVL 19.4 mm); D INPA-H 43945 (SVL 19.0 mm); E INPA-H 43950 (SVL 18.4 mm); F INPA-H 43947 (SVL 18.8 mm); G INPA-H 43949 (SVL 19.6 mm) in dorsal view; and H INPA-H 43949 in ventral view. Photographs: A.T. Mônico.

Dorsal coloration of males in life is highly variable, from brown reddish to yellow greenish (Fig. 5A–5G) with or without interorbital bars. We do not have information on females. Arm is light yellowish. The iris is dichromatic (dark copper metallic down part and cream to silver upper part. Ventral surface is cream translucent (in the belly) and gray translucent (in the pelvic region) with dark melanophores (Fig. 5H).

Advertisement call of Pristimantis ockendeni

We recorded 40 calls of seven males from the underwood vegetation of about 2 m above the ground (air temperatures 23‒25°C; relative humidity 82–99). Descriptive statistics of call parameters are presented in Table 5 and voucher in Appendix 4. The advertisement call of Pristimantis ockendeni has a call duration of 541 ± 151 ms (334–939 ms, n = 40 calls) and 4–8 notes (6.1 ± 0.9, n = 243 notes) with note duration of 24–158 ms (41 ± 31 ms). Calls with 6 and 7 notes were the most common arrangement (37.5% of recorded calls, n = 15, and 32.5% n = 13, respectively), followed by calls with 5 notes (25%, n = 10 calls), and rarely with 4 and 8 notes (2.5% each, n = 1). Inter-note intervals within calls average 62 ± 11 ms (38–87 ms). The spectral structure of the note has average minimum frequency of 2,389 ± 190 Hz (2,046–2,610 Hz), while average maximum frequency is 3,350 ± 201 Hz (2,964–3,641 Hz), and the dominant frequency is 2,864 ± 202 Hz (2,519–3,143 Hz) (Fig. 6A and 6B).

Table 5.

Acoustic variables of the advertisement call of Pristimantis guianensis sp. nov. (n = 111 calls, 34 males) and Pristimantis ockendeni (n = 40 calls, 7 males). Values express mean ± standard deviation (range).

Acoustic variables P. guianensis sp. nov. P. ockendeni
Call duration (ms) 232 ± 42 (158–371) 540 ± 150 (334–940)
Number of notes per call 4.5 ± 0.7 (4–6) 6.1 ± 0.9 (4–8)
Note duration (ms) 18 ± 11 (5–89) 41 ± 31 (27–71)
Inter-note interval (ms) 44 ± 5 (14–56) 68 ± 11 (38–87)
Minimum frequency (Hz) 3,210 ± 152 (2,827–3,695) 2,389 ± 190 (2,047–2,610)
Maximum frequency (Hz) 5,264 ± 639 (4,333–6,688) 3,350 ± 201 (2,965–3,504)
Dominant frequency (Hz) 3,970 ± 179 (3,466–4,521) 2,864 ± 203 (2,519–3,144)
Figure 6. 

Pristimantis ockendeni advertisement calls spectrograms and oscillograms with five and six notes, respectively. A INPA-H 43952 (FNJV 58769) and B INPA-H 43946 (FNJV 58764), air temperature 24.1°C.

Pristimantis guianensis sp. nov.

Figs 7, 8, 9, 10, 11, 15 and 16

Eleutherodactylus ockendeniZimmerman and Rodrigues (1990), Heyer and Hardy (1991), Lima et al. (2006), Menin et al. (2007)

Pristimantis ockendeniRojas-Ahumada and Menin (2010); Lima et al. (2012), Fouquet et al. (2013)

Pristimantis cf. ockendeniVacher et al. (2020), Azevedo et al. (2021), Fouquet et al. (2022b)

Holotype

INPA-H 43918 (field number APL22783), adult male, collected at Reserva Florestal Adolpho Ducke, municipality of Manaus, state of Amazonas, Brazil (2°55′52.0″S; 59°58′25.8″W, 74 m elevation), on 19 November 2019 by A.T. Mônico and I.Y. Fernandes.

Paratopotypes

Seventeen adult specimens (fourteen males and three females), same locality as the holotype: one male INPA-H 43917 (field number APL22782) collected in November 2019; nine males INPA-H 43919, ­INPA-H 439120, INPA-H 439121, INPA-H 43923, ­INPA-H 43924, INPA-H 43925, INPA-H 44248, INPA-H 44249, and INPA-H 44250 (field numbers APL22784–86, 22794–99, respectively) and one female INPA-H 43922 (field number APL22793) collected in December 2019 by A.T. Mônico, I.Y. Fernandes, E.D. Koch and A.P. Lima; two males MPEG 44181 and MPEG 44182 (field numbers APL22804 and 22805, respectively) collected January 2020 by A.T. Mônico and I.Y. Fernandes; two males INPA-H 43942 and INPA-H 43944 (field numbers APL23187 and 23189, respectively) and two females INPA-H 43941 and INPA-H 43943 (field numbers APL23187 and 23189, respectively) collected in 28–29 December 2020 by A.T. Mônico, U.F. Souza and I.Y. Fernandes.

Paratypes

Thirty-eight adult specimens (28 males and 10 females). BRAZIL: AMAZONAS: municipality of Presidente Figueiredo: Km 144 of the BR-174 Highway [six males MPEG 44183, MPEG 44184, INPA-H 43926, INPA-H 44251, INPA-H 43927 and INPA-H 43928 (field numbers APL22806–11, respectively) collected in January 2020 by A.T. Mônico and E.D. Koch], Suçuarana’s Fall at Vila de Balbina [seven males INPA-H 43929, INPA-H 43930, INPA-H 43931, INPA-H 43932, INPA-H 43933, MPEG 44185, MPEG 44186 (field numbers APL22812–18, respectively) and one female MPEG 44187 (field number APL22819) collected in January 2020 by A.T. Mônico and E.D. Koch]; RORAIMA: municipality of São João da Baliza [seven males INPA-H 43935, MPEG 44188, MPEG 44190, INPA-H 43937, INPA-H 43938, INPA-H 43939 and INPA-H 439340 (field numbers APL 22821, 22823, 22825 to 22829, respectively) and three females INPA-H 43934, INPA-H 43936 and MPEG 44189 (field numbers APL22820, 22822, and 22824, respectively) collected in June 2020 by A.T. Mônico, I.Y. Fernandes, M. Ferrão and A.P. Lima]. SURINAME: Sipaliwini [three males MNHN-RA-2020.0115–0117 (field numbers AF2255, 2256 and 2289, respectively) and two females MNHN-RA-2020.0113 and 2020.0114 (field numbers AF2155 and 2258, respectively) collected in April 2014 by A. Fouquet and J.P. Vacher]. GUYANA: Mabura Hill Forest Reserve [four males SMNS11987, 11989, 11993, 11994 and three females SMNS 11990, 11991 and 11995 collected between April 2003 and June 2004; and one male MTD 47769 and one female MTD 47770 collected in August 2010 by. R. Ernst].

Referred material

Available in Appendix 1.

