Description of a species of the genus Paramesotriton (Caudata, Salamandridae) from Guizhou, China, based on morphological and genomic evidence

Description of a species of the genus Paramesotriton (Caudata, Salamandridae) from Guizhou, China, based on morphological and genomic evidence Tao Luo1,2,3*, Jia-Jia Wang2*, Mei Liao2, Ming-Yuan Xiao4, Huai-Qing Deng4, Ning Xiao5, Jiang Zhou2 1 School of Life Sciences, Yunnan University, Kunming, China 2 School of Karst Science, Guizhou Normal University, Guiyang, China 3 Southwest United Graduate School, Kunming, China 4 School of Life Sciences, Guizhou Normal University, Guiyang, China 5 Guiyang Healthcare Vocational University, Guiyang, China https://zoobank.org/8D40ED2F-A2AB-463F-97D3-C97884C8BE1E Corresponding author: Jiang Zhou ([email protected]) * These authors contributed equally to this paper. Academic editor: Umilaela Arifin ♦ Received 11 February 2025 ♦ Accepted 4 September 2025 ♦ Published 1 October 2025 Abstract Species identification and delimitation are critical for biodiversity conservation. Recent mitochondrial-based phylogenetic studies of the genus Paramesotriton, a group of small salamanders inhabiting mountain streams in southern China and northern Vietnam, suggest that its diversity may be underestimated. In this study, we confirm and characterize a new species, Paramesotriton wumengshanensis sp. nov., using morphological, mitochondrial NADH dehydrogenase subunit 2 (ND2), mitogenome, and genomic evidence. This new species can be distinguished from other Paramesotriton species by characteristics such as rough and large body warts, large eyes, the absence of vestigial gills and gill filaments in adults, and distinctive vomerine teeth. Statistical analysis of the morphological data further showed that the new species is significantly different from its close relatives. Phylogenetic reconstruction based on concatenated and coalescent approaches, using both mitochondrial DNA and nuclear single nucleotide polymorphisms (SNPs) generated by restriction site-associated DNA sequencing, revealed that the new species forms a distinct lineage within the genus Paramesotriton, exhibiting a minimum genetic distance of 0.63% in mitochondrial ND2 compared to congeners. The observed cytonuclear discordance and minor mitochondrial differences among species may stem from historical gene flow and/or incomplete lineage sorting, and future studies using genomic evidence are needed to investigate their respective contributions. Based on this discovery, we recommend that independent evidence, especially genomic evidence, be integrated into the classification of closely related species to avoid falling into the trap of mitochondrial introgression. Key Words Guizhou, morphology, mitochondrial introgression, phylogenomics Introduction The Asian newts of the genus Paramesotriton Chang, 1935 (Caudata, Salamandridae) are among the most species-diverse caudate amphibians in China, second only to the genus Tylototriton (AmphibiaChina 2025). Currently, 15 species have been recorded, which are divided into two species groups: the P. caudopunctatus group, which includes P. caudopunctatus (Liu & Hu, 1973), P. longliensis Li, Tian, Gu & Xiong, 2008, P. malipoensis Rao, Liu, Zhu Zoosyst. Evol. 101 (4) 2025, 1933–1947|DOI 10.3897/zse.101.149144 Copyright Luo, T. et al. 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. zse.pensoft.net Luo, T. et al.: Genus Paramesotriton1934 & Ma, 2022, P. maolanensis Gu, Chen, Tian, Li & Ran, 2012, P. wulingensis Wang, Tian & Gu, 2013, and P. zhijinensis Li, Tian & Gu, 2008, and the P. chinensis group, which includes P. aurantius Yuan, Wu, Zhou & Che, 2016, P. chinensis (Gray, 1859), P. deloustali (Bourret, 1934), P. fuzhongensis Wen, 1989, P. guangxiensis (Huang, Tang & Tang, 1983), P. hongkongensis (Myers & Leviton, 1962), P. labiatus (Unterstein, 1930), P. qixilingensis Yan, Zhao, Jiang, Hou, He, Murphy & Che, 2014, and P. yunwuensis Wu, Jiang & Hanken, 2010 (Fei et al. 2006; Fei and Ye 2016; Rao 2022). These species are primarily distributed in southern China and northern Vietnam (Fei et al. 2006; Fei and Ye 2016; AmphibiaChina 2025). The mitochondrial gene-based phylogeny identified two to three cryptic species within the P. caudopunctatus group (Luo et al. 2021; Luo et al. 2022). Luo et al. (2021) identified a Paramesotriton population in Dafang County, Guizhou Province, China, as a potential cryptic species, initially referred to as Paramesotriton sp2 (DF) and later as P. longliensis (DF) (Luo et al. 2022). However, this species was not formally described in either study. This cryptic species exhibits minimal mitochondrial variation (less than 2%) compared to currently recognized species, raising questions regarding its validity. This relatively small mitochondrial variation may be attributed to historical mitochondrial introgression. For instance, mitogenome introgression between