104 urn:lsid:arphahub.com:pub:f2cd1fff-21e4-581f-a7fa-850997197b7f urn:lsid:zoobank.org:pub:B1C81912-2D17-4CD8-8D2C-EFEAAAB2EF75 Vertebrate Zoology VZ 1864-5755 2625-8498 Senckenberg Gesellschaft für Naturforschung 10.3897/vz.72.e90435 90435 Research Article Amphibia Anura Strabomantidae Taxonomy A new species of rain frog (Anura: Strabomantidae: Pristimantis) from the Guiana Shield and amended diagnosis of P. ockendeni (Boulenger, 1912) Mônico Alexander Tamanini alexandermonico@hotmail.com https://orcid.org/0000-0002-2965-8020 1 Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Visualization Writing - original draft Writing - review and editing Ferrão Miquéias https://orcid.org/0000-0001-7035-8276 2 Formal analysis Investigation Methodology Supervision Writing - review and editing Chaparro Juan Carlos 3 4 Data curation Investigation Validation Writing - review and editing Fouquet Antoine 5 Data curation Formal analysis Funding acquisition Investigation Methodology Resources Supervision Writing - review and editing Lima Albertina Pimentel https://orcid.org/0000-0003-4586-5633 1 Conceptualization Data curation Funding acquisition Investigation Methodology Project administration Supervision Writing - review and editing Programa de Pós-Graduação em Biologia (Ecologia), Instituto Nacional de Pesquisas da Amazônia, Manaus, Amazonas, Brazil Programa de Pós-Graduação em Biologia (Ecologia), Instituto Nacional de Pesquisas da Amazônia Manaus Brazil Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts, United States of America Harvard University Cambridge United States of America Museo de Biodiversidad del Perú, Cusco, Peru Museo de Biodiversidad del Perú Cusco Peru Museo de Historia Natural, Universidad Nacional de San Antonio Abad del Cusco, Cusco, Peru Universidad Nacional de San Antonio Abad del Cusco Cusco Peru Laboratoire Evolution et Diversité Biologique, Université Paul Sabatier, Toulouse, France Université Paul Sabatier Toulouse France

Corresponding author: Alexander Tamanini Mônico (alexandermonico@hotmail.com)

Academic editor: Uwe Fritz

 2022 10 11 2022 72 1035 1065 D0416380-CB03-5C2A-B3CF-821DC5767D27 15 07 2022 19 10 2022 Alexander Tamanini Mônico, Miquéias Ferrão, Juan Carlos Chaparro, Antoine Fouquet, Albertina Pimentel Lima This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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.

 Amazonia Amphibia Brachycephaloidea integrative taxonomy natural history Terrarana 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. AF 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)). 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 Amazônia – INPA-H (Manaus, Brazil), Museu Paraense Emílio Goeldi – MPEG (Belém, Brazil), Museum National d’Histoire Naturelle – MNHN-RA (Paris, France), Staatliches Museum für Naturkunde – SMNS (Stuttgart, Germany), and Museum für Tierkunde in Dresden – MTD (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).

Interspecific and intraspecific genetic distances between Pristimantis guianensissp. 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
10.3897/vz.72.e90435.figure1 59B2A5C5-8171-5250-AF77-6990F4569DEF

Part of the phylogenetic reconstruction showing the relationships of Pristimantis guianensissp. 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).

https://binary.pensoft.net/fig/768183

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.

Loadings of 23 morphometric ratios on the first two principal components. Values generated by a principal component analysis based on 43 males of Pristimantis guianensissp. 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

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 guianensissp. 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
10.3897/vz.72.e90435.figure2 15D547ED-94DD-5458-994B-C2BE8987E552

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

https://binary.pensoft.net/fig/768184 Taxonomic account Animalia Anura Strabomantidae 62A5E4C0-7EBC-5352-833F-0A44B5FAD525 Pristimantis ockendeni (Boulenger, 1912) Figs 3 , 4 , 5 Hylodes ockendeni – Boulenger (1912) Eleutherodactylus ockendeni – Lynch (1996) Pristimantis ockendeni – Heinicke 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).

10.3897/vz.72.e90435.figure3 C2C97123-173D-501C-9717-A77F2B263980

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.

https://binary.pensoft.net/fig/768185

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.

