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.76.e167783 167783 Research Article Amphibia Caudata Plethodontidae Molecular systematics Phylogeny Systematics Taxonomy Hidden in the mist: Three new species of salamanders of the genus Pseudoeurycea (Amphibia: Plethodontidae) from Oaxaca, Mexico Aguilar-Herrera Eduardo A. https://orcid.org/0009-0001-5633-307X 1 2 Conceptualization Writing - original draft Writing - review and editing Data curation Formal analysis Investigation Methodology Validation Visualization Sanabria-Urbán Salomón 3 Conceptualization Writing - original draft Writing - review and editing Data curation Formal analysis Funding acquisition Investigation Methodology Resources Software Validation Visualization Flores-Martínez Dulce L. 1 Conceptualization Writing - original draft Writing - review and editing Data curation Formal analysis Methodology Visualization Gómez-Tapia José David 2 3 Conceptualization Writing - original draft Writing - review and editing Data curation Formal analysis Methodology Visualization Seba-Chacha Misael 1 Writing - original draft Writing - review and editing Data curation Formal analysis Methodology Visualization Vázquez-Reyes Leopoldo D. 4 Conceptualization Writing - original draft Writing - review and editing Data curation Formal analysis Investigation Resources Validation Rivera-Ortíz Francisco A. 5 Conceptualization Writing - original draft Writing - review and editing Data curation Formal analysis Investigation Resources Validation Ramírez-Bastida Patricia https://orcid.org/0000-0003-1260-3768 4 Conceptualization Writing - original draft Writing - review and editing Data curation Formal analysis Investigation Resources Validation Cortés-Ortiz Bruno 1 Writing - original draft Data curation Formal analysis Methodology Visualization Calzada-Arciniega R. Alejandro 6 Conceptualization Writing - original draft Writing - review and editing Formal analysis Methodology Validation Visualization Avila-Ortega Etienne U. 1 2 Writing - original draft Data curation Investigation Methodology Validation Víquez-Vega Dónovan I. 1 2 Writing - original draft Data curation Investigation Methodology Validation Hernández-Ordoñez Omar 6 Writing - original draft Data curation Formal analysis Investigation Methodology Validation Blair Christopher 7 8 Writing - original draft Writing - review and editing Data curation Formal analysis Investigation Methodology Validation Jiménez-Arcos Víctor H. victorhja@iztacala.unam.mx https://orcid.org/0000-0001-6948-0071 1 Conceptualization Writing - original draft Writing - review and editing Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Supervision Validation Laboratorio de Herpetología Vivario, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Av. de los Barrios 1, Los Reyes Ixtacala, Tlalnepantla, México, CP 54090, México Department of Biological Sciences, New York City College of Technology, The City University of New York New York United States of America https://ror.org/00453a208 Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México, Coyoacán, CDMX, CP 04510, México Biology PhD Program, CUNY Graduate Center New York United States of America https://ror.org/00awd9g61 Laboratorio de Ecología, UBIPRO, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Av. de los Barrios 1, Los Reyes Ixtacala, Tlalnepantla, México, CP 54090, México Laboratorio de Herpetología Vivario, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México Mexico City Mexico https://ror.org/01tmp8f25 Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Av. de los Barrios 1, Los Reyes Ixtacala, Tlalnepantla, México, CP 54090, México Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México Mexico City Mexico https://ror.org/01tmp8f25 Laboratorio de Ecología Molecular y Evolución, UBIPRO, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Av. de los Barrios 1, Los Reyes Ixtacala, Tlalnepantla, México, CP 54090, México Laboratorio de Ecología, UBIPRO, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México Mexico City Mexico https://ror.org/01tmp8f25 Departamento de Zoología; Instituto de Biología, Universidad Nacional Autónoma de México, Coyoacán, CDMX, CP 04510, México Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México Mexico City Mexico https://ror.org/01tmp8f25 Department of Biological Sciences, New York City College of Technology, The City University of New York, 285 Jay Street, Brooklyn, NY 11201, United States Laboratorio de Ecología Molecular y Evolución, UBIPRO, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México Mexico City Mexico https://ror.org/01tmp8f25 Biology PhD Program, CUNY Graduate Center, 365 5th Avenue, New York, NY 10016, United States Departamento de Zoología; Instituto de Biología, Universidad Nacional Autónoma de México Mexico City Mexico https://ror.org/01tmp8f25

Corresponding author: Víctor H. Jiménez-Arcos (victorhja@iztacala.unam.mx)

Academic editor Deepak Veerappan

 2026 15 05 2026 76 275 299 8E1FE434-C46F-5143-87C5-1E7596519E79 CC64687A-78D7-4930-9F42-4AC168A8D217 05 08 2025 28 04 2026 Eduardo A. Aguilar-Herrera, Salomón Sanabria-Urbán, Dulce L. Flores-Martínez, José David Gómez-Tapia, Misael Seba-Chacha, Leopoldo D. Vázquez-Reyes, Francisco A. Rivera-Ortíz, Patricia Ramírez-Bastida, Bruno Cortés-Ortiz, R. Alejandro Calzada-Arciniega, Etienne U. Avila-Ortega, Dónovan I. Víquez-Vega, Omar Hernández-Ordoñez, Christopher Blair, Víctor H. Jiménez-Arcos 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. https://zoobank.org/CC64687A-78D7-4930-9F42-4AC168A8D217

Abstract

We describe three new species of lungless salamanders (Plethodontidae) in the genus Pseudoeurycea from Cerro Rabon, located on the eastern slopes of the Sierra Mazateca, Oaxaca, Mexico. This mid-elevation mountain, largely surrounded by warmer lowlands, remains relatively understudied. We present molecular and morphological evidence distinguishing the three new species from their congeners. According to our phylogenetic analysis, one of the new species belongs to the P. leprosa group, while the other two are members of the P. juarezi group. Notably, one of the new species represents the fourth worm salamander known from west of the Isthmus of Tehuantepec, and another exhibits morphological features resembling those of cave- or saxicolous salamanders. In addition, we obtain genetic information from Pseudoeurycea werleri and find high divergence between populations of Los Tuxtlas region, Veracruz and Sierra Mazateca, Oaxaca. With the species described herein, the number of recognized species in the genus Pseudoeurycea rises to 44, of which 43 are distributed in Mexico. Due to their occurrence at high elevations and in rainforest (low elevation) on Cerro Rabon, the new species are likely at risk of extinction. However, the protection of communal lands by Mazatec communities could support the conservation of these species and the rest of the biodiversity on a regional scale.

 Bolitoglossini Pseudoeurycea euguiisp. nov. Pseudoeurycea natsiisp. nov. Pseudoeurycea parraoleaesp. nov. Pseudoeurycea werleri DGAPA, PAPIIT, UNAM (No. IA209820) Introduction

Mexico harbors one of the most diverse biotas in the world, whose origin, divergence, and diversification are closely tied to its geographic position and the evolutionary history of its lineages. In particular, the convergence of the Nearctic and Neotropical regions, the country’s complex geological history, Pleistocene climate change (Ramírez-Barahona and Eguiarte 2013), and the diversification processes within the Mexican Transition Zone help explain the uniqueness of the biota (Morrone 2021). This singularity is reflected in the high levels of endemism across numerous animal and plant groups (Escalante et al. 2009; Flores-Villela and García-Vázquez 2014; Parra-Olea et al. 2014; Rodríguez et al. 2018; Vázquez-Reyes et al. 2018). For instance, approximately 70% of amphibian species are endemic to Mexico (AmphibiaWeb 2026), and new species continue to be described each year, often with highly restricted distributions in remote, poorly explored, and isolated regions (Jiménez-Arcos et al. 2019; Hernández-Austria et al. 2024).

One of the groups most characteristic of the Mexican amphibians is the lungless salamanders (family Plethodontidae), because 117 of the 141 species are endemic to the country (84%; AmphibiaWeb 2026). Plethodontids have undergone extensive diversification in two major regions: temperate North America and Middle America (Wake 1987). The known diversity of plethodontid salamanders in Middle America has significantly increased over the past two decades, largely due to the exploration of previously unsurveyed areas and the application of molecular techniques. In Middle America, Mexico currently holds the highest number of described plethodontid species (140), followed by Guatemala (66), Costa Rica (59), Honduras (40), and Panama (31; AmphibiaWeb 2026). Despite sustained and substantial efforts over several decades to catalog plethodontid diversity, the discovery and description of new species remain frequent (e.g., García-Bañuelos et al. 2020; Parra-Olea et al. 2020; Cázares-Hernández et al. 2022).

Unfortunately, plethodontids are also among the most vulnerable amphibian groups in the face of rapid global change. They exemplify the ongoing loss of taxonomic, functional, and phylogenetic diversity within amphibians (Nowakowski et al. 2018; Pyron 2018). Major threats including habitat loss, the panzootic spread of Batrachochytrium dendrobatidis, and climate change have placed plethodontids, along with other amphibians, at the center of the most severe extinction crisis among terrestrial vertebrates (Pottier et al. 2025). These pressures have led to population declines, range contractions, and even amphibian species extinctions (Scheele et al. 2019; Luedtke et al. 2023). Considering this, it is imperative to prioritize the identification and description of evolutionarily distinct lineages, particularly in underexplored regions, to develop conservation strategies for this highly threatened group.

The state of Oaxaca harbors the highest plethodontid richness in Mexico with 48 species, 35 of which are endemic to the state (72.9%; Mata-Silva et al. 2021). Further, several species have a narrow geographic distribution (e.g., Pseudoeurycea saltator). The discovery of new species has been largely driven by the exploration of remote and inaccessible mountainous regions (Parra-Olea et al. 2020). In recent fieldwork aimed at studying patterns of diversity loss in the geographically and socially isolated region of Cerro Rabon, located in the Sierra Mazateca of Oaxaca, we documented a remarkably diverse plethodontid community. Local species richness included eight plethodontid species distributed across rainforest and cloud forest habitats. We were unable to assign four taxa from the cloud forest and one from the rainforest to any previously described species. Subsequent molecular analyses and detailed morphological examination allowed us to quantify population divergence in Pseudoeurycea werleri and determine that three of these salamanders represent undescribed species within the genus Pseudoeurycea, which we formally describe here.

 Materials and methods Field work

Field surveys and collection of specimens were carried out between June 2022 to July 2023 in a cloud forest and rainforest from Cerro Rabon, Oaxaca, specifically in Rancho Guadalupe and San Martín Caballero localities (cloud forest) and Emiliano Zapata (rainforest), all in the municipality of San José Tenango, Oaxaca (Fig. 1). All collected specimens were sacrificed with an overdose of xylocaine ointment (applied to the gular and ventral area), fixed in 10% buffered formalin, preserved in 70% ethanol, and deposited in the Colección Nacional de Anfibios y Reptiles, Universidad Nacional Autónoma de México (CNAR, Mexico City, Mexico). We obtained liver samples for 11 Pseudoeurycea individuals, of which six corresponded to the three species we describe here and five corresponded to Pseudoeurycea cf. werleri. Additionally, we obtained tail tips (muscle; 2 mm tail) from three P. werleri individuals, two from Sierra de Santa Marta, Los Tuxtlas, Veracruz from which there were no tissues previously (July 2022; Ejido Santa Marta, Soteapan municipality, Veracruz, Mexico; 18.3469°N, 94.8934°W; 1275 m a.s.l.) and one from 0.7 km SW of Vista Hermosa, Santiago Comaltepec, Oaxaca (September 2025; 17.6273°N, -96.3466°W; 1513 m a.s.l.). These four samples of P. werleri were included to increase the total number of taxa in our analysis and explore patterns of population divergence within this species.

­10.3897/vz.76.e167783.figure1 BC6876C7-EE23-57D8-A348-6E0DA61F8C8B

Eastern slopes of the Sierra Mazateca, Oaxaca, Mexico at larger geographic scale showing the location of the study area (top). Study area on Cerro Rabon, Oaxaca (bottom). The localities of species in the P. leprosa group (circles) and the P. juarezi group (squares) were obtained from the platform GBIF (www.gbif.org).

https://binary.pensoft.net/fig/1644302 DNA extraction and sequencing

We extracted total genomic DNA from ethanol preserved liver or muscle tissue for 14 Pseudoeurycea specimens (six for the putative three new species and eight from P. werleri) using a Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). We amplified and sequenced fragments of two mitochondrial genes, the large ribosomal RNA subunit (16S, ~ 520 bp) and Cytochrome b (cyt b, ~ 800 bp), which have been widely used in previous systematic studies of Pseudoeurycea (Rovito et al. 2015; Cázares-Hernández et al. 2020; García-Bañuelos et al. 2020). All PCR reactions were conducted using Taq-Platinum DNA Polymerase (Invitrogen, EUA) following the manufacturer protocols and using primers 16SarL and 16SbrH (Palumbi et al. 1991) for 16S, and MVZ 15 and MVZ 16 (Moritz et al. 1992) for cyt b. For both genes, PCR conditions consisted of an initial denaturation step at 94°C for 1 min, followed by 35 cycles of denaturation at 94°C for 45 s, annealing either at 53°C for cyt b or 55°C for 16S for 1 min, and extension at 72°C for 1 min, followed by a final extension step at 72°C for 2 min. We obtained single-band PCR products that were cleaned using 1:5 diluted ExoSAP-IT (USB Corp, Cleveland, OH), cycle sequenced using BigDye v3 terminator chemistry (Applied Biosystems, Foster City, CA) and sequenced on an ABI 3730 capillary sequencer. We obtained sequences from both strands that were analyzed and ensembled using Geneious Prime 2025 (http://www.geneious.com). Moreover, for cyt b sequences, we also verified that no stop codons were present in the final sequences.

 Phylogenetic analyses

We retrieved from GenBank (Benson et al. 2012) 16S and cyt b nucleotide sequences of 36 Pseudoeurycea species, including, when available, multiple individuals per species. We also included genetic information from both genes for Aquiloeurycea cephalica, Isthmura bellii, and Ixalotriton niger that we used as outgroups in our phylogenetic analyses based on the most comprehensive phylogeny of plethodontid salamanders (Rovito et al. 2015). We provide a complete list of taxa and the GenBank accession numbers of all sequences used in Table 1.

GenBank accession numbers for sequences used in phylogenetic analysis.