Odorrana grahami and O. junlianensis in southern China led to minimal differences in mitochondrial cytochrome oxidase subunit I but significant nuclear gene divergence (Yuan et al. 2022). This highlights the importance of genotyping-by-sequencing to obtain thousands of nuclear single nucleotide polymorphisms (SNPs) for accurate species delimitation. Similarly, whole-genome resequencing has been employed to generate large SNP datasets, resolving the taxonomy of closely related species and identifying cryptic diversity (Hu et al. 2020; Yang et al. 2022; Gu et al. 2023). Thus, genome-level SNP data provided critical insights for resolving taxonomic uncertainties among closely related species, particularly those affected by mitochondrial introgression, and offered robust support for species delimitation. Although our previous work using mitochondrial ND2 and combining phylogenetic and species delimitation methods identified the cryptic species Paramesotriton sp2 (DF) or P. longliensis (DF) (Luo et al. 2021; Luo et al. 2022), the smaller mitochondrial differences with closely related species make their validity not strongly supported. An additional problem is that while whole-genome resequencing can yield a large number of SNPs, the P. caudopunctatus group currently has no close reference genome that can be used. Therefore, we performed restriction site-associated DNA sequencing (RADseq), a reduced-representation genome sequencing technique (Miller et al. 2007; Andrews et al. 2016) that is versatile and does not require a reference genome, on 46 samples, thereby obtaining a large amount of SNP data. In this study, our aim is to confirm the validity of this cryptic species and formally describe it in combination with morphology. Materials and methods Sampling and morphological analyses Among the 46 samples collected, nine were from P. caudopunctatus, 10 from P. longliensis, two from P. maolanensis, 10 from P. zhijinensis, eight from Paramesotriton sp2 (DF), and seven from the outgroup (Fig. 1, Suppl. material 1). Tissue samples and specimens of the remaining species were collected before 2018, except for Paramesotriton sp2 (DF), which was collected in July 2018. Muscle samples were collected from the tail tip and immediately preserved in 95% anhydrous ethanol, and specimens were fixed using 75% alcohol immersion and stored indoors in a refrigerator at −80 °C. The field survey followed the rules of the Wildlife Protection Law of the People’s Republic of China. All muscle samples and specimens were preserved in the Animal Ecology Laboratory, Guizhou Normal University, Guiyang, Guizhou Province, China. All animal experiments were approved by the Research Ethics Committee of Guizhou Normal University (approval number: 202504001). In this study, we measured 15 morphological characters of 29 specimens using digital calipers accurate to 0.1 mm (Gu et al. 2012; Suppl. material 1), including new species (four males and three females), P. longliensis (eight males and nine females), and P. zhijinensis (five females). The measured characters were as follows: TOL = total length (from tip of snout to tip of tail); SVL = snout– vent length (from tip of snout to posterior edge of vent); HL = head length (from posterior edge of left parotoid to snout tip); HW = head width (maximum head width); SL = snout length (from tip of snout to the anterior corner of eye); TKL = trunk length (distance between axillae along right body side); IOD = interorbital distance (minimum distance between the eyes); ED = eye diameter (from the anterior corner to the posterior corner of the eye); TAL = tail length (from anterior tip of cloaca to tip of tail); TH = tail height (maximum tail height); TW = tail width (maximum tail width); FLL = forelimb length (from the base of the forelimb to the tip of the longest finger); HLL = hindlimb length (from the base of the hindlimb to the tip of the longest toe); DAG = distance between axilla and groin (from the posterior margin of the base of the forelimb to the anterior margin of the base of the hindlimb); and SW = snout width (maximum snout width). Principal component analysis (PCA) with eigenvalues greater than 1, the maximum variance method, and simple bivariate scatter plots were used to explore and characterize the morphometric differences between the new species and closely related species. A canonical discriminant analysis (CDA) was employed to distinguish between the new species and closely related species, as well as to predict group membership, following methods used in previous studies (Parsons and Jones 2000; Polaszek et al. 2004; Xiong et al. 2015; Shen et al. 2020). To reduce the effect of allometry, each morphometric measurement was corrected by dividing it by the TOL before conducting the Zoosyst. Evol. 101 (4) 2025, 1933–1947 zse.pensoft.net 1935 PCA and CDA analyses. One-way analysis of variance (ANOVA) was conducted to determine the significance