Diagnosis

The new species is characterized by the following unique combination of characters: (1) dorsal skin shagreened, frequently with distinctly tubercles, with or without W-shaped on scapular region; (2) tympanum visible, tympanic membrane poorly differentiated, tympanum diameter 24–34% of eye diameter and annulus partially visible externally; supratympanic black band; (3) snout subacuminate to sub-rounded in dorsal view and rounded in in lateral profile, loreal region concave; (4) upper eyelid tubercles present; dark bar between the eyes, and three oblique black streaks below the eye; cranial crests absent; (5) nostril ovoid, slightly protuberant, directed laterally; interorbital distance 29–37% of head width; (6) tongue cordiform to ovoid; (7) absence of dentigerous processes of vomers (Fig. 7); (8) males with vocal slits, vocal sac median to subgular; nuptial pads absent; (9) Finger I slightly more shorter than II; finger discs ovoid to expanded (finger disc I less expanded compared to finger disc II, III and IV); (10) fingers without lateral fringes; (11) three to five enlarged ulnar tubercles, barely visible in fixed specimens (easily visible in individuals when in life); (12) tibia length 48–56% of SVL; (13) heel tubercle absent; tarsal tubercles aligned, ovoid to elliptical; tarsal fold short, barely visible; (14) thenar tubercle ovoid to elliptical; small palmar tubercle ill-defined, less than 30% of thenar tubercle; (15) toes without lateral fringes; rudiment of web absent; (16) toes I and II almost of the same size; toes discs ovoid (toe disc I and II, especially) to expanded (III, IV and V); (17) belly skin smooth to areolate, and ventral region of the femur externally areolate; (18) in life, translucent groin with small, scattered dark melanophores and absence of bright colored blotches or marks; (19) in life, iris is dichromatic, brown lower part and cream upper part; (20) SVL in adult males of 16.2–20.7 mm (n = 43) and in females of 21.4–25.7 mm (n = 13); and (21) advertisement call with average call duration of 232 ± 42 ms, inter-note interval of 44 ± 5 ms (68 ± 11 ms), minimum frequency of 2,827–3,695 Hz, maximum frequency of 4,333–6,688 Hz and dominant frequency of 3,466–4,521 Hz.

Figure 7. 

Pristimantis guianensis sp. nov. volumetric renderings of μ-CT scans of the skull in ventral views. A Male (Paratype, MNHN-RA-2020.0115) and B Female (Paratype, MNHN-RA-2020.0114). Absence of dentigerous processes of vomers can be noted in the doted red circle.

Comparisons with other species

The new species is compared to other Pristimantis of the P. unistrigatus species group occurring in the Guiana Shield: P. abakapa Rojas-Runjaic, Salerno, Señaris & Pauly, 2013; P. aureoventris Kok, Means & Bossuyt, 2011; P. crepitaculus Fouquet, Peloso, Jairam, Lima, Mônico, Ernst & Kok, 2022; P. espedeus Fouquet, Martinez, Courtois, Dewynter, Pineau, Gaucher, Blanc, Marty & Kok, 2013; P. grandoculis (van Lidth de Jeude, 1904); P. guaiquinimensis (Schlüter & Rödder, 2007); P. imthurni Kok, 2013; P. inguinalis (Parker, 1940); P. jamescameroni Kok, 2013; P. jester Means & Savage, 2007; P. marmoratus (Boulenger, 1900), P. memorans (Myers & Donnelly, 1997), P. pulvinatus (Rivero, 1968); P. saltissimus Means & Savage, 2007; and P. sarisarinama Barrio-Amorós & Brewer-Carias, 2008 and to P. ockendeni, which occurs in southwestern Amazonia. Diagnostic characters of compared species are shown in parentheses unless stated otherwise.

Pristimantis guianensis sp. nov. differs from P. ockendeni by the absence of dentigerous processes of vomers (present) and smaller SVL: male 16.2–20.7 mm (n = 43 specimens) in P. guianensis sp. nov. (18.4–22.8 mm, n = 09 in P. ockendeni) and female 21.4–25.7 mm (n = 13 specimens) in P. guianensis sp. nov. (30.4–30.6 mm, n = 02 in P. ockendeni). Additionally, the advertisement call of P. guianensis sp. nov. has average call duration of 232 ± 42 ms (540 ± 150 ms), inter-note interval of 44 ± 5 ms (68 ± 11 ms) and is emitted at minimum frequency of 2,827–3,695 Hz (2,047–2,610 Hz), maximum frequency of 4,333–6,688 Hz (2,965–3,504 Hz) and dominant frequency of 3,466–4,521 Hz (2,519–3,144 Hz).

The absence of dentigerous processes of vomers easily distinguishes Pristimantis guianensis sp. nov. from P. aureoventris, P. crepitaculus, P. espedeus, P. grandoculis, P. imthurni, P. jamescameroni, P. jester, P. marmoratus and P. saltissimus (present in all species); and the presence of vocal slits in males (absent in P. abakapa, P. aureoventris, P. grandoculis, P. imthurni, P. guaiquinimensis, P. jamescameroni, P. jester and P. saltissimus).

Pristimantis guianensis sp. nov. differs from P. espedeus, P. guaiquinimensis, P. imthurni, P. jamescameroni and P. pulvinatus by having smaller male SVL of 16.2–­20.7 mm (SVL 20.7–24.8 mm in P. espedeus; 33.4–­34.7 mm in P. guaiquinimensis; 22.9 mm in P. imthurni; 22.9 mm in P. jamescameroni; 23.0–26.1 mm in P. pulvinatus; 22.6–25.8 mm in P. sarisarinama); from P. espedeus, P. jamescameroni, P. guaiquinimensis and P. memorans by smaller female SVL of 21.4–25.7 mm (SVL 29.4 mm in P. espedeus; 26.3–27.5 mm in P. jamescameroni; 32.4–33.6 mm in P. guaiquinimensis; 31–32 mm in P. memorans); by having a translucent groin with small, scattered dark melanophores and absence of bright colored blotches or marks in life, P. guianensis sp. nov. can be distinguished from P. abakapa (chocolate brown groin with small white dots), P. aureoventris (black or brown, sometimes with some small golden spots), P. crepitaculus (dark grey groin), P. espedeus (reddish orange groin), P. grandoculis (dark grey groin), P. imthurni (brown groin), P. inguinalis (bright yellow spots on groin), P. jamescameroni (bright orange groin) and P. marmoratus (yellow or pale green wash on groin).

Description of holotype

INPA-H 43918 (field number APL 22783), an adult male (Fig. 8), SVL 17.7 mm; head slightly longer than wide (HL 105% of HW); head width 36.0% of SVL; head length 37.9% of SVL; cranial crest absent; snout subacuminated in dorsal (Fig. 8A) and rounded in lateral (Fig. 8C) views; snout short, END 77.3% of ED; nostril ovoid, slightly protuberant, directed laterally; internarial region slightly concave; canthus rostralis almost straight in dorsal view, rounded in profile; loreal region concave; lips rounded; small tubercles on upper eyelid; interorbital region straight, IOD 32.2% of HW; large eye (ED/TD = 3.4); supratympanic fold distinct, extending from posterior margin of eyelid angling posteroventrally behind tympanic annulus; tympanum visible and rounded, 28.5% of ED; tympanic membrane poorly prominent, directed laterally; tympanic annulus distinct, obscured anteriorly, dorsally, and posteriorly by the supratympanic fold; two small postrictal tubercles, poorly visible; choanae of moderate sized, round, not concealed by palatal sheath of maxilla; dentigerous processes of vomers absent; tongue cordiform, longer than wide; vocal slits present; vocal sac median, simple and subgular.

Figure 8. 

Preserved holotype of Pristimantis guianensis sp. nov. (INPA-H 43918) from Reserva Florestal Adolpho Ducke, Manaus municipality, Amazonas state, Brazil. A dorsal and B views of body; C lateral view of head; and ventral view of D hand and E foot. Photographs: A.T. Mônico.

