Description of a new species of the genus Rana (Anura: Ranidae) from western Guizhou, China, integrating morphological and molecular genetic data

Abstract

The diversity of the brown frog genus Rana may be underestimated as the high similarity of morphological characters. In this study, a new species of Rana from Guizhou Province, China is described, namely Rana zhijinensis Luo, Xiao & Zhou, sp. nov. Molecular phylogenetic analyses clustered the new species into the R. japonica group of Rana and significant morphological characters can be distinguished from the 11 recognized species of the R. japonica group. This description increases the number of recognized Rana to 57 species and the R. japonica group to 12 species, and increases our knowledge of the diversity of the genus Rana.

Full text

Zoological Systematics, 47(4): 275–292 (October 2022), DOI: 10.11865/zs.2022
urn:
Received, accepted
Executive editor: Fuqiang Chen
275
ORIGINAL ARTICLE
Description of a new species of the genus Rana (Anura:
Ranidae) from western Guizhou, China, integrating
morphological and molecular genetic data
Shasha Yan1 †, Qingqing He1 †, Tao Luo2 †, Cheng Xu2, Huaiqing Deng2, Ning Xiao3, Jiang Zhou1 *
1School of Karst Science, Guizhou Normal University, Guiyang 550001, China
2School of Life Sciences, Guizhou Normal University, Guiyang 550001, China
3Guiyang Nursing Vocational College, Guiyang 550003, China
†These authors contributed equally to this paper
*Corresponding author, E-mail: zhoujiang@ioz.ac.cn
Abstract The diversity of the brown frog genus Rana may be underestimated as the high
similarity of morphological characters. In this study, a new species of Rana from Guizhou
Province, China is described, namely Rana zhijinensis Luo, Xiao & Zhou, sp. nov. Molecular
phylogenetic analyses clustered the new species into the R. japonica group of Rana and
significant morphological characters can be distinguished from the 11 recognized species of the
R. japonica group. This description increases the number of recognized Rana to 57 species and
the R. japonica group to 12 species, and increases our knowledge of the diversity of the genus
Rana.
Key words Brown frogs, new species, diversity, taxonomy, morphology.
1 Introduction
The genus Rana sensu lato Linnaeus, 1758 is the third most diverse group in the family Ranidae, after Amolops Cope,
1865 (73 species) and Odorrana Fei, Ye & Huang, 1990 (62 species), widely distributed in Europe, Asia and America (Frost,
2022). The classification of Rana sensu lato has long been controversial because of the high level of morphological similarity
(Fei et al., 1990, 2005, 2009b, 2010, 2012; Dubois, 1992; Frost et al., 2006; Che et al., 2007; Pyron & Wiens, 2011; Yuan
et al., 2016; Jiang et al., 2020; Wang et al., 2020; Wan et al., 2020; Wu et al., 2021; AmphibiaWeb, 2022). A large-scale
phylogenetic framework (Yuan et al., 2016) showed that Rana sensu lato contains nine clades, corresponding to eight
subgenera and an unnamed monophyletic clade (containing only R. sylvatica). These eight subgenera are Rana, Amerana
Dubois, 1992, Liuhurana Fei, Ye, Jiang, Dubois & Ohler, 2010, Aquarana Dubois, 1992, Lithobates Fitzinger, 1843,
Zweifelia Dubois, 1992, Pantherana Dubois, 1992, and Pseudorana Fei, Ye & Huang, 1990. Nevertheless, the controversy
about the taxonomy of Rana still exists. To resolve the taxonomic controversy, Dubois et al. (2021) upgraded seven
subgenera to the genus level and established a new genus Boreorana Dubois, Ohler & Pryon, 2021 with R. sylvatica as the
type species, except Zweifelia (all transferred to Lithobates). However, this classification suggestion was not fully accepted
(Frost, 2022). Currently, 56 species of the genus Rana are recorded, with 28 species distributed in China (AmphibiaChina,
2022; Frost, 2022). In the last five years, new species described within Rana indicated the presence of cryptic species, i.e.,
R. dabieshanensis Wang, Qian, Zhang, Guo, Pan, Wu, Wang & Zhang, 2017, R. luanchuanensis Zhao & Yuan, 2017,

276 Yan et al.
R. jiulingensis Wan, Lyu & Wang, 2020, R. wuyiensis Wu, Shi, Zhang, Chen, Cai, Hoang, Wu & Wang, 2021, and R.
taihangensis Chen, 2022 (Wang et al., 2017; Zhao et al., 2017; Wan et al., 2020; Wu et al., 2021; Shen et al., 2022).
In China, Fei et al. (2009b) classified 14 species of the subgenus Rana into three species groups based on morphological
comparisons and geographical distribution, namely, R. longicrus group, R. chensinensis group, and R. amurensis group.
Subsequent phylogenetic analyses supported the delineation of these species groups and revised the taxonomy of several
species members (Yan et al., 2011; Yuan et al., 2016; Wang et al., 2017; Zhao et al., 2017; Wan et al., 2020; Wu et al., 2021).
The taxonomic revisions merged the R. longicrus group as the R. japonica group (Yang et al. 2017) and proposed a new
species group, R. johnsi group (Wan et al., 2020). These are (1) R. japonica group, 11 species: R. chaochiaoensis Liu, 1946,
R. chevronata Hu & Ye, 1978, R. culaiensis Li, Lu & Li, 2008, R. dabieshanensis, R. hanluica Shen, Jiang & Yang, 2007, R.
japonica Boulenger, 1879, R. jiulingensis, R. jiemuxiensis Yan, Jiang, Chen, Fang, Jin, Li, Wang, Murphy, Che & Zhang,
2011, R. omeimontis Ye & Fei, 1993, R. longicrus Stejneger, 1898, and R. zhenhaiensis Ye, Fei & Matsui, 1995; (2) R.
chensinensis group, five species: R. chensinensis David, 1875, R. dybowskii Günther, 1876, R. huanrenensis Fei, Ye & Huang,
1990, R. kukunoris Nikolskii, 1918, and R. taihangensis; (3) R. amurensis group, three species: R. amurensis Boulenger,
1886, R. coreana Okada, 1928, and R. luanchuanensis; (4) R. johnsi group, three species: R. johnsi Smith, 1921, R.
sangzhiensis Shen, 1986, and R. wuyiensis. However, species groups have not yet been proposed to accommodate the
remaining five species, i.e., R. arvalis Nilsson, 1842, R. asiatica Bedriaga, 1898, R. maoershanensis Lu, Li & Jiang, 2007,
R. sauter Boulenger, 1909, R. shuchinae Liu, 1950, while R. weiningensis (Liu, Hu & Yang, 1962) was replaced in the genus
Pseudorana and recorded as Pseudorana weiningensis (Liu, Hu & Yang, 1962).
During our herpetological surveys in Zhijin County, Guizhou Province, China (Fig. 1), we collected a series of Rana
sensu lato specimens that were recorded as R. chevronata (Fei et al., 2009b). However, morphological examination showed
that these specimens can be distinguished from its congeners. Molecular analysis supports that these specimens form an
independent lineage within Rana. Therefore, based on the differences in molecular and morphological characters, we suggest
the population distributed in Guiguo Town, Zhijin County, Guizhou Province, China as a new species, Rana zhijinensis Luo,
Xiao & Zhou, sp. nov.
Figure 1. Sampling localities of Rana zhijinensis Luo, Xiao & Zhou, sp. nov., R. culaiensis, R. hanluica, and R. omeimontis in Guizhou
Province, China. 1. Guiguo Town, Zhijin County. 2. Supu Town, Qianxi County. 3. Zhujianshan Nature Reserve, Huangping County.
4. Leigongshan National Nature Reserve, Leishan County.