Morphometric measurements in millimeters of adults of Pristimantis guianensissp. 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
10.3897/vz.72.e90435.figure4 4B0987CF-3349-571F-8EB3-84A551350E70

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

https://binary.pensoft.net/fig/768186 Additional comments about <italic><tp:taxon-name><tp:taxon-name-part taxon-name-part-type="genus" reg="Pristimantis">P.</tp:taxon-name-part> <tp:taxon-name-part taxon-name-part-type="species" reg="ockendeni">ockendeni</tp:taxon-name-part></tp:taxon-name></italic> 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).

10.3897/vz.72.e90435.figure5 66DD6217-B433-50C8-8D0A-1B802AA37D9C

Pristimantis ockendeni. AINPA-H 43946 (SVL 20.1 mm); BINPA-H 43952 (SVL 18.9 mm); CINPA-H 43948 (SVL 19.4 mm); DINPA-H 43945 (SVL 19.0 mm); EINPA-H 43950 (SVL 18.4 mm); FINPA-H 43947 (SVL 18.8 mm); GINPA-H 43949 (SVL 19.6 mm) in dorsal view; and HINPA-H 43949 in ventral view. Photographs: A.T. Mônico.

https://binary.pensoft.net/fig/768187

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 <italic><tp:taxon-name><tp:taxon-name-part taxon-name-part-type="genus" reg="Pristimantis">Pristimantis</tp:taxon-name-part> <tp:taxon-name-part taxon-name-part-type="species" reg="ockendeni">ockendeni</tp:taxon-name-part></tp:taxon-name></italic>.

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).

Acoustic variables of the advertisement call of Pristimantis guianensissp. 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)
10.3897/vz.72.e90435.figure6 1FF23542-25B3-5F63-B117-FFA42F5A35BC

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

https://binary.pensoft.net/fig/768188 Animalia Anura Strabomantidae 3192092D-782F-5923-A663-043C342D78E4 Pristimantis guianensis https://zoobank.org/2CD9F0BF-7BB8-4D12-B901-D56F5F489E2B sp. nov. Figs 7 , 8 , 9 , 10 , 11 , 15 and 16 Eleutherodactylus ockendeni – Zimmerman and Rodrigues (1990), Heyer and Hardy (1991), Lima et al. (2006), Menin et al. (2007) Pristimantis ockendeni – Rojas-Ahumada and Menin (2010); Lima et al. (2012), Fouquet et al. (2013) Pristimantis cf. ockendeni – Vacher 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.

10.3897/vz.72.e90435.figure7 30F9AC9F-843E-57A6-B825-7D05980DD1BB

Pristimantis guianensissp. 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.

https://binary.pensoft.net/fig/768189 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 guianensissp. 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. guianensissp. nov. (18.4–22.8 mm, n = 09 in P. ockendeni) and female 21.4–25.7 mm (n = 13 specimens) in P. guianensissp. nov. (30.4–30.6 mm, n = 02 in P. ockendeni). Additionally, the advertisement call of P. guianensissp. 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 guianensissp. 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 guianensissp. 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. guianensissp. 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.

10.3897/vz.72.e90435.figure8 51FCC2AA-90CE-5A1F-A801-E2EF8797762D

Preserved holotype of Pristimantis guianensissp. 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.

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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 guianensissp. 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.

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Preserved Pristimantis guianensissp. nov. dorsal and ventral surface pattern variation. AMPEG 44181; BINPA-H 43929; CINPA-H 43928; DINPA-H 43939; EMPEG 44185; FINPA-H 43940; GMPEG 44181; HINPA-H 43929; and IINPA-H 43940. Photographs: A.T. Mônico.

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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).

10.3897/vz.72.e90435.figure10 7F997DBA-A673-5F43-8631-640C58309E1C

Males of Pristimantis guianensissp. 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.

https://binary.pensoft.net/fig/768192 10.3897/vz.72.e90435.figure11 B62ADE42-3B2C-5063-8455-2D8CC133E8AA

Ventral coloration of Pristimantis guianensissp. 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.

https://binary.pensoft.net/fig/768193 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 guianensissp. 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).

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Pristimantis guianensissp. 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.

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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.

10.3897/vz.72.e90435.figure13 C555EA32-2069-5E70-AF56-7E29AAD40FB9

Pristimantis guianensissp. 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.

https://binary.pensoft.net/fig/768195 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 guianensissp. 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.

10.3897/vz.72.e90435.figure14 56120759-341F-5365-9960-F80A525B44BD

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

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Pristimantis guianensissp. 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. guianensissp. 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).