Species Voucher number GenBank 16S Voucher number GenBank cyt b
Aquiloeurycea cephalica IBH 22603 KP886863 IBH 22603 KP900066
Isthmura bellii MVZ 173433 AY864696 MVZ 173433 AY864692
Ixalotriton niger IBH 29715 KP886874 IBH 29715 KP900077
P. ahuitzotl IBH 30211 MT303858 IBH 30211 MT295473
P. altamontana IBH 22220 KP886861 IBH 22220 KP900064
P. anitae MVZ 137939 AF451227 — —
P. aurantia IBH 20370 KP886844 IBH 20370 KP900048
P. brunnata S1487 (MVZ 137947) AF451232 — —
P. cf. unguidentis GP352 AF380813 GP352 AF380774
P. cochranae IBH 23064 KP886864 IBH 23064 KP900067
P. conanti JAC 21252 DQ283454 — —
P. firscheini IBH 23102 KP886865 IBH 23102 KP900068
P. gadovii IBH 22982 KP886846 IBH 22982 KP900050
P. goebeli CRVA1017 MT303860 CRVA1017 MT295472
P. granitum 1 CARIE 1263 MT303862 CARIE 1263 MT295471
P. granitum 2 IBH 31829 MT303861 IBH 31829 MT295471
P. jaguar MZFCHE 35855 OP605487 MZFCHE 35855 OP617200
P. jaguar IBH 36061 PQ012680 — —
P. jaguar IBH 36062 PQ013681 — —
P. juarezi IBH 29718 KP886848 IBH 29718 KP900052
P. leprosa IBH 22406 KP886866 IBH 22406 KP900069
P. lineola IBH 29719 KP886867 IBH 29719 KP900070
P. longicauda IBH 22247 KP886849 IBH 22247 KP900053
P. lynchi GP160 AF451225 GP160 AF451204
P. melanomolga IBH 22784 KP886868 IBH 22784 KP900071
P. mixcoatl IBH 14194 KP886869 IBH 14194 KP900072
P. mixteca GP289 AF380829 GP289 AF380790
P. mystax GP372 MT955378 GP372 AF380756
P. nigromaculata CARIE 1260 MT303864 CARIE 1260 MT295468
P. obesa MVZ 241574 KP886870 MVZ 241574 KP900073
P. orchileucos (1) JRM 4753 DQ640060 JRM 4753 DQ640022
P. orchileucos (2) IBH 22562 KP886858 IBH 22562 KP900062
P. orchimelas (1) IBH 22999 KP886860 IBH 22999 KP900063
P. orchimelas (2) IBH 29722 KP886859 — —
P. papenfussi IBH 14198 KP886850 IBH 14198 KP900054
P. rex MVZ 263590 KP886852 MVZ 263590 KP900056
P. robertsi IBH 22232 KP886853 IBH 22232 KP900057
P. ruficauda IBH 21646 KP886871 IBH 21646 KP900074
P. saltator IBH 22895 KP886854 IBH 22895 KP900058
P. smithi IBH 29720 KP886855 IBH 29720 KP900059
P. tenchalli IBH 29721 KP886856 IBH 29721 KP900060
P. tlahcuiloh IBH 30233 MT303865 IBH 30233 MT295474
P. unguidentis MVZ 117432 MT303866 — —
Pseudoeurycea sp. IBH IBH 26444 KP886857 IBH 26444 KP900061
P. cf. werleri 1 IBH 37000 PZ317133 IBH 37000 PZ334430
P. cf. werleri 2 IBH 37006 PZ317134 IBH 37006 PZ334431
P. cf. werleri 3 IBH 37007 PZ317135 IBH 37007 PZ334432
P. cf. werleri 4 IBH 37008 PZ317136 IBH 37008 PZ334433
P. cf. werleri 5 IBH 37003 PZ317137 IBH 37003 PZ334434
P. euguiisp. nov. 2 IBH 37011 PZ317143 IBH 37011 PZ334440
P. parraoleaesp. nov. 1 IBH 37013 PZ317139 IBH 37013 PZ334436
P. parraoleaesp. nov. 2 IBH 37019 PZ317140 IBH 37019 PZ334437
P. parraoleaesp. nov. 3 IBH 37020 PZ317141 IBH 37020 PZ334438
P. parraoleaesp. nov. 4 IBH 37014 PZ317142 IBH 37014 PZ334439
P. natsiisp. nov. IBH 37026 PZ317138 IBH 37026 PZ334435
P. werleri (1) Not collected PZ317130 Not collected PZ334427
P. werleri (2) Not collected PZ317131 Not collected PZ334428
P. werleri (3) IBH 22294 KP886872 IBH 22294 KP900075
P. werleri (4) Not collected PZ317132 Not collected PZ334429
P. werleri (5) Not collected — Not collected MW206671

Using MUSCLE (Edgar 2004), we aligned all nucleotide sequences obtained for each locus. For each alignment, we trimmed sequence ends represented by fewer than nine individuals. We then used MESQUITE v.3.51 (Maddison and Maddison 2023) to concatenate the aligned datasets and construct a total-evidence dataset comprising 2206 aligned positions (1402 for 16S; 803 for cyt b) and 60 terminal taxa. This dataset contained 29.6 % missing data because sequence lengths varied within each alignment, primarily due to many GenBank sequences being longer than the fragments we generated. However, longer sequences represented species of the three major clades previously identified in Pseudoeurycea (Rovito et al. 2015). Given this representation, we expected that including all overlapping informative sites would improve phylogenetic signal (Wiens et al. 2007).

To reduce the potential bias introduced by missing data in phylogenetic inference, we generated a second reduced dataset in which more than 95% of the sequences for each gene shared the same length. Notably, in this reduced dataset, 47% of 16S sequences and 89% of cyt b sequences lost up to half of their aligned positions compared with the total-evidence matrix. The reduced dataset retained the same 60 terminal taxa but comprised 871 characters (526 for 16S; 345 for cyt b) and contained 7% missing data, mainly due to indels in the 16S sequences. Both datasets were divided into four partitions: one for 16S and one for each codon position of cyt b. We identified the best-fitting nucleotide substitution models for each partition using the ModelFinder method (Kalyaanamoorthy et al. 2017) and the Bayesian Information Criterion (BIC), as implemented in IQ-Tree v.1.6.5 (Trifinopoulos et al. 2016). According to these analyses, we applied the following models for each partition in both datasets: GTR+I+G for 16S, and HKY+G, HKY+I, and GTR+G for the first, second, and third codon position of cyt b, respectively. These models and partition scheme were used for all our phylogenetic analyses.

For both datasets, we used IQ-Tree to infer a maximum likelihood (ML) phylogeny and assessed branch support using the ultrafast bootstrap (UFB) method (Hoang et al. 2018) with 1000 replicates. The resulting trees were subsequently rooted using I. bellii as the outgroup. Moreover, we inferred phylogenetic relationships based on Bayesian inference (BI) using MrBayes 3.2.6 (Ronquist et al. 2012). These analyses consisted of four independent runs, each with four chains, 10,000,000 generations and sampling every 1000th generation. For all other parameters in the analysis, we used default priors and values. We assessed parameter convergence and proper mixing of independent runs using TRACER 1.6 (Rambaut et al. 2018). All parameter values sampled during the MCMC of the analysis resulted in ESS values >3000. We discarded 25% of the samples obtained prior to stability as burn-in to obtain a final consensus phylogeny. Lastly, we estimated genetic distances among species for each gene using PAUP v4 (Swofford 2002). For these analyses, we considered sequences of cyt b as a single partition, and we specified a GTR substitution model for both 16S and cyt b sequences because it is the most parameter-rich and flexible substitution model implemented in PAUP.

 Morphological revision and comparison

We followed the same basic description format of Lynch and Wake (1989), which had been used recently to describe neotropical plethodontids (Palacios-Aguilar et al. 2020; Cázares-Hernández et al. 2022). We included the same basic characters, all morphological measurements were obtained with a digital caliper (to the nearest 0.01 mm), and we used a stereoscopic microscope for costal grooves, teeth counts, and measurements of fingers and hands of smaller specimens. We used the following characters: distance from snout to the posterior end of the vent or snout-vent length (SVL), tail length (TL; end of vent to tail tip), axilla-groin distance (AX), forelimb length (FLL), hindlimb length (HLL), head length (HL; from tip of snout to gular fold), head width (HW; width of head at angle of jaw), head depth (HD), interocular distance (IO), distance between external nares (IN), right foot width (RFW), length of longest toe (T3; third toe), and length of fifth toe (T5). We counted the premaxillary (PMT), maxillary (MT), and vomerine (VT) teeth from both sides (not including spaces where teeth are missing; Cázares-Hernández et al. 2022). Because the three new species that we describe here were grouped within the P. leprosa and P. juarezi groups based on our phylogenetic analyses, we used the measurements provided by Cázares-Hernández et al. (2022) for comparison with other species, since this work includes the most recent and complete description within the P. juarezi group (morphology, teeth, and color). For the species in the P. leprosa group and I. niger, we reviewed specimens available in the CNAR and obtained measurements and counts from original descriptions, as well as other publications referenced in the corresponding tables. The voucher numbers for measured specimens are presented in Appendix 1.

 Results Phylogenetic relationships and genetic distances

Our phylogenetic analyses were broadly congruent across datasets (total-evidence and reduced-evidence) and inference methods (BI and ML). The main differences among phylogenies involved support values, which were generally higher in total-evidence. Although the placement of a few taxa also varied between datasets (e.g., I. niger and P. aurantia), these alternative relationships received low support across all inferences. We summarize the results of the total-evidence phylogenies in Figure 2, and provide the phylogenies based on the reduced dataset in the Supplemental Material (Figs S1, S2) for comparison.

­10.3897/vz.76.e167783.figure2 EB5C5381-26F6-5ACD-AE30-0B840CB0D8D7

Phylogeny estimated from Bayesian inference of concatenated 16S and cyt b sequence data for Pseudoeurycea. The branches in red denote new species and filled circles indicate posterior probability > 0.97 and ultrafast bootstrap ≥ 95% from the Bayesian and maximum likelihood analyses respectively. Numbers on branches indicate lower posterior probability (above) and ultrafast bootstrap percentage (below).

https://binary.pensoft.net/fig/1644303

Overall, we found that the genus Pseudoeurycea and the P. gadovii, P. leprosa, and P. juarezi species groups were recovered as monophyletic (PP > 97; UB > 85), with the latter two groups being more closely related to each other (PP > 97; UB > 97; Figs 2, S1, S2). Our analysis also revealed that the three putative new Pseudoeurycea species belong to the P. leprosa (Pseudoeurycea euguiisp. nov.) and P. juarezi (Pseudoeurycea parraoleaesp. nov. and Pseudoeurycea natsiisp. nov.) groups. Pseudoeurycea parraoleaesp. nov., which included multiple individuals in the analyses, was strongly recovered as monophyletic. Regarding P. werleri, we found two clades with high population divergence (Fig. 2). The first clade consisted of samples from Cerro Rabon (Pseudoeurycea cf. werleri 1–5; Fig. 2), including one individual sequenced from Cumbres de Tonalixco, Veracruz, in the Sierra de Zongolica region (Pseudoeurycea werleri 5; Fig. 2). The individuals we included from the Santa Marta volcano (Pseudoeurycea werleri 1–2; Fig. 2) and the available sample from the San Martín volcano in the Los Tuxtlas region (Pseudoeurycea werleri 3; Fig. 2) were closer to the sample from the Sierra de Juarez from Oaxaca (Pseudoeurycea werleri 4; Fig. 2).

Within the P. leprosa group, Pseudoeurycea euguiisp. nov. represented an independent lineage most closely related to a clade comprising (((P. werleri + P. conanti) P. obesa) P. mystax)) (Fig. 2; PP ≥ 0.99; UB ≥ 84). Within the P. juarezi group, the relationship between Pseudoeurycea parraoleaesp. nov. and the sister taxon (P. juarezi + P. saltator) or P. aurantia was poorly supported, but close relationships among these four species are seen (Fig. 2). Lastly, Pseudoeurycea natsiisp. nov. was recovered as the sister taxon to a species of uncertain identification (labeled as Pseudoeurycea cf. unguidentis and Pseudoeurycea sp. IBH in GenBank), hereafter referred to as Pseudoeurycea sp., with high support (Fig. 2).

We observed that GTR genetic distances between most newly identified species and their closest relatives are greater or comparable than those observed between currently recognized species within their respective species groups (Tables 2 and 3). In the P. leprosa group, genetic distances between previously recognized species range from 0.022 to 0.113 (16S) and 0.058 to 0.224 (cyt b; Table 2). Genetic distances between Pseudoeurycea euguiisp. nov. and species in its sister clade (P. werleri, P. conanti, P. mystax, and P. obesa) ranged from 0.040 to 0.060 (16S) and 0.132 to 0.200 (cyt b). For the P. juarezi group, genetic distances between previously recognized species ranged from 0.006 to 0.073 (16S) and 0.015 to 0.167 (cyt b; Table 3). The genetic distances between Pseudoeurycea parraoleaesp. nov. and species in its sister clade (P. aurantia, P. juarezi, and P. saltator) ranged from 0.011 to 0.012 (16S) and 0.085 to 0.088 (cyt b). Likewise, genetic distances between Pseudoeurycea natsiisp. nov. and its sister taxon, Pseudoeurycea sp. was 0.016 (16S) and 0.054 (cyt b; Table 3).

Genetic distances between species of the P. leprosa group are based on a generalized time-reversible model (GTR) of nucleotide substitution. Values below and above of the main diagonal represent the genetic distances of 16S and cyt b genes respectively. Values in bold denote the lowest distance values.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1. P. conanti –– NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA
2. P. firscheini 0.113 –– 0.099 0.090 0.164 0.132 0.224 0.153 0.194 0.158 0.168 0.161 0.177 0.186 0.184 0.206 0.201 0.191
3. P. granitum 0.075 0.030 –– 0.141 0.167 0.102 0.193 0.119 0.185 0.158 0.193 0.171 0.183 0.168 0.172 0.170 0.183 0.185
4. P. leprosa 0.109 0.035 0.024 –– 0.184 0.130 0.212 0.167 0.178 0.155 0.166 0.165 0.179 0.188 0.184 0.192 0.185 0.190
5. P. lineola 0.103 0.082 0.049 0.082 –– 0.145 0.197 0.200 0.158 0.161 0.180 0.182 0.175 0.172 0.175 0.167 0.164 0.167
6. P. lynchi 0.069 0.030 0.022 0.026 0.041 –– 0.191 0.146 0.165 0.170 0.189 0.167 0.161 0.167 0.169 0.168 0.169 0.164
7. P. mystax 0.057 0.059 0.053 0.056 0.042 0.045 –– 0.196 0.101 0.176 0.178 0.164 0.149 0.115 0.118 0.114 0.119 0.125
8. P. nigromaculata 0.075 0.033 0.026 0.034 0.053 0.024 0.056 –– 0.191 0.166 0.182 0.174 0.200 0.173 0.174 0.178 0.182 0.174
9. P. obesa 0.079 0.098 0.045 0.089 0.081 0.041 0.043 0.052 –– 0.149 0.157 0.164 0.150 0.110 0.101 0.094 0.096 0.092
10. P. orchileucos (1) 0.088 0.088 0.052 0.085 0.080 0.039 0.037 0.052 0.066 –– 0.079 0.058 0.156 0.151 0.148 0.137 0.139 0.141
11. P. orchileucos (2) 0.095 0.098 0.050 0.087 0.073 0.044 0.043 0.048 0.069 0.039 –– 0.072 0.169 0.146 0.153 0.151 0.154 0.148
12. P. orchimelas 0.094 0.086 0.058 0.086 0.071 0.044 0.044 0.055 0.071 0.029 0.041 –– 0.159 0.154 0.155 0.147 0.155 0.152
13. P. euguiisp. nov. 0.060 0.050 0.049 0.047 0.032 0.038 0.032 0.043 0.038 0.039 0.047 0.043 –– 0.157 0.157 0.139 0.138 0.132
14. P. werleri (1) 0.049 0.055 0.050 0.046 0.040 0.036 0.043 0.050 0.028 0.028 0.033 0.036 0.034 –– 0.018 0.043 0.061 0.058
15. P. werleri (3) 0.075 0.106 0.050 0.097 0.095 0.037 0.044 0.049 0.055 0.066 0.070 0.079 0.040 0.006 –– 0.044 0.061 0.058
16. P. werleri (4) 0.052 0.059 0.052 0.050 0.042 0.038 0.047 0.054 0.024 0.026 0.035 0.033 0.040 0.006 0.008 –– 0.054 0.056
17. P. werleri (5) NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA –– 0.022
18. P. cf. werleri 0.051 0.048 0.043 0.045 0.038 0.030 0.041 0.043 0.027 0.026 0.031 0.034 0.032 0.005 0.010 0.010 NA ––

Genetic distances between species of the P. juarezi group are based on a generalized time-reversible model (GTR) of nucleotide substitution. Values below and above represent the genetic distances of 16S and cyt b genes, respectively. Values in bold denote the lowest distance values.