of differences in morphometric characters between the new species and the aforementioned closely related species. All statistical analyses were performed using SPSS v.21.0 (SPSS, Inc., Chicago, IL, USA), with differences considered statistically significant at p < 0.05. Morphological comparisons of the new species with congeners were based on specimen examination and literature. Sex identification was performed based on Fei et al. (2006) and Fei and Ye (2016)—specifically, males possess a large and low cloaca, with a relatively long longitudinal anal fissure and finger-like papillae on the cloacal wall. In contrast, females have a small and high cloaca, a short oval-shaped anal fissure, and lack finger-like papillae on the cloacal wall. Laboratory protocols, sequencing, and bioinformatic methods The genomic DNA of 46 samples was extracted from 95% ethanol-preserved tissue using a standard CTAB protocol (Hanania et al. 2004). Genomic DNA from each sample was digested using the restriction enzyme EcoRI (GAATTC) following previously published procedures. DNA libraries for each sample were constructed according to the RAD protocol (Baird et al. 2008). PCR products were purified to recover 350–550 bp DNA, and pairedend sequencing was conducted on the MGISEQ-2000. Raw reads were deposited in the NCBI Sequence Read Archive database (https://www.ncbi.nlm.nih.gov/sra) under the BioProject accession number PRJNA1016311. Raw reads for samples were filtered using fastp v.0.23.4 (Chen 2023) with default parameters. Next, we performed further quality control on the cleaned reads using STACKS v.2.61 (Rochette et al. 2019). The process_radtags module was used to clean the tags by discarding low-quality reads, removing reads with uncalled bases, and rescuing barcodes and radtags. Additionally, we used the clone_filter module in STACKS to identify and remove PCR duplicates from the RADseq data. Single nucleotide polymorphism (SNP) calling for each sample was performed using the denovo_map.pl script in STACKS. We used the ustacks, cstacks, sstacks, tsv2bam, and populations modules from the STACKS pipeline to complete the de novo assembly of cleaned reads, generate consensus sequences, and perform SNP calling for each sample. To determine the optimal assembly parameters, we followed previous methods to evaluate two key parameters (Paris et al. 2017; Gundappa et al. 2022): (−M), the maximum number of nucleotide mismatches allowed between stacks, and (−m), the minimum depth of coverage required to create a stack. For our dataset, M ranged from 3 to 8 (in intervals of 1), while m ranged from 4 to 14 (values: 4, 6, 8, 9, 10, 12, 14). For the parameter n Figure 1. Distribution and sampling of the P. caudopunctatus group and outgroup Pachytriton inexpectatus in Guizhou, China. zse.pensoft.net Luo, T. et al.: Genus Paramesotriton1936 (the number of mismatches allowed between sample loci when building the catalog) in the cstacks module, we followed the recommendation of Paris et al. (2017) and set n = M. Based on the r80 rule (Paris et al. 2017), the final optimal parameter combination was M = 6, m = 10, and n = 6. In total, the STACKS program identified 15,581,665 biallelic SNPs across 46 individuals. In this study, we genotyped and filtered the samples to obtain reliable SNPs using the following process: (1) initial genotype filtering was performed based on genotype missing rate and minimum allele frequency using VCFtools v.0.1.16 (Danecek et al. 2011) with the following running parameters: ---maf 0.01 ---max-missing 0.5 ---min-alleles 2 --recode --recode-INFO-all –out. (2) PLINK v.1.90 (Purcell et al. 2007) was then used to filter out samples with a missing genotype rate greater than 50%. (3) Missing genotypes were imputed using Beagle (Browning and Browning 2016). (4) Finally, the SNPs generated in the previous step were further filtered using the R package bigsnpr v.1.9.11 (Privé et al. 2018) based on linkage disequilibrium-based SNP clumping. In this study, only one SNP per locus was retained in the final SNP dataset. Phylogenetic reconstruction and genetic differentiation based on SNP data We used IQ-TREE v.1.6.12 (Nguyen et al. 2015) to reconstruct phylogenetic trees based on maximum likelihood for concatenated sequences from all loci to infer phylogenomic relationships. The analysis was performed with the following parameters: −s −keep-ident −st DNA −nt −m GTR+ASC −bb 1000 −pre. Node support values were assessed using the ultrafast bootstrap approximation (UFBoot) method (Hoang et al. 2018). Species tree inference was performed using SVDquartets (Chifman and Kubatko 2014) implemented in PAUP* 4.0a (Wilgenbusch and Swofford 2003). SVDquartets estimates relationships between taxa under the coalescent model by inferring splits among quartets of randomly sampled taxa. All possible taxon quartets were evaluated, and node support was estimated using 100 standard bootstrap