10.3897/vz.72.e90435.figure15 1FBBECCD-E503-5A03-8DD7-9B8E95B04322

Pristimantis guianensissp. 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.

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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.

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

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As previously mentioned, Pristimantis guianensissp. 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 minutaRojas-Zamora et al. 2014; Dendropsophus brevifronsFouquet et al. 2015; Boana geographicaFouquet 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 marmoratusKok et al. 2018; Anomaloglossus baeobatrachusFouquet et al. 2019; Pristimantis grandoculisFouquet 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 guianensissp. 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).

 References AltschulSFMaddenTLSchäfferAAZhangJZhangZMillerWLipmanDJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Research25: 3389–3402. ArteagaAPyronRAPenafielNRomero-BarretoPCulebrasJBustamanteLYanez-MunozMHGuayasaminJM (2016) Comparative Phylogeography Reveals Cryptic Diversity and Repeated Patterns of Cladogenesis for Amphibians and Reptiles in Northwestern Ecuador. PLoS One 11(4): e0151746. https://doi.org/10.1371/journal.pone.0151746 AzevedoWOliveiraAMCostaER (2021) Herpetofauna from two locations in the state of Roraima, Amazon Rainforest, Brazil.Herpetology Notes14: 1417–1428. Bioacoustics Research Program (2014) Raven Pro: interactive sound analysis software. Version 1.5. Ithaca, New York: The Cornell Lab of Ornithology. https://ravensoundsoftware.com/software/raven-pro [Acessed 18 May 2022]. BoulengerGA (1912) Descriptions of new batrachians from the Andes of South America, preserved in the British Museum.Annals and Magazine of Natural History8(10): 185–191. CaldwellJPLimaAPKellerC (2002) Redescription of Colostethus marchesianus (Melin, 1941) from its type locality. Copeia 2002: 157–165. https://doi.org/10.1643/0045-8511(2002)002[0157:ROCMMF]2.0.CO;2 de CarvalhoTRMoraesLJCLLimaAPFouquetAPelosoPLVPavanDDrummondLORodriguesMTGiarettaAAGordoMNeckel-OliveiraSHaddadCFB (2021) Systematics and historical biogeography of Neotropical foam-nesting frogs of the Adenomera heyeri clade (Leptodactylidae), with the description of six new Amazonian species.Zoological Journal of the Linnean Society191: 395–433. https://doi.org/10.1093/zoolinnean/zlaa051 CatenazziALehrE (2018) Pristimantis antisuyu sp. n. and Pristimantis erythroinguinis sp. n., two new species of terrestrial-breeding frogs (Anura, Strabomantidae) from the eastern slopes of the Andes in Manu National Park, Peru.Zootaxa13(2): 185–206. https://doi.org/10.11646/zootaxa.4394.2.2 ChávesGCatenazziA (2016) A new species of frog of the genus Pristimantis from Tingo María National Park, Huánuco Department, central Peru (Anura, Craugastoridae).ZooKeys610: 113–130. https://doi.org/10.3897/zookeys.610.8507 CheJChenHMYangJXJinJQJiangKYuanZYMurphyRWZhangYP (2012) Universal COI primers for DNA barcoding amphibians.Molecular Ecology Resources12: 247–258. https://doi.org/10.1111/j.1755-0998.2011.03090.x CrawfordAJLipsKRBerminghamE (2010) Epidemic disease decimates amphibian abundance, species diversity, and evolutionary history in the highlands of central Panama.Proceedings of the National Academy of Sciences107(31): 13777–13782. https://doi.org/10.1073/pnas.0914115107 De OliveiraEAPenhacekMGuimarãesKLAdo NascimentoGARodriguesLRRHernández-RuzEJ (2019) Pristimantis in the Eastern Brazilian Amazon: DNA barcoding reveals underestimated diversity in a megadiverse genus. Mitochondrial DNA Part A: 