﻿A new species of Odorrana Fei, Ye & Huang, 1990 (Amphibia, Anura, Ranidae) from central Guangxi, China with a discussion of the taxonomy of Odorrana (Bamburana)

﻿Abstract A new species of odorous frog, Odorranadamingshanensissp. nov., was found at the Damingshan National Nature Reserve in Guangxi, China. This species can be distinguished from its congeners by a combination of the following characters: medium body size (SVL 52.3–54.8 mm in males and 74.8–81.2 mm in females), sawtooth spinules on the upper lip, obtusely rounded snout that extends beyond the lower margin, distinct dorsolateral folds, horny tubercles on the rear of the back, presence of outer metatarsal tubercles, dilated nuptial pad with velvety spinules, distinct maxillary gland with tiny spines, and external lateral vocal sac. Through analysis of the 16S mitochondria gene, the new species is closely related to O.nasica and O.yentuensis, but the genetic divergence between the new species and the latter exceeds 7% (uncorrected p-distance). Currently, the new species is only known from its original discovery site. Furthermore, a discussion on the taxonomy of Odorrana (Bamburana) was conducted, identifying seven species within the subgenus Odorrana (Bamburana).


Introduction
The genus Odorrana Fei, Ye & Huang, 1990 is currently known to consist of 62 species, primarily found in East and Southeast Asia (AmphibiaChina 2023; Frost 2023).Within China, there have been records of 40 species, with 18 species identified in Guangxi (AmphibiaChina 2023).Among these, Odorrana versabilis (Liu & Hu, 1962) was previously believed to have a wide distribution across southern and central China, including provinces such as Zhejiang, Guizhou, Anhui, Fujiang, Jiangxi, Hunan, Guangdong, Guangxi, and Hainan (Guo et al. 1966;Hu et al. 1973Hu et al. , 1978;;Liu et al. 1973;Ma et al. 1982;Zou 1983;Pan et al. 1985;Zong and Ma 1985;Wu et al. 1986;Huang et al. 1990;Chen 1991;Sichuan Institute of Biology 1974;Fei et al. 2009;Mo et al. 2014;AmphibiaChina 2023) Li et al. (2001) conducted a comparison of specimens from different geographic populations and observed distinct morphological divergences, indicating the presence of a species complex within O. versabilis.Based on both morphological and molecular data, Li et al. (2001) proposed that the O. versabilis species complex consisted of three separate species: O. exiliversabilis Li, Ye &Fei, 2001, O. nasuta Li, Ye &Fei, 2001, andO. versabilis.Odorrana exiliversabilis is found in Fujiang, Zhejiang, Anhui, and Jiangxi provinces, with Huangkeng County in Fujiang serving as the type locality (Li et al. 2001;AmphibiaChina 2023).Odorrana nasuta is restricted to Hainan Island.Odorrana versabilis, on the other hand, occurs in Guizhou, Anhui, Jiangxi, Hunan, Guangdong, and Guangxi, with Longsheng and Jinxiu counties in Guangxi as the type locality.Subsequently, Fei et al. (2005) assigned these three species to the subgenus Bamburana within the genus Odorrana, based on several distinguishing characters.These included the presence of dorsolateral folds in the subgenus Bamburana (absent in the subgenus Odorrana), the upper lip adorned with sawtooth spinules (absent in the subgenus Odorrana), a xiphisternum without a notch (deeply notched in the subgenus Odorrana), and a widened posterior sternum (sternum not widened posteriorly in the subgenus Odorrana).
In 2010, Fei et al. conducted a revision of the genus Odorrana, reorganizing it into four separate and valid genera within the tribe Odorranini: Bamburana, Eburana, Matsuirana, and Odorrana.Fei et al. (2010) proposed that the genus Bamburana consisted of seven species: B. exiliversabilis, B. montivaga (Smith, 1921), B. nasica (Boulenger, 1903), B. nasuta, B. trankieni (Orlov, Le & Ho, 2003), B. tormota (Wu, 1977), and B. versabilis.However, the idea of dividing the tribe Odorranini into four genera did not gain widespread acceptance.In 2012, Fei et al. followed up on their previous classification (Fei et al. 2005), which had divided the genus Odorrana into the subgenera Odorrana (Odorrana) and Odorrana (Bamburana).Chen et al. (2013) subsequently confirmed the monophyly of the genus Odorrana and divided it into seven distinct clades (clades A-G).Clade F included O. exiliversabilis, O. nasica, O. nasuta, O. tormota, and O. versabilis, and corresponded to the subgenus Odorrana (Bamburana).However, Chen et al. (2013) did not specifically address the validity of Odorrana (Bamburana) but only confirmed that this group forms a monophyletic cluster.Furthermore, there was a lack of molecular data available to support the inclusion of O. trankieni in the subgenus Odorrana (Bamburana).To date, no further research has discussed the validity of Odorrana (Bamburana) or its constituent species.
The distribution of O. versabilis in Guangxi has been previously discussed by Fei et al. (2005) and Mo et al. (2014).They argued that it was widely present in counties such as Wuming, Shangsi, Longshen, Jinxiu, and Ziyuan.Previous studies have also indicated the occurrence of O. nasuta and O. nasica in Guangxi (Zhang and Wen 2000;Fei et al. 2005;Chen 2018;Huang et al. 2020).However, O. nasuta was specifically documented in Shiwandashan and Damingshan National Nature Reserves (Chen 2018;Huang et al. 2020), while information on the distribution of O. nasica is only available on AmphibiaChina (2023) without specifying its precise location.It is important to note that these findings are solely based on morphological descriptions and lack molecular evidence.
In recent years, we conducted herpetological surveys in various nature reserves in Guangxi (Fig. 1), where we collected a series of specimens resembling O. versabilis.However, through phylogenetic analyses, we discovered that these specimens did not form a monophyletic group.Instead, they were distributed across five distinct branches, suggesting the presence of cryptic species within the O. versabilis species complex.The objective of this study is to investigate the species diversity within the O. versabilis species complex, describe the potential new species that have been identified, and assess the validity of the subgenus Odorrana (Bamburana) and its constituent species.