© Zoological Systematics, 47(4): 275–292 A new species of Rana from China 277
2 Materials and methods
2.1 Sampling
A total of 24 specimens were collected from Guizhou Province in this study: 14 specimens of the describing species;
four of R. culaiensis, from Zhujiashan Nature Reserve, Huangping County; one of R. omeimontis, from Supu Town, Qianxi
County; five of R. omeimontis, from Fangxiang Town, Leishan County (Fig. 1). All of the specimens were fixed in 10%
buffered formalin and later transferred to 75% ethanol for preservation. The muscles used for molecular analysis were
preserved in 95% alcohol at -20°C. All of the specimens are deposited at Guizhou Normal University, Guiyang, China.
2.2 DNA Extraction, PCR amplification, and sequencing
Genomic DNA was extracted from muscular tissue using DNA extraction kit from Tiangen Biotech Co., Ltd. (Beijing,
China). Three mitochondrial genes and six nuclear genes were sequenced on six samples by Zhijin, Guizhou. The
mitochondrial genes included partial sequence of the 12S ribosomal RNA gene, complete sequence of tRNAVa l and partial
sequences of 16S ribosomal RNA (12S-tRNAVa l -16S), cytochrome b (Cyt b), and NADH dehydrogenase subunit 2 (ND2).
The nuclear DNA markers included partial sequences of recombinase activating 1 protein gene (RAG1), recombinase
activating 2 protein gene (RAG2), brain-derived neurotrophic factor gene (BDNF), solute carrier family 8 member 3
(SLC8A3), exon 1 of the tyrosine precursor gene (Tyr), and pro-opiomelanocortin A gene (POMC) (see Table S1 for all
primer sequences)
.
PCR amplifications were performed in a 25
μl reaction volume with the following cycling conditions: an initial
denaturing step at 95°C for five min; 36 cycles of denaturing at 95°C for 40 s; annealing at 50°C (for 12S-tRNAVa l -16S, Cyt b,
and Tyr)/48°C (for POMC and SLC8A3)/42°C (for ND2)/41°C (for BDNF)/52°C (for RAG1)/57°C (for RAG2) for 40 s and
extension at 72°C for 1 min, and a final extension step at 72°C for 10 min. PCR products were sequenced on an ABI Prism 3730
automated DNA sequencer at Chengdu TSING KE Biological Technology Co., Ltd. (Chengdu, China). All sequences have been
deposited in GenBank (Table 1).
2.3 Molecular phylogenetic analysis
In total, we used 251 sequences for molecular analysis, including 197 sequences representing 23 species of the genus
Rana and outgroups downloaded from GenBank (Table 1). All of the sequences were assembled and aligned using the MUSCLE
(Edgar, 2004) module in MEGA 7.0 (Kumar et al., 2016) with default settings. In phylogenetic analysis, the construction of
mitochondrial datasets, nuclear gene datasets and concatenated mitochondrial and nuclear gene datasets were used independently
to reconstruct phylogenetic trees.
The best partition scheme and evolutionary models for phylogenetic analysis were estimated within PartitionFinder 2 (Lanfear
et al., 2017) based on Bayesian information criteria and defined for 12S-tRNAVal -16S, Cyt b, ND2 genes as well as six nuclear
genes (Table S2). Phylogenetic analysis using maximum likelihood (ML) and Bayesian inference (BI) methods was
implemented in IQ-tree 2.0.4 (Nguyen et al., 2015) and MrBayes 3.2.1 (Ronquist et al., 2012), respectively. The ML analysis
was conducted in IQ-tree 2.0.4 (Nguyen et al., 2015) with 20000 ultrafast bootstrapping replicates (Hoang et al., 2018) and was
run until a correlation coefficient of at least 0.99 was reached. The BI analysis was performed in MrBayes 3.2.1 (Ronquist et al.,
2012). Two independent runs were conducted in the BI analysis, each of which was performed for 1×108 generations and
sampled every 1000 generations, with the first 25% of the samples discarded as a burn-in, resulting a potential scale reduction
factor of less than 0.01. Genetic distance between species the uncorrected p-distance model for the 16S and Cyt b genes was
estimated using MEGA 7.0, respectively. In addition, we also used nuclear genes in PopART 1.7 (Leigh & Bryant, 2015)
based on the Median Joining method (Bandelt et al., 1999) to obtain haplotypes for assessing differences between new
species and genetically close species.
2.4 Species delimitation
We used two different methods to assess whether the new species represented a valid species. First, a Bayesian hypothesis-
testing approach (Bayes Factor Delimitation, BFD) was implemented to statistically test alternate hypotheses of species
delimitation (Grummer et al., 2014). Two species models were tested: 24 species (i.e., containing the new species) and 23 species
(lumping the new species with sister species). All analyses were performed in *BEAST using BEAST 1.8.2 (Drummond et
al., 2012) under an uncorrelated lognormal relaxed molecular clock. A Yule process was used for the species tree prior, and
a piecewise linear and constant root was used for the population size model. The *BEAST was run each time for 5×108

Table 1. Localities, voucher information, and GenBank numbers for all samples used in this study.
ID Species Voucher Locality* 12S-16S CYTB ND2 RAG1 RAG2 TYR BDNF POMC SLC8A3
1 R. zhijinensis sp. nov. GZNU2018081604 Guiguo, Zhijin, Guizhou, China* OP577493 OP589317 OP589311 OP589335 OP589341 OP589353 OP589323 OP589329 OP589347
2 R. zhijinensis sp. nov. GZNU2018081605 Guiguo, Zhijin, Guizhou, China* OP577494 OP589318 OP589312 OP589336 OP589342 OP589354 OP589324 OP589330 OP589348
3 R. zhijinensis sp. nov. GZNU2018081606 Guiguo, Zhijin, Guizhou, China* OP577495 OP589319 OP589313 OP589337 OP589343 OP589355 OP589325 OP589331 OP589349
4 R. zhijinensis sp. nov. GZNU2018081607 Guiguo, Zhijin, Guizhou, China* OP577496 OP589320 OP589314 OP589338 OP589344 OP589356 OP589326 OP589332 OP589350
5 R. zhijinensis sp. nov. GZNU2018081608 Guiguo, Zhijin, Guizhou, China* OP577497 OP589321 OP589315 OP589339 OP589345 OP589357 OP589327 OP589333 OP589351
6 R. zhijinensis sp. nov. GZNU2018081609 Guiguo, Zhijin, Guizhou, China* OP577498 OP589322 OP589316 OP589340 OP589346 OP589358 OP589328 OP589334 OP589352
7 R. chaochiaoensis SCUM0405170CJ Zhaojue, Sichuan, China* KX269192 KX269339 KX269408 KX269557 KX269632 KX269800 KX269267 KX269481 KX269709
8 R. culaiensis KIZ-SD080501 Culaishan Shandong, China* KX269190 KX269337 KX269406 KX269555 KX269630 KX269783 KX269265 KX269479 KX269707
9 R. zhenhaiensis KIZ0803271 Zhenhai, Zhejiang, China* KX269218 JF939105 KX269433 KX269583 KX269658 KX269809 KX269293 KX269507 KX269734
10 R. longicrus NMNS15022 Miaosu, Xiangtianhu, Taiwan, China KX269189 KX269336 KX269405 KX269554 KX269629 KX269782 KX269264 KX269478 KX269706
11 R. jiemuxiensis KIZ-HUN0708013 Jiemuxi, Hunan, China* KX269221 KX269365 MN958809 KX269586 KX269661 KX269812 KX269296 KX269510 KX269737
12 R. dabieshanensis AHU2016R001 Dabie Mountains area, Anhui, China* MF172963 MF172964 MF172974 MF172971 / MF172973 MF172972 / /
13 R. jiulingensis SYS a005519 Mt. Guanshan, Jiangxi, China MT408985 / / / / / / / /
14 R. omeimontis SCUM0405196CJ Hongya, Zhangcun, Sichuan, China* KX269193 KX269340 KX269409 KX269558 KX269633 KX269785 KX269268 KX269482 KX269710
15 R. hanluica KIZGX07112915 Maoershan shan, Guangxi, China KX269191 KX269338 KX269407 KX269556 KX269631 KX269784 KX269266 KX269480 KX269708
16 R. japonica KIZ-YPX11775 Chiba Prefecture, Isumi-shi, Japan KX269220 KX269364 KX269434 KX269585 KX269660 KX269811 KX269295 KX269509 KX269736
17 R. chensinensis KIZ-RD05SHX01 Huxian, Shaanxi, China KX269186 KX269333 KX269402 KX269551 KX269626 KX269779 KX269261 KX269475 KX269703
18 R. huanrenensis MMS 231 South Korea KX269183 KX269330 KX269400 KX269548 JN984611 KX269776 JN984557 JN984587 KX269700
19 R. kukunoris KIZ-CJ06102001 Qinghai Lake, Qinghai, China KX269185 KX269332 KX269401 KX269550 KX269625 KX269778 KX269260 KX269474 KX269702
20 R. dybowskii MSUZP-IVM-1d Khasanskii District, Primorye, Russia KX269188 KX269335 KX269404 KX269553 KX269628 KX269781 KX269263 KX269477 KX269705
21 R. amurensis MSUZP-SLK-RUS49 Teguldetskii District, Tomskaya, Russia KX269203 KX269349 KX269418 KX269568 KX269643 KX269795 KX269278 KX269492 KX269720
22 R. coreana MMS 223 South Korea KX269202 KX269348 KX269417 KX269567 KX269642 KX269794 KX269277 KX269491 KX269719
23 R. sauteri SCUM0405175CJ Kaohsiung, Taiwan, China KX269204 KX269350 KX269419 KX269569 KX269644 KX269796 KX269279 KX269493 KX269721
24 R. arvalis MSUZP-SLK-MKR21 Chamzinskii District, Mordovia, Russia KX269197 KX269344 KX269413 KX269562 KX269637 KX269789 KX269272 KX269486 KX269714
25 R. asiatica KIZ-XJ0251 47tuan, Xinjiang, China KX269200 KX269346 KX269415 KX269565 KX269640 KX269792 KX269275 KX269489 KX269717
26 R. johnsi ABV 00203 Loc Bao, Lam Dong, Vietnam KX269182 KX269328 KX269398 KX269546 KX269622 KX269774 KX269257 KX269471 KX269698
27 R. sangzhiensis SCUM0405190CJ Zhangcun, Hongya, Sichuan, China KX269206 KX269352 KX269421 KX269571 KX269646 KX269798 KX269281 KX269495 KX269723
28 R. shuchinae CIB-HUI040009 Zhaojue, Sichuan, China KX269210 KX269356 KX269425 KX269575 KX269650 DQ360057 KX269285 KX269499 KX269727
29 R. wuyiensis CIB WY20201106016 Wuyi Mountain, Fujiang, China* MZ337980 MZ355497 MZ355540 MZ355426 / MZ355465 MZ355396 / /
30 P. weiningensis SCUM0405171 Weining, Sichuan, China KX269217 KX269362 KX269432 KX269582 KX269657 KX269808 KX269292 KX269506 KX269733
*Type locality.

