﻿A new odorous frog species of Odorrana (Amphibia, Anura, Ranidae) from Guizhou Province, China

﻿Abstract The frog genus Odorrana is distributed across east and southeastern Asia. Based on morphological differences and molecular phylogenetics, a new species of the genus occurring from Leigong Mountain in Guizhou Province, China is described. Phylogenetic analyses based on DNA sequences of the mitochondrial 12S rRNA, 16S rRNA, and ND2 genes supported the new species as an independent lineage. The uncorrected genetic distances between the 12S rRNA, 16S rRNA, and ND2 genes between the new species and its closest congener were 5.0%, 4.9%, and 16.3%, respectively. The new species is distinguished from its congeners by a combination of the following characters: body size moderate (SVL 39.1–49.4 mm in males, 49.7 mm in female); head width larger than head length; tympanum distinctly visible; small rounded granules scattered all over dorsal body and limbs; dorsolateral folds absent; heels overlapping when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level between eye to nostril when leg stretched forward; vocal sacs absent in male and nuptial pads present on the base of finger I.


Introduction
The odorous frogs of the genus Odorrana Fei, Ye & Huang, 1990 inhabit mountain streams at elevations of about 200-2000 m and can also be found on rocks or branches near the riverbed, ranging from Japan, southern China and Indochina, northeastern India, Myanmar and Thai-Malay Peninsula, Java, Sumatra, and Borneo (Frost 2024).Phylogenetic studies indicate that Odorrana is monophyletic (Chen et al. 2013).The genus currently consists of 65 species (Frost 2024), of which 42 occur in China and 27 species are endemic to China (Fei et al. 2012;Amphibia China 2024;Frost 2024).
Systematic arrangements in this genus have been controversial.Ye and Fei (2001) suggested four species groups (O.andersonii, O. kuangwuensis, O. schmackeri, and O. livida species groups) based on a morphological study.Fei et al. (2005) established two subgenera (Odorrana Fei, Ye &Huang, 1990 andBamburana Fei, Ye, Huang, Jiang &Xie, 2005) within Odorrana.Molecular phylogenetic studies support the division of species groups within Odorrana but not the two subgenera (Che et al. 2007).Subsequently, Fei et al. (2009) divided the Chinese Odorrana species into six species groups (O. tormota, O. taiwaniana, O. graminea, O. margaretae, O. schmackeri, and O. andersonii species groups).These divisions have been accepted by some researchers (Pham et al. 2016a, b;Li et al. 2018a) but others have rejected the monophyly of the O. margaretae, O. schmackeri, and O. andersonii species groups (Chen et al. 2013).The species diversity in the genus is also indicated as underestimated in these phylogenetic frameworks.
Guizhou Province is one of the areas of the most abundant amphibians in China, and in the last five years a series of new frog species have been described from this region (Frost 2024;Amphibia China 2024).During fieldwork in Leigongshan Nature Reserve, Leishan County, Guizhou Province, China, between March to October 2023, seven Odorrana specimens were collected.Morphologically, these specimens most closely O. huanggangensis Chen, Zhou & Zheng, 2010, and O. wuchuanensis (Xu, 1983), but differs from these two species by the presence of small, rounded granules scattered all over the dorsal body and limbs, and the vocal sacs are absent in the male.To further distinguish these specimens, we conducted phylogenetic analyses based on mitochondrial DNA and morphological comparisons.All analyses consistently indicated that the specimens from Leigongshan Nature Reserve are a new taxon, described herein as a new species.
86 Sequences were assembled and aligned using the Clustalw module in BioEdit v. 7.0.9.0 (Hall 1999) with default settings.The datasets were checked by eye and revised manually if necessary.Based on the 12S rRNA, 16S rRNA, ND2, and 12S rRNA +16S rRNA + ND2 concatenated dataset, phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian inference (BI) methods, implemented in PhyML 3.0 (Guindon et al. 2010)   3.12 (Ronquist and Huelsenbeck 2003), respectively, and the best-fit model was obtained by the Bayesian inference criteria (BIC) computed with PartitionFinder 2 (Lanfear et al. 2012).The analysis suggested that the best partition scheme was 12S rRNA/16S rRNA/ND2 genes.We selected GTR+R as the best model for 12S rRNA and 16S rRNA and the TN93 + I + G as the best model for the ND2 gene.For ML analyses conducted in PhyML 3.0, the bootstrap consensus tree inferred from 1000 replicates was used to estimate nodal supports of inferred relationships on phylogenetic trees.For Bayesian analyses conducted in MrBayes 3.12, four Markov chains were run for 50 million generations with sampling every 1000 generations until the trees reach convergence (split frequency < 0.05).The first 25% of trees were removed as the "burn-in" stage followed by calculation of Bayesian posterior probabilities and the 50% majority-rule consensus of the post burn-in trees sampled at stationarity.Finally, uncorrected p-distances (1000 replicates) between species based on 12S rRNA (45 species), 16S rRNA (51 species), and ND2 (23 species) were calculated in MEGA 6.06 (Tamura et al. 2013).ED eye diameter (distance from the anterior corner to the posterior corner of the eye); FL foot length (distance from tarsus to the tip of fourth toe); HDL head length (distance from the tip of the snout to the articulation of jaw); HDW maximum head width (greatest width between the left and right articulations of jaw); HLL hindlimb length (maximum length from the vent to the distal tip of the Toe IV); IND internasal distance (minimum distance between the inner margins of the external nares); IOD interorbital distance (minimum distance between the inner edges of the upper eyelids); LAL length of lower arm and hand (distance from the elbow to the distal end of the Finger IV); ML manus length (distance from tip of third digit to proximal edge of inner palmar tubercle); NED nasal to eye distance (distance between the nasal and the anterior corner of the eye); NSD nasal to snout distance (distance between the nasal the posterior edge of the vent); LW lower arm width (maximum width of the lower arm); SVL snout-vent length (distance from the tip of the snout to the posterior edge of the vent); SL snout length (distance from the tip of the snout to the anterior corner of the eye); TFL length of foot and tarsus (distance from the tibiotarsal articulation to the distal end of the Toe IV); THL thigh length (distance from vent to knee); TL tibia length (distance from knee to tarsus); TW maximal tibia width; TYD maximal tympanum diameter; UEW upper eyelid width (greatest width of the upper eyelid margins measured perpendicular to the anterior-posterior axis).