1 2 3 4 5 6 7 8 9
1. P. aurantia –– NA 0.135 0.039 0.147 0.039 0.163 0.087 0.153
2. P. jaguar (1) 0.067 –– NA NA NA NA NA NA NA
3. P. jaguar (3) 0.035 0.008 –– 0.138 0.109 0.142 0.121 0.152 0.126
4. P. juarezi 0.010 0.073 0.038 –– 0.137 0.015 0.159 0.085 0.147
5. P. ruficauda 0.012 0.068 0.035 0.053 –– 0.145 0.121 0.168 0.146
6. P. saltator 0.010 0.077 0.038 0.006 0.057 –– 0.167 0.088 0.148
7. Pseudoeurycea sp. 0.036 0.044 0.032 0.034 0.038 0.034 –– 0.159 0.054
8. P. parraoleaesp. nov. 0.012 0.055 0.041 0.011 0.036 0.011 0.036 –– 0.169
9. P. natsiisp. nov. 0.040 0.046 0.032 0.042 0.016 0.042 0.016 0.046 ––

Notably, genetic distances between populations of P. werleri were 0.005 to 0.010 (16S) and 0.022 to 0.058 (cyt b). Specifically, the samples from Cerro Rabon showed greater genetic distances in cyt b from conspecific samples from Los Tuxtlas and Sierra de Juarez (0.056 to 0.058) than the sample from Sierra de Zongolica (0.022). Regarding the genetic distance of the 16S gene, we were only able to compare our samples from Cerro Rabon with the Los Tuxtlas region and Sierra de Juarez (0.005 to 0.010 respectively), since there is no available sequence of this gene for the Sierra de Zongolica specimen.

Based on phylogenetic evidence, genetic distances, and morphological comparisons, we presented the description of the three new species:

 Systematics Animalia Caudata Plethodontidae A59E2984-56B1-57A9-A6EE-6F45730FEDD5 Pseudoeurycea euguii https://zoobank.org/52A09B5F-BF66-4E93-9BE6-4EC587C21BDD Jiménez-Arcos, Calzada-Arciniega, Cortés-Ortiz & Aguilar-Herrera sp. nov. Figure 3 ; Table 4 Suggest English name: Eugui’s worm salamander Suggest Spanish name: Salamandra de Eugui Holotype.

IBH 37012. An adult female from 1.36 km SW of Emiliano Zapata, San José Tenango municipality, Oaxaca, Mexico (18°10.50’N, 96°36.41’W, 731 m elevation, WGS84 datum), collected by Etienne Uriel Avila Ortega on 21 January 2023.

 Paratype.

One. One immature male: IBH 37010 (22 January 2023). Same locality as the holotype.

 Referred specimen.

One juvenile: IBH 37011 (22 July 2022). Same locality as the holotype.

 Diagnosis.

Pseudoeurycea euguiisp. nov. is assigned to the genus Pseudoeurycea based on the presence of a sublingual fold, an elongate body and tail, attenuated limbs, substantially shorter fifth toe than the fourth, as well as mtDNA sequences. Pseudoeurycea euguiisp. nov. is a member of the P. leprosa group as an independent lineage most closely related to a clade comprising ((P. werleri + P. conanti) + P. obesa + P. mystax) (Fig. 2). Pseudoeurycea euguiisp. nov. only resembles other forms of elongated and fossorial salamanders from west of the Isthmus of Tehuantepec: Pseudoeurycea lineola, P. orchileucos, and P. orchimelas. Pseudoeurycea euguiisp. nov. is different from the three species of worm salamanders by the following combination of characters: Pseudoeurycea euguiisp. nov. male having fewer vomerine teeth than P. lineo­la and P. orchileucos (VT males: Pseudoeurycea euguiisp. nov. 10, P. lineola 15–21, P. orchileucos 14–16), the female with fewer vomerine teeth than P. lineola and P. orchimelas (VT females: Pseudoeurycea euguiisp. nov. 8, P. lineola 14–22, P. orchimelas 16–19), as well as fewer premaxillary teeth (PMT females: Pseudoeurycea euguiisp. nov. 2, P. lineola 3–10, P. orchimelas 6–12). Both sexes differ from the three species of worm salamanders described by have a shorter distance between external nares (IN males: Pseudoeurycea euguiisp. nov. 0.7, P. lineola 1.0–1.4, P. orchileucos 1.0, P. orchimelas 0.9–1.0; IN females: Pseudoeurycea euguiisp. nov. 0.7, P. lineola 1.0–1.2, P. orchimelas 0.8–1.0). Table 4 provides the available morphological measurements and contents for worm salamanders from Mexico.

 Description of holotype.

An adult female (SVL 34.7 mm), body slender and elongate, head long and broad (HL/SVL = 0.18; HW/SVL = 0.09), slightly wider than body, neck region defined. Snout rounded in dorsal and lateral views. Nostrils are small and circular. Eyes protuberant, narrowly visible beyond margin of jaw in dorsal view. Postorbital groove present. Costal folds 13, counting one each between axilla and groin. Tail 1.7 times larger than the body, tapering gradually in the last quarter, base of tail round, with no noticeable constriction. Limbs short, slender, separated by 9 costal folds when appressed to side of body grooves. Hands and feet straight, rounded digits, without webbing, digits in order of decreasing length: III–II–IV–I on hands; III–IV–II–V–I on feet. Phalangeal formulae 1–2–3–2 for hands and 1–2–3–3–2 for feet. Two premaxillary teeth, same size as maxillary, 41 maxillary teeth, 8 vomerine teeth extend beyond margin of choanae; parasphenoid teeth in two patches as an inverted “V” shape.

­10.3897/vz.76.e167783.figure3 63B5C7C7-950A-5DFD-BD82-7F3E0DDC89B9

Live specimens of Pseudoeurycea euguiisp. nov. A Adult female holotype (IBH 37012). B full body and C close-up of the head of an immature male paratype (IBH 37010). D full body of juvenile (IBH 37011). Photos: Víctor H. Jiménez-Arcos.

https://binary.pensoft.net/fig/1644304

Mean, standard deviation, and range of 16 morphological characters of slender salamanders of the genus Pseudoeurycea for males (♂) and females (♀). Values for P. lineola, P. orchileucos (only males available), and P. orchimelas were obtained from Brodie et al. (2002) and complemented with specimens listed in the Appendix 1. Abbreviations correspond to those presented in Materials and Methods.

Character Sex P. euguii sp. nov. (n = 1, 1) P. lineola (n = 22, 19) P. orchileucos (n = 4) P. orchimelas (n = 14, 7)
SVL ♂ 30.8 36.2±2.4 (33.5–43.0) 31.0±0.64 (30.0–33.0) 32.3±1.9 (31.0–33.7)
♀ 34.7 39.5±3.2 (34.0–44.0) — 31.4±4.8 (28.0–34.8)
TL ♂ 63.0 67.3±3.5 (45.0–91.0) 59.8±5.0 (46.0–68.0) 56.4±8.2 (50.6–62.3)
♀ 57.3 72.2±8.5 (62.0–90.0) — 57.6±14.0 (47.6–67.5)
AX ♂ 20.3 23.2±1.8 (21.2–28.0) 19.3±0.7 (18.0–21.0) 20.0±0.8 (19.4–20.6)
♀ 22.1 25.9±2.4 (22.0–29.0) — 20.6±2.2 (19.0–22.2)
FLL ♂ 2.5 3.4±0.2 (3.1–3.7) n = 8 3.5 n = 2 3.3±0.4 (2.7–3.6) n = 7
♀ 2.7 3.3±0.2 (2.9–3.6) n = 7 — 3.1±0.2 (2.9–3.4) n = 5
HLL ♂ 2.5 3.5±0.3 (3.0–3.8) n = 8 3.8 n = 2 3.5±0.4 (2.9–3.8) n = 7
♀ 2.9 3.5±0.3 (3.2–3.9) n = 7 — 3.4±0.3 (3.0–3.7) n = 5
HL ♂ 6.2 6.1±0.4 (5.7–7.5) 5.9±0.1 (5.7–6.1) 5.9±0.3 (5.7–6.2)
♀ 6.2 6.4±0.2 (5.4–7.3) — 5.8±0.5 (5.4–6.1)
HW ♂ 3.1 3.7±0.1 (3.3–4.3) 3.4±0.1 (3.2–3.6) 3.1±0.2 (2.9–3.3)
♀ 3.1 3.6±0.2 (3.1–4.2) — 3.1±0.5 (2.8–3.5)
HD ♂ 2.0 1.9±0.1 (1.6–2.1) n = 8 1.8±0.2 (1.7–1.9) n = 2 1.7±0.1 (1.5–1.9) n = 7
♀ 1.9 1.9±0.2 (1.7–2.0) n = 7 — 1.7±0.1 (1.5–1.8) n = 5
IO ♂ 1.2 1.2±0.1 (1.0–1.5) n = 20 1.0±0.0 (1.0–1.1) 1.1±0.0 (0.9–1.2)
♀ 1.0 1.1±0.0 (1.0–1.5) n = 17 — 1.1±0.0 (1.0–1.2) n = 6
IN ♂ 0.7 1.1±0.0 (1.0–1.4) 1.0±0.0 (1.0) 1.0±0.0 (0.9–1.0)
♀ 0.7 1.1±0.0 (1.0–1.2) — 1.0±0.0 (0.8–1.0) n = 6
RFW ♂ 0.7 0.9±0.1 (0.7–1.0) n = 8 0.9±0.1 (0.9–1.0) n = 2 0.8±0.0 (0.8–0.9) n = 7
♀ 0.7 0.7±0.1 (0.5–0.9) n = 7 — 0.7±0.1 (0.5–0.9) n = 5
T3 ♂ 0.5 0.6±0.1 (0.4–0.7) n = 8 0.2 n = 2 0.8±0.0 (0.8–0.9) n = 7
♀ 0.6 0.5±0.1 (0.3–0.7) n = 7 — 0.4±0.2 (0.3–0.7) n = 5
T5 ♂ 0.3 0.4±0.1 (0.2–0.5) n = 8 0.7±0.1 (0.7–0.8) n = 2 0.5±0.2 (0.2–0.7) n = 7
♀ 0.3 0.4±0.1 (0.3–0.5) n = 7 — 0.3±0.2 (0.2–0.5) n = 5
PMT ♂ 4 2.8±1.6 (1–5) n = 8 4.5±0.7 (4–5) n = 2 3.0±0.7 (2–4) n = 7
♀ 2 6.3±2.5 (3–10) n = 7 — 10.0±2.8 (6–12) n = 5
MT ♂ 35 29.2±4.4 (23–35) n = 8 39.5±2.1 (38–41) n = 2 38.8±6.9 (32–48) n = 7
♀ 41 35.3±9.7 (22–48) n = 7 — 46.5±4.1 (41–51) n = 5
VT ♂ 10 18.0±0.5 (15–21) 15.0±0.4 (14–16) 16.0±1.0 (10–22)
♀ 8 18.0±0.6 (14–22) — 18.0±0.5 (16–19)
Variation and sexual dimorphism.

The type series includes two specimens, an adult female (holotype) and immature male. The female is larger than the male (SVL male 30.8 mm; female: 34.7 mm). The tail is shorter in the female (TL female: 57.3 mm, male: 63.0 mm), and relatively longer in the male (TL/SVL male: 2.05; female 1.65). The head is similar size, despite the difference in SVL, suggesting that males may have proportionally longer heads than females. Male with greater number of premaxillary and vomerine teeth (MT male: 4; female 2; VT male 10; female: 8), and fewer maxillary teeth (male: 19/16, female 20/21). The juvenile individual (referred specimen) is smaller (SVL 29.9 mm) with a relatively smaller tail (TL/SVL 1.09). We do not observe a mental gland in the male of Pseudoeurycea euguiisp. nov. However, in mature males of P. orchimelas the gland is extremely small and only visible after dissection while in P. orchileucos it is visible externally (Brodie et al. 2002). However, we suggest caution because the male is an immature specimen, and likely adult males of this species reach larger size.

 Coloration of the holotype.

In life, dorsum and dorsal surface of the tail dark chocolate brown, becoming darker on the tail. Highest percentage of shade of brown stipples on the dorsum, decreasing towards the tail; light gray dots on the lateral surface, increasing toward the dorsal area of the tail up to the middle of the body. The limb coloration is similar as body, with a higher percentage of light brown stipples on the forearm and thighs. Light yellow eyelids with small black dots and the iris is dark with a copper hue (Fig. 3A). In preservative, body and tail dark brown, almost black, limbs slightly lighter and ventral coloration, including limbs, dark gray.

 Distribution and natural history.