replicates. In cases where node support was low, the SVDquartets analysis helped identify potential incomplete lineage sorting or introgression among species. Both analyses used 1,475,246 unlinked SNPs. The fixation index (Fst) between the new species and its closely related species was assessed using VCFtools v.0.1.16 (Danecek et al. 2011) to quantify the degree of genetic variation among different species. Phylogenetic reconstruction, genetic distance, and haplotype network based on mitochondrial sequences The 48 mitochondrial ND2 sequences and 19 mitochondrial genomes used for phylogenetic reconstruction were primarily sourced from our previously published data (Table 1) (Luo et al. 2021; Luo et al. 2022). Multiple sequence alignment was performed using MUSCLE (Edgar 2004) within MEGA v.7.0 (Kumar et al. 2016), with default settings. Based on the Bayesian information criterion, we evaluated the best-fit partitioning schemes and corresponding nucleotide substitution models for ND2 and mitochondrial genomes using PartitionFinder v.2.1.1 (Lanfear et al. 2017). Phylogenetic trees were constructed using both maximum likelihood (ML) and Bayesian inference (BI) methods. ML analysis was run in IQ-TREE v.1.6.1 (Nguyen et al. 2015) with the best model and 20,000 ultrafast bootstrap (UBP) replicates. The BI tree was reconstructed using MrBayes v.3.2.1 (Ronquist et al. 2012). Two independent runs were conducted in the BI analysis, each of which was performed for 5 million generations and sampled every 1000 generations. The first 25% of the samples were discarded as a burn-in. Nodes were considered well supported when the Bayesian posterior probability (BPP) was greater than 0.95 and the ML ultrafast bootstrap value (UBP) was greater than 95%. Uncorrected p-distances (1000 replicates) based on the ND2 gene were calculated using MEGA v.7.0 (Kumar et al. 2016). Additionally, we reconstructed the mitochondrial ND2 gene haplotype network using PopART v.1.7 (Leigh et al. 2015) based on the median-joining method (Bandelt et al. 1999). Data availability statement The datasets containing the accession numbers for the P. caudopunctatus group generated in this study are available as supplemental information. Original sequence reads of genome data are available at the National Center for Biotechnology Information (accession numbers SAMN37379588–SAMN37379633). Results Morphological differences based on measured data We measured 15 morphological characters in 29 specimens (Suppl. material 1) and performed PCA, CDA, and ANOVA analyses to assess differences between P. wumengshanensis sp. nov. and closely related species. For males and females, three and three principal components were extracted, respectively. The first two components accounted for 72.87% and 73.84% of the total variation (Suppl. material 2). In the PC1 versus PC2 scatterplot, P. wumengshanensis sp. nov., P. longliensis, and P. zhijinensis formed nearly distinct clusters, with slight separation along the PC1 axis (Fig. 2A, B). For males, traits that loaded on the PC1 axis included total length, snout–vent length, head length, head width, eye diameter, tail width, forelimb length, and distance between axilla and groin (Suppl. material 2). For females, traits Zoosyst. Evol. 101 (4) 2025, 1933–1947 zse.pensoft.net 1937 that loaded on the PC1 axis included total length, snout– vent length, snout length, trunk length, interorbital distance, tail length, forelimb length, hindlimb length, and distance between axilla and groin (Suppl. material 2). The CDA based on the 15 morphological traits clearly classified all individuals into specific taxa, with CAN1 and CAN2 explaining 87.2% and 12.8% of the total variance, respectively. Distinct clusters were formed along the CAN1 axis (Fig. 2C; Suppl. material 2). Univariate ANOVA revealed significant morphological differentiation between the new species and its congeners (Table 2). Among males, P. wumengshanensis sp. nov. and P. longliensis showed statistically significant differences (p < 0.05) in snout length, tail width, and snout width (Table 2). In contrast, P. wumengshanensis sp. nov. and P. zhijinensis exhibited significant morphological divergence across nine measured traits: total length, trunk length, interorbital distance, tail length, tail height, tail width, forelimb length, hindlimb length, and snout width (p < 0.05). Among females, P. wumengshanensis sp. nov. and P. longliensis showed significant differentiation in eight morphological characters (Table 2). Table 1. Localities, voucher information, and GenBank numbers for all samples used. For the voucher number and locality of the mitogenome, see Luo et al. (2022b). ID Species Voucher number Locality (* type localities) ND2 Mitogenome 1P. longliensis GZNU 20070421001 Longli, Guizhou, China* FJ169608 ON357921 2P. longliensis KIZ-GZH 081025 Longli, Guizhou, China* GU980576 3P. longliensis GZNU 20070421004 