Morphological comparisons
To reduce the impact of allometry, a size-corrected value from the ratio of each character to SVL was calculated for the following morphometric analyses.Principal component analysis (PCA) of size-corrected variables and simple bivariate scatterplots was used to explore and reflect the morphometric differences between the undescribed taxon and the phylogenetic relationships closely and sympatric species contains Odorrana hejiangensis, O. huanggangensis, O. ichangensis, O. kweichowensis, O. schmackeri, and O. wuchuanensis.One-way analysis of variance (ANOVA) was used to test the significance of differences on morphometric characters between the undescribed taxon and O. hejiangensis, O. huanggangensis, O. ichangensis, O. kweichowensis, O. schmackeri, and O. wuchuanensis in the males.The statistical analyses were performed using SPSS 21.0 (SPSS, Inc., Chicago.IL, USA), and differences were considered to be significant at p < 0.05.
Sex was determined by direct observation of calling behavior and the presence of internal vocal sac openings for males, as well as the presence of eggs on the abdomen for females.The presence or absence of nuptial pads/spines was examined by optical microscopy.
We compared the morphological characters of the undescribed taxon with other species of Odorrana.Comparative data were obtained from the literature for 65 species of Odorrana (all of the authorities of the 65 species were shown in Table 2).For comparison, we examined the type and/or topotype materials for O. hejiangensis, O. huanggangensis, O. ichangensis, O. kweichowensis, O. schmackeri, and O. wuchuanensis (Suppl.material 1).