Pseudoeurycea euguiisp. nov. is only known from the type locality in Emiliano Zapata, San Jose Tenango municipality, located in the eastern slopes of Cerro Rabon in the Sierra Mazateca, Oaxaca at 731 m elevation. The three individuals were found in the vicinity of a corn plantation (≈640 m2) in a rainforest remanent. The first individual (IBH 37011) was found among the leaf litter at the edge of the corn field, whereas two specimens (male and female) were found in loose soil that had accumulated between rocks probably due to rain erosion. The area has a high percentage of rocks in the soil, and in surroundings, the presence of limestone rocks up to 2 m high. The juvenile individual was found on July 2022 at 12:35 hrs, with a body temperature of 23.2°C (taken in the groin for all individuals to not alter body temperature due to its small size; Jiménez-Arcos et al. 2022), air temperature of 21.7°C, and substrate temperature of 22°C. In January 2023, at 66 m from the collecting site of the first specimen we located the male at 00:02 hrs, 54 mm below the loose soil, with body temperature: 23.2°C, air temperature: 17.1°C; and substrate temperature: 18.3°C with relative humidity of 98.3%. The female holotype was found at 14:14 hrs, 34 cm from the male paratype under a rock 170 mm underground. The female exhibited caudal luring after being discovered, presumably as a defense mechanism (distraction). In the microhabitat we recorded a high abundance of earthworms, ants, as well as one tarantula. Close to the type locality (between 150 to 250 m in a straight line) we have recorded the presence of other sympatric lungless salamanders, Bolitoglossa platydactyla and Bolitoglossa rufescens, and other anuran species including Agalychnis moreletii, Craugastor loki, Craugastor spatulatus, Smilisca cyanosticta, and Triprion spinosus.

 Etymology.

Pseudoeurycea euguiisp. nov. is a patronym in honor of Eugui Roy Martínez Pérez, a dear friend and collaborator of some co-authors of this study. He was a biology student originally from Oaxaca with a bright future in herpetology that was interrupted by defending the cloud forest of the Sierra Sur of Oaxaca.

 Animalia Caudata Plethodontidae 0612F77F-16AC-5F24-9F01-91D1B19772AB Pseudoeurycea parraoleae https://zoobank.org/CC65D18A-4015-419B-B8DF-C6F54CD7A989 Jimenez-Arcos, Aguilar-Herrera, Flores-Martínez & Calzada-Arciniega sp. nov. Figure 4 , 5 ; Table 5 Suggested English name: Gaby’s salamander Suggested Spanish name: Salamandra de Gaby Holotype.

IBH 37013. An adult female from 1.1 km W of San Martín Caballero, San José Tenango municipality, Oaxaca, Mexico (18° 06.29’N, 96°38.48’W, 1590 m elevation, WGS84 datum), collected by Víctor H. Jiménez Arcos on 4 October 2022.

 Paratypes.

Five males: IBH 37014–37018 (8 October 2022); five females: IBH 37019 (8 October 2022); IBH 37020–37023 (10 October 2022). Same locality as the holotype.

 Referred specimens.

Two juveniles IBH 37024–37025 (10 October 2022). Same locality as the holotype.

 Diagnosis.

Pseudoeurycea parraoleaesp. nov. is assigned to the genus Pseudoeurycea based on the presence of a sublingual fold, substantially shorter fifth toe compared to the fourth, and mtDNA. Pseudoeurycea parraoleaesp. nov. is member of the P. juarezi group as sister species of a clade containing (P. aurantia (P. juarezi + P. saltator)). It is a medium-sized species in the genus Pseudoeurycea and in the P. juarezi group, with maximum SVL = 49.8 mm in females and 44.6 mm in males (range 33.7–49.8 mm; Table 5). Pseudoeurycea parraoleaesp. nov. shows a homogeneous black to dark brown dorsal coloration (Fig. 5) which allows it to be distinguished from all other species in the P. juarezi group (except some specimens of P. jaguar with which it shares the most morphological similarity, see below). From its closest relatives, P. aurantia has a brown to orange dorsal coloration with lighter orange or whitish splotches, and in some specimens a few black spots on the tail. Also, Pseudoeurycea parraoleaesp. nov. has a greater number of maxillary teeth (MT males: Pseudoeurycea aurantia 52–66, Pseudoeurycea parraoleaesp. nov. 82–89; females: Pseudoeurycea aurantia 49–66, Pseudoeurycea parraoleaesp. nov. 68–98). Pseudoeurycea juarezi shows a uniform brown to yellow dorsal coloration; in some specimens with dorsal black blotches or splotches of different sizes and abundance, the dorsolateral coloration is gray to brown, and Pseudoeurycea parraoleaesp. nov. has a greater number of maxillary teeth (MT males: Pseudoeurycea juarezi 39–58, Pseudoeurycea parraoleaesp. nov. 82–89; females: Pseudoeurycea juarezi 54–58, Pseudoeurycea parraoleaesp. nov. 68–98). Pseudoeurycea saltator has an orange to yellow coloration on the back, delimited by a black coloration and the dorsolateral area of ​​light to dark brown. In addition, Pseudoeurycea parraoleaesp. nov. tends to have a greater number of maxillary teeth (MT males: Pseudoeurycea parraoleaesp. nov. 82–89, P. saltator 58–67; females: Pseudoeurycea parraoleaesp. nov. 68–98, P. saltator 47–56). From P. jaguar where some individuals may exhibit a uniform dark coloration (Cázares-Hernández et al. 2022; Peralta-Hernández 2023), the main morphological difference is that adult specimens of Pseudoeurycea parraoleaesp. nov. tend to have smaller body size (SVL males: Pseudoeurycea jaguar 58.0–58.7 mm, Pseudoeurycea parraoleaesp. nov. 33.7–44.6 mm; females: Pseudoeurycea jaguar 42.4–71.0 mm, Pseudoeurycea parraoleaesp. nov. 38.5–49.8 mm), and therefore smaller head, shorter interocular distance and distance between nostrils (Table 5).

Mean, standard deviation, and range of 16 morphological characters of males (♂) and females (♀) of species in the Pseudoeurycea juarezi group. Values for P. jaguar and P. juarezi were obtained from Cázares-Hernández et al. (2022), except premaxillary and maxillary counts. Abbreviations correspond to those presented in Materials and Methods.

Character Sex P. aurantia (n=4, 4) P. jaguar (n=2, 6) P. juarezi (n=11, 11) P. natsii sp. nov. (n=1, 1) P. parraoleae sp. nov. (n=5, 7) P. ruficauda (n=2, 3) P. saltator (n=4, 3) Pseudoeurycea sp. (n=2, 9)
SVL ♂ 42.5±2.0 (40.2–45.0) 58.3±0.5 (58.0–58.7) 45.5±2.8 (38.5–49.2) 54.5 38.8±4.5 (33.7–44.6) 24.7±0.8 (24.2–25.3) 38.8±1.9 (36.8–40.5) 51.1±3.5 (48.6–53.6)
♀ 43.6±4.0 (39.8–49.1) 59.0±11.8 (42.4–71.0) 47.5±2.5 (44.0–51.3) 41.3 43.0±5.0 (38.5–49.8) 41.7±7.5 (36.6–50.36) 38.1±0.7 (37.6–38.6) 46.7±6.0 (38.6–57.5)
TL ♂ 41.3±4.5 (36.6–47.0) 54.5±18.6 (41.4–67.6) 46.1±6.6 (29.3–52.6) 60.7 44.8±8.5 (35.0–56.5) 21.7±1.1 (21.0–22.5) 41.2±4.9 (36.4–46.2) 48.3±2.3 (46.7–50.0)
♀ 40.0±4.4 (37.3–46.6) 66.1±14.2 (46.4–87.9) 43.9±5.1 (33.1–52.1) 48.8 42.9±4.8 (36.9–49.9) 46.0±12.5 (37.1–60.3) 33.9±2.2 (32.3–35.5) 39.8±13.8 (23.9–64.3) n=7
AX ♂ 21.3±1.4 (19.8–23.2) 29.5±0.1 (29.4–29.6) 22.2±1.6 (19.4–24.6) 29.7 19.2±2.5 (16.4–22.6) 12.2±1.5 (11.2–13.3) 20.3±1.3 (19.1–21.6) 26.2±3.4 (23.8–28.6)
♀ 22.9±3.3 (20.5–27.5) 30.8±6.8 (22.0–39.6) 24.1±1.0 (22.3–25.6) 21.2 23.5±3.2 (19.2–27.6) 22.3±4.5 (18.7–27.5) 20.7±0.1 (20.6–20.8) 24.9±3.6 (20.5–31.4)
FLL ♂ 11.4±1.1 (10.0–12.7) 15.3±4.0 (12.5–18.2) 12.6±0.8 (11.2–13.5) 18.2 14.0±1.9 (11.7–16.6) 7.1±0.2 (7.0–7.3) 11.5±1.2 (10.2–12.4) 15.5±0.5 (15.2–15.8)
♀ 11.8±0.9 (10.7–12.9) 15.1±4.7 (10.1–22.4) 12.0±0.6 (11.5–12.9) 13.4 14.8±1.6 (12.8–17.4) 12.6±3.0 (10.4–16.0) 9.3±0.4 (9.0–9.6) 14.9±1.9 (12.4–18.5)
HLL ♂ 13.2±0.9 (12.4–14.3) 16.6±3.4 (14.2–19.0) 14.0±0.9 (13.1–15.1) 18.6 14.9±1.7 (13.0–17.5) 7.3±0.8 (6.7–7.9) 11.6±0.3 (11.2–11.8) 15.7±1.5 (14.6–16.8)
♀ 12.2±0.8 (11.5–13.2) 15.3±4.7 (9.9–22.3) 13.6±0.7 (12.4–14.7) 14.2 15.7±2.0 (13.0–17.9) 11.2±1.9 (9.9–13.4) 9.0±1.9 (7.6–10.3) 15.1±2.5 (12.4–20.0)
HL ♂ 10.4±0.6 (9.9–11.2) 14.4±1.0 (13.7–15.1) 11.4±0.8 (9.4–12.5) 11.7 9.7±1.7 (8.2–12.3) 6.7±0.0 (6.7–6.7) 8.3±0.8 (7.8–9.2) 11.7±1.0 (11.0–12.4)
♀ 9.5±1.2 (8.0–10.7) 14.6±2.7 (11.0–17.6) 11.4±0.9 (10.0–12.8) 10.4 9.5±1.3 (8.2–11.7) 8.5±1.1 (7.8–9.7) 7.2±0.4 (6.9–7.4) 10.7±1.8 (9.2–14.3)
HW ♂ 6.6±0.2 (6.5–6.8) 10.0±0.4 (9.8–10.3) 6.9±0.4 (5.9–7.8) 8.7 6.9±1.0 (5.8–8.4) 4.5±0.1 (4.4–4.6) 6.2±0.5 (5.9–6.8) 8.0±0.3 (7.7–8.2)
♀ 6.3±0.4 (5.9–6.7) 10.2±1.7 (7.8–12.0) 7.1±0.5 (6.2–7.9) 7.2 7.3±0.9 (6.2–8.5) 5.9±0.7 (5.3–6.6) 5.9±0.1 (5.9–6.0) 7.4±0.7 (6.3–8.7)
HD ♂ 3.7±0.2 (3.5–3.9) 4.5±0.3 (4.3–4.7) 3.7±0.4 (3.1–4.6) 3.9 2.2±0.6 (3.1–3.7) 2.5 n=1 3.0±0.2 (2.8–3.2) 3.4±0.2 (3.2–3.5)
♀ 3.5±0.5 (2.8–3.9) 4.5±0.8 (3.6–5.3) 4.0±0.2 (3.6–4.4) 3.3 3.5±0.4 (2.9–4.0) 3.0±0.3 (2.7–3.3) 2.8±0.1 (2.7–2.8) 3.2±0.5 (2.6–4.3)
IO ♂ 2.4±0.2 (2.2–2.7) 2.9±0.0 (2.9) 2.4±0.2 (2.1–2.7) 2.8 1.97±0.43 (1.3–2.4) 3.0 n=1 2.7±0.6 (2.0–3.1) 3.9±0.2 (3.8–4.0)
♀ 2.6±0.6 (2.1–3.2) 3.4±0.8 (2.4–4.3) 2.4±0.4 (1.7–3.0) 1.9 2.3±0.3 (1.9–2.6) 3.1±0.3 (2.8–3.5) 2.3±0.4 (2.0–2.5) 3.5±0.3 (3.1–3.8)
IN ♂ 2.1±0.2 (1.8–2.4) 3.2±0.2 (3.1–3.4) 2.5±0.8 (1.9–2.9) 2.5 1.8±0.3 (1.5–2.2) 1.3±0.3 (1.1–1.6) 2.0±0.1 (1.9–2.1) 3.5±0.2 (3.4–3.6)
♀ 1.9±0.2 (1.7–2.1) 3.2±0.5 (2.7–3.8) 2.1±0.1 (1.9–2.4) 2.1 1.8±0.3 (1.5–2.3) 1.8±0.3 (1.4–2.0) 2.0±0.4 (1.7–2.5) 2.5±0.3 (2.1–2.9)
RFW ♂ 4.7±0.4 (4.2–5.2) 7.8±0.2 (7.7–8.0) 5.1±0.4 (4.3–5.6) 5.9 3.6±1.0 (2.7–4.6) 2.5±0.6 (2.1–3.0) 3.4±0.3 (3.2–3.7) 5.1±0.1 (5.0–5.2)
♀ 3.8±0.6 (2.9–4.3) 7.5±2.1 (4.0–9.3) 5.0±0.6 (4.0–6.1) 4.0 3.8±0.9 (2.6–5.3) 3.4±0.4 (3.1–3.9) 3.6±0.3 (3.4–3.8) 4.2±0.4 (3.7–4.8)
T3 ♂ 2.3±0.4 (1.8–2.8) 2.9±0.7 (2.4–3.4) 2.5±0.3 (2.0–2.9) 2.9 2.2±0.4 (1.7–2.8) 1.1±0.1 (1.1–1.2) 2.2±0.3 (2.0–2.4) n=2 2.3±0.0 (2.3–2.3)
♀ 2.4±0.3 (2.0–2.7) 3.1±0.4 (2.7–3.9) 2.5±0.3 (2.0–3.0) 2.1 2.5±0.4 (2.1–3.1) 2.8±0.9 (1.9–3.7) 2.1±0.4 (1.8–2.4) 3.3±0.9 (2.5–4.8)
T5 ♂ 1.0±0.1 (0.8–1.1) 1.0±0.1 (1.0–1.1) 1.2±0.2 (0.8–1.5) 1.8 1.4±0.4 (0.9–2.0) 0.5±0.1 (0.5–0.6) 1.4±0.1 (1.4–1.5) n=2 1.7±0.0 (1.7–1.7)
♀ 1.2±0.3 (0.8–1.6) 1.0±0.0 (0.7–1.6) 1.1±0.2 (0.9–1.4) 1.1 1.3±0.3 (0.8–1.7) 1.7±0.5 (1.4-2.3) 1.2±0.6 (0.8–1.6) 1.9±0.4 (1.3–2.4)
PMT ♂ 4.7±1.2 (4–6) n=3 3.0 n=2 (3-4) 3.0±0.7 (2–4) n=5 5 2.4±1.1 (1–4) 9.7±1.5 (8–11) 7.5±2.4 (5–10) 5.5±2.1 (4–7)
♀ 9.9±4.2 (6–18) n=7 17 (13–20) n=1 13.8±3.9 (10–19) n=4 12 10.0±1.8 (8–13) 7.0±5.2 (4–13) 12.7±3.1 (10–16) 13.7±1.5 (12–15)
MT ♂ 59.0±7.0 (52–66) n=3 81.0±15.6 (70–92) n=2 50.6±7.7 (39–58) n=5 96 84.8±2.7 (82–89) 45.0±11.3 (38–58) 62.5±4.2 (58–67) 76.0±22.6 (60–92)
♀ 57.0±5.4 (49–66) n=7 66 n=2 56.5±1.7 (54–58) n=4 78 81.1±11.1 (68–98) 45.3±27.4 (29–77) 52.0±4.6 (47–56) 77.3±9.3 (71–88)
VT ♂ 23.0±4.5 (17–27) 30.5±7.8 (25–36) 22.0±2.9 (17–27) 63 25.4±3.8 (22–29) 15.5±2.1 (14–17) 24.3±3.2 (22–28) 23.5±2.1 (22–25)
♀ 18.5±5.3 (11–23) 30.3±7.7 (18–37) 23.2±2.8 (19–26) 43 27.0±4.9 (21–35) 12.3±3.8 (8–15) 20.5±2.1 (19–22) 22.6±6.2 (11–31) n=8

Morphologically, Pseudoeurycea parraoleaesp. nov. could also resemble I. niger by having protuberant eyes, long legs, hands, feet, and dark body coloration. However, Pseudoeurycea parraoleaesp. nov. has black pigmentation in the subocular groove while in I. niger it is unpigmented, a proportionally shorter tail in Pseudoeurycea parraoleaesp. nov. (TL/SVL males = 1.15; females = 1.00) versus I. niger (males = 1.45; females = 1.33; Rovito et al. 2017), and I. niger is slender and more gracile compared to the new species. In addition, Pseudoeurycea parraoleaesp. nov. has premaxillary teeth larger than the maxillary teeth, whereas premaxillary and maxillary teeth usually of the same size in I. niger (Wake and Johnson 1989). Moreover, I. niger is distributed east of the Isthmus of Tehuantepec where it is known from two localities in Chiapas, Mexico, more than 300 km from the type locality of Pseudoeurycea parraoleaesp. nov. (Rovito et al. 2017).

