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.

Keywords

Bolitoglossini, Pseudoeurycea euguii sp. nov., Pseudoeurycea natsii sp. nov., Pseudoeurycea parraoleae sp. nov., Pseudoeurycea werleri

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.

Figure 1.

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).

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.

Table 1.

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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. euguii sp. nov. 2	IBH 37011	PZ317143	IBH 37011	PZ334440
P. parraoleae sp. nov. 1	IBH 37013	PZ317139	IBH 37013	PZ334436
P. parraoleae sp. nov. 2	IBH 37019	PZ317140	IBH 37019	PZ334437
P. parraoleae sp. nov. 3	IBH 37020	PZ317141	IBH 37020	PZ334438
P. parraoleae sp. nov. 4	IBH 37014	PZ317142	IBH 37014	PZ334439
P. natsii sp. 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 1000^th 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.

Figure 2.

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).

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 euguii sp. nov.) and P. juarezi (Pseudoeurycea parraoleae sp. nov. and Pseudoeurycea natsii sp. nov.) groups. Pseudoeurycea parraoleae sp. 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 euguii sp. 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 parraoleae sp. 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 natsii sp. 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 euguii sp. 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 parraoleae sp. 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 natsii sp. nov. and its sister taxon, Pseudoeurycea sp. was 0.016 (16S) and 0.054 (cyt b; Table 3).

Table 2.

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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. euguii sp. 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	––

Table 3.

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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. parraoleae sp. nov.	0.012	0.055	0.041	0.011	0.036	0.011	0.036	––	0.169
9. P. natsii sp. 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

Pseudoeurycea euguii Jiménez-Arcos, Calzada-Arciniega, Cortés-Ortiz & Aguilar-Herrera, sp. nov.

https://zoobank.org/52A09B5F-BF66-4E93-9BE6-4EC587C21BDD

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 euguii sp. 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 euguii sp. 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 euguii sp. 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 euguii sp. nov. is different from the three species of worm salamanders by the following combination of characters: Pseudoeurycea euguii sp. nov. male having fewer vomerine teeth than P. lineola and P. orchileucos (VT males: Pseudoeurycea euguii sp. 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 euguii sp. nov. 8, P. lineola 14–22, P. orchimelas 16–19), as well as fewer premaxillary teeth (PMT females: Pseudoeurycea euguii sp. 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 euguii sp. nov. 0.7, P. lineola 1.0–1.4, P. orchileucos 1.0, P. orchimelas 0.9–1.0; IN females: Pseudoeurycea euguii sp. 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.

Figure 3.

Live specimens of Pseudoeurycea euguii sp. 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.

Table 4.

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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)
SVL	♀	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)
TL	♀	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)
AX	♀	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
FLL	♀	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
HLL	♀	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)
HL	♀	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)
HW	♀	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
HD	♀	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)
IO	♀	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)
IN	♀	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
RFW	♀	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
T3	♀	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
T5	♀	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
PMT	♀	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
MT	♀	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)
VT	♀	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 euguii sp. 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 euguii sp. 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 euguii sp. 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.

Pseudoeurycea parraoleae Jimenez-Arcos, Aguilar-Herrera, Flores-Martínez & Calzada-Arciniega, sp. nov.

https://zoobank.org/CC65D18A-4015-419B-B8DF-C6F54CD7A989

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 parraoleae sp. 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 parraoleae sp. 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 parraoleae sp. 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 parraoleae sp. nov. has a greater number of maxillary teeth (MT males: Pseudoeurycea aurantia 52–66, Pseudoeurycea parraoleae sp. nov. 82–89; females: Pseudoeurycea aurantia 49–66, Pseudoeurycea parraoleae sp. 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 parraoleae sp. nov. has a greater number of maxillary teeth (MT males: Pseudoeurycea juarezi 39–58, Pseudoeurycea parraoleae sp. nov. 82–89; females: Pseudoeurycea juarezi 54–58, Pseudoeurycea parraoleae sp. 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 parraoleae sp. nov. tends to have a greater number of maxillary teeth (MT males: Pseudoeurycea parraoleae sp. nov. 82–89, P. saltator 58–67; females: Pseudoeurycea parraoleae sp. 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 parraoleae sp. nov. tend to have smaller body size (SVL males: Pseudoeurycea jaguar 58.0–58.7 mm, Pseudoeurycea parraoleae sp. nov. 33.7–44.6 mm; females: Pseudoeurycea jaguar 42.4–71.0 mm, Pseudoeurycea parraoleae sp. nov. 38.5–49.8 mm), and therefore smaller head, shorter interocular distance and distance between nostrils (Table 5).