1–8. https://doi.org/10.1080/24701394.2019.1634696 DuellmanWE (2005) Cusco Amazonico, The lives of amphibians and reptiles in an Amazonian rainforest.Comstock Publishing Associates, Cornell University, New York, 483 pp. DuellmanWELehrE (2009) Terrestrial breeding frogs (Strabomantidae) in Peru.NTV Science, Germany, 382 pp. ElmerKRCannatellaDC (2008) Three new species of leaflitter frogs from the upper Amazon forests: cryptic diversity within Pristimantis “ockendeni” (Anura: Strabomantidae) in Ecuador.Zootaxa38: 11–38. https://doi.org/10.11646/zootaxa.1784.1.2 ElmerKRDávilaJALougheedSC (2007a) Cryptic diversity and deep divergence in an upper Amazonian leaflitter frog, Eleutherodactylus ockendeni.BMC Evolutionary Biology7(1): 247. https://doi.org/10.1186/1471-2148-7-247 ElmerKRDávilaJALougheedSC (2007b) Applying new inter-individual approaches to assess fine-scale population genetic diversity in a neotropical frog, Eleutherodactylus ockendeni. Heredity 99: 506–515. https://doi.org/10.1038/sj.hdy.6801025 FerrãoMSouzaRAColatreliOHankenJLimaAP (2022) Hidden in the litter: cryptic diversity of the leaf-litter toad Rhinella castaneotica-proboscidea complex revealed through integrative taxonomy, with description of a new species from southwestern Amazonia.Systematics and Biodiversity20(1): 1–24. https://doi.org/10.1080/14772000.2022.2039317 FouquetALeblancKFabreACRodriguesMTMeninMCourtoisEADewynterMHöltingMErnstRPelosoPLVKokPJR (2021a) Comparative osteology of the fossorial frogs of the genus Synapturanus (Anura, Microhylidae) with the description of three new species from the Eastern Guiana Shield.Zoologischer Anzeiger293: 46–73. https://doi.org/10.1016/j.jcz.2021.05.003 FouquetALeblancKFramitMRéjaudARodriguesMTCastroviejo-FisherSPelosoPLPratesIManziSSuescunUBaroniSMoraesLJCLRecoderRSouzaSMVecchioFDCamachoAGhellereJMRojas-RunjaicFJMGagliardi-UrrutiaGCarvalhoVTGordoMMeninMKokPJRHrbekTWerneckFPCrawfordAJRonSRMueses-CisnerosJJRojas-ZamoraRRPavanDSimõesPIErnstRFabreA-C (2021b) Species diversity and biogeography of an ancient frog clade from the Guiana Shield (Anura: Microhylidae: Adelastes, Otophryne, Synapturanus) exhibiting spectacular phenotypic diversification.Biological Journal of the Linnean Society,132(2): 233–256. https://doi.org/10.1093/biolinnean/blaa204 FouquetAMartinezQCourtoisEADewynterMPineauKGaucherPBlancMMartyCKokPJR (2013) A new species of the genus Pristimantis (Amphibia, Craugastoridae) associated with the moderately elevated massifs of French Guiana.Zootaxa3750: 569–586. https://doi.org/10.11646/zootaxa.3750.5.8 FouquetAMartinezQZeidlerLCourtoisEAGaucherPBlancMLimaJDMarques-SouzaSRodriguesMTKokPJR (2016) Cryptic diversity in the Hypsiboas semilineatus species group (Amphibia, Anura) with the description of a new species from the eastern Guiana Shield.Zootaxa4084: 79–104. https://doi.org/10.11646/zootaxa.4084.1.3 FouquetANoonanBPRodriguesMTPechNGillesAGemmellNJ (2012) Multiple Quaternary Refugia in the Eastern Guiana Shield Revealed by Comparative Phylogeography of 12 Frog Species.­Systematic Biology61(3): 461–489. https://doi.org/10.1080/14772000.2022.2039317 FouquetAOrricoVGDErnstRBlancMMartinezQVacherJ-PRodriguesMTOuboterPEJairamRRonSR (2015) A new Dendropsophus Fitzinger, 1843 (Anura: Hylidae) of the parviceps group from the lowlands of the Guiana Shield.Zootaxa4052: 39–64. https://doi.org/10.11646/zootaxa.4052.1.2 FouquetARéjaudARodriguesMTRonSRChaparroJCOsornoMWerneckFPHrbekTLimaAPCamacho-BadaniTJaramillo-MartinezAFChaveJ (2022a) Diversification of the Pristimantis conspicillatus group (Anura: Craugastoridae) within distinct neotropical areas throughout the Neogene. Systematics and