 Description of holotype.

An adult female (49.8 SVL) which is the largest specimen of the type series. Body slender (shoulder width = 5.9 mm), head relatively broad (HW/SVL = 0.16) and wider than the body with a well-defined neck. Mouth truncates in dorsal view with a slightly marked canthus rostralis closer to the eye. Eyes protruding, exceeding margin of jaw when viewed from above. Nostrils circular. Costal folds 13, counting one each in axilla and groin. Tail as long as body (TL/SVL = 1.00), slender, roughly rectangular at base, and tapering gradually along length ending in a point. Large limbs (FLL + HLL/SVL = 0.70). Adpressed limbs overlap by 3 costal interspaces. Hands and feet are broad, digits long and relatively slender, blunt with distinct subterminal pads. Hands and feet are highly webbed compared to most other members of Pseudoeurycea (moderately webbed compared with other genera), with webbing extending to the end of the penultimate phalanx on third toe of foot. First toe short. Order of digits by increasing length: manus I–IV–III–II, pes I–V–II–IV–III. Phalangeal formulae: manus 1–2–3–2, pes 1–2–3–3–2. Vomerine teeth one on each side, hook-shaped; teeth closest to the parasphenoid teeth are larger than those closest to the premaxillary. Parasphenoid teeth arranged as an inverted “V” shape and with similar size as vomerine teeth.

 Measurements of the holotype.

(in mm) Snout to posterior angle of vent 49.8; head width 7.9; head length 10.6; head depth at angle of jaw 3.6; eyelid length 3.0; eyelid width 2.4; anterior rim of orbit to snout 3.4; eye diameter 1.4; interorbital distance 1.9; snout to forelimb 15.5; internarial distance 2.3; intercanthal distance 4.1; nostril diameter 0.6; snout projection beyond mandible 0.7; snout to anterior angle of vent 44.7; axilla to groin 27.2; shoulder width 5.9; tail length 49.9; tail width at base 2.7; tail depth at base 3.3; forelimb length 17.4; hind limb length 17.6; hand width 3.9; foot width 5.3; length of the longest (third) toe 2.9; length of fifth toe 1.7. Tooth counts: premaxillary13; maxillary 49/45; vomerine 14/11.

 Coloration of the holotype.

In life, it is a black salamander, dorsal uniform black or dark brown almost black throughout the body. Ventral coloration dark brown, slightly lighter in the throat and from the middle of the body towards the cloaca, and black tail. The tips of the digits are reddish. Black eyelids, eyes almost black, iris more visible near the eyelids, with yellowish copper coloration. In preservative, dorsal coloration dark brown, in some parts almost black. Venter dark gray, and ventral surface of hands, feet, and gular region dark gray, but lighter than the rest of the ventral coloration.

 Variation and sexual dimorphism.

The type series includes 11 specimens, of which six are adult females, five adult males, and two more juveniles of indeterminate sex. The sexual size dimorphism is evident, where females can reach 49.8 mm (43.0±5.0) and males 44.6 mm (38.8±4.5) in SVL. The younger specimens reach 22.1 mm and 20.5 mm in SVL. In addition, sexual dimorphism is observed in tail size (TL/SVL males: 1.20±0.06; females: 1.00±0.09), intercostal distance (AX/SVL males: 0.49±0.02; females: 0.54±0.02), and the nasolabial protuberances are more evident in males (Fig. 4B, D). Round and prominent almost circular mental gland (2.1–2.5 mm n = 5; wide), almost the same color as the rest of the gular area—the smallest specimen with a visible gland reached 38.8 mm of SVL. The body coloration is similar between juveniles and adults, however, smaller specimens have golden splotches on dorsum and tail, whereas in larger individuals these are less frequent or absent.

­10.3897/vz.76.e167783.figure4 1CB81667-5FAA-5256-978B-E1DDB76BCF05

Live specimens of Pseudoeurycea parraoleaesp. nov. A full body and B close-up of the head of the adult female holotype (IBH 37013). C full body and D close-up of the head of an adult male paratype (IBH 37014). Photos: Víctor H. Jiménez-Arcos.

https://binary.pensoft.net/fig/1644305 ­10.3897/vz.76.e167783.figure5 8B3B284D-0D79-5512-9CE1-A48F58AC63C7

Dorsal and ventral view of the holotype of Pseudoeurycea parraoleaesp. nov. (IBH 37013) to the left. Ventral view of right hand and right foot to the right. Photos: Víctor H. Jiménez-Arco.

https://binary.pensoft.net/fig/1644306 Distribution and natural history.

Pseudoeurycea pa­rra­oleaesp. nov. is only known from San Martín Caballero, specifically in the cloud forest of Cerro Rabon, located in the eastern portion of Sierra Mazateca, Oaxa­ca. Therein the species inhabits conserved cloud forest from 1590 m elevation. On 7 October 2022, we recorded an average body temperature of 19.0°C for 10 individuals found active between 20:00 to 00:00 hrs, an average air temperature of 18.2°C, and average substrate temperature of 18.5°C, with a relative humidity of 99%. During six trips documenting amphibian and reptile diversity in the area, Pseudoeurycea parraoleaesp. nov. was recorded only in October (2022 and 2023), absent in the months of January, April, June, July, November, and December. We located individuals after 20:00 hrs during light rain, which suggests that its activity is restricted to the season with high environmental humidity (September and October). We consider this species as saxicolous, since most specimens were recorded on rocks, emerging from crevices, and between 15 cm to more than 200 cm from the substrate in walls of limestone rocks. The species occurs in sympatry with Parvimolge townsendi, Pseudoeurycea cf. werleri, and Thorius sp, as well as anuran species such as C. polymniae and T. spinosus.

 Etymology.

Pseudoeurycea parraoleaesp. nov. is named in honor of Gabriela Parra Olea, a prominent Mexican researcher, in recognition of her invaluable contributions to the systematics of amphibians, specifically salamanders of the family Plethodontidae. She has participated in the description of 32 species of plethodontids in seven genera and has also described seven species of frogs. Finally, we recognize her extensive work in the conservation of amphibians worldwide.

 Animalia Caudata Plethodontidae C4C5872F-6B8C-5E67-865A-EB994D48AE88 Pseudoeurycea natsii https://zoobank.org/336D3CDF-46DC-40C9-AFCE-EF4B7FB0A146 Aguilar-Herrera, Flores-Martínez, Seba-Chacha & Jiménez-Arcos sp. nov. Figure 6 , 7 ; Table 5 Suggested English name: Mazatec tree salamander Suggested Spanish name: Salamandra arborícola mazateca Holotype.

IBH 37026. An adult male from 1.16 km of San Martín Caballero, San José Tenango municipality, Oaxaca, Mexico (18°05.87'N, 96°38.03'W, 1611 m elevation, WGS84 datum), collected by Misael Seba-Chacha on 9 October 2022.

 Paratype.

One. An adult female IBH 37027 (16 July 2023). Same locality as the holotype.

 Diagnosis.

Pseudoeurycea natsiisp. nov. was assigned to the genus Pseudoeurycea based on the presence of a sublingual fold, substantially shorter fifth toe than the fourth, relatively large size compared with other species, and mtDNA sequences. Pseudoeurycea natsiisp. nov. is a large to medium-sized species with a SVL = 54.5 mm in the male and 41.3 mm in the only known female. Pseudoeurycea natsiisp. nov. can be easily distinguished from all species in the P. juarezi group as well as in the genus Pseudoeurycea by having a greater number of vomerine teeth, 63 in the male holotype and 43 in the female paratype (­Table 5). From its sister species, Pseudoeurycea sp., by the arrangement of parasphenoid teeth. Pseudoeurycea natsiisp. nov. has parasphenoid teeth arranged in a single patch while in Pseudoeurycea sp. they are arranged in two separate patches, and the only know male is larger than males of Pseudoeurycea sp. (SVLPseudoeurycea natsiisp. nov. 54.5 mm, Pseudoeurycea sp. 48.6–53.6 mm). Further, both species differ in coloration. Numerous brownish and dark cream blotches with a pale yellowish ground color can be distinguished in preserved specimens of Pseudoeurycea natsiisp. nov. (prepared in October 2022 and July 2023) while Pseudoeurycea sp. specimens have a uniform dark purple coloration in dorsal view of body, head, limbs, and tail with few scattered whitish splotches.

In addition to the higher number of vomerine teeth compared to all other known species in the P. juarezi group, Pseudoeurycea natsiisp. nov. can be distinguished easily from all the species described by the color pattern, having a dorsum with yellowish-green coloration with dark and yellow scattered blotches (Fig. 6), while the ventral surface is cream with yellow specks. Pseudoeurycea juarezi has a light dorsum with darker markings and dark sides; P. saltator has a dark gray dorsal ground color with a paler mid-dorsal stripe; P. aurantia has a distinctive orange coloration with an orange mid-dorsal stripe from the scapular region to the tip of the tail with more concentrated yellow spots, and its venter is pale yellow; P. ruficauda has an orange-tan ground color with an irregular dorsal stripe and the tail is orange with black spots with a red-orange to yellow-orange tip and the venter is grayish with occasional light spots; P. jaguar has irregular yellow specks on a dark brown or almost black ground color (green-yellowish ground color in Pseudoeurycea natsiisp. nov.); and Pseudoeurycea parraoleaesp. nov., described herein, has a uniform black to dark brown dorsum.

­10.3897/vz.76.e167783.figure6 1A57FFE7-DF48-5B00-ADE3-9D8AF6E29AFE

Live specimens of Pseudoeurycea natsiisp. nov. A full body and B close-up of the head of the adult male holotype (IBH 37026). C full body and D close-up of the head of an adult female paratype (IBH 37027). Photos: Víctor H. Jiménez-Arco.

https://binary.pensoft.net/fig/1644307

Pseudoeurycea natsiisp. nov. may resemble coloration of P. granitum, P. lynchi, and P. nigromaculata, and the distribution of these latter species is relatively close to this new species. Therefore, we present the comparison with these species. For morphology we compare with data from García-Bañuelos et al. (2020) and for coloration based on photographs available on AmphibiaWeb (2026). In addition to a greater number of vomerine teeth, both sexes of Pseudoeurycea natsiisp. nov. have a greater number of premaxillary and maxillary teeth (sum of the PMT and MT from Table 5 for Pseudoeurycea natsiisp. nov. for comparison; PMT+MT males: Pseudoeurycea granitum 57–73, P. lynchi 43–92, Pseudoeurycea natsiisp. nov. 101, P. nigromaculata 62–91; females: Pseudoeurycea granitum 57–73, P. lynchi 58–96, Pseudoeurycea natsiisp. nov. 101, P. nigromaculata 58–96), longer fore and hind limbs in males (FLLP. granitum 8.3–11.9 mm, P. lynchi 9.1–13.1 mm, Pseudoeurycea natsiisp. nov. 18.2 mm, P. nigromaculata 9.9–12.7 mm; HLLP. granitum 9.3–12.0 mm, P. lynchi 11.9–14.9 mm, Pseudoeurycea natsiisp. nov. 18.6 mm, P. nigromaculata 11.8–13.9 mm) and in females only P. nigromaculata with longer fore limbs (FLLP. granitum 7.8–12.8 mm, P. lynchi 8.1–12.9, Pseudoeurycea natsiisp. nov. 13.4 mm). In coloration, P. granitum only exhibits the greenish-yellow color with black dots on dorsum at the level of the front limbs towards the tail, the neck and face area being dark gray; P. lynchi has black coloration in the ventral region while in the new species the ventral color is cream with yellow spots; P. nigromaculata has a dorsal coloration ranging from brown-orange to dark brown, becoming lighter towards the tail, and has black dots on the body and tail, as well as lighter (yellow-orange) dots on the tail.

 Description of holotype.

A relatively medium sized adult male (SVL 54.5 mm) with relatively slender body. The head is relatively long and broad (HW/SVL = 0.16), wider than body with clearly defined neck. Mouth bluntly in lateral view and slightly truncate in dorsal view. Eyes only slightly protuberant reaching the margin of jaw when viewed from above. External nares small and slightly oval-shaped. Nearly round mental gland (2.2 mm wide, 2.0 mm length). Slightly marked supranasal ridge (canthus rostralis). Nasolabial protuberances present but relatively weakly developed. Costal folds 13, counting one each in axilla and groin. Tail longer than body (TL/SVL = 1.11), slender, roughly rectangular at base, and tapering gradually along length ending in a point. Limbs large (FLL+HLL/SVL = 0.67) adpressed limbs overlap by 1 costal interspace. Hands and feet are broad, digits long and relatively slender, blunt with distinct subterminal pads. Hands and feet are highly webbed compared to most other members of Pseudoeurycea (moderately webbed compared with other genera), with webbing extending to the base of the penultimate phalanx on the third toe of foot. First toe short. Order of digits by increasing length: manus I–IV–III–II, pes I–V–II–IV–III. Phalangeal formulae: manus 1–2–3–2, pes 1–2–3–3–2. Premaxillary teeth monocuspid, larger than the other teeth; maxillary teeth smaller and hook shaped and inward oriented; vomerine teeth arranged in one line in each side, curved near the edge of the choanae; parasphenoid teeth arranged in two series as inverted “V” shape forming a patch.