Longli, Guizhou, China* JF438975 4P. longliensis GZNU 20070421003 Longli, Guizhou, China* JF438974 5P. longliensis GZNU 20070421002 Longli, Guizhou, China* JF438973 6P. longliensis GZNU 2018061813 Huishui, Guizhou, China MW147285 ON357922 7P. longliensis GZNU 2018061814 Huishui, Guizhou, China MW147283 MW524141 8P. longliensis GZNU 20180618016 Huishui, Guizhou, China MW147286 9P. longliensis GZNU 20180618017 Huishui, Guizhou, China MW147284 10 P. maolanensis GZNU 2006030003 Libo, Guizhou, China* FJ169607 ON357924 11 P. maolanensis GZNU 2006030006 Libo, Guizhou, China* JF438972 12 P. maolanensis GZNU 2006030004 Libo, Guizhou, China* JF438993 13 P. maolanensis GZNU 2006030005 Libo, Guizhou, China* JF438994 14 P. wumengshanensis sp. nov. GZNU 20180709024 Dafang, Guizhou, China* MT811029 MW524142 15 P. wumengshanensis sp. nov. GZNU 2018070904 Dafang, Guizhou, China* MT811030 ON357926 16 P. wumengshanensis sp. nov. GZNU 2018070905 Dafang, Guizhou, China* MT811031 17 P. wumengshanensis sp. nov. GZNU 2018070903 Dafang, Guizhou, China* MT811028 18 P. zhijinensis GZNU 20070415002 Zhijin, Guizhou, China* JF438976 ON357925 19 P. zhijinensis GZNU 20070415003 Zhijin, Guizhou, China* JF438977 20 P. zhijinensis GZNU 20070415004 Zhijin, Guizhou, China* JF438978 21 P. zhijinensis GZNU 20070415001 Zhijin, Guizhou, China* FJ169609 22 P. zhijinensis KIZ-GZH 081026 Zhijin, Guizhou, China* GU980575 23 P. wulingensis GZNU 07072001 Jiangkou, Guizhou, China FJ938040 ON357928 24 P. wulingensis GZNU 2007071002 Jiangkou, Guizhou, China JF438987 25 P. wulingensis GZNU 2007071004 Jiangkou, Guizhou, China JF438989 26 P. wulingensis GZNU 08072603 Youyang, Chongqing, China* JF438991 27 P. wulingensis GZNU 08072602 Youyang, Chongqing, China* JF438990 28 P. wulingensis GZNU 2007071003 Youyang, Chongqing, China* JF438988 29 P. wulingensis GZNU 08072604 Youyang, Chongqing, China* JF438992 30 P. wulingensis KIZ 21898 Fanjingshan, Guizhou, China KJ650055 31 P. wulingensis KIZ 21899 Fanjingshan, Guizhou, China KJ650056 32 P. caudopunctatus GZNU 2007071001 Leishan, Guizhou, China* FJ169606 ON357930 33 P. caudopunctatus GZNU 200904251 Leishan, Guizhou, China* MW147288 34 P. caudopunctatus GZNU 200904252 Leishan, Guizhou, China* MW147287 35 P. caudopunctatus GZNU 2007072005 Leishan, Guizhou, China* JF438986 36 P. caudopunctatus GZNU 2009042501 Leishan, Guizhou, China* JF438983 37 P. caudopunctatus GZNU 20050727001 Leishan, Guizhou, China* JF438985 38 P. caudopunctatus GZNU 20050727002 Leishan, Guizhou, China* JF438984 39 P. deloustali Tissue ID: GZNU35 Hekou County, Yunnan, China ON357931 ON357931 40 P. guangxiensis Tissue ID: GZNU36 Shiwandashan, Guangxi, China* ON357932 ON357932 41 P. yunwuensis GZNU20201018003 Xinyi City, Guangdong, China* ON357933 ON357933 42 P. labiatus GZNU20210531001 Jinxiu County, Guangxi, China* ON357934 ON357934 43 P. fuzhongensis GZNU20070520002 Zhongshan, Guangxi, China* ON357935 ON357935 44 P. hongkongensis Tissue ID: GZNU37 Hong Kong, China* ON357937 ON357937 45 P. qixilingensis Tissue ID: GZNU38 Yongxin County, Jiangxi, China* ON357938 ON357938 46 P. aurantius GZNU20201018001 Jingning County, Zhejiang, China* ON357939 ON357939 47 P. chinensis Tissue ID: GZNU39 Ningbo City, Zhejiang, China* ON357940 ON357940 48 Pachytriton inexpectatus GZNU20180706001 Danzhai County, Guizhou, China ON422325 ON422325 zse.pensoft.net Luo, T. et al.: Genus Paramesotriton1938 Genetic data information In this study, the full length of the mitochondrial ND2 used for phylogenetic reconstruction was 1407 base pairs (bp), of which 409 were variable and 277 were parsimony-informative. The model selection analysis suggested a division into three partitions, i.e., the first codon, the second codon, and the third codon of ND2, corresponding to the models HKY+G, HKY+I, and HKY+I. For the mitochondrial genome of 15,467 bp, the following evolutionary models were identified as optimal for the corresponding gene segments: TVM+I+G for 12S rRNA and 16S rRNA, HKY+I+G for tRNAs, GTR+G for protein-coding genes (COX2, COX1, COX3, Cyt b, ATP8, ND2, ND3, ND4, ATP6, ND1, ND5, and ND4L), and HKY+G for ND6. A total of 46 samples generated approximately 2948.5 Gb of raw data and 1004 million paired-end reads, with an average of 64.09 Gb of data and 21.8 million paired-end reads per sample. A total of 15,581,665 biallelic SNPs were generated from the STACKS-cleaned reads. After filtering for low-quality individuals using PLINK, 1,475,250 SNPs remained. Table 2. Morphometric statistics and results of analysis of variance from P. wumengshanensis sp. nov. (PW), P. longliensis (PL), and P. zhijinensis (PZ). All units are in mm. P-values are at 95% significance. Measurements P. wumengshanensis sp. nov. P. longliensis P. zhijinensis P-value from ANOVA Male (n = 4) Female (n = 3) Male (n = 8) Female (n = 9) Male (n = 5) Male Female Range (Mean ± SD) Range (Mean ± SD) Range (Mean ± SD) Range (Mean ± SD) Range (Mean ± SD) PW vs. PL