Forearm slightly longer than hand (FAL 102.6% of HAND); four ulnar tubercles enlarged and aligned, poorly defined; size Finger I<II<IV<III (Fig. 8D); finger discs rounded (Finger I and II) to expanded (Finger III and IV); thenar tubercle ovoid; palmar tubercle bifid and poorly defined, almost twice the width of elongate thenar tubercle; subarticular tubercles well defined, most prominent on fingers I and IV, rounded in dorsal and lateral view; all fingers with ventral pads well defined by circumferential grooves.

Hindlimbs slender, with transversal bars complementary, being four on thigh, three on tibia and two on tarsus; tibia length 54% of SVL; heel without tubercles; tarsus with row of small, low tubercles, poorly visible; tarsal folds absent; foot length 41% of SVL; subarticular tubercles well defined (except in Toe IV), round in dorsal and lateral view; toes lacking lateral fringes and webbed; toes lengths I < II < III < V < IV (Fig. 8E); Toe I only slightly shorter than II; Toe V slightly longer than Toe III (Toe III extending to border of toe discs V); discs well defined by circumferential grooves; toe discs ovoid (toes I, II and III) to expanded (toes IV and V); thenar tubercle ovoid and palmar tubercle small and ill-defined; all toes with ventral pads well defined, most prominent in toes IV and V.

Dorsal skin shagreened, with a W-shaped mark on scapular region, a dark blotch between the eyes and other ill-defined near the groin (Fig. 8A); upper eyelid shagreened, with several distinctly enlarged tubercles on each eyelid; upper and posterior surfaces of hindlimbs smooth; skin on flanks and chest smooth; slightly areolate on belly; and ventral region of the femur externally areolate; dorsolateral folds absent; pectoral and discoidal folds not visible; cloaca not protuberant, cloacal region without tubercles.

Morphometric measurements are presented in Table 4.

Coloration of holotype in preservative : After 30 months in 70% ethanol, colours of the holotype faded, notably glandular supracarpal pad (Fig. 8). The snout is dorsally dark brown and well-delimited by a transversal light gray interorbital band extending on half of the eyelids; dark bands below the eyes; supratympanic black band; black blotches in the tympanic region (Fig. 8C); a dark brown mark is well-defined on the scapular region posteriorly followed by two oblique traversal dark brown bands. Similar, transversal dark brown bands are present on the arms, hands and legs and feet. Arms, belly and ventral surfaces of thighs, tibia and tarsus are cream with brown melanophores, arm and belly skin are the lightest, with melanophores sparsest, while ventral surfaces of hindlimbs have a greater amount of melanophores (Fig. 8B); upper arm without bands (only few melanophores) and lower arm with one dark transversal band; black blotch in the cloaca region; more colors details are shown in Fig. 8.

Coloration of holotype in life : In life, yellow dorsal surface with orange blotches and tubercles all over the dorsal surface; three horizontal dark bands on dorsum, the anterior one on interorbital region, the medial one bordering the W-shaped mark on the scapular region, and posterior one ill-defined near the groin (Fig. 10A). Black blotches located in the tympanic region and dark bands below the eyes. Upper surface of legs and arms with light brown transversal band. Ventral surface is cream covered with minute gray melanophores, while the throat region is slightly yellow. The arm and belly skin are the lightest and the melanophores are the sparsest. Ventral surfaces of thighs, tibia and tarsus are gray, due to the high amount of melanophores. During the day the coloration turns darker. The iris is dichromatic: lower part brown and upper part golden color with dark brown reticulation. More colors details are shown in Fig. 8A.

Intraspecific variation

Pristimantis guianensis sp. nov. is small, SVL in adult males of 16.2–20.7 mm (n = 43; Table 4) and in females of 21.4–25.7 mm (n = 13; Table 4). Dorsal pattern of males is variable: most specimens of the type series (73% of type specimens) show irregular dark markings present on dorsum, similar to holotype (Fig. 9A), slightly dark middorsal and other ill-defined near the groin (12% of types; Fig. 9B), with snout-vent stripe (only one specimen; Fig. 9C), dorsal view coloration strongly delimited and different from sideview (13% of types; Fig. 9D, 9E and 9F), lighter (Fig. 9D) or darker (Fig. 9F), with or without dark transverse stripes on dorsum (Fig. 8E). Specimens of dorsal pattern with irregular dark markings (Fig. 9A and 9C) and slightly dark middorsal (Fig. 9B) has interorbital region is covered by a dark band (85.7% of specimens). In addition, these two patterns are closely linked to the presence of a W-shaped on scapular region (66% of types), even if not very evident (16% of types). Dorsal skin texture is fairly variable too (probably depending on activity, as observed in many species of the genus (e.g., Guayasamin et al. 2015; Kok et al. 2018), from shagreened to granular, with or without tubercles.

Figure 9. 

Preserved Pristimantis guianensis sp. nov. dorsal and ventral surface pattern variation. A MPEG 44181; B INPA-H 43929; C INPA-H 43928; D INPA-H 43939; E MPEG 44185; F INPA-H 43940; G MPEG 44181; H INPA-H 43929; and I INPA-H 43940. Photographs: A.T. Mônico.

Dark bands and blotches located below the eyes are present in the most specimens of type series, not very evident or absent (91%, 4%, and 5% of types, respectively). Transversal dark brown bands are present on the arms and hands, intermediary, or absent (89%, 4%, and 7% of types, respectively). In legs and feet, transversal dark bands are present in most specimens of the type series, intermediary, or absent (80%, 9%, and 11% of types, respectively). The three to five ulnar tubercles are small in 9% of types. Supratympanic black band in all species.

Ventral surface can have different shades, varying according to the concentration of melanophores: light (Fig. 9G), intermediary (Fig. 9H) or dark (Fig. 9I). Most type specimens (57%) had the intermediate concentration, while light and dark were less frequent (29% and 14% of types).

Dorsal background coloration in life is widely variable, from greenish yellow (Fig. 10A, 10B and 10F) and light orange (Fig. 10C, 10F and 10G) to brown (Fig. 10E, 10H, 10I), while females do not vary as much. Snout-vent stripe can be present (Fig. 9G). The iris is dichromatic: brown to dark copper metallic lower part and cream to silver upper part with dark brown reticulation (Fig. 10). When active, the ventral surface is with cream background, cover by white granules and brown melanophores (nocturnal, Fig. 11A), and gray at the day, which is the result of melanophores expansion (diurnal, Fig. 11B).

Figure 10. 

Males of Pristimantis guianensis sp. nov. in life. A Holotype INPA-H 43918 (SVL 17.7 mm); B Paratype MPEG 44188 (SVL 18.4 mm); C Paratopotype INPA-H 44250 (SVL 17.6 mm); D Paratype INPA-H 43939(SVL 17.6 mm); E Paratype MPEG 44185 (SVL 17.9 mm); F Paratopotype MPEG 44181 (SVL 17.8 mm); G Paratype INPA-H 43928 (SVL 17.9 mm); H Paratype INPA-H 43931 (SVL 17.7 mm); and I Paratype INPA-H 43926 (SVL 16.9 mm). Photographs: A.T. Mônico, except (C) E.D. Koch.

Figure 11. 

Ventral coloration of Pristimantis guianensis sp. nov. in life. A Paratopotype INPA-H 43944 (nocturnal); and B Paratype MNHN-RA-2020.0115 (diurnal). Photographs: (A) A.T. Mônico and (B) A. Fouquet.