Material and methods
Fifty-four specimens from five different species were collected from various national nature reserves in Guangxi, China, including Shiwandashan (SWDS), Dayaoshan (DYS), Damingshan (DMS), Huaping (HP), and Diding (DD) reserves, between 2013 and 2022 (Fig. 1, Appendix 1).The specimens were euthanized using isoflurane, then fixed in 10% formalin and stored in 75% ethanol.Muscle tissues were obtained from each specimen before formalin fixation and preserved in 100% ethanol for molecular analyses (Luo et al. 2021).Morphological measurements were taken to the nearest 0.1 mm using digital calipers, following the methods described by Fei et al. (2009) and Luo et al. (2021).Abbreviations of characters used in the paper are as follows:

SVL
snout-vent length, distance from the tip of snout to the posterior margin of vent; HDL head length, distance from the tip of snout to the rear of jaw; HDW maximum head width, the greatest width between the left and right articulations of jaw; SNT snout length, distance from the tip of snout to the anterior corner of eye diameter of the fourth toe disc.
Genomic DNA was extracted from muscle tissues using DNeasy tissue extraction kits (Qiagen).Three fragments of mitochondrial DNA (mtDNA) were amplified, targeting segments of the 12S (~ 750 bp) and 16S (~ 1000 bp) ribosomal RNA genes, as well as the COI (~ 630 bp) gene.The primer sequences and PCR conditions followed the protocols outlined by Chen et al. (2013) for the 12S and 16S regions, and by Che et al. (2012) for the COI region.To confirm successful amplification, the PCR products were directly sequenced using an ABI 3730 automated DNA sequencer.The obtained sequences were then validated for accuracy and specificity through BLAST searches (Altschul et al. 1997) and deposited in GenBank (Table 1).
The DNA sequences obtained were aligned using the ClustalW algorithm implemented in Mega v. 7 (Kumar et al. 2016) with default settings.Homologous DNA sequences from GenBank were downloaded for phylogenetic analyses (Table 1).The uncorrected paired divergence (p-distance) was calculated using Mega v. 7. The best-fitting models of DNA substitution for the molecular data were determined using the Akaike Information Criterion (AIC) implemented in