© Zoological Systematics, 47(4): 275–292 A new species of Rana from China 279
generations of the MCMC algorithm, sampling every 5000 generations and discarding the first 25% of the iterations as a
burn-in. After *BEAST analyses, two methods of marginal-likelihood estimation, path-sampling (PS; Baele et al., 2012)
and stepping-stone analysis (SS; Xie et al., 2011), were performed. PS and SS analyses were each run for a chain length of
1×106 generations for 100 path steps. We followed the suggestions provided by Grummer et al. (2014) to assess the strength
of support for a species delimitation hypothesis.
In addition to the Bayesian methods tested, we applied three tree-based species-delimitation methods, i.e., Bayesian
implementation of the Poisson Tree Processes model (bPTP; Zhang et al., 2013). The parameters of these three analyses
were set as follows: 1×105 generations, a thinning of 100, and a burn-in of 10%. Convergence of models was assessed by
visualizing plots of the MCMC iterations vs. the log likelihood results. The bPTP analysis was conducted on the bPTP web
server (http://species.h-its.org/ptp/) using concatenated mitochondrial and nuclear genes ML trees as input.
2.5 Morphometrics
Morphometric data were taken for 14 well-preserved specimens. Measurements were recorded to the nearest 0.1
mm
with digital calipers by Tao Luo following Fei et al. (2009a). The measurements were as follows:
ED—eye diameter (diameter of exposed portion of the eye);
FL—foot length (from the proximal end of the inner metatarsal tubercle to the tip of toe IV);
HAL—hand length (from distal end of radioulna to tip of distal phalanx III);
HL—head length (from posterior corner of mandible to tip of snout);
HLL—hind limb length (distance from tip of fourth toe to vent);
HW—head width (the greatest cranial width);
IMTL—inner metatarsal tubercle length (measured as the maximal distance from proximal to distal ends of the inner
metatarsal tubercle);
IND—internarial distance (distance between nares);
IOD—interorbital distance (the minimal distance between upper eyelids);
LAHL—lower-arm and hand length (from the tip of finger III to the elbow joint);
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);
SVL—snout-vent length (from tip of snout to vent);
SL—snout length (from tip of snout to the anterior corner of the eye);
TD—tympanum diameter (horizontal tympanic diameter);
TED—tympanum-eye distance (from anterior edge of tympanum to posterior corner of the eye);
TFL—length of tarsus and foot (from the proximal end of tarsus to the tip of the toe IV);
TIB—tibia length (distance from knee to heel);
TW—tibia width (the greatest width of tibia);
UEW—upper eyelid width (the maximal width of the upper eyelid).
We determined sex by secondary sexual characteristic, i.e., the presence of nuptial pads in males, following Fei et al.
(2009a), and compared the morphological characteristics of the new species with other species of the R. japonica group, and
the 11 species comparison data were obtained from the literature (Fei et al., 2009b; Shen et al., 2007; Li et al., 2008; Yan et
al., 2011; Wang et al., 2017; Wan et al., 2020). In addition, for comparison, we also examined the topotype materials for R.
culaiensis and R. omeimontis (see Appendix 2).
3 Results
3.1 Phylogenetic analyses and genetic divergence
The nucleotide sequences used to reconstruct the phylogenetic tree after alignment included mitochondrial 12S-
tRNAVal-16S (1997 bp), Cyt b (834 bp), ND2 (988 bp) and nuclear genes RAG1 (1190 bp), RAG2 (447 bp), Tyr (456 bp),
BDNF (453 bp), and POMC (285 bp).
The phylogenetic trees reconstructed using the mitochondrial dataset and the nuclear gene dataset exhibit topological
inconsistencies (Fig. 2), but all support R. johnsi group as a basal bra nch in the four species groups, R. johnsi, R. chensinensis,
R. amurensis, and R. japonica, within the subgenus Rana. Another major difference is that the relationship between

280 Yan et al.
Figure 2. Phylogenetic tree based on three mitochondrial genes and six nuclear genes. A. Maternal tree; B. Nuclear gene tree. In both
phylogenetic tree, ultrafast bootstrap support (UFB) values from ML analyses/Bayesian posterior probabilities (BPP) from BI analyses
are given beside nodes. Scale bars denote nucleotide substitutions per sites for mitochondrial and nuclear genes.
0.003
Rana culaiensis
Rana kukunoris
Rana dybowskii
Rana huanrenensis
Rana shuchinae
Rana arvalis
Rana dabieshanensis
Rana hanluica
Rana sauteri
Pseudorana weiningensis
Rana johnsi
Rana japonica
Rana omeimonti s
Rana core ana
Rana wuyien sis
Rana zhenhaiensis
Rana sangzhiensis
Rana jiemuxiensis
Rana longicru s
Rana asiatica
Rana chensinensis
Rana amurensis
Rana chaochiaoensis
0.74/ 48
1.00/100
1.00/100
0.90/ 57
0.79/ 94
1.00/100
0.80/ 80
1.00/100
0.78/ 49
0.90/65
1.00/100
1.00/99
1.00/100
1.00/96
1.00/100
1.00/76
0.96/ 68
1.00/96
1.00/100
1.00/100
1.00/99
Rana zhijinensis sp. nov.
BPP/UBP
R
L
Rh
Ra
Rc
Rj
subgenus Rana
subgenus Liuhurana
R. japonica group
R. amurensis group
R. chensinensis group
R. johnsi group
L
Rh
Rc
Ra
Rj
R

© Zoological Systematics, 47(4): 275–292 A new species of Rana from China 281
R. chensinensis, R. amurensis, and R. japonica groups is not resolved, with R. japonica group as sister group to R.
chensinensis group in the mitochondrial tree (Fig. 2A) but R. japonica group as sister group to R. amurensis group in the
nuclear gene tree (Fig. 2B), but both phylogenetic relationships are not highly supported. The reconstructed phylogenetic
tree of concatenated mitochondrial and nuclear genes yielded results that were inconsistent with the mitochondrial tree and
nuclear genes, i.e., the R. japonica group as sister group to the R. amurensis group +R. chensinensis group (Fig. 3).
The results of all ML and BI analyses were largely consistent, as shown in Figures 2 and 3, with the Rana samples from
Zhijin County, Guizhou, forming a highly supported sister species lineage with R. chaochiaoensis (Bayesian posterior
probability (BPP)/ultrafast bootstrap support (UBP) = 1.00/100; Fig. 2). Haplotype networks based on POMC, Tyr and
RAG1 indicated no common haplotypes between the new species and the closely related species, indicating genetic
differences between the new species and the closely related species (Figs 4A–B, E). On the SLC8A3 and RAG2 haplotype
networks, the new species shared haplotypes with R. culaiensis and R. chaochiaoensis (Figs 4C–D). Notably, for the nuclear
gene BDNF, the new species fully shared haplotypes with closely related species, R. chaochiaoensis, R. culaiensis, R.
longicrus, R. dabieshanensis, and R. hanluica, indicating that there is genetic similarity between these species in the BDNF
gene (Fig. 4F).
The genetic distance between individuals of undescribed species was 0.0% for 16S (ranges 0.0%–0.3% for Cyt b). The
smallest p-distance between the undescribed species and its closely related (R. chaochiaoensis) species was 1.1% in the 16S
and 12.5% in the Cyt b; these are significantly greater than the genetic distances between recognized species (Tables 2–3).
3.2 Species delimitation
The *BEAST analysis of the candidate species model showed that the Bayes factor values SS (79.90) and PS (82.72)
of the 25 species model were much greater than five, indicating support for the new species as a valid species (Table 4). In
Figure 3. Phylogenetic tree based on four mitochondrial genes and six nuclear genes. In this phylogenetic tree, UFB from ML analyses/
BPP from BI analyses are given beside nodes. The scale bar represents 0.03 nucleotide substitutions per site. Red lines represent
species delimitation results of bPTP and BPP.
0.03
Rana sangzhiensis
Rana asiatica
Rana omeimontis
Rana jiemuxiensis
Rana amurensis
R
ana culaien
s
i
s
Pseudorana wein ingensis
Rana japonica
Rana hanluica
Rana saute ri
Rana zhenhaiensis
Rana core ana
Rana dabieshanensis
Rana huanrenensis
Rana kukunori s
Rana jiulingensis
Rana chensin ensis
Rana shuchinae
Rana johnsi
Rana dybowskii
Rana wuyiens is
Rana longicru s
Rana chaochiaoensis
Rana arval is
1.00/100
0.65/ 45
100/100
1.00/100
0.78/ 79
0.65/ 8 0
0.98/ 94
0.97/ 60
1.00/98
1.00/100
1.00/100
1.00/99
1.00/100 1.00/100
1.00/100
1.00/100
1.00/96
1.00/100
1.00/100
1.00/100
1.00/100
1.00/86
1.00/100
R
L
Rh
Ra
Rc
Rj
subgenus Rana
subgenus Liuhurana
R. japonica group
R. amurensis group
R. chensinensis group
R. johnsi group
R
L
Rh
Rc
Ra
Rj