 Measurements of the holotype.

(in mm) Snout to posterior angle of vent 54.5; head width 8.7; head length 11.7; head depth at angle of jaw 3.9; eyelid length 3.5; eyelid width 1.8; anterior rim of orbit to snout 3.7; eye diameter 1.6; interorbital distance 3.8; snout to forelimb 17.3; internarial distance 2.5; intercanthal distance 4.4; nostril diameter 0.4; snout projection beyond mandible 0.5; snout to anterior angle of vent 45.8; axilla to groin 29.7; shoulder width 5.6; tail length 60.7; tail width at base 3.0; tail depth at base 3.4; forelimb length 18.2; hind limb length 18.6; hand width 4.2; foot width 5.9; length of the longest (third) toe 2.9; length of fifth toe 1.8; mental gland width 2.2; mental gland length 2.0. Tooth counts: premaxillary 5; maxillary 45/51; vomerine 33/30.

 Coloration of the holotype.

In life, dorsum and dorsal surface of the tail yellow, becoming more intense towards the end of body and tail. Black and dark gray stipples on the dorsal area of the body, with lighter and more intense yellow dots and their proportion increase towards the tail; limb coloration is similar as body (Fig. 6). Ventral surface of throat relatively opaque yellow-orange with lighter yellows splotches; body up to the end of the cloaca with little pigmentation, more intense at the beginning of the tail and chest, also with yellow splotches although in lesser proportion than in the throat; tail with background color like the throat, with small dark gray blotches; limbs of similar coloration as dark areas of the body. Eyelids with a nearly black or dark brown background, with numerous yellow splotches; black eyes with a very noticeable yellow iris with greenish tints. In preservative, pale yellowish ground coloration of entire body with evident dark blotches and smaller cream splotches in tail and diffused in dorsum, a cream ventral coloration with lighter yellow or cream splotches, with a higher percentage in the throat, chest, and mid-ventral area (Fig. 7).

­10.3897/vz.76.e167783.figure7 E5BC6A63-FEE7-5B87-A1D8-0314462F744D

Dorsal and ventral view of the holotype of Pseudoeurycea natsiisp. nov. (IBH 37026) to the left. Ventral view of right hand and right foot to the right. Photos: Víctor H. Jiménez-Arco.

https://binary.pensoft.net/fig/1644308 Variation and sexual dimorphism.

The type series includes two specimens, an adult male (holotype) and an adult female. The male is larger than the female (SVL 54.5 mm in male and 41.2 mm in female) with a longer, wider, and deeper head. The tail is relatively longer in the female (TL/SVL female: 1.18 vs 1.11 in male). Male with evident mental gland (width 2.2 mm). The male has more maxillary teeth (MT male: 45+51; female: 40+38) and vomerine teeth (VT male: 63; female: 43), the female has more premaxillary teeth (PMT male: 5; female: 12). Hands and feet are broader in the male (hand width 4.2 mm in male; 3.1 in female; RFW male: 5.9 mm; female: 4.0 mm). Nasolabial protuberance slightly more developed in the male. However, we consider caution in the measurements between sexes considering the general trend of a larger body size in females within the family Plethodontidae (Kupfer 2007). The single female may be a younger female adult and may not have reached the maximum size. Premaxillary teeth are larger in the male and the same size as maxillary teeth in the female. Maxillary teeth smaller, hook-shaped and inward oriented; vomerine teeth arranged in two lines, one line on each side, curved near the edge of the choanae; parasphenoid teeth arranged in two series as inverted “V” shape forming a patch.

 Distribution and natural history.

Pseudoeurycea natsiisp. nov. is only known from San Martín Caballero, specifically in the cloud forest of Cerro Rabon, located in the eastern portion of the Sierra Mazateca in Oaxaca. The species inhabits conserved cloud forest at 1611 m elevation. The two individuals were encountered during different survey events, and none were found during April, June, November, and December, suggesting that the species is rare at the locality. The holotype was found active in October at 23:00 hrs after rain above a clean dry tree branch about 60 mm diameter, 60 cm above the substrate. The paratype was also found on a dry branch about 20 mm diameter, 76 cm above the floor on July 16 at 21:50 hrs on a trail in the forest near a shade-grown coffee plantation. Because both individuals were found on branches and were relatively uncommon in our ecological study, we suggest that Pseudoeurycea natsiisp. nov. may be an arboreal species, which may explain the lower detection rate as compared to the other species described here. This is consistent with the species’ morphology (elongated body and limbs). We observed predation from the female (paratype) on two flies (Diptera) about 2 mm in size that flew about 15 mm near its head. Also, the paratype showed escape behavior after being touched by a stick, consisting of sudden movement of the body, followed by immobility, and as we continued the stimulus the organism jumped from the branch to the ground and then moved away. The body temperature (cloacal) of the two individuals was 16.1°C and 19.2°C for the holotype and paratype respectively, air temperatures were 16.2°C and 18.9°C, and the substrate temperatures were 16°C and 19.1°C respectively. Bolitoglossa rufescens, Parvimolge townsendi, Pseudoeurycea cf. werleri, and Thorius sp., as well as anuran species including C. polymniae and C. loki were found active around the area of the holotype collecting site.

 Etymology.

The specific epithet is taken from the Mazateco language word “Na tsií” which can be translated as “Queen of the rain”. This is in recognition to the San Martín Caballero and Rancho Guadalupe communities for the protection of communal areas, designed for the conservation of natural resources.

 Discussion

We find both molecular and morphological evidence supporting the recognition of the three new species described herein. In the P. leprosa group, we recover the two clades previously recognized (Rovito et al. 2015; García-Bañuelos et al. 2020; Cázares-Hernández et al. 2022). These studies have recovered the species pairs P. obesa + P. werleri and P. conanti + P. mystax (García-Bañuelos et al. 2020; Cázares-Hernández et al. 2022). In our analysis we recover P. conanti as sister species to P. werleri (although with low support). Our analysis comprises more taxa with one new species in the group, the new samples of P. werleri from Santa Marta (Veracruz) and Sierra de Juarez (Oaxaca), and the new two species in P. juarezi group. Hence, the relationships of P. conanti, P. mystax, and P. obesa remain ambiguous. Inclusion of cyt b sequences for P. conanti and nuclear or genomic data could help clarify our understanding of the relationships inside this clade, specifically among species that show problematic relationships.

Pseudoeurycea euguiisp. nov. represents the fourth described species of worm salamander within the genus Pseudoeurycea. Presumably due to their fossorial habits, the probability of detection in the wild is low. For example, the female of P. orchileucos has not been formally described (Brodie et al. 2002). We cannot rule out that it may be a naturally rare or difficult-to-detect species. However, our ecological sampling included locations between 740 and 1600 m a.s.l. at the east of Cerro Rabon. The specimens recorded in our study may be at the limit of their geographic range, with the majority of the distribution towards the western area of this mountain. Also, due to their small size, diagnostic characteristics are usually few or internal, something that also happens in other miniature salamanders (e.g., Thorius; Parra-Olea et al. 2016). It is necessary to increase taxonomic work, since according to our data, at least one lineage of worm salamanders (labeled as Pseudoeurycea orchileucos (2) in Fig. 2) from Peña Verde, Santa Maria Papalo, Oaxaca (Rovito et al. 2015) remains to be described, as previously suggested (Lamoreux et al. 2015). Our results confirm that it is an evolutionarily independent lineage of P. orchileucos. Moreover, one record from Sierra Negra, Puebla was identified as P. lineola (Canseco-Márquez et al. 2000) and this population requires a review to clarify its status.

Our phylogeny is also consistent with previous studies in recognizing two major clades within the P. juarezi group (Rovito et al. 2015, Cázares-Hernández et al. 2022; Peralta-Hernández et al. 2024), although they differ because I. niger is grouped as sister linage of the P. juarezi and P. leprosa groups with low support in our analyses. Including Ixalotriton parvus, nuclear markers and sequences of the new species may probably clarify the position of the genus Ixalotriton (Rovito et al. 2015). In the internal relationships of the P. juarezi group, the first clade includes ((Pseudoeurycea parraoleaesp. nov. + (P. juarezi + P. saltator)) + P. aurantia) and the second clade comprises (((Pseudoeurycea sp. + Pseudoeurycea natsiisp. nov.) + P. ruficauda) + P. jaguar). The species composition and even their phylogenetic relationships are consistent with recent studies (Cázares-Hernández et al. 2022; Peralta-Hernández et al. 2024). Notably, we find a considerable level of divergence between the P. jaguar (1) population from Veracruz (type locality) and the P. jaguar (2) population from Puebla as previously recorded (Peralta-Hernández et al. 2024). Although only the 16S marker is available for the Puebla samples, it would be interesting to analyze this population in more detail to determine their evolutionary history.

Pseudoeurycea parraoleaesp. nov. is readily distinguishable from all other species of Pseudoeurycea by its unique coloration and morphology, which are associated with saxicolous habitat use. Like fossorial species, saxicolous salamanders exhibit distinctive morphological traits, including slender and elongated bodies, long limbs, broad heads, and expanded hands and feet. These features are also observed in other plethodontids, such as I. niger (Rovito et al. 2017), Nyctanolis pernix (Elias and Wake 1983; Barrio-Amorós et al. 2016), and Chiropterotriton magnipes (Rabb 1965). Pseudoeurycea parraoleaesp. nov. is morphologically and ecologically similar to I. niger as their habitat descriptions are strikingly concordant. Both species inhabit cloud forests with a high prevalence of limestone outcrops (Fig. 8), lack surface water (e.g., streams), and are typically found on rocks, rock walls, and crevices (Wake and Johnson 1989). These shared traits among saxicolous species from different genera suggest a case of convergent evolution in response to life in rocky substrates.

­10.3897/vz.76.e167783.figure8 1F09B7FF-862A-5BCC-9C04-B2AD86D7C5EE

Habitat at the type locality of APseudoeurycea euguiisp. nov., BPseudoeurycea parraoleaesp. nov., and CPseudoeurycea natsiisp. nov. Photos: Leopoldo D. Váquez-Reyes.

https://binary.pensoft.net/fig/1644309

Pseudoeurycea natsiisp. nov. was the rarest species encountered in the cloud forest of Cerro Rabon. This species is also easily distinguishable from all other species in the genus by having a greater number of vomerine teeth. Perhaps a greater number of teeth favors the capture of small or slippery prey, although the explanation may not be adaptive; in any case, specific studies are required. The only two known specimens of this species were found on branches, suggesting an arboreal lifestyle. In general, arboreal species are notoriously difficult to detect due to their cryptic behavior, the vertical complexity of forest canopies and high density of epiphytic plants, which limit visibility and detectability during sampling (Wells 2007; Haysom et al. 2021). Alternatively, its rarity may be because the locality is near the limits of its distribution. However, this explanation might be unlikely, given that there are considerable sampling efforts to the northwest of the Sierra Mazateca (mainly from Huautla de Jimenez northwards) and to the south in the Sierra de Juarez where it has not been found (see Fig. 1). However, this species, as well as the other two species described here, may have a wider distribution towards the western area of Cerro Rabon. Lastly, Pseudoeurycea natsiisp. nov. is recovered as the sister species to an undescribed taxon, Pseudoeurycea sp. This taxon was initially proposed to be P. unguidentis (Parra-Olea and Wake 2001; Parra-Olea et al. 2004), later recognized as an undescribed species (Rovito et al. 2015), and ultimately excluded from subsequent studies on the P. juarezi group (Cázares-Hernández et al. 2022; Peralta-Hernández et al. 2024). As such, this lineage remains formally undescribed.

The genetic divergence between populations of P. werleri is interesting. The cyt b sequence available from Cumbres de Tonalixco, Veracruz (labeled as Pseudoeurycea werleri (5) in Fig. 2; Peralta-Hernández et al. 2020) clusters with P. cf. werleri (our samples from Cerro Rabon) with strong support and is located ~90 km north of the Cerro Rabon in the same mountain system (Sierra Mazateca–Sierra de Zongolica). In contrast, the Sierra de Juarez, Oaxaca sample (labeled as Pseudoeurycea werleri (4) in Fig. 2) lies ~60 km south of our samples from Cerro Rabon in the same mountain system, yet groups with P. werleri from Los Tuxtlas, Veracruz, despite being ~144 km northeast. The lowest genetic distances between the two P. werleri clades were 0.005 (16S) and 0.056 (cyt b) between P. cf. werleri versus P. werleri 1 (from Santa Marta volcano, Los Tuxtlas) and P. werleri 4 respectively. Although these values are lower in the P. leprosa group (Table 2), they are comparable with other pairs of species in the genus such as P. juarezi / P. saltator (0.006 for 16S and 0.015 for cyt b). Furthermore, the preliminary morphological review suggests a degree of divergence, although further examination of specimens, including other intermediate populations, is necessary. Also, including nuclear markers and phylogeographic analyses, would be interesting and necessary to discern the evolutionary history of this widely distributed species.

Cerro Rabon is a mountain on the eastern slopes of the Sierra Mazateca in Oaxaca below 2400 m elevation. While the surroundings areas have been comparatively better sampled, this sampling has been concentrated mostly in higher elevations in the Sierra de Juarez and in the western portion of the Sierra Mazateca (Fig. 1). Cerro Rabon is almost entirely surrounded by lowlands, but it connects to the west with the road (Highway 182) to Huautla de Jimenez at elevations between 1600 and 1800 m a.s.l. In Mexican amphibians, mid-elevation areas harbor the greatest species richness (Rivera-Reyes et al. 2025). Pseudoeurycea generally exhibits the highest species richness above 2000 m (Cázares-Hernández et al. 2022), although the species we describe here are located at intermediate elevations (731–1611 m elevation). Therefore, it is necessary to increase sampling effort throughout mid-elevation areas including Cerro Rabon and other surrounding mountains that have not been explored as they likely harbor additional undescribed species.