PW vs. PZ PW vs. PL TOL 95.5–123.6 (110.2 ± 13.6) 150.1–160.0 (155.8 ± 5.1) 97.2–143.6 (117.0 ± 15.5) 114.6–136.3 (126.2 ± 8.4) 128.1–142.4 (136.7 ± 5.4) 0.610 0.014 0.013 SVL 53.0–72.6 (63.4 ± 8.9) 82.3–87.8 (85.6 ± 2.9) 47.7–78.3 (60.7 ± 9.9) 65.4–91.8 (71.5 ± 8.3) 63.7–68.3 (66.3 ± 1.8) 0.610 1.000 0.052 HL 16.5–23.9 (20.8 ± 3.3) 23.5–25.2 (24.6 ± 0.9) 16.3–22.1 (19.4 ± 1.8) 18.5–43.1 (22.7 ± 7.7) 20.9–23.9 (22.3 ± 1.1) 0.308 0.806 0.052 HW 11.8–18.9 (16.0 ± 3.3) 18.3–19.2 (18.7 ± 0.5) 12.5–16.6 (13.9 ± 1.5) 13.2–37.5 (17.8 ± 7.6) 15.1–17.9 (17.1 ± 1.1) 0.308 0.902 0.052 SL 6.3–8.6 (7.1 ± 1.1) 7.1–8.3 (7.9 ± 0.7) 5.7–7.6 (6.2 ± 0.6) 5.8–7.3 (6.5 ± 0.5) 7.0–8.5 (7.6 ± 0.6) 0.042 0.327 0.033 TKL 37.5–52.5 (45.6 ± 6.5) 61.0–66.5 (63.6 ± 2.7) 36.2–57.9 (46.7 ± 6.7) 46.9–59.8 (51.6 ± 4.3) 52.2–56.8 (54.6 ± 1.7) 0.865 0.027 0.013 IOD 4.5–6.8 (5.8 ± 1.1) 6.6–7.7 (7.3 ± 0.6) 4.5–7.7 (6.5 ± 0.9) 3.6–5.3 (4.4 ± 0.6) 8.3–9.5 (8.7 ± 0.6) 0.308 0.014 0.013 ED 2.9–3.9 (3.4 ± 0.5) 3.6–4.8 (4.1 ± 0.6) 2.3–3.8 (2.9 ± 0.5) 2.9–4.5 (3.6 ± 0.5) 2.7–3.6 (2.9 ± 0.4) 0.126 0.050 0.116 TL 40.8–55.1 (48.6 ± 6.1) 66.6–74.4 (70.0 ± 4.0) 45.8–63.8 (55.8 ± 6.1) 48.3–60.2 (54.7 ± 4.4) 63.8–71.9 (69.0 ± 3.5) 0.089 0.014 0.012 TH 6.3–9.9 (7.6 ± 1.6) 6.6–11.2 (9.1 ± 2.3) 6.7–10.0 (8.1 ± 1.0) 7.6–11.2 (9.0 ± 1.4) 8.4–20.0 (12.5 ± 4.5) 0.234 0.027 0.926 TW 2.4–3.3 (2.9 ± 0.4) 3.5–5.8 (4.7 ± 1.1) 3.4–5.1 (4.3 ± 0.5) 3.5–4.4 (3.9 ± 0.3) 4.5–7.3 (5.8 ± 1.1) 0.007 0.014 0.309 FLL 13.9–24.5 (18.4 ± 5.1) 26.0–28.2 (27.2 ± 1.1) 17.5–23.0 (20.4 ± 1.8) 19.4–23.6 (20.8 ± 1.3) 22.7–31.5 (27.5 ± 3.2) 0.497 0.027 0.013 HLL 14.8–26.2 (21.1 ± 4.8) 26.2–31.0 (28.0 ± 2.6) 17.5–27.1 (21.1 ± 2.9) 21.5–24.9 (23.2 ± 1.3) 24.2–30.9 (27.4 ± 2.4) 0.734 0.027 0.013 DAG 23.1–35.4 (29.9 ± 5.2) 41.3–41.9 (41.6 ± 0.3) 23.2–38.2 (28.7 ± 4.9) 23.0–40.5 (31.6 ± 5.7) 30.1–35.2 (32.2 ± 2.1) 0.865 0.462 0.013 SW 4.4–4.7 (4.6 ± 0.2) 4.1–5.8 (4.9 ± 0.9) 4.4–7.3 (5.5 ± 0.8) 4.1–5.8 (4.8 ± 0.7) 5.5–6.1 (5.8 ± 0.2) 0.027 0.014 0.926 Figure 2. Plots of principal component analysis (A, B) and canonical discriminant analysis (C) scores of P. wumengshanensis sp. nov., P. longliensis, and P. zhijinensis based on morphological characters. A. Male; B. Female; C. Male and female. Zoosyst. Evol. 101 (4) 2025, 1933–1947 zse.pensoft.net 1939 Phylogenetic reconstruction, genetic distance, and haplotype Phylogenetic reconstruction based on the mitochondrial ND2 gene and mitogenome revealed that Paramesotriton can be divided into Clade I and Clade II (Fig. 3A, B). Clade I corresponds to the P. chinensis group, with a large genetic divergence between the species, and the interspecific relationships are initially resolved. Clade II corresponds to the P. caudopunctatus group, which exhibits relatively low mitochondrial divergence yet encompasses six distinct evolutionary lineages (Fig. 3A). The four samples from Dafang, Guizhou, form a distinct evolutionary lineage within Clade II, which is close to P. longliensis and P. maolanensis (BPP/UFB = 0.84/72; Fig. 3A). However, the phylogenetic tree reconstructed from the mitogenome showed that the two Dafang samples from Guizhou clustered close to P. longliensis with low support (BPP/UFB = 0.47/42; Fig. 3B). The new species had small mitochondrial genetic distances from relatives, ranging from 0.63 to 9.39% (Table 3). The haplotype network based on ND2 showed no shared haplotypes within the P. caudopunctatus group, with the new species forming a unique haplotype (Fig. 3C). Using both concatenation and coalescent-based approaches, we reconstructed maximum-likelihood (ML) (Fig. 4A) and species trees (Fig. 4B) based on SNP data through IQ-TREE and SVDquartets analyses, respectively. Both analyses strongly supported the samples from Dafang, Guizhou, as a distinct evolutionary lineage. However, we observed phylogenetic discordance between the ML and species tree topologies regarding the placement of the new species and P. zhijinensis, suggesting potential influences of incomplete lineage sorting and gene flow events on phylogenetic reconstruction. The new species exhibits moderate genetic differentiation Table 3. Uncorrected p-distance (%) between six species of the P. caudopunctatus group based on mitochondrial ND2. ID Species 1 2 3 4 5 1P. wumengshanensis sp. nov. 2P. caudopunctatus 9.39 3P. longliensis 1.03 9.68 4P. maolanensis 0.63 9.49 0.61 5P. wulingensis 9.39 2.70 9.69 9.50 6P. zhijinensis 1.84 9.64 1.82 1.42 9.65 Figure 3. Reconstructed phylogeny and haplotype network. A. Phylogenetic tree reconstructed based on the mitochondrial ND2 gene; B. Phylogenetic tree reconstructed based on the mitochondrial genome. In phylogenetic trees, Bayesian posterior probabilities (BPP) from BI analyses/ultra-fast bootstrap supports (UFB) from ML analyses were noted beside nodes. The scale bar represents 0.02 nucleotide substitutions per site; C. ND2-based mitochondrial haplotype network. 