282 Yan et al.
addition, the results of the maximum likelihood and highest Bayesian supported solution (both support values are 0.748,
acceptance rate 0.068) of the bPTP analysis also supported a 25 species taxonomy model, also supports the new species as
a valid species (Fig. S1).
Therefore, integrating evidence for morphological, genetic differences, and the results of species delimitation, we
describe the specimens collected from Zhijin County as a new species, Rana zhijinensis Luo, Xiao & Zhou, sp. nov.
Rana zhijinensis Luo, Xiao & Zhou, sp. nov. (Figs 5–6, Table 5)
Holotype. ♂, adult, GZNU2018081606 (Fig. 5), Guiguo Town (26°33'12.75"N, 105°50'26.90"E; elev. 1525
m), Zhijin
County, Guizhou Province, China, 16 August 2018, coll. Tao Luo.
Paratypes. 6♂1♀, adult, GZNU2018081601–1605, GZNU2018081607–1608, same data as holotype, coll. Tao Luo,
Kai Gao and Huan He; 3♂3♀, adult, GZNU2019080401–0402, GZNU2019080501–0504, same locality as holotype, 4
August 2019, coll. Tao Luo.
Etymology. The specific epithet “zhijinensis” is in reference to the type locality, Zhijin County, Guizhou, China. For
the common name, we suggest “Zhijin Brown Frog”, and for the Chinese name “Zhi Jin Lin Wa (织金林蛙)”.
Diagnosis. The new species is assigned to the genus Rana based on the morphological characteristics typical for this
genus, including the possession of a prominent dorsolateral fold, a dark temporal mask, and a body that is counter-shaded in
various shades of brown.
The species can be distinguished from its congeners by the following combination of morphological characteristics: (1)
body medium-sized, SVL
=
46.1–53.7
mm in adult males (n
=
8), 54.5–58.6
mm in adult females (n
=
4); (2) head length equal
to head width, HL/HW 1.01 in males and 1.00 in females; (3) internarial distance equal to interorbital distance, HL/HW 1.04
in males females; (4) supratympanic fold present; (5) dorsolateral fold present and thin, extending straight from posterior
margin of the upper eyelid to above the groin; (6) tympanum diameter significantly smaller than eye diameter, ED/TD
=
0.64
in males and 0.71 in females; (7) fingers circummarginal grooves and webbed absent, relative finger lengths I
<
II
<
IV
<
III;
(8) toes circummarginal grooves absent, toe webbing present, toe webbing formula I 1½–2 II 1⅓–2⅔ III 2–2⅔ IV 2⅔–1 V,
Figure 4. Haplotype networks of Rana zhijinensis Luo, Xiao & Zhou, sp. nov. and its related species constructed based on the nuclear
gene sequences. Different species of the R. japonica group are shown as different colors.
(POMC) (Tyr) (SLC8A3)
(RAG2) (RAG1) (BDNF)
ABC
DE F
Rana zhijinensis sp. nov.
Rana chaoch iaoensis Rana japonica
Rana culaiensis
Rana zhenhaiensis
Rana lon gicrus
Rana jiemuxiensis
Rana da bieshanensis
Rana ome imontis
Rana hanluica
1
6
Number of
sampl e

© Zoological Systematics, 47(4): 275–292 A new species of Rana from China 283
relative toe lengths I
<
II
<
III
<
V
<
IV; (9) supernumerary tubercles present only below the base of fingers III and IV; (10)
breeding males possess dark gray-blackish nuptial pad separated into three parts with tiny dark gray-blackish tiny spines on
finger I; (11) tibiotarsal articulation reaching far forward beyond tip of the snout.
Description (holotype, GZNU2018081606). Body size large (SVL 53.4
mm); head length slightly larger than head width
(HL/HW
=
1.02); snout short, slightly protruding, and projecting beyond lower jaw in dorsal view, significantly larger than
diameter of eye (SL/ED
=
1.25); nostril rounded, distinct, slightly closer to snout tip than eye (NEL/NSL
=
1.05); canthus
rostralis distinct; internasal distance slightly larger than interorbital distance (IND/IOD
=
1.06) and equal to upper eyelid
width (IND/UEW
=
1.00); tympanum rounded, tympanic rim slightly elevated relative to tympanum, tympanum diameter
significantly less than diameter of eye (TD/ ED
=
0.63); vomerine teeth present, moderately developed, on two oblique ridges;
tongue cordiform, deeply notched posteriorly; vocal sacs absent; eyes large, slightly protuberant in dorsal view, eye diameter
Figure 5. Morphological features of the live adult male holotype GZNU2018081606 of Rana zhijinensis Luo, Xiao & Zhou, sp. nov.
A. Dorsolateral view; B. Dorsal view; C. Ventral view; D. Egg cluster; E. Ventral view of hand and dark gray-blackish nuptial pad;
F. Ventral view of foot.

284 Yan et al.
significantly less than head length (ED/HL
=
0.32); dorsolateral fold present and thin, extending straight from posterior
margin of upper eyelid to above groin, not curved outward above tympanum supratympanic fold distinct and connected to
maxillary gland.
Forelimbs short and robust, lower-arm 0.20 of SVL and hand 0.25 of SVL; fingers slender, webbing and lateral fringes
absent; tip of fingers rounded, not expanded, circummarginal grooves absent; relative finger lengths: I
<
II
<
IV
<
III;
subarticular tubercles slightly prominent, rounded: 1, 1, 2, 2; distinct, small, rounded supernumerary tubercles present only
below base of fingers III and IV; inner metacarpal tubercle indistinct, ovoid, mostly covered by nuptial pad; two outer
metacarpal tubercles distinctly separated and small; nuptial pad with tiny dark gray-blackish spines on finger I, divided into
three parts, prior two parts completely joined together.
Hindlimbs long and slender, tibia 0.64 of SVL and foot 0.63 of SVL; heels overlapping when hindlimbs flexed at right
angles to axis of body; tibiotarsal articulation reaching forward far beyond tip of snout when hindlimb stretched forward
alongside of body; foot length slightly less than tibia length (FL/TIB
=
0.98); circummarginal grooves absent; subarticular
tubercles prominent 1, 1, 2, 3, 2; toes one and two webbed, toes webbing formula I 1½–2 II 1⅓–2⅔ III 2–2⅔ IV 2⅔–1 V;
relative toe lengths I
<
II
<
III
<
V
<
IV; absence of lateral fringes on lateral edges of toes I and V; two metatarsal tubercles,
inner metatarsal tubercle large, ovoid; outer metatarsal tubercle small.
Dorsal skin smooth, with sparse tiny granules; several small granules on flank; maxillary glands relatively developed;
ventral surface smooth; tiny granules on lateral body, dorsal surface of limbs, upper edge of eyelid, temporal region, and
anterior and posterior edges of tympanum; throat, chest, and belly of thighs being smooth.
Coloration (Fig. 5). In life, dorsal parts of head and dorsum, flank, forelimb, thigh, tibia, and foot brownish with a few
Figure 6. Variation of the live adult male holotype GZNU20220705001 of Rana zhijinensis Luo, Xiao & Zhou, sp. nov. A. Dorsolateral
view; B. Dorsal view; C. Ventral view.