Biodiversity 20: 1. https://doi.org/10.1080/14772000.2022.2130464 FouquetAPelosoPJairamRLimaAPMônicoATErnstRKokPJR (2022b) Back from the deaf: integrative taxonomy revalidates an earless and mute species, Hylodes grandoculis van Lidth de Jeude, 1904, and confirms a new species of Pristimantis Jiménez de la Espada, 1870 (Anura: Craugastoridae) from the Eastern Guiana Shield.Organisms Diversity & Evolution,2022(2): 1–34. https://doi.org/10.1007/s13127-022-00564-w FouquetAVacherJ-PCourtoisEADeschampsCOuboterPEJairamRGaucherPDuboisAKokPJR (2019) A new species of Anomaloglossus (Anura: Aromobatidae) of the stepheni group with the redescription of A. baeobatrachus (Boistel and de Massary, 1999), and an amended definition of >A. leopardus Ouboter and Jairam, 2012.Zootaxa4576: 439–460. https://doi.org/10.11646/zootaxa.4576.3.2 FoxJWeisbergS (2019) An {R} Companion to Applied Regression. Thousand Oaks CA: Sage. www.socialsciences.mcmaster.ca/jfox/Books/Companion [acessed 10 Apr. 2022]. FrostDR (2022) Amphibian Species of the World: an Online Reference. Version 6.1. https://research.amnh.org/herpetology/amphibia/index.html [Acessed 11 Jul. 2022] GuarnizoCEPazAMunoz-OrtizAFlechasSVMendez-NarvaezJCrawfordAJ (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 GuayasaminJMKrynakTKrynakKCulebrasJHutterCR (2015) Phenotypic plasticity raises questions for taxonomically important traits: a remarkable new Andean rainfrog (Pristimantis) with the ability to change skin texture.Zoological Journal of the Linnean Society173: 913–928. https://doi.org/10.1111/zoj.12222 HedgesSBDuellmanWEHeinickeMP (2008) New World direct-developing frogs (Anura: Terrarana): molecular phylogeny, classification, biogeography, and conservation.Zootaxa1737: 1–182. https://doi.org/10.11646/zootaxa.1737.1.1 HeinickeMPDuellmanWEHedgesSB (2007) Major Caribbean and Central American frog faunas originated by ancient oceanic dispersal.Proceedings of the National Academy of Sciences104(24): 10092–10097. https://doi.org/10.1073/pnas.0611051104 HeyerWRRandASCruzCAGPeixotoOLNelsonCE (1990) Frogs of Boracéia.Arquivos de Zoologia31(4): 231–410. HeyerWRHardyLM (1991) A new species of frog of the Eleutherodactylus lacrimosus assembly from Amazonia, South America (Amphibia: Anura: Leptodactylidae).Proceedings of the Biological Society of Washington104: 436–447. HoangDTChernomorOvon HaeselerAMinhBQVinhLS (2018) UFBoot2: improving the ultrafast bootstrap approximation.Molecular Biology and Evolution35: 518–522. https://doi.org/10.1093/molbev/msx281 HorikoshiMTangY (2016) ggfortify: Data Visualization Tools for Statistical Analysis Results. https://CRAN.R-project.org/package=ggfortify [Acessed 14 Apr. 2022] KalyaanamoorthySMinhBQWongTKFvon HaeselerAJermiinLS (2017) ModelFinder: fast model selection for accurate phylogenetic estimates.Nature Methods14(6): 587–589. https://doi.org/10.1038/nmeth.4285 KatohKStandleyDM (2013) MAFFT Multiple sequence alignment software version 7: improvements in performance and usability.Molecular Biology and Evolution30: 772–780. https://doi.org/10.1093/molbev/mst010 KimuraM (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences.Journal of Molecular Evolution16: 111–120. KöhlerJJansenMRodriguezAKokPJRToledoLFEmmrichMGlawFHaddadCFBRödelMOVencesM (2017) The use of bioacoustics in anuran taxonomy: theory, terminology, methods and recommendations for best practice.Zootaxa4251: 1–124. https://doi.org/10.11646/zootaxa.4251.1.1 KokPJRDezfoulianRMeansDBFouquetA.Barrio-AmorósCL (2018) Amended diagnosis and redescription of Pristimantis marmoratus (Boulenger, 1900) (Amphibia: Craugastoridae), with a description of its advertisement call