A new species of odorous frog, Odorrana damingshanensis sp. 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).

Cryptic species, morphology, Odorous frog, phylogeny, taxonomy

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. 1973, 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, and O. 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.

Figure 1. 

A distributions of the subgenus Odorrana (Bamburana), and B habitat of O. (B.) damingshanensis sp. nov. Abbreviations: DMS = Damingshan, DYS = Dayaoshan, SWDS = Shiwandashan, HP = Huaping, DD = Diding.

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;

EN eye-nostril distance, distance from the anterior of eye to nostril;

EYE eye diameter, horizontally from the anterior to posterior corner of eye;

IN internarial space, the shortest distance between the inner margins of nostrils;

IOD interorbital distance, the shortest distance between the anterior corners of orbits;

TMP tympanum diameter;

TEY tympanum-eye distance, from the anterior edge of tympanum to the posterior corner of eye;

TIB tibia length, distance from knee to tarsus;

THL thigh length, distance from vent to knee;

PL pes length, distance from the tip of the fourth toe to the base of the inner metatarsal tubercles;

FLL forelimb length, distance from elbow to the tip of the third finger;

ML manus length, distance from the tip of the third digit to the base of tubercle on prepollex;

FD₃ diameter of the third finger disc;

TD₄ 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 MrModeltest v. 2.3 (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; Miller et al. 2010) with the estimation of the proportion of invariable sites and 1000 bootstrap pseudo replicates.

The specimens in our study were classified into five species: O. damingshanensis sp. nov., O. nasica, O. trankieni, O. versabilis, and O. yentuensis, indicating a significant diversity of species in Guangxi, China. Our findings challenge previous research (Zhang and Wen 2000; Fei et al. 2005; Chen 2018; 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) and Huang 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) and Huang et al. (2020) misidentified their specimens (Voucher nos.: NHMG 1303003, NHMG 141103–04 for Chen; Huang 201808296–98 for Huang et al.). Zhang and Wen (2000) proposed the occurrence of O. nasica in Debao County and DMS (voucher no. 830354, female, SVL 60.0 mm, collected in Debao County, Guangxi, China). 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 (Table 2; Zhang and Wen 2000). Considering the geographical proximity of Debao County to DD, we conclude that the Debao specimen and DD specimen represent the same species, O. nasica.

Upon examination of the series of specimens collected across Guangxi, we discovered that O. damingshanensis sp. nov., O. nasica, O. nasuta, O. trankieni, and O. versabilis exhibit similar body sizes and morphological characteristics in females, but not in males (Table 2). These factors have often led to misidentification of these species. Fig. 1 indicates that O. exiliversabilis and O. versabilis are present in the northern region of the Xi River, while O. damingshanensis sp. nov., O. nasica, O. nasuta, and O. trankieni are found in the southern region of the Xi River. However, further research is required to determine the extent to which the Xi River plays a significant role in the separation of these species. Currently, we know that O. exiliversabilis occurs in Southeastern China (Fujian, Zhejiang, Anhui, and Jiangxi provinces), O. nasica is found in the Sino-Vietnamese region (Yunnan, Guangxi, and Northern Vietnam), O. nasuta is limited to Hannan Island, O. trankieni is distributed in Northern Vietnam (Son La, Hoa Binh, and Bac Giang provinces) and Guangxi, China (this study), representing a new country record for China. O. versabilis inhabits Southern China (Guizhou, Guangxi, Guangdong, Hunan, and Jiangxi provinces), and O. yentuensis occurs in the Sino-Vietnamese region (AmphibiaChina 2023; Frost 2023). Odorrana damingshanensis sp. nov. is only known from DMS. It should be noted that O. damingshanensis sp. nov. and O. trankieni are sympatric species. However, O. trankieni is typically found at lower altitudes ranging from 200 to 900 m, while O. damingshanensis sp. nov. is found at higher altitudes exceeding 1000 m. Odorrana trankieni is commonly encountered in small cascade streams or wide streams with a slow current, whereas O. damingshanensis sp. nov. was specifically found in small, slow-flowing rocky streams (Fig. 1B).

Fei et al. (2005) initially proposed dividing the genus Odorrana into two subgenera, Odorrana (Bamburana) and Odorrana (Odorrana), based on several distinguishing characters. These characters included the presence of dorsolateral folds (absent in the latter), upper lip with sawtooth spinules (absent in the latter), xiphisternum without a notch (deeply notched in the latter), and posterior widening of the sternum (not widened in the latter). In a subsequent revision by Fei et al. (2010), the genus Odorrana was elevated to the generic level and divided into four genera within the tribe Odorranini: Bamburana, Eburana, Matsuirana, and Odorrana. Fei et al. (2010) suggested that the genus Bamburana comprised seven species: B. exiliversabilis, B. montivaga, B. nasica, B. nasuta, B. tormota, B. trankieni, and B. versabilis. However, this revision has not been widely accepted (AmphibiaChina 2023; Frost 2023). In a subsequent study by Fei et al. (2012), they did not adopt this revision and instead followed their original proposal of Odorrana (Bamburana) and Odorrana (Odorrana) (Fei et al. 2005). Phylogenetically, Chen et al. (2013) confirmed the monophyly of the genus Odorrana and divided it into seven major branches (clades A–G). They did not support the genera Bamburana, Eburana, Matsuirana, and Odorrana proposed by Fei et al. (2010): Bamburana, Eburana, and Matsuirana formed monophyletic groups, while Odorrana was paraphyletic. Furthermore, except for Bamburana, no diagnostic characters corresponded to the four genera proposed by Fei et al. (2010). According to Fei et al. (2005), the genus Odorrana can be divided into two subgenera: Odorrana (Bamburana) and Odorrana (Odorrana). By following this classification, differentiating Odorrana (Bamburana) from Odorrana (Odorrana) becomes easier based on four distinct characteristics: distinct dorsolateral folds, sawtooth spinules on the upper lip, absence of a notch in the xiphisternum, and posterior widening of the sternum. Our phylogenetic trees also provide support for the monophyly of Odorrana (Bamburana) species (BS = 100; PP = 1.00), which include eight species: O. (B.) damingshanensis sp. nov., O. (B.) exiliversabilis, O. (B.) nasica, O. (B.) nasuta, O. (B.) tormota, O. (B.) trankieni, O. (B.) versabilis, and O. (B.) yentuensis. However, it is important to note that O. (B.) tormota has a deeply sunk tympanum forming an external auditory canal (Wu 1977). Furthermore, O. (B.) tormota lacks sawtooth spinules on the upper lip and has a deep notch on the xiphisternum. Therefore, we suggest excluding O. (B.) tormota from the subgenus Odorrana (Bamburana). Further investigation is necessary to determine if the subgenus Odorrana (Odorrana) can be further subdivided.

In this study, we identified five of seven species of the subgenus Odorrana (Bamburana) that occur in Guangxi: O. (B.) damingshanensis sp. nov., O. (B.) nasica, O. (B.) trankieni, O. (B.) versabilis, and O. (B.) yentuensis. This finding highlights the significant species diversity of Odorrana (Bamburana) in Guangxi. It is worth noting that the presence of O. (B.) trankieni is a new record for China.

We express our gratitude to the staff of Shiwandashan, Dayaoshan, Damingshan, Huaping, and Diding National Nature Reserves for their valuable assistance during the fieldwork. Additionally, we would like to acknowledge Tao Thien Nguyen for enhancing the manuscript.