Advertisement call

We recorded 111 calls of 34 males from 2 m above the ground underwood vegetation at temperatures between 23‒25°C and 82–99% relative air humidity. The list of call recordings is disponible in Appendix 4, and descriptive statistics of call parameters are in Table 5. The advertisement call of Pristimantis guianensis sp. nov. is short (mean 232 ms ± standard deviation 42, range 158–371 ms) and composed of 4–6 notes (4.6 ± 0.7). Calls with four notes were the most common arrangement, corresponding to 51% of all analysed calls (n = 57 calls), followed by calls with five notes (38%; n = 42 calls) and six notes (11%; n = 12 calls). Note duration averaged 16 ± 5 ms (5–30 ms; n = 510 notes). Inter-note intervals are homogeneous within calls (average = 44 ± 5 ms; range 14–56 ms; n = 399 inter-note intervals), i.e., inter-note the same temporal structure. Notes lack true harmonics. The average minimum frequency is 3,210 ± 152 Hz (2,827–3,695 Hz), while average maximum frequency is 5,264 ± 639 Hz (4,333–6,688 Hz), and the dominant frequency is 3,970 ± 179 Hz (3,466–4,521 Hz) (n = 111 calls) (Fig. 12A and 12B, Table 5).

Figure 12. 

Pristimantis guianensis sp. nov. advertisement calls spectrograms and oscillograms with five and six notes, respectively. A Holotype INPA-H 43918 (FNJV 58730); B Paratopotype INPA-H 43919 (FNJV 58731), air temperature 23.2°C.

We provide additional acoustic variables in Appendix 5.

Courtship call repertoire

We observed that when a female is present, males calls consist of 7–8 notes (7.75 ± 0.5; n = 4 calls of two males), with call duration of 359 ± 31 ms (321–391 ms), note duration of 13 ± 2 ms (10–14 ms) with inter-note intervals of 39 ± 1 ms (38–40 ms). The average minimum frequency is 3,097 ± 55 Hz (3,025–3,158 Hz), while average maximum frequency is 5,080 ± 678 Hz (4,134–5,861 Hz), and the dominant frequency is 3,914 ± 191 Hz (3,790–4,199 Hz).

We recorded the vocal repertoire of a male (INPA-H 43944, FNJV 58762) until the amplexus with a female (INPA-H 43943) for ~80 s (Fig. 13). The male jumped and positioned close to the female (approximately 20 cm) and then emitted one call with 8 notes (Fig. 13A), two-harmonic structure and more high-pitched than advertisements calls (maximum frequency reaching 8,182 Hz). The call duration was 386 s, where notes lasted 15 ± 1 ms (13–17 ms) with inter-note intervals of 38 ± 2 ms (35–41 ms). The call was emitted with a dominant frequency of 3,790 ± 172 Hz, minimum frequency of 3,231 ± 168 Hz and maximum frequency of 5,489 ± 602 Hz. The male came even closer to female and, after 47 s emitting the previous call, emitted a second call (Fig. 13B), less high-pitched and longer than the previous one (Fig. 9A): 11 notes, call duration 757 ms, note duration 20 ± 5 ms (17–33 ms), inter-note intervals 54 ± 19 ms (28–85 ms), minimum, maximum and dominant frequency of 3,144 ± 111 Hz, 4,780 ± 158 Hz and 3,704 ± 151 Hz, respectively. Finally, the male emitted two calls with inter-call interval of 860 ms between them (Fig. 13C): the former call has call duration of 761 ms and consisted of 13 notes with note duration of 19 ± 3 ms (14–22 ms), inter-note interval of 45 ± 16 ms (31–87 ms) s, and frequencies were similar to previous call (minimum 3,032 ± 104 Hz, maximum 4,845 ± 245 Hz and dominant of 3,876 ± 213 Hz); while the latter call lasted 547 ms and was composed of 9 notes, with note duration of 18 ± 3 ms (15–25 ms), inter-note interval of 39 ± 4 ms (32–45), except for last note (107 ms), dominant frequency was 3,962 ± 219 Hz, minimum frequency 2,995 ± 76 Hz and maximum frequency 4,705 ± 214 Hz. The female then bent down and formed the amplexus with the male.

Figure 13. 

Pristimantis guianensis sp. nov. courtship call repertoire spectrograms for 80 seconds before the amplexus. Male INPA-H 43944, FNJV 58762; air temperature 24.8°C and humidity 97%. A First call after approaching the female; B second call, issued after 47 s of previous call; C last two calls before forming the amplexus.

Etymology

The specific epithet “guianensis” refers to the region of occurrence of the new species, widely distributed in the western Guiana region (the lowlands of the eastern part of the Guiana Shield).

Distribution, Natural history and Conservation

Pristimantis guianensis sp. nov. is likely endemic to the western portion of the Guiana region (Fig. 14) which is bounded by the Amazon and the Negro Rivers on the South, the Branco River on the southwest. Similarly distributed species are Allobates sumtuosus (Morales, 2002) (Simões et al. 2013), Synapturanus mirandaribeiroi Nelson & Lescure, 1975 (Fouquet et al. 2021a), Amazophrynella manaos Rojas, Carvalho, Ávila, Farias & Hrbek, 2014, and Anomaloglossus stepheni (Martins, 1989) (Vacher et al. 2017). In Brazil, it is known from Manaus and Presidente Figueiredo (Amazonas), São João da Baliza (Roraima) and Oriximiná (Pará); in Guyana from the Mabura Hill Reserve, and in Suriname from Sipaliwini.

Figure 14. 

Geographic distribution of individuals of Pristimantis guianensis sp. nov. (red circles) and Pristimantis ockendeni sensu stricto (yellow triangles) used in our analyses. Stars indicate type localities of each species.

Pristimantis guianensis sp. nov. is crepuscular/nocturnal and inhabits the understory of unflooded (terra firme) forests. Its breeding activity takes place in the rainy season, between November and February in the state of Amazonas, and between May and August in the state of Roraima, both in Brazil. Calling males of P. guianensis sp. nov. aggregate in groups of about eight, separated by 2–3 m distance. At Reserva Ducke (Manaus, Brazil), understory areas rich in Marantaceae are preferentially used (Fig. 15A). In other localities (e.g., São João da Baliza, Brazil), the species is found mainly on the edge of forest fragments. The new species occurs in sympatry with congeneric P. koehleri Padial & De la Riva, 2009 and P. zimmermanae (Heyer & Hardy, 1991) in Manaus and Presidente Figueredo, P. zeuctotylus (Lynch & Hoogmoed, 1977) in São João da Baliza, P. chiastonotus (Lynch & Hoogmoed, 1977), P. zeuctotylus and P. inguinalis in Sipaliwini (Fouquet et al. 2015).

Figure 15. 

Pristimantis guianensis sp. nov. natural history and breeding aspects. A Example of habitat used by the new species; B calling male (INPA-H 43942); C amplectant pair (female INPA-H 43943, male INPA-H 43944), from RFAD, Manaus, Amazonas; D suspended litter spawning about 1 m above the ground, the two clutches are at different stages (left, more developed; right, recent oviposition), from São João da Baliza municipality, Roraima, Brazil. Photographs: A.T. Mônico, except (B) I.Y. Fernandes.

Males call perched on vegetation (Fig. 15B) about 2 m above the ground. They start calling at dusk (~18:00h) and remain active until ~19:00h, their activity then remains sporadical until around midnight. When the weather is rainy, the activity is sustained throughout the night. The amplexus (n = 2) is axillary (Fig. 15C). Juveniles and females are rarely found. We registered three clutches in situ in São João da Baliza (Roraima, Brazil), all within dead leaves hung in the vegetation above the ground. Two clutches were laid in the same nest (Fig. 14D) about 1 m above the ground, one was recent and the other in a more advanced developmental stage; the third clutch (about 40 cm above the ground) contained dead eggs, apparently due to fungal infestation. The first two clutches contained 11 and 13 large eggs. In the surroundings of the nests (up to 5 m), we observed adult males (n= 6 individuals) and females (n = 3).