and notes on its breeding ecology and phylogenetic relationships.European Journal of Taxonomy397: 1–30. https://doi.org/10.5852/ejt.2018.397 KokPJRKalamandeenM (2008) Introduction to the taxonomy of the amphibians of Kaieteur National Park, Guyana.Abc Taxa5: 1–278. KokPJMacCullochRDMeansDBRoelantsKVan BocxlaerIBossuytF (2012) Low genetic diversity in tepui summit vertebrates.Current Biology22(15): 589–590. https://doi.org/10.1016/j.cub.2012.06.034 KokPJRMeansDBBossuytF (2011) A new highland species of Pristimantis Jiménez de la Espada, 1871 (Anura: Strabomantidae) from the Pantepui region, northern South America.Zootaxa2934: 1–19. https://doi.org/10.11646/zootaxa.2934.1.1 LêSJosseJHussonF (2008) FactoMineR: an R package for multivariate analysis.Journal of Statistical Software25: 1–18. https://doi.org/10.18637/jss.v025.i01 LimaAPFerrãoM.SilvaDL (2020) Not as widespread as thought: Integrative taxonomy reveals cryptic diversity in the Amazonian nurse frog Allobates tinae Melo-Sampaio, Oliveira and Prates, 2018 and description of a new species.Journal of Zoological Systematics and Evolutionary Research58: 1173–1194. https://doi.org/10.1111/jzs.12406 LimaAPMagnussonWEMeninMErdtmannLKRodriguesDJKellerCHödlW (2006) Guia dos Sapos da Reserva Adolpho Ducke – Amazônia Central / Guide to the frogs of Reserva Adolpho Ducke – Central Amazonia. 1. ed. Manaus: Attama, 168 pp. LimaAPMagnussonWEMeninMErdtmannLKRodriguesDJKellerC.HödlW (2012) Guia dos Sapos da Reserva Adolpho Ducke – Amazônia Central / Guide to the frogs of Reserva Adolpho Ducke – Central Amazonia. 2. ed. Manaus: Attama, 188 pp. LimaAPSanchezDEASouzaJRD (2007) A new Amazonian species of the frog genus Colostethus (Dendrobatidae) that lays its eggs on undersides of leaves.Copeia2007: 114–122. https://doi.org/10.1643/0045-8511(2007)7%5b114:ANASOT%5d2.0.CO;2 LöselPDvan de KampTJaymeAErshovAFaragóTPichlerOJeromeNTAadepuNBremerSChilingaryanSAHeethoffM (2020) Introducing Biomedisa as an open-source online platform for biomedical image segmentation.Nature Communications11(1): 1–14. https://doi.org/10.1038/s41467-020-19303-w LynchJD (1996) The relationships of the Hispaniolan frogs of the subgenus Pelorius (Eleutherodactylus: Leptodactylidae). In: PowellRHendersonRW (Eds) Contributions to West Indian Herpetology: A Tribute to Albert Schwartz.Oxford, Ohio, Society for the Study of Amphibians and Reptiles, 141–155. LyraMLHaddadCFBAzeredo-EspinAML (2016) Meeting the challenge of DNA barcoding Neotropical amphibians: Polymerase chain reaction optimization and new COI primers.Molecular Ecology Resources17(5): 966–980. https://doi.org/10.1111/1755-0998.12648 MaddisonWPMaddisonDR (2021) Mesquite: a modular system for evolutionary analysis. Version 3.70. http://www.mesquiteproject.org[accessed 21 Apr. 2022]. MeninMLimaAPMagnussonWWaldezF (2007) Topographic and edaphic effects on the distribution of terrestrially reproducing anurans in Central Amazonia: Mesoscale spatial patterns.Journal of Tropical Ecology23(5): 539–547. https://doi.org/10.1017/S0266467407004269 NguyenLTSchmidtHAvon HaeselerAMinhBQ (2015) IQ-TREE: a fast and effective stochastic algorithm for estimating Maximum-Likelihood phylogenies.Molecular Biology and Evolution32: 268–274. https://doi.org/10.1093/molbev/msu300 OcampoMAparicioJWallaceR (2016) Southernmost record for the leaflitter frog Pristimantis ockendeni (Boulenger, 1912) (Anura: Craugastoridae).Check List13(1): 1–3. https://doi.org/10.15­5­60/13.1.2031 OksanenJSimpsonGLBlanchetFGKindtRLegendrePMinchinPRO’HaraRBSolymosPHenryMStevensHSzoecsEWagnerHBarbourMBedwardMBolkerBBorcardDCarvalhoGChiricoMde