0.02 Paramesotriton qixilingensis Paramesotriton yunwuensis Paramesotriton hongkongensis Paramesotriton aurantius Paramesotriton guanxiensis Paramesotriton chinensis Pachytriton inexpectatus Paramesotriton deloustali Paramesotriton labiatus Paramesotriton fuzhongensis 1.00/99 100/1.00 1.00/94 0.71/65 0.99/89 0.52/71 0.97/96 1.00/100 1.00/100 1.00/100 1.00/100 0.41/77 1.00/98 0.84/72 P. longliensis Paramesotriton maolanensis Paramesotriton wumengshanensis sp. nov. Paramesotriton zhijinensis Paramesotriton wulingensis Paramesotriton caudopunctatus Paramesotriton Outgroup BPP/UFB A I II P. caudopunctatus P. wulingensis P. wumengshanensis sp. nov. P. maolanensis P. longliensis P. zhijinensis 1 6 C B Paramesotriton Outgroup I II 0.02 Paramesotriton labiatus Pachytriton inexpectatus Paramesotriton hongkongensis Paramesotriton deloustali Paramesotriton fuzhongensis Paramesotriton qixilingensis Paramesotriton guanxiensis Paramesotriton yunwuensis Paramesotriton caudopunctatus Paramesotriton chinensis Paramesotriton zhijinensis Paramesotriton maolanensis Paramesotriton wulingensis Paramesotriton aurantius 0.47/42 1.00/100 1.00/100 1.00/100 1.00/98 0.65/46 1.00/100 1.00/100 1.00/100 1.00/74 1.00/100 1.00/100 1.00/98 Paramesotriton longliensis Paramesotriton wumengshanensis sp. nov. BPP/UFB 0.99/99 1.00/99 1.00/100 1.00/100 zse.pensoft.net Luo, T. et al.: Genus Paramesotriton1940 from its closely related congeners, with Fst values ranging from 0.16 to 0.19 (Fig. 4C), suggesting partial but significant species divergence. Based on morphological, mitochondrial, and nuclear genetic differences, there is clear support that the Paramesotriton geographic population from Dafang, Guizhou, China, should be treated as a new species. Taxonomic account Paramesotriton wumengshanensis Luo & Zhou, sp. nov. https://zoobank.org/72A9ACFC-B93C-4727-BA3F-ACA804B7B041 Figs 5, 6, Suppl. material 1 Chresonymy. Paramesotriton sp2 (DF): Luo et al. 2021 (Dafang, Guizhou, China); Paramesotriton longliensis (DF): Luo et al. 2022 (Dafang County, Guizhou, China). Holotype. Adult male, GZNU20180711, collected by Tao Luo on 11 July 2018, in Yuchong Township, Dafang County, Guizhou Province, China, 27.44933332°N, 105.95403671°E, elevation 1137 m a.s.l. Paratypes. Three adult males: GZNU2018070903, GZNU2018070904, and GZNU2018070905. Three adult females: GZNU2018070901, GZNU2018070902, and GZNU20180709011. The locality and date of all paratypes are same as holotype. Etymology. The specific epithet wumengshanensis refers to the type locality, located within the Wumeng Mountains, Guizhou, China. The suggested English name is the Wumeng Mountains Warty Newt, and the Chinese name is Wū Mēng Shān Luǒ Yuán (乌蒙山瘰螈). Diagnosis. (1) large body size, TOL 95.5–123.6 mm in adult males, TOL 150.1–160.0 mm in adult females; (2) absence of distinct colored stripes on dorsolateral ridges, consistent with body coloration; (3) absence of vestigial gills and gill filaments; (4) external eyes normal, eye diameter 14–20% of head length; (5) absence of colored spots on the tails of males, a single red-orange stripe from the posterior margin of the cloaca to the 4/5 parts of tail on ventral side; (6) rough skin, body warts rough and large, and longitudinal grooves distinct on the body flanks; (7) yellow spot below the posterior margin of the eye absent; (8) vomerine teeth distinct, middle part depressed outward, having a slightly wider and shallow groove at middle base of each choana, and a distinctly prominent bony plate is present posterior to the choana; (9) anterior end of the epibranchial bone of the hyoid apparatus is near T-shaped. Description of the holotype. Adult male, TOL 123.6 mm, SVL 72.6 mm. Measurements are presented in Suppl. material 1. Body relatively slender. Head flat, distinctly longer than wide (length/width = 1.4), distinct scent glands on side of head; knob-like (Fig. 6D), with a fleshy protuberance present in the branchial region. Snout short; snout arris evident, truncated, extending beyond lower lip; snout length distinctly longer than eye diameter; nostrils short, close to snout tip; eyes large; labial fold developed, extending from lower eye to tip of snout; gular fold developed; glandular ridge on each side of the head, slightly developed; tongue short, elliptical, both lateral sides dissociated; one premaxilla with tiny teeth on the upper and lower jaws. Vomerine teeth distinct, Ʌ-shaped, middle part depressed outward; dentition slowly compressed from posterior to anterior-to-anterior margin 0.02 D5 A8 A5 Y2 C2 C9 H9 A6 C4 A2 H3 Y1 H10 C10 Y5 A7 A4 H7 C6 C5 C8 D2 A1 Y7 D3 A3 H6 H5 H2 D6 Y4 A10 Y6 H4 D1 D4 A9 Y3 C7 D7 C1 H8 H1 M1 M2 C3 100 100 100 100 100 100 100 100 98 100 99 100 100 97 100 98 100 100 100 96 100 100 100 98 100 P. longliensis P. caudopunctatus UFBoot = 100 95≤UFBoot<100 UFBoot<95 P. maolanensis P. zhijinensis P. wumengshanensis sp. nov. 0.6 100 100 100 Outgroup Clade I Clade II A B PLPC PW PZ PM 0.16 0.17 0.19 0.16 C Pachytriton inexpectatus Figure 4. Phylogeny and interspecific differentiation. Maximum likelihood tree reconstructed (A) and species tree generated from SVDquartets (B) analysis based on 1,475,246 SNPs. Support values are displayed on nodes, and dashed lines indicate topology-conflicting species. Tip labels: branch tip numbers are sample numbers for the corresponding species. Note: Sample C1 represents the new species but retains its original label due to initial misidentification and for consistency with NCBI records. (C) Species differentiation between new species and other species. The numbers above and below the dashed line represent Fst values. Abbreviation: PC = P. caudopunctatus, photo modified from Fei et al. (2006); PW = P. wumengshanensis sp. nov.; PZ = P. zhijinensis; PL = P. longliensis; PM = P. maolanensis, photo modified from Gu et al. (2012). Zoosyst. Evol. 101 (4) 2025, 1933–1947 zse.pensoft.net 1941 meeting between two nares; having a slightly wider and shallow groove at middle base of each choana; a distinctly prominent bony plate is present posterior to the choana (Fig. 7A). Anterior end of the epibranchial bone of the hyoid apparatus is near T-shaped, connected with the first and second ceratobranchials of the bone; ceratohyal knife-like, with the anterior two-thirds in cartilage, left and right unconnected (Fig. 7D). Vertebral ridge developed, light tan, conspicuous, unintermittent, from occiput to tail. Forelimbs short, reaching to tip of snout when adpressed forward; hind limbs longer and sturdier than forelimbs; palm and sole overlap when adpressed forelimb backward and hind limb forward along body flank; without metacarpal or metatarsal tubercles; four fingers and five toes without webbing or fringe; relative finger length 1 < 4 < 2 < 3; relative toe length 1 < 5 < 2 < 4 < 3. Tail relatively long; dorsal and ventral caudal fins evident, expanding on posterior half of tail; tail-tip rounded. Cloaca a large slit, swollen into a small mound, villous mastoid around cloaca, many pubes on gaps of cloaca. Skin rough, especially on the back of the body; many warts and granular glands on dorsal skin, dorsal surface of the limbs, and lateral sides of tail. Longitudinal flank grooves distinct on the body flanks, approximately 17 (Fig. 6E). Ventral skin wrinkled, densely covered with small warts (Fig. 6C, H). Sexual dimorphism. Apophysis of cloaca of the male large and low, and the counterpart of the female small and high (Fig. 5C, F). Female cloaca small and oval without gland in inside and lack finger-like papillae on the cloacal wall. Coloration. In life, dorsal surface of body blackbrown and light olive color. Head, body, and ventral surfaces of limbs dark brown overall with irregular reddish-orange markings. Absence of distinct colored stripes on dorsolateral ridges, consistent with body coloration. A single red-orange or tangerine yellow stripe extends from the posterior margin of cloaca to the 4/5 parts of the tail on the ventral side (Fig. 6H). Finger and toe tips grayish brown. Color lightens in 75% ethanol preservation. Comparison. The new species can be assigned to the P. caudopunctatus group on the basis of phylogeny and can be distinguished from other species within that group by the following morphological characteristics (Table 4). Paramesotriton wumengshanensis sp. nov. differs from P. caudopunctatus and P. wulingensis by absence of colored spots on the tails of males and colored stripes on dorsolateral ridges (vs. presence of purplish red spots on the tails of males and three yellowish brown longitudinal stripes very distinct on dorsolateral ridges). Paramesotriton wumengshanensis sp. nov. differs from P. malipoensis by absence of vestigial gills and gill filaments in adults (vs. presence), snout-vent length greater than tail length (vs. almost equal), and absence of distinct colored stripes on dorsolateral ridges, consistent with body coloration (vs. dorsolateral ridges scattered with distinctive orange-red or brownish-yellow spots). Paramesotriton wumengshanensis sp. nov. differs from P. zhijinensis by absence of distinct colored stripes on dorsolateral ridges, consistent with body coloration (vs. two or three near-continuous yellow stripes on the dorsolateral ridge), yellow spot below the posterior margin of the eye absent (vs. present), absence of vestigial gills and gill filaments in adults (vs. neoteny is common with most adult specimens having vestigial gills and gill filaments), vomerine teeth distinct, middle protruding outward, dentition slowly compressed from posterior to anterior to anterior margin meeting between two nares, having a slightly wider and shallow groove at middle base Figure 5. Holotype GZNU20180711 (A–C) and female paratype GZNU20180709011 (D–F) of Paramesotriton wumengshanensis sp. nov. in preservative. (A, D) Lateral view. (B, E) Dorsolateral view. (C, F) Ventral view.