© Zoological Systematics, 47(4): 275–292 A new species of Rana from China 285
and larger irregular black patches laterally; black speckles forming an indistinct linear stripe between eyelids; black edge
intermittently present on anterior two-thirds of both sides of dorsolateral fold; loreal region brownish; canthus rostralis below
brownish-black; maxillary gland brownish-black; tympanic rim light purple, central light yellow-brown; forelimbs dorsally
same color as dorsal surface of body, with four brown-black transverse bars in forearm; dorsum of thigh and tibia present a
grayish brown, with 12 distinct dark brown-black transverse bars; an distinct inverted V-shaped black glandular ridge in
scapular region and extending backward to middle of dorsal surface of body to form a distinct inverted V-shaped black
glandular ridge at posterior end; throat creamy white; chest and anterior third of belly yellowish; ventral surfaces of forelimbs,
inner surface of tarsals, inner surface of thighs, and inner surface of shanks red-orange; nuptial pad dark gray-blackish;
tubercles around vent yellowish-white. After preservation in 75% ethanol, dorsal surface of body coloration changed to light
grey patches; transverse bands on limbs and digits distinct, and coloration changed to lighter colors. Ventral surface white,
with greyish-white on chest and throat; ventral surfaces of forelimbs and hindlimbs creamy yellow with brown mottling;
hands and toe webs dark grey.
Measurements (in mm). SVL 53.4, HL 18.7, HW 18.3, SL 7.4, IND 3.8, IOD 3.6, UEW 3.8, ED 5.9, TD 3.7, LAHL
24.1, HAL 13.6, HLL 103.9, TIB 34.0, TFL 44.6, FL 33.2, TW 7.6, NED 3.9, NSD 3.7, TED 1.1, IMTL 2.6.
Variations. The basic statistics for measurements present as in Table 2. Females (SVL 56.8
±
1.8
mm, n
=
4) have slightly
larger body size than males (SVL 50.4
±
2.9
mm, n
=
10). Some individual have smaller irregular black patches on the flank,
dorsum of the thigh and tibia has seven brown transverse bars, some of which are indistinct (Fig. 6). Adult males lack vocal
sacs. In breeding, dark gray-blackish nuptial pads are present on finger I in males.
Comparisons. The new species is assigned to the R. japonica group based on the following morphological
characteristics, i.e., digits without circummarginal grooves, and dorsolateral fold distinct, extending straight from the
posterior margin of the upper eyelid to above the groin (Wan et al., 2020). Here, the morphology of the new species with 11
species of the R. japonica group were compared.
The new species is phylogenetically closest to R. chaochiaoensis, but can be morphologically distinguished from the
latter by combining the following morphological characteristics: head length equal to head width, HW/HL 1.01 in males and
1.00 in females (vs. smaller, HW/HL 0.96 in males and females); internarial distances equal to interorbital distances,
IND/IOD 1.04 in males and females (vs. significantly smaller, IND/IOD 0.55 in males and females); relative finger lengths
I
<
II
<
IV
<
III (vs. II
<
IV
<
I
<
III); relative toe lengths I
<
II
<
III
<
V
<
IV (vs. I
<
II
<
V = III
<
IV), toe webbing formula I 1½–
2 II 1⅓–2⅔ III 2–2⅔ IV 2⅔–1 V (vs. I 1–1⅔ II 1⅓–2 III 1½–2½ IV 2⅔–1 V); presence of lateral fringes on the inner and
outer edges of toes II, III and IV (vs. absence of all toes); tibiotarsal articulation reaching forward far beyond the tip of the
snout (vs. beyond nostrils); inverted V-shaped black glandular ridge in the scapular region and body dorsal mid-side (vs.
only in the scapular region), nuptial pad dark gray-blackish divided into three parts in breeding males (vs. gray, divided into
four parts).
Compared to the ten remaining species in the R. japonica group, the new species differs from R. hanluica as follows:
head length equal to width, HL/HW 1.01 in males and 1.00 in females (vs. larger, HL/HW 1.11 in males and 1.13 in females),
relative finger lengths I
<
II
<
IV
<
III (vs. II
<
I
<
IV
<
III), toe webbing formula I 1½–2 II 1⅓–2⅔ III 2–2⅔ IV 2⅔–1 V (vs. I
1⅓–1⅔ II 1–2 III 1⅓–2½ IV 2⅓–1 V), supernumerary tubercles present only below the base of fingers III and IV (vs. each
finger), nuptial pad dark gray-blackish and divided into three parts (vs. nuptial pad gray and divided into two parts); from R.
omeimontis by having a relatively medium body size, SVL
=
46.1–53.7
mm in adult males, 54.5–58.6
mm in adult females
(vs. 56.7–63.7
mm in males, 61.7–70.3
mm in females), one inverted V-shaped markings on the dorsal surface (vs. absent),
head length equal to width, HL/HW 1.01 in males and 1.00 in females (vs. slightly smaller, 0.98 in males and females),
supratympanic fold present (vs. absent), relative finger lengths I
<
II
<
IV
<
III (vs. II
<
IV
<
I
<
III), relative toe lengths I
<
II
<
III
<
V
<
IV (vs. I
<
II
<
V
=
III
<
IV), supernumerary tubercles present only below the base of finger IV (vs. absent), tibiotarsal
articulation reaching forward far beyond tip of the snout (vs. reaching the front of nostrils); from R. jiulingensis by head
length equal to width, HL/HW 1.01 in males and 1.00 in females (vs. significantly smaller, HW/HL 0.82 in males and 0.85
in females), internarial distances equal to interorbital distances, IND/IOD 1.04 in males and females (vs. significantly larger,
IND/IOD 1.20 in males and 1.22 females), supratympanic fold present (vs. absent), one inverted V-shaped markings on the
dorsal surface (vs. absent), supernumerary tubercles present only below the base of fingers III and IV (vs. each finger), toe
webbing formula I 1½–2 II 1⅓–2⅔ III 2–2⅔ IV 2⅔–1 V (vs. I 1⅓–2 II 1⅓–2⅓ III 1 ½–2⅔ IV 3–1⅓ V), nuptial pad dark
gray-blackish (vs. creamy white); from R. japonica by two outer metacarpal tubercles distinctly separated (vs. absent),
inverted V-shaped black glandular ridge in the scapular region and body dorsal mid-side (vs. only in the scapular region),
tibiotarsal articulation reaching forward far beyond tip of the snout (vs. reaching or beyond tip of the snout in males, reaching
the center of the eye or beyond the nostril in females), nuptial pad dark gray-blackish and divided into three parts (vs. nuptial
pad grayish brown or yellowish brown and divided into two parts); from R. dabieshanensis by supratympanic fold present

Table 2. Mean p-distance (%) of 16S gene among the genus Rana species used in this study.
ID Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1 R. zhijinensis sp. nov.
2 R. chaochiaoensis 1.1
3 R. culaiensis 6.5 6.0
4 R. zhenhaiensis 6.0 5.5 0.5
5 R. longicrus 6.5 6.3 1.1 0.7
6 R. jiemuxiensis 7.0 6.1 2.3 1.9 2.6
7 R. dabieshanensis 6.4 6.2 3.9 3.5 4.1 3.6
8 R. jiulingensis 6.8 6.6 4.1 3.7 4.4 4.1 1.7
9 R. omeimontis 6.6 6.4 3.2 2.8 3.5 2.9 1.2 1.8
10 R. hanluica 6.8 6.6 3.5 3.1 3.8 3.3 4.2 4.7 3.8
11 R. japonica 6.5 6.5 8.1 7.5 8.5 7.3 8.6 8.4 8.6 8.8
12 R. chensinensis 12.5 11.8 13.5 13.213.814.814.915.614.815.812.1
13 R. huanrenensis 13.0 12.2 12.4 12.212.713.614.014.713.914.412.61.8
14 R. kukunoris 12.1 11.8 14.0 13.813.814.814.314.514.215.812.92.0 2.0
15 R. dybowskii 12.7 12.0 13.2 12.713.712.614.515.515.314.413.07.9 7.1 7.5
16 R. amurensis 12.0 10.8 12.2 11.9 12.7 11.7 10.8 11.0 11.7 13.3 14.1 15.2 13.0 14.1 15.4
17 R. coreana 17.2 15.8 17.3 17.0 17.0 15.8 16.7 17.5 16.9 18.7 18.3 15.4 15.4 14.9 17.97.4
18 R. sauteri 12.0 11.1 13.0 12.8 13.3 11.8 13.3 14.0 13.2 13.7 11.2 11.7 10.5 10.6 11.913.915.8
19 R. arvalis 12.1 11.1 11.9 11.6 11.6 11.1 12.6 14.4 13.1 13.2 10.3 9.3 9.5 10.1 11.5 13.114.310.4
20 R. asiatica 10.9 10.0 11.7 11.9 12.9 11.0 11.7 13.6 12.4 13.8 11.0 11.7 10.0 11.2 11.211.512.77.4 8.1
21 R. johnsi 13.1 12.1 12.7 12.7 13.7 13.4 13.7 14.1 14.1 14.9 12.0 11.9 11.2 11.0 14.715.216.912.511.612.1
22 R. sangzhiensis 14.1 13.0 13.1 13.1 13.6 13.3 12.6 13.0 13.0 15.9 12.4 12.6 10.9 10.8 14.1 14.1 16.6 11.5 11.5 11.1 2.0
23 R. shuchinae 13.2 12.7 14.5 14.2 14.7 13.1 15.3 17.3 15.2 15.2 11.3 14.3 13.7 14.9 15.1 14.7 16.3 13.4 10.3 10.4 13.2 12.7
24 R. wuyiensis 8.7 8.3 7.4 6.7 8.2 7.1 8.2 7.4 7.8 8.5 6.1 8.7 6.9 6.6 9.7 8.2 9.4 7.7 4.6 6.1 1.5 1.5 7.2
25 P. weiningensis 30.4 30.8 33.6 32.1 31.2 31.1 30.7 30.8 29.9 30.4 31.8 37.0 34.1 31.7 33.4 33.8 33.9 32.0 31.0 32.4 34.8 32.6 27.9 23.5
Table 3. Mean p-distance (%) of Cyt b gene among the genus Rana species used in this study.
ID Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
1 R. zhijinensis sp. nov.
2 R. chaochiaoensis 12.5
3 R. culaiensis 20.9 24.9