On 29 December 2020 we found an amplecting pair (Fig. 14C) from Reserva Florestal Adolpho Ducke (Tinga basin), Manaus municipality. Male (INPA-H 43944) and female (INPA-H 43943) remained amplected overnight in the same collection container. Oviposition of 13 eggs took place at ~9:30 on the next day. In laboratory, we observed egg development until hatching (Fig. 16) in a sterile and moist container (at room temperature, between 24.8 and 28.3°C). Egg diameter increased from ~4.1 mm at oviposition to 5.8 mm before froglet hatching. The first froglet has hatched on the 25th day, and it lasted seven days (33rd day) to all the remnant froglets hatch. The SVL of froglets at hatching was ~4.3 mm.

Figure 16. 

Pristimantis guianensis sp. nov. eggs and embryos from Reserva Florestal Adolpho Ducke (Manaus, Brazil). Photographs: A.T. Mônico.

As previously mentioned, Pristimantis guianensis sp. nov. has a wide geographic distribution, from Manaus, Brazil in the South to Mabura Hill Reserve, Guyana in the North (i.e., 800 km linear distance), and potentially 256 thousand km². The species inhabits primary and secondary forests, as well as edge areas. Furthermore, their populations are locally abundant. Therefore, we believe that the species fits into the category “Least Concern” of the International Union for Conservation of Nature (IUCN).

Discussion

The integration of molecular, acoustic and morphological data can help decipher the large proportion of still hidden amazonian biodiversity (e.g., Paéz and Ron 2019; Fouquet et al. 2021b), and lead to the discovery and description of new taxa (e.g., Lima et al. 2020; Carvalho et al. 2021; Fouquet et al. 2021a; Ferrão et al. 2022). However, taxonomic acts are not trivial since many Amazonian species have been historically misidentified, often lumped within putatively widespread taxa (e.g., Amazophrynella minuta Rojas-Zamora et al. 2014; Dendropsophus brevifrons Fouquet et al. 2015; Boana geographica Fouquet et al. 2016). Therefore, tackling these taxonomic issues also requires examining types, and topotypical material and often redescribing or redefining these supposedly widespread species (e.g., Pristimantis marmoratus Kok et al. 2018; Anomaloglossus baeobatrachus Fouquet et al. 2019; Pristimantis grandoculis Fouquet et al. 2022b).

The case of Pristimantis guianensis exemplifies these taxonomic challenges. Populations of P. guianensis were misidentified as P. ockendeni (in Brazil) or P. marmoratus (in Guyana) for over 30 years (Zimmerman and Rodrigues 1990; Heyer and Hardy 1991), although Fouquet et al. (2013) previously questioned the identity of individuals from Manaus. The existence of many morphologically superficially similar species of the P. unistrigatus species group remaining undescribed in Amazonia, associated with the difficulties to clarify the phylogenetic positions and the acoustic characteristics of P. ockendeni (as well as of P. marmoratus, recently resolved by Kok et al. 2018, and P. grandoculis, recently resolved by Fouquet et al. 2022b), represented challenges that needed to be overcome before being able to describe P. guianensis. We demonstrate that the new species is easily diagnosable based on characters that are widely used for Pristimantis.

The distribution of Pristimantis guianensis sp. nov. is restricted to the southwestern portion of the Guiana region sensu Vacher et al. (2020), where the range boundaries of many species overlap (e.g., Allobates sumtuosus, Anomaloglossus stepheni and Amazophynella manaos), geographically bounded southward by the Amazonas River and the Negro River and westward by the Rupununi savanna and the Branco River. We do not expect that P. guianensis to occur on the opposite margins of these rivers where other related species occur, precisely as our ongoing investigations have detected morphological, acoustic and genetic differences between them. We do not expect P. guianensis to reach the Branco River, because it lacks suitable habitat in the open environments of savannas/lavrados in the Guyana and Roraima state, Brazil. Northwestward, the new species is bounded by the Pantepui region in Guyana. However, the landscape features that bound the distribution of P. guianensis and of this bioregion eastward and northward in Para and Suriname are much less clear.

Fouquet et al. (2022b) suggested that Pristimantis guianensis (treated as “P. cf. ockendeni”) might belong to a lineage apart from the other species of the Guiana Shield that evolved from western Amazonian ancestors and posteriorly dispersed to the Guiana Shield across the Amazonas River. Other species hidden under the name “Pristimantis ockendeni” likely occur in the Brazilian Amazon, since at least five lineages forming a “trans-Amazon complex”, proposed by Vacher et al. (2020) and Fouquet et al. (2022b). The taxonomic status of these candidate species and their historical relationships deserve to be clarified.

Funding

This study was funded by GreenPeace Brazil through the program Tatiana de Carvalho de Pesquisa e Conservação da Biodiversidade da Amazônia and by the Brazilian National Council for Scientific and Technological Development (CNPq Universal Grant nº: 401120/2016-3 to A.P.L.). Ale­xander T. Mônico received a PhD fellowship from CNPq (process nº 142153/2019-2). Miquéias Ferrão received an Edward O. Wilson Biodiversity Postdoctoral Fellowship from the Harvard Museum of Comparative Zoology and a fellowship from the David Rockefeller Center for Latin American Studies of Harvard University. Antoine Fouquet benefited from an “Investissement d’Avenir” grant managed by the Agence Nationale de la Recherche (CEBA, ref. ANR-10- LABX-25-01; TULIP, ref. ANR-10-LABX-0041; ANAEE-France: ANR-11- INBS-0001). We thank the μ-CT facilities of the MRI platform member of the National Infrastructure France-BioImaging (supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»), the Labex CEMEB (ANR-10-LABX-0004), and NUMEV (ANR-10-LABX-0020)).

Acknowledgements

We thank Igor Y. Fernandes, Esteban D. Koch, Ubiratã F. Souza, Carlos H. O. Nogueira, Afonso S.O. Meneses, Lucas R. Mendonça and Bryan C. Martins for assistance with fieldwork; to Vanessa Marino, Gilmar Klein and Jassimara Lima for logistic support in Presidente Figueiredo, Balbina and São João da Baliza, respectively; to Jean-Pierre Vacher, Raffael Ernst, Jhon Jairo López-Rojas, Anthony S. Ferreira, Jackeline Delgado, Consuelo Alarcon, Gilberto Huaro and Frank Peter Condori Ccarhuarupay for collected others specimens utilized in this study; to Instituto Nacional de Pesquisas da Amazônia (INPA) for logistic assistance (specially to Andresa Viana) and for supporting molecular data acquisition in the Laboratório Temático de Biologia Molecular (LTBM, specially to Paula Barbosa and Giselle M. Moura) facility; to Fernanda P. Werneck and Ariane Silva for access to the INPA-H collection and to Camila C. Ribas and Marlene Freitas for access to the collection of Genetic Resources; to reviewers (Edgar Lehr, Alessandro Catenazzi and another anonymous reviewer) for important suggestions about the manuscript; to Natural History Museum of London (specially to Jeff Streicher) for making available the photos of the syntypes of Pristimantis ockendeni; to Laboratório de Entomologia Sistemática Urbana e Forense (LESUF – INPA; specially to Marcelo Cutrim) for assistance with photos of the lateral head, hand and foot of holotype; to Instituto Chico Mendes de Conservação da Biodiversidade/Sistema de Autorização e Informação em Biodiversidade for sampling permit (Process n° 73647-3); and to Ethics Committee on the Use of Animals from Instituto Nacional de Pesquisas da Amazônia (CEUA-INPA) by permission (Process n° 35/2020, SEI 01280.001134/2020-63).

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Appendix 1

Referred material

Pristimantis ockendeni

BMNH 1947.2.16.88, Syntype, Female, PERU: ­Departamento Puno: Carabaya: Rio Huacamayo: La Union [13°31′58.3″S; 69°45′06.7″W], 07-May-1907.

BMNH 1947.2.16.89, Syntype, Female, PERU: ­Departamento Puno: Carabaya: Rio Huacamayo: La Union [13°31′58.3″S; 69°45′06.7″W], 07-May-1907.

BMNH 1947.2.16.90, Syntype, Male, PERU: Departamento Puno: Carabaya: Rio Huacamayo: La Union [13°31′58.3″S; 69°45′06.7″W], 07-May-1907.

INPA-H 43945, Male, BRAZIL: Acre: Manoel Urbano: Floresta Estadual do Afluente [8°42′16.6″S; 69°32′02.6″W], 23-Nov-2020 by A.T. Mônico, I.Y. Fernandes, U.F. Souza and C.H.O. Nogueira.

INPA-H 43946, Male, BRAZIL: Acre: Manoel Urbano: Floresta Estadual do Afluente [8°42′16.6″S; 69°32′02.6″W], 23-Nov-2020 by A.T. Mônico, I.Y. Fernandes, U.F. Souza and C.H.O. Nogueira.

INPA-H 43947, Male, BRAZIL: Acre: Manoel Urbano: Floresta Estadual do Afluente [8°42′16.6″S; 69°32′02.6″W], 24-Nov-2020 by A.T. Mônico, I.Y. Fernandes, U.F. Souza and C.H.O. Nogueira.

INPA-H 43948, Male, BRAZIL: Acre: Manoel Urbano: Floresta Estadual do Afluente [8°42′16.6″S; 69°32′02.6″W], 24-Nov-2020 by A.T. Mônico, I.Y. Fernandes, U.F. Souza and C.H.O. Nogueira.

INPA-H 43949, Male, BRAZIL: Acre: Feijó [8°38′46.6″S; 69°43′29.8″W], 25-Nov-2020 by A.T. Mônico, I.Y. Fernandes, U.F. Souza and C.H.O. Nogueira.

INPA-H 43950, Male, BRAZIL: Acre: Manoel Urbano: Floresta Estadual do Afluente [8°42′16.6″S; 69°32′02.6″W], 26-Nov-2020 by A.T. Mônico, I.Y. Fernandes, U.F. Souza and C.H.O. Nogueira.

INPA-H 43951, Male, BRAZIL: Acre: Manoel Urbano: Floresta Estadual do Afluente [8°42′16.6″S; 69°32′02.6″W], 26-Nov-2020 by A.T. Mônico, I.Y. Fernandes, U.F. Souza and C.H.O. Nogueira.

INPA-H 43952, Male, BRAZIL: Acre: Feijó [8°38′46.6″S; 69°43′29.8″W], 29-Nov-2020 by A.T. Mônico, I.Y. Fernandes, U.F. Souza and C.H.O. Nogueira.

Pristimantis ockendeni specimens used exclusively in molecular analyses

MUBI 10538, Unknown sex, PERU: Departamento Madre de Dios: Manu Province: Reserva Comunal Amarakaeri [12°59′25.7″S; 71°00′37.6″W], 25-May-2011 by J.A.D.C. and S.R.M.T.

MUBI13049, Juvenile, PERU: Departamento Cusco: Paucartambo Province: Kosñipata [12°53′10.7″S; 71°26′41.8″W], 28-Aug-2013 by Frank P.C. Ccarhuarupay.

MUBI14568, Juvenile, PERU: Departamento Madre de Dios: Manu Province: Reserva Comunal Amarakaeri [12°46′37.9″S; 70°56′57.8″W], 06-Feb-2015 by J.C. Chaparro, C. Alarcon and G. Huaro.

Pristimantis sp. specimen used exclusively in ­molecular analyses

MUBI17035, Unknown sex, PERU: La Convencion Province: Cusco: Echarate District: Estacion Ticumpinia [11°59′37.8″S 72°59′08.0″W], 09-Seb-2018 by J.C. Chaparro and F.P. Condori.

Pristimantis guianensis sp. nov. specimens used ­exclusively in molecular analyses

MPEG 33750, Male, BRAZIL: Pará: Oriximiná [1°46′07.9″S; 56°12′45.5″W], 27-Sep-2009.

JJLR 007, Male, BRAZIL: Amazonas: Presidente Figueiredo: Reserva Biológica do Uatumã [1°32′19.5″S; 59°32′19.3″W], 28-Nov-2015 by J.J. López-Rojas and A. Ferreira.

JJLR 010, Male, BRAZIL: Amazonas: Presidente Figueiredo: Reserva Biológica do Uatumã [1°32′19.5″S; 59°32′19.3″W], 28-Nov-2015 by J.J. López-Rojas and A. Ferreira.

JJLR 016, Male, BRAZIL: Amazonas: Presidente Figueiredo: Reserva Biológica do Uatumã [1°32′19.5″S; 59°32′19.3″W], 30-Nov-2015 by J.J. López-Rojas and A. Ferreira.

AF2257, Male, SURINAME: Sipaliwini: Savanna Nature Preserve [2°01′27.8″N; 56°07′30.4″W], 27-Apr-2014 by A. Fouquet and J.P. Vacher.

Appendix 2

Species of Pristimantis and Oreobates used in phylogenetic analyses, with respective voucher, Genbank acession number and references.