Molecular analyses
Both BI and ML analyses produced similar results, which align with previous studies conducted by Chen et al. (2013) and Luo et al. (2021).Our specimens were categorized into five distinct lineages based on preliminary phylogenetic analyses (Fig. 2).Firstly, the specimens from DMS were divided into two separate lineages.2).The uncorrected p-distances for the 16S fragments within O. versabilis species complex are remarkably low, ranging from 1.3% to 3.1%.For instance, the genetic distances between O. versabilis and O. nasuta range from 1.8% to 2.4%, while those between O. versabilis and O. nasica range from 0.7% to 2.8% (Suppl.material 1: table S1).However, the DMS_II specimens show significant genetic divergences (> 5.0%) from their congeners.When examining the COI fragments, the smallest genetic distance among them is greater than 4.0% (Suppl.material 1: table S2).Specifically, the genetic distances between the specimens from DYS and SWDS range from 5.1% to 6.3%.On the other hand, the genetic divergences between the specimens from DMS and SWDS are very low, ranging from 0.3% to 0.8%, suggesting that these specimens are congeners.Nevertheless, the proposed new species (DMS_II specimens) exhibits distinct genetic differences from other similar species, with divergence values exceeding 5.0% for 16S and 10.0% for COI (Suppl.material 1: tables S1, S2).Tran et al. 2008;Lu et al. 2016 characteristics listed in Table 2, such as SVL, vocal sac, horny tubercles on the rear of the back, and sawtooth spinules on the upper lip.For further information, please refer to the Taxonomic account section below.

Taxonomic results
Based on the analysis of morphological characters and molecular data, it has been determined that the specimens from SWDS, DMS_I, and those previously identified as O. trankieni from Vietnam, belong to the same species, namely O. trankieni.The specimens from HP and DYS have been identified as O. versabilis.The DD specimen has been classified as O. nasica.Furthermore, the DMS_II specimens have been found to represent an undescribed species of Odorrana, which will be described below.
Diagnosis.Based on both molecular analyses and specific morphological traits, these specimens were assigned to the genus Odorrana.The distinguishing morphological characteristics of these species include dilated and tapering tips of the digits, disks with circummarginal grooves and a longer vertical diameter than horizontal diameter, fully webbed toes, the absence of a tarsal fold, a thick first finger with a distinct nuptial pad, sawtooth spinules on the upper lip, and well-defined dorsolateral folds (Fei et al. 2001(Fei et al. , 2005)).
Odorrana damingshanensis sp.nov.can be distinguished from other species in its genus by the following combination of characters: (1) medium body size 2 mm in females); (2) SVL of female/ SVL of male = 1.46; (3) sawtooth spinules on the upper lip; (4) snout obtusely rounded without significantly extending beyond the lower jaw; (5) well-defined dorsolateral folds; (6) horny tubercles on the rear of the back; (7) presence of outer metatarsal tubercles; ( 8) absence of a supratympanic fold; ( 9) highly dilated nuptial pad with velvety spinules on the dorsolateral surface of Finger I; (10) distinct expansion of the tips of the second, third, and fourth fingers, with the first slightly enlarged; expanded tips of the toes with distinct circummarginal grooves; (11) distinct maxillary gland with tiny spines; and ( 12) external lateral vocal sac (Fig. 3A-I).
Body surface shagreened; rear of the back with horny tubercles; ventral surface of venter, forelimbs, and thighs smooth; flanks shagreened; dorsal of forelimbs and hindlimbs shagreened, and hindlimbs with sparse tubercles; two distinct maxillary glands with tiny spinules (Fig. 3I).
Coloration in life.Dorsum grey-beige, with irregular grass-green blotches; a discrete darker brown stripe from tip of snout, across canthus rostral, along the inferior dorsolateral fold, finally ending at the anterior of groin; pineal gland grass-green; cream white stripe from anterior of upper lip to maxillary glands; tympanic region brown, some creamy white tubercles around the tympanum; the upper part of belly, chest, and throat with irregular grey cloud, but the lower part of belly creamy white without spots; ventral surface of thighs and forelimbs incarnadine without spots; forelimbs and hindlimbs with pale brown crossbars, three on lower arm, four on thigh and four on tibia; pupil black with orange border; iris creamy yellow, but the posterior iris pale jacinth; and velvety nuptial pad creamy white (Fig. 3A-I).
Coloration in preservative.Dorsum brown; bars on forelimbs and hindlimbs darker brown; horny tubercles on the rear of the back turned into creamy white; creamy white nuptial pad turned into grey; the upper part of belly, throat, and chest with brown cloud; the lower part of belly immaculate creamy white; the ventral surface of the hindlimbs creamy yellow; external lateral vocal sac pale green and projecting distinctly (Fig. 3I).
Etymology.The specific name of this species, damingshanensis, is derived from its discovery locality, Damingshan National Nature Reserve.In English, it is suggested to be called the Damingshan Bamboo-leaf Frog.In Chinese, it is known as大明山竹叶蛙(Dà Míng Shān Zhú Yè Wā).
Distribution and ecology.Odorrana damingshanensis sp.nov. is a newly discovered species found in the Damingshan National Nature Reserve, located in Wuming District, Nanning City, Guangxi, China.This species was observed near slow-flowing rocky streams, which were ~ 2-3 m wide and 10-30 cm deep (Fig. 1B).The frogs were often seen sitting on rocks, and interestingly, one specimen (NNU 00691) was found on a leaf near a stream, while another specimen (NNU 00693) was perched on a dry branch above a stream.The surrounding vegetation in the area consists of evergreen forest, and the elevation is ~ 1200 m with an ambient temperature of 20 °C in April.During the survey, no advertisement calls were heard; however, both female specimens were gravid, carrying creamy yellow eggs without black poles (Fig. 4A).Additionally, amplexus behavior was observed when males encountered females indoors (Fig. 4B).The breeding season of O. damingshanensis sp.nov. is speculated to occur between April and May.Other sympatric species in the area include Gracixalus jinxiuensis and Quasipaa shini.
Sexual dimorphism and variation.The measurements of O. damingshanensis sp.nov.are provided in Table 3. Females of this species were observed to be significantly larger than males in terms of SVL, with a ratio of 1.46.The specimens show variation in dorsal colors: NNU 00691 displays a grass green coloration with brown blotches (Fig. 4C), while NNU 00689 exhibits a pale beige coloration with grass green blotches (Fig. 4D).Males have a higher density of horny tubercles on the rear of their backs compared to females (Fig. 4C-F).