Table 3 (continued)
ID Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
4 R. zhenhaiensis 21.2 26.5 2.7
5 R. longicrus 28.8 28.2 5.8 6.4
6 R. jiemuxiensis 26.3 27.4 17.0 15.6 18.0
7 R. dabieshanensis 22.6 27.1 11.3 12.0 16.0 15.9
8 R. omeimontis 26.2 28.7 12.0 12.8 16.1 16.6 5.0
9 R. hanluica 24.9 20.7 13.0 12.6 13.8 13.0 12.2 10.8
10 R. japonica 27.3 26.2 23.1 22.3 32.7 25.1 24.9 26.7 24.5
11 R. chensinensis 34.4 26.3 36.1 31.9 42.7 36.7 37.5 40.1 36.0 33.0
12 R. huanrenensis 32.3 31.2 43.2 38.3 49.9 41.7 32.7 39.1 37.7 34.0 8.0
13 R. kukunoris 31.3 26.9 32.9 33.5 35.5 35.0 32.7 35.0 32.9 27.6 9.1 7.5
14 R. dybowskii 29.9 35.5 34.5 34.3 39.1 34.3 32.0 40.2 36.1 31.0 23.5 18.7 15.5
15 R. amurensis 56.3 48.1 48.6 49.5 57.6 40.4 39.5 32.7 35.8 45.5 43.5 48.6 40.8 46.1
16 R. coreana 45.5 41.5 36.1 39.1 42.4 39.7 41.8 39.4 31.1 48.1 38.6 51.4 36.3 48.3 29.9
17 R. sauteri 39.8 46.4 36.9 39.1 46.5 41.9 39.1 42.8 35.5 35.6 39.3 42.7 32.7 36.1 38.537.5
18 R. arvalis 40.8 42.7 41.5 43.2 53.7 46.5 40.4 45.6 42.8 28.9 39.6 34.7 31.0 33.8 38.6 42.6 38.5
19 R. asiatica 28.3 37.0 25.4 27.0 32.9 30.3 26.7 29.6 28.0 28.5 30.8 31.0 24.5 36.6 30.3 38.6 26.4 23.3
20 R. johnsi 39.1 37.4 29.8 30.3 32.9 35.0 32.2 37.8 38.3 33.2 35.5 47.2 33.9 35.0 47.2 49.3 47.7 38.3 42.1
21 R. sangzhiensis 41.5 37.1 32.7 31.7 37.8 37.7 37.7 43.3 39.1 41.5 36.6 43.4 35.0 42.1 51.6 54.0 50.9 39.1 45.1 5.8
22 R. shuchinae 45.3 49.3 38.5 38.3 55.8 49.1 37.4 41.0 42.5 45.6 45.1 42.0 36.7 40.3 45.7 41.3 43.8 45.5 36.3 44.2 47.3
23 R. wuyiensis 37.0 38.2 27.9 28.3 34.8 36.2 34.0 36.5 36.2 39.0 46.5 55.7 40.4 41.7 46.5 52.3 42.9 41.8 31.9 7.6 5.9 43.8
24 P. weiningensis 45.4 52.6 66.2 63.4 52.0 60.7 59.0 60.4 55.3 56.1 61.5 63.1 55.1 61.4 59.5 59.8 56.6 65.9 50.2 74.1 63.5 61.7 64.5
Table 4. The species delimitation results of new species and several closely related species in BF method.
Model Species delimitation MLE Path
Sampling (PS)
MLE Stepping
Stone (SS)
BF (PS) BF (SS)
M1 25 species: Rzj + Rcc + Ram + Rar + Ras + Rch + Rco + Rcu + Rdb + Rdy + Rha + Rhu + Rja + Rji + Rjl + Rlo
+ Rzh + Rjo + Rkk + Rwy+Rom + Rsn + Rsu + Rsh +Lwn
−32696.58 −32697.56 7.19 7.99
M2 24 species:{Rzj + Rcc} + Ram + Rar + Ras + Rch + Rco + Rcu + Rdb + Rdy + Rha + Rhu + Rja + Rji + Rjl +
Rlo + Rzh + Rjo + Rkk + Rwy+Rom + Rsn + Rsu + Rsh + Lwn
−32692.98 −32693.56 - -
Abbreviations: Rzj—R. zhijinensis sp. nov.; Rcc—R. chaochiaoensis; Ram—R. amurensis; Rar—R. arvalis; Ras—R. asiatic; Rch—R. chensinensis; Rco—R. core; Rcu—R. culaiensis; Rdb—R.
dabieshanensis; Rdy—R. dybowskii; Rha—R. hanluica; Rhu—R. huanrenensis; Rja—R. japonica; Rji—R. jiemuxiensis; Rjl—R. jiulingensis; Rlo—R. longicrus; Rzh—R. zhenhaiensis; Rjo—R.
johnsi; Rkk—R. kukunoris; Rwy—R. omeimontis; Rom—R. wuyiensis; Rsn—R. sangzhiensis; Rsu—R. sauteri; Rsh—R. shuchinae; Pwn—P. weiningensis.