Morphological analyses
The results of ANOVA indicated that in male, the undescribed taxon was significantly different from Odorrana hejiangensis, O. huanggangensis, O. ichangensis, O. kweichowensis, O. schmackeri, and O. wuchuanensis in many morphometric characters (all P values < 0.05; Table 3).In PCA for males, the total variation of the first two principal components was 43.3%, on the two-dimensional plots of PC1 vs PC2, the undescribed taxon could be separated from O. hejiangensis, O. huanggangensis, O. ichangensis, O. kweichowensis, O. schmackeri, and O. wuchuanensis (Fig. 3).Detailed morphological comparisons revealed discrete diagnostic characters between the undescribed taxon and its congeners.Therefore, adopt integrative taxonomy approaches with evidence from molecular and morphology to take the decision to describe the unidentified taxon as new species described herein.Diagnosis.Odorrana leishanensis sp.nov.can be distinguished from its congeners by the following characters: (1) body size moderate (SVL♂ (n = 6) = 39.1-49.4mm, SVL♀ (n = 1) = 49.7 mm in female); (2) head width larger than head length; (3) tympanum distinctly visible; (4) small rounded granules scattered all over dorsal body and limbs; (5) dorsolateral folds absent; (6) heels overlapping when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level between eye to nostril when leg stretched forward; (7) vocal sacs in male absent, and nuptial pads in male present on base of finger I.  4, 5).Adult male, body size moderate (SVL 49.4 mm); head width larger than head length (HDW/HDL = 1.14); snout short, rounded in dorsal view, projecting beyond lower jaw; eye large and convex, ED 0.73 SL; nostril rounded, closer to tip of snout than to eye; internasal distance larger than interorbital distance; tympanum distinct, approximately 0.68 ED; vomerine teeth on well-developed ridges; tongue deeply notched posteriorly; pupil horizontally oval; vocal sac absent.

Description of holotype (Figs
Forelimbs slender (LW/SVL = 0.09); lower arm and hand not reach one-second of body length (LAL/SVL = 0.42); fingers slender, relative finger lengths II < I < IV < III; finger tips on I-IV dilated to wide cordiform disks with circum-marginal grooves, without webbing and lateral fringes; subarticular tubercle prominent; supernumerary tubercle indistinct; inner metacarpal tubercle oval, elongate; outer metacarpal tubercles absent; light yellow glandular nuptial pad on finger I.
Hindlimbs long; tibio-tarsal articulation reaching between eye to nostril when hindlimb adpressed along the side of the body; heels overlapped; tibia longer than thigh length; toes slender, relative lengths I < II < III < V < IV; toes entirely webbed; tips of toes expanded into disc with circummarginal grooves; outer metatarsal tubercle absent; inner metatarsal tubercle present.
Dorsal rough, there are small, rounded granules scattered all over dorsal body and limbs, ventral surfaces of the head, body, and limbs smooth; weak supratympanic fold from the posterior edge of the eye to the posterior edge of the tympanum; dorsolateral folds absent.
Coloration of holotype in life (Fig. 4).Dorsum grass-green with a small amount of brown spots; flanks pale yellow with several black spots; dorsal surfaces of anterior forelimbs pale yellow, anterior forelimbs olive-brown, with black bands and irregular grass-green spots; dorsal surfaces of hindlimbs grass-green with black bands; upper jaw with a ring of brown spots; lower jaw yellow with black spots; grass-green and black spotted mosaic on the loreal region; tympanum brown-black; ventral surface of throat and chest brown, belly pale yellow.
Coloration of holotype in preservation (Fig. 5).After three months in 75% ethanol, the dorsal surface of the body faded to dark olive; the dorsal surface of the head changed to darker; the transverse bands on limbs and digits were not distinct; ventral surface of throat brown, gradually dark brown on chest, the belly was pale yellow; palm color faded to white.
Variation.Morphological measurements of all specimens are presented in Table 4 and Suppl.material 1.All specimens were very similar in morphology and color pattern, but in MT LS20230805001 the skin from the corner of the eye to the base of the thigh was noticeably pale brown with green patches mixed in and the flank of the ventral surface was white with dark brown spots (Fig. 6A,  B); in MT LS20230806010 the dorsum was green and the ventral surface of the throat and chest darker (Fig. 6C, D); in MT LS20230811024 the granulation on the dorsolateral surface was covered with black spots and the ventral surface of the throat and chest were white with darker spots (Fig. 6E, F).
Secondary sexual characters.Adult females slightly larger than adult males; adult males lack vocal sacs.During breeding season, pale yellow glandular nuptial pads in males present on finger I (Figs 4C, 5D).
Comparisons.The molecular phylogenetic analyses placed the new species in an independent clade and sharing a sister relationship with the clade