Conservation

Due to their location at the top of Cerro Rabon, two of the three species (Pseudoeurycea parraoleaesp. nov. and Pseudoeurycea natsiisp. nov.) are likely at significant risk of extinction. We conservatively estimate the area of ​​Cerro Rabon above 1000 m elevation to be just over 100 km² and we assume that it could include the habitat of these species even though they have been found at higher elevations. With this, we estimate the extent of occurrence (EOO; <5000 km2, criteria B1; IUCN Standards and Petitions Committee 2024). Furthermore, both species are known only from a single locality very close to each other and to the town of San Martín Caballero (condition a). Also, in just over three years of working in the area, we have observed a small reduction in forest cover in some areas near these localities, mainly due to human activities such as agriculture and livestock, which can be inferred as a reduction in the quantity or quality of the habitat (condition b(iii)). Also, in June 2024, a fire broke out approximately 500 m from the San Martín Caballero localities. This fire was quickly contained by Oaxaca environmental authorities and residents of San Martin Caballero, preventing it from reaching the areas where the new species were recorded. According to the residents, a fire of this magnitude has not been recorded in as long as they can remember, so the effects of climate change may begin to be noticeable and threaten these species. In summary, this suggests that these two species with very restricted distributions could be categorized provisionally as Endangered (EN) under the criteria B1ab(iii)+B2ab(iii). Similarly, P. euguiisp. nov. has been recorded in lowland areas, and its distribution may be larger due to the lack of clear geographic or climatic barriers at elevations below 1000 m. However, habitat loss and degradation in rainforest have been among the most intense in Mexico (Martínez-Ruiz et al. 2025). In fact, the specimens we found were recorded in a corn-growing area (Fig. 8). We consider that this species could tentatively be categorized as Vulnerable (VU) under the criteria B1ab(iii)+B2ab(iii) with a relatively wider distribution than the previous species.

The discovery of the new species emerged from an assessment of taxonomic, functional, and phylogenetic diversity loss in Cerro Rabon. In addition to the species described here, at least one more distinct plethodontid lineage (Thorius sp.) and one lizard (Anolis sp.) represent new species, both of which are currently in the process of being formally described. Noteworthy distributional records were also obtained such as for Parvimolge townsendi, which represents the first record for the state of Oaxaca (Mata-Silva et al. 2021). Many additional distribution records are pending publication. Together, the Sierra Mazateca, Sierra de Zongolica, and Sierra de Juarez form a complex mountainous system. However, they do not constitute a single natural unit as they harbor groups with diverse evolutionary origins and histories (Morrone 2021). Thus, this region comprises one of the most biologically diverse areas in Mexico. Previous studies documenting amphibian and reptile diversity in the eastern Sierra Mazateca are incipient (Campbell and Duellman 2000; Villegas-García et al. 2015), highlighting the need for further research on amphibians, reptiles, and other biological groups to accurately catalogue the region’s biodiversity. The protection of communal lands by Mazatec communities, particularly in San Martín Caballero, Rancho Guadalupe, and Emiliano Zapata, as well as in other parts of Oaxaca (e.g., Sierra de Juarez; Simón-Salvador et al. 2021), could facilitate the conservation of the species described here, as well as promote the care of all biodiversity at a regional scale.

 Acknowledgments

We especially thank to Irene Castillo Castañeda, Eleuterio Zenteno Ramírez, Zuleima Tovar Castillo, Abad Tovar Castillo (San Felipe Tilpam), Jonathan Herrera Canseco and his family (Emiliano Zapata), Esteban García García, David García Tejada and his family (San Martín Caballero) for providing guidance, logistic support, shelter, and food during our fieldwork. We thank the community authorities of San Martín Caballero, Rancho Guadalupe, and Emiliano Zapata (Angelo Martínez Lazaro), as well as the municipal authorities of San José Tenango, specifically Luis L. Carrera Juárez, for allowing us to work in their territory and providing us with logistical support during the sampling. We thank Osvaldo Cervantes Zamudio, Laboratorio de Microscopia, FES Iztacala, UNAM for the provision of tools and advice during the specimen’s review and Eduardo Cid Méndez, Laboratorio de Herpetología Vivario for their invaluable support in reviewing specimens. Luis A. Cadena Escobar for their help in fieldwork. We thank Oscar Flores and Gustavo Campillo from the Museo de Zoología de la Facultad de Ciencias (MZCF) for the loan of specimens. We especially thank Gabriela Parra Olea and Joseph Mendelson III for their help in reviewing morphological characters. We thank to C. Marisol Gómez Hurtado for their help in image editing. We thank Karime Unda Harp, head of the Secretaria de Medio Ambiente, Biodiversidad, Energias y Sostenibilidad of the State of Oaxaca for her attention in seeking help to contain the fires in Cerro Rabon in 2024. Collecting permit provided by SEMARNAT No. FAU0374/2022. This project was supported by equipment and material from the PAPIIT, UNAM project No. IA207524 and Ciencia de Frontera program project No. CF-2023-I-2445 assigned to VHJA. MSC thanks the Posgrado en Ciencias Biológicas de la Universidad Autónoma de Tlaxcala y Centro Tlaxcala Biología de la Conducta (CTBC) for the academic training. EAAH and JDGT thanks the Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México. EAAH, JDGT, and MSC thanks to Secretaria de Ciencias, Humanidades, Tecnología e Innovación for the scholarships for postgraduate studies (CVU 1280683, 1281753, 1232698 respectively). We especially appreciate the comments and suggestions of two anonymous reviewers and Deepak Veerappan, which improved the quality of previous versions of this manuscript.

 References AmphibiaWeb (2026) University of California, Berkeley, CA. https://amphibiaweb.org [accessed 10 April 2026]. Barrio-Amorós C, Grünwald CI, Franz-Chávez H, La Forest BT (2016) A new Mexican locality for the endangered salamander Nyctanolis pernix (Caudata: Plethodontidae). Mesoamerican Herpetology 3: 534–536. Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW (2012) GenBank. Nucleic Acids Research 41: 1–7. https://doi.org/10.1093/nar/gks1195 Brodie Jr, ED, Mendelson III, JR, Campbell JA (2002) Taxonomic revision of the Mexican plethodontid salamanders of the genus Lineatriton, with the description of two new species. Herpetologica 58: 194–204. https://doi.org/10.1655/0018-0831(2002)058[0194:TROTMP]2.0.CO;2 Campbell, JA, Duellman WE (2000) New species of stream-breeding hylid frogs from the northern versant of the highlands of Oaxaca, Mexico. Scientific Papers Natural History Museum University of Kansas 16: 1–28. https://doi.org/10.5962/bhl.title.16165 Canseco-Márquez L, Gutierrez-Mayen G, Salazar-Arenas J (2000) New records and range extensions for Amphibians and Reptiles from Puebla, Mexico. Herpetological Review 31: 259–263. Cázares-Hernández E, Jimeno-Sevilla HD, Rovito SM, López-Luna MA, Canseco-Márquez L (2022) A new arboreal Pseudoeurycea (Caudata: Plethodontidae) from the Sierra de Zongolica, Veracruz, Mexico. Vertebrate Zoology 72: 937–950. https://doi.org/10.3897/vz.72.e87275 Edgar RC (2004) MUSCLE: A multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5: 1–19. https://doi.org/10.1186/1471-2105-5-113 Elias P, Wake DB (1983) Nyctanolis pernix, a new genus and species of plethodontid salamander from northwestern Guatemala and Chapas, Mexico. In: Rhodin AGJ, Miyata K (Eds) Advances in Herpetology and Evolutionary Biology: Essays in Honor of Ernest E. Williams. Museum of Comparative Zoology, Cambridge, MA, 1–12. https://biostor.org/reference/133156 Escalante T, Szumik C, Morrone JJ (2009) Areas of endemism of Mexican mammals: Reanalysis applying the optimality criterion. Biological Journal of the Linnean Society 98: 468–478. https://doi.org/10.1111/j.1095-8312.2009.01293.x Flores-Villela O, García-Vázquez UO (2014) Biodiversidad de reptiles en México. Revista Mexicana de Biodiversidad 85: S467–S475. https://doi.org/10.7550/rmb.43236 García-Bañuelos P, Aguilar-López JL, Kelly-Hernández A, Vásquez-Cruz V, Pineda E, Rovito SM (2020) A new species of Pseudoeurycea (Amphibia: Caudata) from the mountains of central Veracruz, Mexico. Journal of Herpetology 54: 258–267. https://doi.org/10.1670/19-052 Global Biodiversity Information Facility [GBIF] (2026) https://www.gbif.org [accessed 27 February 2026]. Haysom JK, Deere NJ, Wearn OR, Mahyudin A, Jami JB, Reynolds G, Struebig MJ (2021) Life in the canopy: Using camera-traps to inventory arboreal rainforest mammals in Borneo. Frontiers in Forests and Global Change 4: 673071. https://doi.org/10.3389/ffgc.2021.673071 Hernández-Austria R, García-Castillo MG, Parra-Olea G (2024) A new species of direct-developing frog of the genus Eleutherodactylus (Anura: Eleutherodactylidae) from Tamaulipas, Mexico. Zootaxa 5471: 433–450. https://doi.org/10.11646/zootaxa.5471.4.3 Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS (2018) UFBoot2: Improving the Ultrafast Bootstrap Approximation. Molecular Biology and Evolution 35: 518–522. https://doi.org/10.1093/molbev/msx281 IUCN Standards and Petitions Committee (2024) Guidelines for Using the IUCN Red List Categories and Criteria. Version 16. Prepared by the Standards and Petitions Committee. https://www.iucnredlist.org/documents/RedListGuidelines.pdf Jiménez-Arcos VH, Calzada-Arciniega RA, Alfaro-Juantorena LA, Vázquez-Reyes LD, Blair C, Parra-Olea G (2019) A new species of Charadrahyla (Anura: Hylidae) from the cloud forest of western Oaxaca, Mexico. Zootaxa 4554: 371–385. https://doi.org/10.11646/zootaxa.4554.2.3 Jiménez-Arcos VH, Cortés-Ortiz B, Gómez-Hurtado CM, Calzada-Arciniega RA (2022) Agalychnis callidryas (red-eyed treefrog). Field body temperature and morphology. Herpetological Review 53: 646–­647. Kalyaanamoorthy S, Minh B, Wong T, von Haeseler A, Jermiin LS (2017) ModelFinder: Fast model selection for accurate phylogenetic estimates. Nature Methods 14: 587–589. https://doi.org/10.1038/nmeth.4285 Kupfer A (2007) Sexual size dimorphism in amphibians: An overview. In: Fairbairn DJ, Blanckenhorn WU, Székely T (Eds) Sex, Size and Gender Roles: Evolutionary Studies of Sexual Size Dimorphism. Oxford University Press, Oxford, 50–59. Lamoreux JF, McKnight MW, Cabrera Hernandez R (2015) Amphibian Alliance for Zero Extinction Sites in Chiapas and Oaxaca. IUCN, Gland, xxiv, 320 pp. https://iucn.org/es/node/31042 Luedtke JA, Chanson J, Neam K, Hobin L, Maciel AO, Catenazzi A, Borzée A, Hamidy A, Aowphol A, Jean A, Sosa-Bartuano Á, Fong GA, de Silva A, Fouquet A, Angulo A, Kidov AA, Muñoz Saravia A, Diesmos AC, Tominaga A, Shrestha B, Gratwicke B, Tjaturadi B, Martínez Rivera CC, Vásquez Almazán CR, Señaris C, S R C, Strüssmann C, Cortez Fernández CF, Azat C, Hoskin CJ, Hilton-Taylor C, Whyte DL, Gower DJ, Olson DH, Cisneros-Heredia DF, Santana DJ, Nagombi E, Najafi-Majd E, Quah ESH, Bolaños F, Xie F, Brusquetti F, Álvarez FS, Andreone F, Glaw F, Castañeda FE, Kraus F, Parra-Olea G, Chaves G, Medina-Rangel GF, González-Durán G, Ortega-Andrade HM, Machado IF, Das I, Ribeiro Dias I, Urbina-Cardona JN, Crnobrnja-Isailović J, Yang JH, Jianping J, Wangyal JT, Rowley JJL, Measey J, Vasudevan K, Chan KO, Gururaja KV, Ovaska K, Warr LC, Canseco-Márquez L, Toledo LF, Díaz LM, Khan MMH, Meegaskumbura M, Acevedo ME, Napoli MF, Ponce MA, Vaira M, Lampo M, Yánez-Muñoz MH, Scherz MD, Rödel MO, Matsui M, Fildor M, Kusrini MD, Ahmed MF, Rais M, Kouamé NG, García N, Gonwouo NL, Burrowes PA, Imbun PY, Wagner P, Kok PJR, Joglar RL, Auguste RJ, Albuquerque Brandão R, Ibáñez R, von May R, Hedges SB, Biju SD, Ganesh SR, Wren S, Das S, Flechas SV, Ashpole SL, Robleto-Hernández SJ, Loader SP, Incháustegui SJ, Garg S, Phimmachak S, Richards SJ, Slimani T, Osborne-Naikatini T, Abreu-Jardim TPF, Condez TH, De Carvalho TR, Cutajar TP, Pierson TW, Nguyen TQ, Kaya U, Yuan Z, Long B, Langhammer P, Stuart SN (2023) Ongoing declines for the world’s amphibians in the face of emerging threats. Nature 622: 308–314. https://doi.org/10.1038/s41586 Lynch JF, Wake DB (1989) Two new species of Pseudoeurycea (Amphibia: Caudata) from Oaxaca, Mexico. Contributions in Science, Los Angeles County Museum of Natural History 411: 11–22. https://doi.org/10.5962/p.208133 Maddison WP, Maddison DR (2023) Mesquite: A modular system for evolutionary analysis. Version 3.81. http://www.mesquiteproject.org Martínez-Ruiz M, Arroyo-Rodríguez V, San-José M, Arce-Peña N, Cudney-Valenzuela S, Galán-Acedo C (2025) Effect of forest loss and fragmentation per se on arboreal and ground mammals of the Lacandon rainforest, Mexico. Biodiversity Conservation 34: 1365–1383. https://doi.org/10.1007/s10531-025-03022-z Mata-Silva V, García-Padilla E, Rocha A, Desantis DL, Johnson JD, Ramírez-Bautista A, Wilson LD (2021) A reexamination of the herpetofauna of Oaxaca, Mexico: Composition update, physiographic distribution, and conservation commentary. Zootaxa 4996: 201–252. https://doi.org/10.11646/zootaxa.4996.2.1 Moritz C, Schneider CJ, Wake DB (1992) Evolutionary relationships within the Ensatina eschscholtzii complex confirm the ring species interpretation. Systematic Biology 41: 273–291. https://doi.org/10-093/sysbio/41.3.273 Morrone JJ (2021) The Mexican Transition Zone. Springer, Cham, 191 pp. https://doi.org/10.1007/978-3-030-47917-6 Nowakowski AJ, Frishkoff LO, Thompson ME, Smith TM, Todd BD (2018) Phylogenetic homogenization of amphibian assemblages in human-altered habitats across the globe. Proceedings of the National Academy of Sciences of the USA 115: E3454–E3462. https://doi.org/10.1073/pnas.1714891115 Palacios-Aguilar R, Cisneros-Bernal AY, Arias-Montiel JD, Parra-Olea G (2020) A new species of Bolitoglossa (Amphibia: Plethodontidae) from the central highlands of Guerrero, Mexico. Canadian Journal of Zoology 98: 359–365. https://doi.org/10.1139/cjz-2019-0244 Palumbi SR, Martin AP, Romano S, McMillan WO, Stice L, Grabowski G (1991) The Simple Fool’s Guide to PCR. Department of Zoology, University of Hawaii, Honolulu, HI, 45 pp. Parra-Olea G, Flores-Villela O, Mendoza-Almeralla C (2014) Biodiversidad de anfibios en México. Revista Mexicana de Biodiversidad 85: S460–S466. https://doi.org/10.7550/rmb.32027 Parra-Olea G, Garcia-Castillo MG, Rovito SM, Maisano JA, Hanken J, Wake DB (2020) Descriptions of five new species of the salamander genus Chiropterotriton (Caudata: Plethodontidae) from eastern Mexico and the status of three currently recognized taxa. PeerJ 8: e8800. https://doi.org/10.7717/peerj.8800 Parra-Olea G, García-París M, Hanken J, Wake DB (2004) A new species of arboreal salamander (Caudata: Plethodontidae: Pseudoeurycea) from the mountains of Oaxaca, Mexico. Journal of Natural History 38: 2119–2131. https://doi.org/10.1080/00222930310001617724 Parra-Olea G, Rovito SM, García-París M, Maisano JA, Wake DB, Hanken J (2016) Biology of tiny animals: three new species of minute salamanders (Plethodontidae: Thorius) from Oaxaca, Mexico. PeerJ 4:e2694 https://doi.org/10.7717/peerj.2694 Parra-Olea G, Wake DB (2001) Extreme morphological and ecological homoplasy in tropical salamanders. Proceedings of the National Academy of Sciences of the USA 98: 7888–7891. https://doi.org/10.1073/pnas.131203598 Peralta-Hernández RO, Parra-Olea G, López-Velázquez A, García-Castillo MG (2020) Amphibians and reptiles from cloud forest at Cumbre de Tonalixco in the central Veracruz highlands of Mexico. Reptiles & Amphibians 27: 501–505. https://doi.org/10.17161/randa.v27i3.14898 Peralta-Hernández R (2023) New data on occurrence, ecology, and color variation of Pseudoeurycea jaguar (Caudata: Plethodontidae), an endemic salamander from Mexico. Phyllomedusa 22: 43–47. https://doi.org/10.11606/issn.2316-9079.v22i1p43-47 Peralta-Hernández R, García-Castillo MG, Hernández-Monasterio S, Parra-Olea G (2024) First records of occurrence and predation of Pseudoeurycea jaguar (Caudata: Plethodontidae) in the state of Puebla, Mexico. North-Western Journal of Zoology 20: 192–195. Pottier P, Kearney MR, Wu NC, Gunderson AR, Rej JE, Rivera-Villanueva AN, Pollo P, Burke S, Drobniak SM, Nakagawa S (2025) Vulnerability of amphibians to global warming. Nature 639: 954–961. https://doi.org/10.1038/s41586-025-08665-0 Pyron RA(2018) Global amphibian declines have winners and losers. Proceedings of the National Academy of Sciences of the USA 115: 3739–3741. https://doi.org/10.1073/pnas.1803477115 Rabb GB (1965) A new salamander of the genus Chiropterotriton (Caudata: Plethodontidae) from Mexico. Breviora 235: 1–8. https://biostor.org/reference/4304 Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA (2018) Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Systematic Biology 67: 901–904. https://doi.org/10.1093/sysbio/syy032 Ramírez-Barahona S, Eguiarte LE (2013) The role of glacial cycles in promoting genetic diversity in the Neotropics: The case of cloud forests during the Last Glacial Maximum. Ecology and Evolution 3: 725–738. https://doi.org/10.1002/ece3.483 Rivera-Reyes R, Goyenechea Mayer-Goyenechea I, Ochoa-Ochoa LM (2025) Elevational diversity gradients of amphibians in Mexican mountain ranges: Patterns, environmental factors, and spatial scale effects. Biogeographia 40: a046. https://doi.org/10.21426/B6.39854 Rodríguez A, Castro-Castro A, Vargas-Amado G, Vargas-Ponce O, Zamora-Tavares P, González-Gallegos J, Carrillo-Reyes P, Anguiano-Constante M, Carrasco-Ortiz M, García-Martínez M, Gutiérrez-Rodríguez B, Aragón-Parada J, Valdes-Ibarra C, Munguía-Lino G (2018) Richness, geographic distribution patterns, and areas of endemism of selected angiosperm groups in Mexico. Journal of Systematics and Evolution 56: 537–549. https://doi.org/10.1111/jse.12457 Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539–542. https://doi.org/10.1093/sysbio/sys029. Rovito SM, Muñoz Alonso LA, Lamoreux JF, McKnight MW, Parra-Olea G (2017) Taxonomic status of a population of Black Ixalo­triton from Cerro Baúl, Chiapas, México. Journal of Herpetology 51: 590–594. https://doi.org/10.1670/16-103 Rovito SM, Parra-Olea G, Recuero E, Wake DB (2015) Diversification and biogeographical history of Neotropical plethodontid salamanders. Zoological Journal of the Linnean Society 175: 167–188. https://doi.org/10.1111/zoj.12271 Scheele BC, Pasmans F, Skerratt LF, Berger L, Martel A, Beukema W, Acevedo AA, Burrowes PA, Carvalho T, Catenazzi A, de la Riva I, Fisher MC, Flechas SV, Foster CN, Frías-Álvarez P, Garner TWJ, Gratwicke B, Guayasamin JM, Hirschfeld M, Kolby JE, Kosch TA, La Marca E, Lindenmayer DB, Lips KR, Longo AV, Maneyro R, McDonald CA, Mendelson J, Palacios-Rodriguez P, Parra-Olea G, Richards-Zawacki CL, Rödel M-O, Rovito SM, Soto-Azat C, Toledo LF, Voyles J, Weldon C, Whitfield SM, Wilkinson M, Zamudio KR, Canessa S (2019) Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity. Science 363:1459–1463. https://www.science.org/doi/10.1126/science.aav0379 Simón-Salvador PR, Arreortúa M, Flores CA, Santiago-Dionicio H, González-Bernal E (2021) The role of Indigenous and Community Conservation Areas in herpetofauna conservation: A preliminary list for Santa Cruz Tepetotutla, Oaxaca Mexico. ZooKeys 1029: 185–208. https://doi.org/10.3897/zookeys.1029.62205 Swofford D (2002) PAUP*: Phylogenetic Analysis Using Parsimony (* and other Methods), Version 4. Sinauer Associates, Sunderland, MA. Trifinopoulos J, Nguyen LT, von Haeseler A, Minh BQ (2016) W-IQ-TREE: A fast online phylogenetic tool for maximum likelihood ana­lysis. Nucleic Acids Research 8: W1–W232. https://doi.org/10.1093/nar/gkw256 Vázquez-Reyes LD, Jiménez-Arcos VH, Santa Cruz-Padilla SA, García-Aguilera R, Aguirre-Romero A, Arizmendi MDC, Navarro-Sigüenza AG (2018) Aves del Alto Balsas de Guerrero: diversidad e identidad ecológica de una región prioritaria para la conservación. Revista Mexicana de Biodiversidad 89: 873–897. https://doi.org/10.22201/ib.20078706e.2018.3.2314 Villegas-García R, Vázquez-Vega LF, Caviedes-Solis IW, Solano-Zavaleta I, Flores-Villela O (2015) Estudio herpetofaunístico de la Sierra Mazateca, Oaxaca, México. Universidad Nacional Autónoma de México. Facultad de Ciencias. Informe final SNIB-CONABIO, proyecto No. JF058. México D.F. http://www.conabio.gob.mx/institucion/proyectos/resultados/InfJF058.pdf Wake DB (1987) Adaptive radiation of salamanders in Middle American cloud forests. Annals of the Missouri Botanical Garden 74: 242–264. https://doi.org/10.2307/2399397 Wake DB, Johnson JD (1989) A new genus and species of plethodontid salamander from Chiapas, Mexico. Contributions in Science Natural History Museum of Los Angeles County 411: 1–10. https://doi.org/10.5962/p.208132 Wells KD (2007) The Ecology and Behavior of Amphibians. University of Chicago Press, Chicago, IL, 1148 pp. Wiens JJ, Parra-Olea G, García-París M, Wake DB (2007) Phylogenetic history underlies elevational biodiversity patterns in tropical salamanders. Proceedings of the Royal Society B 274: 919–928. https://doi.org/10.1098/rspb.2006.0301 Appendix 1