Table 5.

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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)
SVL	♀	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)
TL	♀	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)
AX	♀	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)
FLL	♀	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)
HLL	♀	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)
HL	♀	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)
HW	♀	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)
HD	♀	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)
IO	♀	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)
IN	♀	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)
RFW	♀	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)
T3	♀	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)
T5	♀	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)
PMT	♀	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)
MT	♀	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)
VT	♀	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 parraoleae sp. nov. could also resemble I. niger by having protuberant eyes, long legs, hands, feet, and dark body coloration. However, Pseudoeurycea parraoleae sp. nov. has black pigmentation in the subocular groove while in I. niger it is unpigmented, a proportionally shorter tail in Pseudoeurycea parraoleae sp. 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 parraoleae sp. 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 parraoleae sp. 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.

Figure 4.

Live specimens of Pseudoeurycea parraoleae sp. 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.

Figure 5.

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

Distribution and natural history.

Pseudoeurycea parraoleae sp. 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 parraoleae sp. 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 parraoleae sp. 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.

Pseudoeurycea natsii Aguilar-Herrera, Flores-Martínez, Seba-Chacha & Jiménez-Arcos, sp. nov.

https://zoobank.org/336D3CDF-46DC-40C9-AFCE-EF4B7FB0A146

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 natsii sp. 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 natsii sp. 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 natsii sp. 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 natsii sp. 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. (SVL Pseudoeurycea natsii sp. 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 natsii sp. 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 natsii sp. 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 natsii sp. nov.); and Pseudoeurycea parraoleae sp. nov., described herein, has a uniform black to dark brown dorsum.

Figure 6.

Live specimens of Pseudoeurycea natsii sp. 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.

Pseudoeurycea natsii sp. 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 natsii sp. nov. have a greater number of premaxillary and maxillary teeth (sum of the PMT and MT from Table 5 for Pseudoeurycea natsii sp. nov. for comparison; PMT+MT males: Pseudoeurycea granitum 57–73, P. lynchi 43–92, Pseudoeurycea natsii sp. nov. 101, P. nigromaculata 62–91; females: Pseudoeurycea granitum 57–73, P. lynchi 58–96, Pseudoeurycea natsii sp. nov. 101, P. nigromaculata 58–96), longer fore and hind limbs in males (FLL P. granitum 8.3–11.9 mm, P. lynchi 9.1–13.1 mm, Pseudoeurycea natsii sp. nov. 18.2 mm, P. nigromaculata 9.9–12.7 mm; HLL P. granitum 9.3–12.0 mm, P. lynchi 11.9–14.9 mm, Pseudoeurycea natsii sp. nov. 18.6 mm, P. nigromaculata 11.8–13.9 mm) and in females only P. nigromaculata with longer fore limbs (FLL P. granitum 7.8–12.8 mm, P. lynchi 8.1–12.9, Pseudoeurycea natsii sp. 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).

Figure 7.

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

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 natsii sp. 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 natsii sp. 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 euguii sp. 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 parraoleae sp. nov. + (P. juarezi + P. saltator)) + P. aurantia) and the second clade comprises (((Pseudoeurycea sp. + Pseudoeurycea natsii sp. 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 parraoleae sp. 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 parraoleae sp. 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.

Figure 8.

Habitat at the type locality of A Pseudoeurycea euguii sp. nov., B Pseudoeurycea parraoleae sp. nov., and C Pseudoeurycea natsii sp. nov. Photos: Leopoldo D. Váquez-Reyes.

Pseudoeurycea natsii sp. 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 natsii sp. 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 parraoleae sp. nov. and Pseudoeurycea natsii sp. 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. euguii sp. 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.

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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 material

Supplementary material 1

Figures S1, S2

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)

Data type: .docx

Explanation notes: 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.

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.

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