CaceresMDurandSEvangelistaHBAFitzJohnRFriendlyMFurneauxBHanniganGHillMOLahtiLMcGlinnDOuelletteMHCunhaERSmithTStierATer BraakCJFWeedonJ (2022) vegan: Community Ecology Package. R package version 2.6-2. https://CRAN.R-project.org/package=vegan[accessed 11 Apr. 2022]. Ortega-AndradeHMDeichmannJLChaparroJC (2021) Two new cryptic Pristimantis (Anura, Craugastoridae) from the Southern Amazon Basin of Peru with taxonomic comments on Pristimantis imitatrix (Duellman, 1978).South American Journal of Herpetology21: 41–64. https://doi.org/10.2994/SAJH-D-17-00068.1 Ortega-AndradeHMRojas-SotoOREspinosa de los MonterosAValenciaJHReadMRonS (2017) Revalidation of Pristimantis brevicrus (Anura, Craugastoridae) with taxonomic comments on a widespread Amazonian direct-developing frog.Herpetological Journal27(1): 87–103. Ortega-AndradeHMRojas–SotoORValenciaJHEspinosa de los MonterosAMorroneJJRonSRCannatellaDC (2015) Insights from integrative systematics reveal Cryptic Diversity in Pristimantis frog (Anura: Craugastoridae) from the upper Amazon basin. PLoS One 10(11): e0143392. https://doi.org/10.1371/journal.pone.0143392 Ortega-AndradeHMVenegasPJ (2014) A new synonym for Pristimantis luscombei (Duellman and Mendelson 1995) and the description of a new species of Pristimantis from the upper Amazon basin (Amphibia: Craugastoridae).Zootaxa3895(1): 31–57. https://doi.org/10.11646/zootaxa.3895.1.2 PadialJMDe La RivaI (2009) Integrative taxonomy reveals cryptic Amazonian species of Pristimantis (Anura: Strabomantidae).Zoological Journal of the Linnean Society155: 97–122. https://doi.org/10.1111/j.1096-3642.2008.00424.x PadialJMGonzales-ÁlvarezLReichleSAguayo-VediaCRDe la RivaI (2004) First records of five species of the genus Eleutherodactylus Dumeril and Bibron, 1841 (Anura, Leptodactylidae) for Bolivia.Graellsia60: 167–174. https://doi.org/10.3989/graellsia.2004.v60.i2.212 PadialJMGrantTFrostDR (2014) Molecular systematics of terraranas (Anura: Brachycephaloidea) with an assessment of the effects of alignment and optimality criteria.Zootaxa3825: 1–132. https://doi.org/10.11646/zootaxa.3825.1.1 PaézBPRonSR (2019) Systematics of Huicundomantis, a new subgenus of Pristimantis (Anura, Strabomantidae) with extraordinary cryptic diversity and eleven new species.ZooKeys868: 1–112. https://doi.org/10.3897/zookeys.868.26766 PalumbiSR (1996) Nucleic acids II: the polymerase chain reaction. In: HillisDMMoritzCMableBK (Eds) Molecular Systematics, Sinauer & Associates Inc., Sunderland, Massachusetts, 205–247. Pinto-SanchezNRIbanezRMadrinanSSanjurOIBerminghamECrawfordAJ (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 Evolution62(3): 954–72. https://doi.org.10.1016/j.ympev.2011.11.022 R Core Team (2019) A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing. http://www.r-project.org [acessed 04 Jan. 2022]. Rojas-AhumadaDPMeninM (2010) Composition and abundance of anurans in riparian and non-riparian areas in a forest in Central Amazonia, Brazil.South American Journal of Herpetology5(2): 157–167. https://doi.org/10.2994/057.005.0210 Rojas-ZamoraRRCarvalhoVTGordoMÁvilaRWFariasIPHrbekT (2014) A new species of Amazophrynella (Anura: Bufonidae) from the southwestern part of the Brazilian Guiana Shield.Zootaxa3753: 79–95. https://doi.org/10.11646/zootaxa.3753.1.7 SambrookJRussellDW (2001) Molecular cloning: a laboratory manual. 