Species Voucher Genbank acession numbers Reference
16S COI RAG1
P. abakapa VUB3749 JQ742162 Kok et al. (2012)
P. abakapa VUB3750 JQ742163 Kok et al. (2012)
P. acerus KU217786 EF493678 Heinicke et al. (2007)
P. altae AJC0398 JN991361 JQ025174 Pinto-Sanchez et al. (2012)
P. ardalonychus KU212301 EU186664 Hedges et al. (2008)
P. altamazonicus QCAZ44700 MF118685 MF118717 MF118735 Ortega-Andrade et al. (2017)
P. altamazonicus QCAZ20781 MF118699 MF118707 MF118734 Ortega-Andrade et al. (2017)
P. altamazonicus QCAZ51104 MF118678 MF118720 MF118738 Ortega-Andrade et al. (2017)
P. altamnis QCAZ53031 KP064155 KP064164 Ortega-Andrade and Venegas (2014)
P. aureoventris VUB3747 JQ742154 Kok et al. (2012)
P. aureoventris VUB3748 JQ742152 Kok et al. (2012)
P. bogotensis NRPS0033 JN991432 JN991362 Pinto-Sanchez et al. (2012)
P. brevicrus QCAZ52997 MF118697 MF118726 MF118744 Ortega-Andrade et al. (2017)
P. brevicrus QCAZ40964 MF118700 MF118715 MF118751 Ortega-Andrade et al. (2017)
P. buenaventura MZUTI3270 KU999169 Arteaga et al. (2016)
P. cajamarcensis KU217845 EF493663 Heinicke et al. (2007)
P. carvalhoi MUBI13202 OL989869 Ortega-Andrade et al. (2021)
P. carvalhoi EB14.13 MG820152 MG820177 Catenazzi and Lehr (2018)
P. ceuthospilus KU212216 EF493520 Heinicke et al. (2007)
P. chalceus KU177638 EF493675 Heinicke et al. (2007)
P. crepitaculus AF2786 KDQF01001103 Fouquet et al. (2022)
P. crepitaculus 21AF JN691315 Fouquet et al. (2022)
P. crepitaculus UNIFAP 1072 ON908889 ON954784 ON963983 This study
P. crepitaculus UNIFAP 3375 ON908890 ON954785 ON963984 This study
P. crepitaculus UNIFAP 3376 ON908891 ON954786 ON963985 This study.
P. croceoinguinis QCAZ53532 KP064144 KP064157 Ortega-Andrade and Venegas (2014)
P. cruciocularis MTD45716 MG820161 MG820186 Catenazzi and Lehr (2018)
P. cruciocularis MTD45908 MG820162 MG820187 Catenazzi and Lehr (2018)
P. daquilemai QCAZ71332 MZ430056 Zabata-Brito et al. (2021)
P. daquilemai QCAZ71333 MZ430057 Zabata-Brito et al. (2021)
P. delius QCAZ53035 KP064150 KP064162 MF118753 Ortega-Andrade and Venegas (2014)
P. diadematus QCAZ18014 MH516177 Waddell et al. (2018)
P. diadematus QCAZ18015 MH516178 Waddell et al. (2018)
P. espedeus CM395 JN691314 Fouquet et al. (2013)
P. gagliardi CORBIDI13166 OL989857 OL960444 Ortega-Andrade et al. (2021)
P. gagliardi MUBI13205 OL989868 OL960435 Ortega-Andrade et al. (2021)
P. grandoculis MPEG 30085 KDQF01002880 Fouquet et al. (2022)
P. grandoculis MPEG 30088 KDQF01002881 Fouquet et al. (2022)
P. grandoculis MPEG 30084 ON908894 ON964531 ON963988 This study
P. grandoculis MPEG 30091 ON908895 ON964532 ON963989 This study
P. guianensis sp. nov. INPA-H 43918 ON897772 ON898573 ON920937 This study
P. guianensis sp. nov. INPA-H 43919 ON897773 ON898574 ON920938 This study
P. guianensis sp. nov. INPA-H 43920 ON897774 ON898575 ON920939 This study
P. guianensis sp. nov. INPA-H 43921 ON897775 ON898576 ON920940 This study
P. guianensis sp. nov. INPA-H 43923 ON897776 ON898577 ON920941 This study
P. guianensis sp. nov. INPA-H 43924 ON897777 ON898578 ON920942 This study
P. guianensis sp. nov. INPA-H 43925 ON897778 ON898579 ON920943 This study
P. guianensis sp. nov. INPA-H 43943 ON897779 ON898580 ON920944 This study
P. guianensis sp. nov. MPEG 44183 ON897780 ON898581 ON920945 This study
P. guianensis sp. nov. MPEG 44184 ON897781 ON898582 ON920946 This study
P. guianensis sp. nov. MPEG 44188 ON897782 ON898583 ON920947 This study
P. guianensis sp. nov. MPEG 44190 ON897783 ON898584 ON920948 This study
P. guianensis sp. nov. INPA-H 43926 ON897784 ON898585 ON920949 This study
P. guianensis sp. nov. INPA-H 43929 ON897785 ON898586 ON920950 This study
P. guianensis sp. nov. INPA-H 43930 ON897786 ON898587 ON920951 This study
P. guianensis sp. nov. INPA-H 43931 ON897787 ON898588 ON920952 This study
P. guianensis sp. nov. INPA-H 43935 ON897788 ON898589 ON920953 This study
P. guianensis sp. nov. MPEG 33750 ON897789 ON898590 This study
P. guianensis sp. nov. JJRL 0007 ON897790 ON898591 This study
P. guianensis sp. nov. JJRL 0010 ON897791 ON898592 This study
P. guianensis sp. nov. JJRL 0016 ON897792 ON898593 This study
P. guianensis sp. nov. MNHN-RA-2020.0114 KDQF01000865 Fouquet et al. (2022)
P. guianensis sp. nov. AF2257 KDQF01000889 Fouquet et al. (2022)
P. guianensis sp. nov. SMNS 11989 KDQF01004221 Fouquet et al. (2022)
P. guianensis sp. nov. SMNS 11990 KDQF01004222 Fouquet et al. (2022)
P. guianensis sp. nov. SMNS 11994 KDQF01004223 Fouquet et al. (2022)
P. imitatrix CORBIDI7451 OL989854 Ortega-Andrade et al. (2021)
P. imitatrix CORBIDI8735 OL989855 Ortega-Andrade et al. (2021)
P. inguinalis 204BM JN691317 Fouquet et al. (2012)
P. inguinalis AM015 KDQF01001691 Vacher et al. (2020)
P. inguinalis AF2054 KDQF01000814 Vacher et al. (2020)
P. inguinalis MPEG 30059 ON908892 ON964824 ON963986 This study
P. inguinalis MPEG 30060 ON908893 ON964825 ON963987 This study
P. jamescameroni SBH268110 EU186721 Hedges et al. (2008)
P. jester VUB3493 JQ742169 Kok et al. (2012)
P. kichwarum QCAZ52975 KP064154 Ortega-Andrade and Venegas (2014)
P. lirellus KU212226 EF493521 Heinicke et al. (2007)
P. luscombei QCAZ53268 KP064156 Ortega-Andrade and Venegas (2014)
P. luscombei QCAZ25457 EU130618 MH481368 Elmer et al. (2007a)
P. luteolateralis MZUTI3182 KU999208 Arteaga et al. (2016)
P. luteolateralis MZUTI1404 KU999205 Arteaga et al. (2016)
P. marmoratus ROM43302 EU186716 Kok et al. (2018)
P. marmoratus VUB3491 JQ742167 Kok et al. (2018)
P. martiae QCAZ52983 KP064148 KP064160 MF118754 Ortega-Andrade and Venegas (2014)
P. martiae QCAZ52984 KP064149 KP064161 MF118755 Ortega-Andrade and Venegas (2014)
P. matidiktyo QCAZ 53021 KP064147 KP064159 Ortega-Andrade and Venegas (2014)
P. matidiktyo QCAZ 52779 KP064146 KP064158 Ortega-Andrade and Venegas (2014)
P. miktos QCAZ 53531 KP064153 Ortega-Andrade and Venegas (2014)
P. miktos GGU807 KP064151 Ortega-Andrade and Venegas (2014)
P. miktos GGU808 KP064152 KP064163 Ortega-Andrade and Venegas (2014)
P. miyatai AJC3475 KP149490 KP149276 Guarnizo et al. (2015)
P. nietoi MZUTI3050 KU999214 Arteaga et al. (2016)
P. nietoi MZUTI3001 KU999212 Arteaga et al. (2016)
P. ockendeni KU222023 EF493519 EF493434 Heinicke et al. (2007)
P. ockendeni SS RvM5 12 KY652654 KY672986 KY672970 von May et al. (2017)
P. ockendeni SS INPA-H 43945 ON897793 ON898594 ON920954 This study
P. ockendeni SS INPA-H 43946 ON897794 ON898595 ON920955 This study
P. ockendeni SS INPA-H 43947 ON897795 ON898596 ON920956 This study
P. ockendeni SS INPA-H 43949 ON897796 ON898597 ON920957 This study
P. ockendeni SS INPA-H 43952 ON897797 ON898598 ON920958 This study
P. ockendeni SS MUBI 10538 ON907779 This study
P. ockendeni SS MUBI 13049 ON907778 This study
P. ockendeni SS MUBI 14568 ON907780 This study
P. orcus IIAP1063 OL989865 OL960437 Ortega-Andrade et al. (2021)
P. okmoi CORBIDI16294 KY652651 KY672983 KY672967 Ortega-Andrade et al. (2021)
P. pardalis KRL0690 FJ784336 FJ766804 JQ025198 Crawford et al. (2010)
P. pardalis CH6284 JN991460 JN991390 Pinto-Sanchez et al. (2012)
P. parvillus MZUTI483 KU999215 Arteaga et al. (2016)
P. parvillus MZUTI2121 KU999216 Arteaga et al. (2016)
P. pirrensis AJC0594 JN991462 JN991393 JQ025199 Pinto-Sanchez et al. (2012)
P. pulvinatus VUB3751 JQ742164 Kok et al. (2018)
P. pulvinatus VUB3674 JQ742165 Kok et al. (2018)
P. saltissimus VUB3490 JQ742168 Kok et al. (2012)
P. saltissimus ROM43310 EU186693 Hedges et al. (2008)
P. sp. PicoNeblina1 MTR15532 KDQF01003322 Vacher et al. (2020)
P. sp. PicoNeblina1 MTR15534 KDQF01003323 Vacher et al. (2020)
P. sp. PicoNeblina2 MTR15536 KDQF01003323 Vacher et al. (2020)
P. sp. MUBI 17035 ON907781 This study
P. sp. MTR 12855 KDQF01003224 Fouquet et al. (2022)
P. sp. JOG 847 KDQF01002705 Fouquet et al. (2022)
P. sp. BM 153 KDQF01001829 Fouquet et al. (2022)
P. sp. MTR 12690 KDQF01003210 Fouquet et al. (2022)
P. sp. H2773 KDQF01002605 Fouquet et al. (2022)
P. sp. SMS155 KDQF01004256 Fouquet et al. (2022)
P. sp. SMS156 KDQF01004257 Fouquet et al. (2022)
P. taeniatus AJC1839 JN991470 JN991403 JQ025208 Pinto-Sanchez et al. (2012)
P. taeniatus AJC1126 JN991472 JN991406 JQ025206 Pinto-Sanchez et al. (2012)
P. stictogaster KU291659 EF493704 EF493445 Heinicke et al. (2007)
P. unistrigatus KU218057 EF493387 EF493444 Heinicke et al. (2007)
P. walkeri MZUTI3183 KU999230 Arteaga et al. (2016)
P. yuruaniensis VUB3717 JQ742160 Kok et al. (2012)
P. yuruaniensis VUB3720 JQ742161 Kok et al. (2012)
P. zophus NRPS0060 JN991479 JN991414 JQ025213 Pinto-Sanchez et al. (2012)
P. zophus NRPS0072 JN991478 JN991413 JQ025214 Pinto-Sanchez et al. (2012)
O. quixensis ALCX186P53 KU495404 KU494611 Lyra et al. (2016)
O. granulosus AC94_07 KY652649 KY672982 KY672965 von May et al. (2017)