Table 5. Measurements (in mm) of the adult specimens of R. dorbiapicem sp. nov. See abbreviations for the morphological characters in the materials and methods section.
Specimen GZNU
2018
081606
GZNU
2018
081607
GZNU
2018
081608
GZNU
2019
080401
GZNU
2019
080402
GZNU
2019
080501
GZNU
2018
081602
GZNU
2018
081603
GZNU
2018
081604
GZNU
2018
081605
GZNU
2018
081601
GZNU
2019
080502
GZNU
2019
080503
GZNU
2019
080504
Range (Mean ± SD)
Sex Male Male Male Male Male Male Male Male Male Male Female Female Female Female Males Females
SVL 53.4 49.9 53.6 49.8 53.7 49.2 46.5 49.1 46.1 53.1 54.5 56.6 58.6 57.6 46.1–53.7 (50.4 ± 2.9) 54.5–58.6 (56.8 ± 1.8)
HL 18.7 18.0 19.9 17.6 18.3 18.8 17.6 18.9 16.9 18.8 19.5 21.1 22.4 22.3 16.9–19.9 (18.4 ± 0.9) 19.5–22.4 (21.3 ± 1.4)
HW 18.3 17.1 19.6 16.5 16.4 15.9 16.0 16.8 16.8 17.9 18.3 18.6 19.9 18.9 15.9–19.6 (17.1 ± 1.2) 18.3–19.9 (18.9 ± 0.7)
SL 7.4 8.4 7.3 7.4 7.5 7.6 6.2 7.6 7.8 7.8 7.2 8.3 8.6 8.9 6.2–8.4 (7.5 ± 0.6) 7.2–8.9 (8.3 ± 0.7)
IND 3.8 3.7 4.4 3.8 4.8 3.5 3.9 3.6 3.5 4.3 4.1 4.0 4.5 4.5 3.5–4.8 (3.9 ± 0.4) 4.0–4.5 (4.3 ± 0.3)
IOD 3.2 3.7 3.9 3.8 4.4 3.2 3.5 3.5 3.2 3.9 4.8 4.4 4.6 4.8 3.2–4.4 (3.6 ± 0.4) 4.4–4.8 (4.7 ± 0.2)
UEW 3.8 3.2 3.7 3.9 3.8 3.1 3.7 3.9 3.3 3.7 5.0 3.4 5.2 5.6 3.1–3.9 (3.6 ± 0.3) 3.4–5.6 (4.8 ± 1.0)
ED 5.9 5.5 5.9 5.9 6.1 5.2 5.2 5.4 5.6 5.6 6.0 5.9 6.5 6.9 5.2–6.1 (5.6 ± 0.3) 5.9–6.9 (6.3 ± 0.5)
TD 3.7 3.7 3.3 3.7 3.2 3.7 3.8 3.8 3.3 3.6 4.5 3.8 4.9 4.8 3.2–3.8 (3.6 ± 0.2) 3.8–4.9 (4.5 ± 0.5)
LAHL 24.1 23.7 23.4 21.1 23.8 23.9 22.2 22.9 21.2 24.0 24.3 24.6 25.6 25.4 21.1–24.1 (23.0 ± 1.1) 24.3–25.6 (25.0 ± 0.6)
HAL 13.6 13.8 14.3 12.8 14.5 14.2 12.3 14.3 12.7 14.5 15.7 14.8 15.7 16.2 12.3–14.5 (13.7 ± 0.8) 14.8–16.2 (15.6 ± 0.6)
HLL 103.9 106.6 107.8 102.1 106.6 106.6 100.9 101.9 101.4 103.2 106.2 111.6 121.6 119.8 100.9–107.8 (104.1 ± 2.6) 106.2–121.6 (114.8 ± 7.2)
TIB 34.0 32.9 33.3 30.2 33.6 32.1 30.5 31.7 29.5 31.8 36.2 34.7 41.6 42.5 29.5–34 (32.0 ± 1.5) 34.7–42.5 (38.8 ± 3.9)
TFL 44.6 45.9 45.9 42.9 44.4 45.9 43.7 46.8 42.1 44.5 45.8 45.9 49.6 19.5 42.1–46.8 (44.7 ± 1.5) 19.5–49.6 (40.2 ± 13.9)
FL 33.2 31.1 33.4 27.9 32.5 33.5 30.5 31.9 28.3 31.7 33.0 33.7 25.3 25.3 27.9–33.5 (31.4 ± 2.0) 25.3–33.7 (29.3 ± 4.7)
TW 7.6 6.9 6.0 5.0 5.7 5.6 4.1 5.7 5.1 7.4 5.8 5.4 6.2 6.1 4.1–7.6 (5.9 ± 1.1) 5.4–6.2 (5.9 ± 0.4)
NED 3.9 4.4 4.1 4.1 3.9 4.1 3.9 4.3 4.3 4.2 4.4 4.6 5.5 5.4 3.9–4.4 (4.1 ± 0.2) 4.4–5.5 (5.0 ± 0.6)
NSD 3.7 3.9 3.3 3.4 3.5 3.6 2.7 3.3 3.5 3.5 2.8 3.8 4.7 4.3 2.7–3.9 (3.4 ± 0.3) 2.8–4.7 (3.9 ± 0.8)
TED 1.1 1.7 1.7 1.5 1.8 1.8 1.4 1.7 1.6 1.8 1.1 2.4 3.1 2.9 1.1–1.8 (1.6 ± 0.2) 1.1–3.1 (2.4 ± 0.9)
IMTL 2.6 2.2 2.3 1.9 2.3 1.7 1.4 1.8 1.7 2.1 2.0 2.5 2.5 2.4 1.4–2.6 (2.0 ± 0.4) 2.0–2.5 (2.4 ± 0.2)
HL/SVL 0.35 0.36 0.37 0.35 0.34 0.38 0.38 0.38 0.37 0.35 0.36 0.37 0.38 0.39 0.30–0.40 (0.36 ± 0.01) 0.4–0.4 (0.38 ± 0.01)
HW/SVL 0.34 0.34 0.37 0.33 0.31 0.32 0.34 0.34 0.36 0.34 0.34 0.33 0.34 0.33 0.31–0.37 (0.34 ± 0.02) 0.33–0.34 (0.34 ± 0.01)
HL/HW 1.02 1.05 1.02 1.07 1.12 1.18 1.10 1.13 1.01 1.05 1.07 1.13 1.13 1.18 1.01–1.18 (1.08 ± 0.06) 1.07–1.18 (1.13 ± 0.05)
SL/SVL 0.14 0.17 0.14 0.15 0.14 0.15 0.13 0.15 0.17 0.15 0.13 0.15 0.15 0.15 0.13–0.17 (0.15 ± 0.01) 0.13–0.15 (0.15 ± 0.01)
SL/HL 0.40 0.47 0.37 0.42 0.41 0.40 0.35 0.40 0.46 0.41 0.37 0.39 0.38 0.40 0.35–0.47 (0.41 ± 0.04) 0.37–0.4 (0.39 ± 0.01)
IOD/HW 0.17 0.22 0.20 0.23 0.27 0.20 0.22 0.21 0.19 0.22 0.26 0.24 0.23 0.25 0.17–0.27 (0.21 ± 0.03) 0.23–0.26 (0.25 ± 0.01)
IND/IOD 1.19 1.00 1.13 1.00 1.09 1.09 1.11 1.03 1.09 1.10 0.85 0.91 0.98 0.94 1.00–1.19 (1.08 ± 0.06) 0.85–0.98 (0.92 ± 0.05)
ED/TD 1.59 1.49 1.79 1.59 1.91 1.41 1.37 1.42 1.70 1.56 1.33 1.55 1.33 1.44 1.37–1.91 (1.58 ± 0.17) 1.33–1.55 (1.41 ± 0.11)
ED/HL 0.32 0.31 0.30 0.34 0.33 0.28 0.30 0.29 0.33 0.30 0.31 0.28 0.29 0.31 0.28–0.34 (0.31 ± 0.02) 0.28–0.31 (0.30 ± 0.02)
TD/HL 0.20 0.21 0.17 0.21 0.17 0.20 0.22 0.20 0.20 0.19 0.23 0.18 0.22 0.22 0.17–0.22 (0.20 ± 0.02) 0.18–0.23 (0.21 ± 0.02)
HAL/SVL 0.25 0.28 0.27 0.26 0.27 0.29 0.26 0.29 0.28 0.27 0.29 0.26 0.27 0.28 0.25–0.29 (0.27 ± 0.01) 0.26–0.29 (0.28 ± 0.01)
HLL/SVL 1.95 2.14 2.01 2.05 1.99 2.17 2.17 2.08 2.20 1.94 1.95 1.97 2.08 2.08 1.94–2.20 (2.07 ± 0.10) 1.95–2.08 (2.02 ± 0.07)