Discussion
In recent years, new species of Odorrana have been discovered almost every year (Frost 2024).Within the genus, O. schmackeri has been considered as the most widespread species in China, covering Henan, Sichuan, Chongqing, Guizhou, Hubei, Anhui, Jiangsu, Zhejiang, Hunan, Fujian, Guangdong, and Guangxi provinces (Fei et al. 2012).In recent years O. schmackeri was indicated as a complex of species, probably containing some cryptic species (Chen et al. 2013;Li et al. 2015; Zhu 2016), and have been described one after another (Wang et al. 2015;Li et al. 2018a;Shen et al. 2020;Zhang et al. 2021).Molecular phylogenetic analyses indicated that Odorrana leishanensis sp.nov.was revealed as the sister to the clade corresponding to the O. schmackeri complex, and is morphologically distinct from the latter (vocal sacs absent, and smaller body size in female).This may indicate that the new species has probably experienced an independent evolutionary history.
Leigong Mountain in Guizhou Province, China is the main summit of the Miaoling mountain range.Since the 1980s, many scholars have investigated the amphibians in this area and several species were described, i.e., Paramesotriton caudopunctatus (Liu & Hu, 1973), Boulenophrys spinata, Leptobrachium leishanense (Liu &Hu, 1973), B. leishanensis, andNidirana leishanensis Li, Wei, Xu, Cui, Fei, Jiang, Liu &Wang, 2019.Among them, B. leishanensis and N. leishanensis had previously been misidentified as B. minor (Stejneger, 1926) and N. adenopleura (Boulenger, 1909) (Hu et al. 1973;Li et al. 2018b;Li et al. 2019).From 2014 to July 2023 we conducted several surveys in this region but the new species has only just been discovered, with only seven adult specimens found in a small area at elevations of 1600-1800 m.Therefore, we infer that the population of the new species is small, and we recommend classifying the new species as vulnerable (VU) according to the evaluation criteria of the IUCN Red List of threatened Species (IUCN 2012).Future research should focus on determining the distribution and elevational range of the species.

Figure 4 .
Figure 4. Photographs of the holotype MTLS20230729013 of Odorrana leishanensis sp.nov. in life A dorsal view B ventral view C dorsal view of hand D ventral view of hand E ventral view of foot.

Figure 5 .
Figure 5.The holotype specimen MTLS20230729013 of Odorrana leishanensis sp.nov.(preserved) in A dorsal view B ventral view C lateral view D dorsal view of hand E ventral view of hand F ventral view of foot.

Figure 6 .
Figure 6.Color variation in Odorrana leishanensis sp.nov.A dorsolateral view of the male specimen MTLS20230805001 B ventral view of the male specimen MTLS20230805001 C dorsolateral view of the male specimen LS20230806010 D ventral view of the male specimen MTLS20230806010 E dorsolateral view of the female specimen LS20230811024 F ventral view of the female specimen MTLS20230811024.

Figure 7 .
Figure 7. Habitats of Odorrana leishanensis sp.nov. at the type locality, Leishan County, Guizhou Province, China (inset: the holotype on bush stems beside the stream).

Table 1 .
Information of samples used in molecular phylogenetic analyses in this study; a slash (/) indicates information absent.

Table 2 .
References for morphological characters for congeners of the genus Odorrana.