Appendix 3

Phylogenetic reconstruction showing the relationships of Pristimantis guianensis sp. nov. and P. ockendeni. Maximum likelihood tree based on genes 16S, COI and RAG1. Non-parametric bootstrap support is shown near branches. Species names are preceded by the specimen voucher number.

Appendix 4

Call recordings list by species and localities: voucher (call deposit number).

Pristimantis guianensis sp. nov.: BRAZIL, Amazonas, Manaus, Reserva Florestal Adolpho Ducke: INPA-H 43918 (FNJV 58730), INPA-H 43919 (FNJV 58731), INPA-H 43920 (FNJV 58732), INPA-H 43921 (FNJV 58733), INPA-H 43923 (FNJV 58734), INPA-H 43924 (FNJV 58735), INPA-H 43925 (FNJV 58736), APL 22797 (FNJV 58737), APL 22798 (FNJV 58738), APL 22799 (FNJV 58739), INPA-H 43942 (FNJV 58761) and INPA-H 43944 (FNJV 58762). — BRAZIL, Amazonas, Presidente Figueiredo, Km 144 of the BR-174 Highway: MPEG 44183 (FNJV 58740), MPEG 44184 (FNJV 58741), INPA-H 43926 (FNJV 58742), APL 22809 (FNJV 58743), INPA-H 43927 (FNJV 58744) and INPA-H 43928 (FNJV 58745). — BRAZIL, Amazonas, Presidente Figueiredo, Balbina: INPA-H 43929 (FNJV 58746-48), INPA-H 43930 (FNJV 58749), INPA-H 43931 (FNJV 58750), INPA-H 43932 (FNJV 58751), INPA-H 43933 (FNJV 58752), MPEG 44185 (FNJV 58753) and MPEG 44186 (FNJV 58754). — BRAZIL, Roraima, São João da Baliza: MPEG 44188 (FNJV 58755), MPEG 44190 (FNJV 58756), INPA-H 43937 (FNJV 58757-58), INPA-H 43938 (FNJV 58759) and INPA-H 43940 (FNJV 58760). — GUYANA, Mabura Hill Forest Reserve: SMNS 11988 (MNHN-SO-2022-784). — SURINAME, Sipaliwini: MNHN-RA-2020.0115 (MNHN-SO-2022-785) and MNHN-RA-2020.0117 (MNHN-SO-2022-786).

Pristimantis ockendeni : BRAZIL, Acre, Manoel Urbano: INPA-H 43945 (FNJV 58763), INPA-H 43946 (FNJV 58764), INPA-H 43947 (FNJV 58765), INPA-H 43948 (FNJV 58766) and INPA-H 43950 (FNJV 58768). — BRAZIL, Acre, Feijó: INPA-H 43949 (FNJV 58767) and INPA-H 43952 (FNJV 58769).

Appendix 5

Acoustic variables of Pristimantis guianensis sp. nov. and Pristimantis ockendeni summarized according to call arrangement. Abbreviation: SP, species; CD, call duration; ND, note duration (ms); NN, number of notes per call, INI, inter-note interval (ms); LF, HF and DF, minimum, maximum, and dominant frequencies (Hz), respectively.

SP NN CD ND INI LF HF DF
P. guianensis sp. nov. 4 (n = 57) mean 204 18 44 3,195 5,492 3,918
SD 22 10 7 163 368 171
min 158 5 14 2,827 4,334 3,467
max 261 79 54 3,696 6,481 4,436
5 (n = 42) mean 246 17 43 3,261 5,197 4,025
SD 21 14 5 132 287 171
min 188 6 32 2,997 4,414 3,811
max 284 89 56 3,558 6,688 4,522
6 (n = 12) mean 313 14 46 3,098 5,062 4,030
SD 34 6 3 79 618 184
min 265 5 39 2,965 4,521 3,709
max 371 23 51 3,204 6,428 4,414
P. ockendeni 4 (n = 1) 334 43 54 2,610 3,504 3,079
5 (n = 10) mean 385 31 58 2,453 3,383 2,933
SD 31 6 5 25 14 22
min 365 24 50 2,407 3,355 2,885
max 467 41 65 2,493 3,398 2,950
6 (n = 15) mean 506 36 65 2,461 3,405 2,943
SD 37 25 8 165 178 176
min 457 24 52 2,080 2,965 2,519
max 576 125 74 2,609 3,530 3,079
7 (n = 13) mean 700 56 63 2,265 3,278 2,730
SD 121 44 17 215 267 229
min 544 32 38 2,047 2,984 2,519
max 940 158 87 2,610 3,642 3,144
8 (n = 1) 74 40 61 2,072 2,985 2,519
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