Discussion
The specimens in our study were classified into five species: However, they did not provide any supporting evidence for their findings.Upon examining the description provided by Zhang and Wen (2000), we observed that our DMS_I specimens exhibited similar body size, color pattern, and other diagnostic features to their specimen (

Figure 3 .
Figure 3.The holotype of O. (B.) damingshanensis sp.nov.(NNU 00690) A dorsal view B ventral view C dorsolateral view D rear of the back with horny tubercles and dorsal view of thighs E ventral view of hand F nuptial pad with velvety spinules G ventral view of snout H ventral view of foot, and I external lateral vocal sac and tiny spines on maxillary glands.

Figure 4 .
Figure 4.A female with creamy yellow eggs without pigmented poles B amplexus C dorsal view of NNU 00691 D dorsal view of NNU 00689 E dorsal view of NNU 00692 F dorsal view of NNU 00693 G dorsolateral view of O. (B.) yentuensis (NHMG1401036, adult male) H dorsolateral view of O. (B.) versabilis (NNU00638, adult male) I dorsal view of O. (B.) nasica (NNU00663, adult female) J dorsolateral view of O. (B.) trankieni (NHMG141107, adult male).

Table 1 .
Miller et al. 2010)12)formation and GenBank accession numbers for all samples used in this study.Nylander 2004), resulting in the selection of the GTR + I + G model.Phylogenetic relationships within the genus Odorrana were reconstructed using Bayesian inference (BI) with MrBayes v. 3.2(Ronquist et al. 2012).A majority-rule consensus tree was constructed to calculate the Bayesian posterior probabilities (BPP) for the nodes in the tree.Maximum likelihood (ML) trees were inferred using the CIPRES Science Gateway server (https://www.phylo.org/portal2;Milleretal. 2010)with the estimation of the proportion of invariable sites and 1000 bootstrap pseudo replicates.

Table 3 .
Measurements of O. (B.) damingshanensis sp.nov.(in mm).Abbreviations are defined in the text.
Huang et al. (2020)that identified SWDS and DMS_I specimens as O. trankieni instead of O. nasuta or O. nasica, based on morphology and phylogeny (Fig.2).Chen (2018) andHuang et al. (2020)suggested the presence of O. nasuta in SWDS and DMS but lacked molecular data to support their claims.By combining phylogenetic and morphological data, we confirm that Chen (2018) andHuang et al.