Specimens examined

Institutional abbreviations: IBH – Colección Nacional de Anfibios y Reptiles, Instituto de Biología, UNAM; MZFC – Museo de Zoología “Alfonso L. Herrera”, Facultad de Ciencias, UNAM.

Pseudoeurycea aurantia (n = 7). México: Oaxaca: 19 km E Concepcion Papalo, Carr. a Peña Verde, Oaxaca (IBH 22283); 11.8km E (by air) of Concepcion Papalo, Sierra de Juarez (IBH 23137–38, 23140); Concepción Papalo, 11 km ESE (by air) of Concepcion Papalo, Sierra de Juarez (IBH 23139); Santa María Pápalo, 1.3 km N (by rd) of turnoff to Santa María Pápalo on road to Peña Verde (IBH 32245, 32326).

Pseudoeurycea euguii sp. nov. (n = 3). México: Oaxaca: San José Tenango: 1.36 km SW of Emiliano Zapata (IBH 37010–12).

Pseudoeurycea jaguar (n = 2). México: Veracruz: Cerro Gentil, Huioapan de Cuauhtémoc (IBH 36061); PUEBLA: Cerro Tsitsintepetl, Santa Maria Coyomeapan (IBH 36062).

Pseudoeurycea juarezi (n = 9). México: Oaxaca: Calpulalpam de Méndez: Calpulalpan arroyo/encinar húmedo (IBH 24780, 24784, 24787, 24789, 24790); Santiago Comaltepec: 3.7 km N (by rd) of Cerro Pelón mirador on MX Hwy 175 (IBH 32369); 3.3 km north (by rd) of Cerro Pelon mirador on MX Hwy 175 (IBH 32394); 1.1 km N (by rd) of Cerro Pelón mirador on MX Hwy 175 (IBH 32420); Totontepec Villa de Morelos: 5.8 km NW (by rd) of junction with road to Zacatepec on MX Hwy 179 to Totontepec Villa de Morelos (IBH 32404).

Pseudoeurycea lineola (n = 13). México: Veracruz: Atoyac: Progreso, municipio de Atoyac, Ver. MX (IBH 26540); Fortín: Barranca San Miguel, Fortín de Las Flores, Ver. Mex. (IBH 22998); Tezonapa: Rincón de las Flores, 10.4 km W (by rd) of highway from Omealca to Tezonapa (IBH 32082, 32126, 32212, 32218, 32240, 32269, 32282, 32284, 32314, 32324); Cerro Chicahuaxtla (IBH 22289, 22999).

Pseudoeurycea natsii sp. nov. (n = 2). México: Oaxaca: San José Tenango: 1.16 km of San Martín Caballero (IBH 37026–27).

Pseudoeurycea orchileucos (n = 2). México: Oaxaca: Along trail running E of Hwy 175 at point 0.5 rd mi S Vista Hermosa (MZFC 14057–58).

Pseudoeurycea orchimelas (n = 9). México: Veracruz: Catemaco, Buena Vista, Volcan San Martín Tuxtla (IBH 18812); San Andrés Tuxtla, Estación de Biología Tropical “Los Tuxtlas” UNAM ((IBH 18814, El Bastonal, Sierra de Santa Marta (IBH 18827)); W side of La Perla de San Martín, 18.6 km NE (by rd) of San Andrés Tuxtla (IBH 32112, 32114, 32117, 32244, 32295); SW slope Volcán San Martín, Rancho Primero de Mayo, aprox., 12.3 rd km. ENE Tapalapan (MZFC 14056).

Pseudoeurycea parraoleae sp. nov. (n = 13). México: Oaxaca: San José Tenango: 1.1 km W of San Martín Caballero (IBH 37013–25).

Pseudoeurycea ruficauda (n = 3): México: Oaxaca: Teotitlán, 2 km W Puerto de la Soledad, Villanueva (IBH 22207); Santa María Tlalixtac, 4.0 km NE (by rd) of Peña Verde on road to Tlalixtac Viejo, Sierra de Juarez (IBH 22610); San Bernardino, 1.6 km S (by rd) of Puerto Soledad on road to San Bernardino, Sierra Mazateca (IBH 30229).

Pseudoeurycea saltator (n = 6). México: Oaxaca: Calpulalpam de Méndez: Calpulalpan arroyo (IBH 24779); San Isidro Yolox: ca. 200 m from MX Hwy 175 on road to San Isidro Yolox (IBH 30220, 30244); San Pedro Yolox: La Galera, 11.0 km SW (by rd) of La Esperanza on MX Hwy 175 (IBH 32193, 32402, 32425).

Pseudoeurycea sp. (as Pseudoeurycea cf. unguidentis) (n = 10). México: Oaxaca: 2.5-5 km W Cerro Machin road to Comaltepec (IBH 6443–53); ca. 200 m from MX Hwy 175 on road to San Isidro Yolox (IBH 30213).

Pseudoeurycea werleri (n = 6). México: Oaxaca: La Esperanza, San Bernardo, 4.8 km SW (by rd) of La Esperanza on MX176 (IBH 30214); VERACRUZ: Catemaco, Buena Vista Volcan San Martín Tuxtla (IBH 19334); San Andrés Tuxtla, Volcan San Martín Tuxtla (IBH 19341, 23208–09); Catemaco, Tres Caminos a la Azufrera, camino entre, Sierra de Santa Marta (IBH 19345).

Pseudoeurycea cf. werleri (n = 10). México: Oaxaca: San José Tenango: 1.96 km SE of Rancho Guadalupe (IBH 37000); 1.16 km SE of San Martín Caballero (IBH 37001–05); 1.1 km W of San Martín Caballero (IBH 37006–09).

 Supplementary materials ­10.3897/vz.76.e167783.suppl1 8B0A1B12-D3C2-56BE-9DD7-7F501E10B23A

Figures S1, S2

: .docx

Figure S1. Phylogeny estimated from Bayesian inference of concatenated 16S and cyt b sequence data for Pseudoeurycea using the reduced dataset (see Materials and Methods). Numbers on branches indicate posterior probability. — Figure S2. Phylogeny estimated from Maximum likelihood inference of concatenated 16S and cyt b sequence data for Pseudoeurycea using the reduced dataset (see Materials and Methods). Numbers on branches indicate ultrafast bootstrap values.

 https://binary.pensoft.net/file/1644310 This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Aguilar-Herrera EA, Sanabria-Urbán S, Flores-Martínez DL, Gómez-Tapia JD, Seba-Chacha M, Vázquez-Reyes LD, Rivera-Ortíz FA, Ramírez-Bastida P, Cortés-Ortiz B, Calzada-Arciniega RA, Avila-Ortega EU, Víquez-Vega DI, Hernández-Ordoñez O, Blair C, Jiménez-Arcos VH (2026)