3rd edition. Cold Spring Harbor Laboratory Press, New York. Silva-e-SilvaYBCosta-CamposCEValentimDSS (2015) Geographic distribution: Pristimantis ockendeni. Herpetological Review 46: 58. SimõesPIKaeferILFariasIPLimaAP (2013) An integrative appraisal of the diagnosis and distribution of Allobates sumtuosus (Morales, 2002) (Anura, Aromobatidae).Zootaxa3746: 401–421. https://doi.org/10.11646/zootaxa.3746.3.1 SueurJAubinTSimonisC (2008) Seewave, a free modular tool for sound analysis and synthesis.Bioacoustics18: 213–226. https://doi.org/10.1080/09524622.2008.9753600 TamuraKStecherGKumarS (2021) MEGA11: Molecular Evolutionary Genetics Analysis Version 11.Molecular Biology and Evolution38(7): 3022–3027. https://doi.org/10.1093/molbev/msab120 TangYHorikoshiMLiW (2016) “ggfortify: Unified Interface to Visualize Statistical Result of Popular R Packages”.The R Journal8(2): 478–489. https://doi.org/10.32614/RJ-2016-060 TorralvoKCFragaRLimaAPDayrellJMagnussonWE (2021) Environmental filtering and deforestation shape frog assemblages in Amazonia: An empirical approach assessing species abundances and functional traits.Biotropica54: 226–238. https://doi.org/10.1111/btp.13053 TrifinopoulosJNguyenLTvon HaeselerAMinhBQ (2016) W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis.Nucleic Acids Research44: 232–235. https://doi.org/10.1093/nar/gkw256 VacherJ-PChaveJFicetolaFGSommeria-KleinGTaoSThébaudCBlancMCamachoACassimiroJColstonTJDewynterMErnstRGaucherPGomesJOJairamRKokPJRLimaJDMartinezQMartyCNoonanBPNunesPMSOuboterPRecoderRRodriguesMTSnyderAMarques-SouzaSFouquetA (2020) Large scale DNA-based survey of frogs in Amazonia suggests a vast underestimation of species richness and endemism.Journal of Biogeography47: 1781–1791. https://doi.org/10.1111/jbi.13847 VacherJ-PKokPJRRodriguesMTLimaJDLorenziniAMartinezQFalletMCourtoisEABlancMGaucherPDewynterMJairamROuboterPEThébaudCFouquetA (2017) Cryptic diversity in Amazonian frogs: Integrative taxonomy of the genus Anomaloglossus (Amphibia: Anura: Aromobatidae) reveals a unique case of diversification within the Guiana Shield.Molecular Phylo­genetics and Evolution112: 158–173. https://doi.org/10.1016/j.ympev.2017.04.017 VillacampaJSerrano-RojasSWhitworthA (2017) Amphibians of the Manu Learning Centre and other areas of the Manu region. The Crees Foundation.Cusco, Peru, 282 pp. von MayRCatenazziACorlASanta-CruzRCarnavalACMoritzC (2017) Divergence of thermal physiological traits in terrestrial breeding frogs along a tropical elevational gradient.Ecology and Evolution7(9): 3257–3267. https://doi.org/10.1002/ece3.2929 WaddellEHCrottiMLougheedSCCannatellaDCElmerKR (2018) Hierarchies of evolutionary radiation in the world’s most species rich vertebrate group, the Neotropical Pristimantis leaf litter frogs.Systematics and Biodiversity16: 807–819. https://doi.org/10.1080/14772000.2018.1503202 WalesiakMDudekA (2020) The choice of variable normalization method in cluster analysis. In: SolimanKS (Ed.) Education excellence and innovation management: A 2025 vision to sustain economic development during global challenges.IBIMA, 325–340. ZimmermanBLRodriguesMT (1990) Frogs, snakes, and lizards or the INPA-WWF reserves near Manaus, Brazil. In: GentryAH (Ed.) Four Neotropical Rainforest.Yale University Press, New Haven, 426–454. Zabata-BritoDReyes-PuigCCisneros-HerediaDZumelDRonSR (2021) Description of a new minute frog of the genus Pristimantis (Anura: Strabomantidae) from Cordillera del Condor, Ecuador.Zootaxa5072(4): 351–372. https://doi.org/10.11646/zootaxa.5072.4.3 ZumelDBuckleyDRonSR (2022) The Pristimantis trachyblepharis species group, a clade of miniaturized frogs: description of four new species and insights into the evolution of body size.Zoological Journal of the Linnean Society195(1): 315–354. https://doi.org/10.1093/zoolinnean/zlab044 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 Appendix 3 10.3897/vz.72.e90435.spnov 6BC538EF-4E72-529D-90B9-62961762B61D 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