© Zoological Systematics, 47(4): 275–292 A new species of Rana from China 289
(vs. absent), relative finger lengths I
<
II
<
IV
<
III (vs. II
<
IV
<
I
<
III), relative toe lengths I
<
II
<
III
<
V
<
IV (vs. I
<
II
<
V
<
III
<
IV), one inverted V-shaped markings on the dorsal surface (vs. absent), supernumerary tubercles present only below the
base of fingers III and IV (vs. absent), toe webbing formula I 1½–2 II 1⅓–2⅔ III 2–2⅔ IV 2⅔–1 V (vs. I 2–1 II 2+–1+ III 3–
2 IV 2–2+ V), black edge intermittently present on the anterior two-thirds of both sides of the dorsolateral fold (vs. golden),
nuptial pad divided into three parts (vs. two parts); from R. jiemuxiensis by head length equal to head width, HL/HW 1.01
in males and 1.00 in females (vs. slightly smaller, HL/HW 0.96 in males and females), one inverted V-shaped markings on
the dorsal surface (vs. absent), dorsolateral fold extending straight from posterior margin of the upper eyelid to above the
groin (vs. slightly curved above the tympanum), relative finger lengths I
<
II
<
IV
<
III (vs. II
<
IV
<
I
<
III), nuptial pad dark
gray-blackish and divided into three parts (vs. gray and divided into two parts), two outer metacarpal tubercles distinctly
separated (vs. merged at base); from R. longicrus by head length equal to head width, HL/HW 1.01 in males and 1.00 in
females (vs. larger, HL/HW 1.18 in males and 1.07 females), dorsolateral fold extending straight from posterior margin of
the upper eyelid to above the groin (vs. slightly curved above the tympanum), relative finger lengths I
<
II
<
IV
<
III (vs. II
<
I
<
IV
<
III), supernumerary tubercles present only below the base of fingers III and IV (vs. absent), toe webbing formula I
1½–2 II 1⅓–2⅔ III 2–2⅔ IV 2⅔–1 V (vs. I 1⅔–2⅓ II 1½–2⅔ III 1⅔–3½ IV 3⅓–1½ V); from R. zhenhaiensis by head length
equal to head width, HL/HW 1.01 in males and 1.00 in females (vs. slightly larger, HL/HW 0.98 in males and females),
internarial distances equal to interorbital distances, IND/IOD 1.04 in males and females (vs. significantly smaller, IND/IOD
0.58 in males and females), dorsolateral fold extending straight from posterior margin of the upper eyelid to above the groin
(vs. slightly curved above the tympanum), relative finger lengths I
<
II
<
IV
<
III (vs. II
<
IV
<
I
<
III), relative toe lengths I
<
II
<
III
<
V
<
IV (vs. I
<
II
<
V
=
III
<
IV), two outer metacarpal tubercles distinctly separated (vs. merged at the base), tibiotarsal
articulation reaching forward beyond tip of the snout (vs. reaching to the nostril); from R. culaiensis by head length equal to
head width, HL/HW 1.01 in males and 1.00 in females (vs. larger, HL/HW 1.40 in males), dorsolateral fold extending straight
from posterior margin of the upper eyelid to above the groin (vs. slightly curved to temporal fold above tympanum),
tibiotarsal articulation reaching forward far beyond tip of the snout (vs. reaching to the nostril), one inverted V-shaped
markings on the dorsal surface (vs. absent), nuptial pad dark gray-blackish and divided into three parts (vs. nuptial pad dark
brown and divided into two parts); from R. chevronta by head length equal to head width, HL/HW 1.01 in males and 1.00
in females (vs. larger, HL/HW 0.97 in males and 0.96 in females), internarial distances equal to interorbital distances,
IND/IOD 1.04 in males and females (vs. larger, IND/IOD 1.18 in males and 1.38 females), relative finger lengths I
<
II
<
IV
<
III (vs. II
<
IV
<
I
<
III), relative toe lengths I
<
II
<
III
<
V
<
IV (vs. I
<
II
<
V
<
III
<
IV), nuptial pad dark gray-blackish and
divided into three parts (vs. nuptial pad purplish gray, undivided, and complete).
Distribution. China (Guizhou).
Ecology. Currently, the new species is known only from the type locality, Guiguo Town, at elev. 645–728
m. The new
species has only been found in or near a pool of water outside a cave in an area located far from the village. After three years of
survey data showed that adults and egg (Fig. 5D) were collected and discovered only from late July to mid-August each year, we
presumed that this was their breeding season. Therefore, we speculated that the breeding period begins in late June and lasts until
about mid-August.
4 Discussion
In this study, a group of specimens of the genus Rana sensu lato were collected and described as a new species based
on morphological data and genetic markers, namely mitochondrial and nuclear genes. The reconstructed phylogenetic tree
obtained tree topologies that are inconsistent with recent studies (Wan et al., 2020; Wu et al., 2021), but all support the
monophyly of R. japonica group, R. chensinensis group, R. amurensis group, and R. johnsi group within the subgenus Rana
(Figs 2–3). This inconsistent topology may result from the number and type of genetic markers and evolutionary models, as
different genes may have different rates of evolution (Degnan & Rosenberg, 2009) and phylogenetic analyses may yield
conflicting results. As a result, phylogenetic studies using a certain number of mitochondrial and nuclear genes can help to
resolve the phylogeny and taxonomy of the genus Rana (Yuan et al., 2016).
Morphologically, the new species has morphological characteristics that distinguish it from all species in the R. japonica
group (see comparison section for details). Genetically, within the R. japonica group, the genetic differences between the
three species R. culaiensis, R. zhenhaiensis and R. longicrus are closer than the others (0.5%–1.1% for 16S, 2.7%–6.4% for
Cyt b), but the validity of these three species has been supported by morphological examination (Li et al., 2008; Fei et al.,
2009b, 2012). Genetic variation between the new species and its close relative R. chaochiaoensis was 1.1% for 16S and
12.5% for Cyt b, greater than that between R. culaiensis and R. zhenhaiensis (0.5% for 16S, 2.7% for Cyt b), and between

290 Yan et al.
R. zhenhaiensis and R. longicrus (0.7% for 16S, 6.4% for Cyt b) (Tables 2–3). Hence, the combined morphology and genetics
confirmed the validity of the new species.
Currently, six species of the genus Rana sensu lato are distributed in Guizhou Province, namely P. weiningensis, R.
omeimontis, R. chaochiaoensis, R. zhenhaiensis, R. hanluica, R. culaiensis, and R. dabieshanensis (Wu et al., 1986; Fei et
al., 2009b, 2012; Zhang et al., 2010; Chen et al., 2017; Xiao et al., 2019; Cheng et al., 2021), but the detailed geographical
distribution of these species is unclear. A clear species diversity list can help provide important basic data for making
scientific decisions concerning protected area construction, ecological conservation, and species diversity. However, nobody
has systematically studied the species of Rana recorded in Guizhou Province. Therefore, the range and the validity of the
distribution records of these six species in Guizhou need to be further studied.
Funding This work was supported by the programs of the Guizhou Province Top Discipline Construction Program Project
(Qianjiao Keyan Fa [2019]125), the Postgraduate Education Innovation Programme of Guizhou Province (Qianjiaohe
YJSKYJJ [2021]091), the Strategic Priority Research Program B of the Chinese Academy of Sciences (CAS) (XDB
31000000), the National Animal Collection Resource Center, China (2005DKA21402), the Application of Amphibian
Natural Antioxidant Peptides as Cosmetic Raw Material Antioxidants (QKZYD [2020]4002).
Acknowledgements We thank Xingrui Zhao, Xingliang Wang, Huan He, Kai Gao, Xiangdi Fan, and others for their help
with sample collection. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this
manuscript.
References
AmphibiaChina. 2022. The database of Chinese amphibians. Kunming Institute of Zoology (CAS), Kunming, Yunnan, China. Available
from http://www.amphibiachina.org/ (accessed 30 September 2022).
AmphibiaWeb. 2022. AmphibiaWeb. University of California, Berkeley, CA, USA. Available from http://amphibiaweb.org (accessed 30
September 2022).
Bandelt, H.J., Forster, P., Röhl, A. 1999. Median-joining networks for inferring intraspecific phylogenies. Molecular Biology and
Evolution, 16(1): 37–48. doi: 10.1093/oxfordjournals.molbev.a026036
Baele, G., Lemey, P., Bedford, T., Rambaut, A., Suchard, M.A., Alekseyenko, A. V. 2012. Improving the accuracy of demographic and
molecular clock model comparison while accommodating phylogenetic uncertainty. Molecular Biology and Evolution, 29(9): 2157–
2167. doi: 10.1093/molbev/mss084
Bedriaga, J.V. 1898. Amphibien und Reptilien. Wissenschaftliche Resultate der von N. M. Przewalski nach Central-Asien unternommenen
Reisen, & c.-Nauchnuie Rezul’tatui puteshestvii N. M. Przheval’skagho po tzentral’noi Azii, & c. Volume 3, Zoologischer Theil, Part
1. Akadamie der Wissenschaften, St. Petersburg. pp. 71–278.
Boulenger, G.A. 1886. Notes sur les grenouilles rousses d’Asie. Bulletin de la Société Zoologique de France, 11: 595–600. doi: 10.5962/
bhl.part.24010
Boulenger, G.A. 1909. Descriptions of four new frogs and a new snake discovered by Mr. H. Sauter in Formosa. Annals and Magazine of
Natural History Series, 8(4): 492–495. doi: 10.1080/00222930908692704
Che, J., Pang, J.F., Zhao, E.M., Matsui, M., Zhang, Y.P. 2007. Phylogenetic relationships of the Chinese brown frogs (genus Rana)
inferred from partial mitochondrial 12S and 16S rRNA gene sequences. Zoological Science, 24: 71–80. doi: 10.2108/zsj.24.71
Chen, J.J., Gu, D.H., Hou, D.H., Song, Z.H. 2017. Hanlui brown frog (Rana hanluica) found in Guizhou Province. Chinese Journal of
Zoology, 52(1): 155–157. doi: 10.13859/j.cjz.201701020
Cheng, Y.L., Liu, J., Chen, L., Lei, S.R., Zou, J.W., Zhang, X., Li, S.Z. 2021. Identification of Rana dabieshanensis in Tongzi, Guizhou
Province. Sichuan Journal of Zoology, 40(1): 59–65. doi: 10.11984/j.issn.1000-7083.20200206
Cope, E.D. 1865. Sketch of the primary groups of Batrachia Salientia. Natural History Review, 5: 97–120.
David, A. 1875. Journal de mon Troisième Voyage d’Exploration dans l’Empire Chinoise. Vol. 1. Hachette et cie, Paris. 383pp. doi:
10.5962/bhl.title.118647
Degnan, J.H., Rosenberg, N.A. 2009. Gene tree discordance, phylogenetic inference and the multispecies coalescent. Trends in Ecology
and Evolution, 24(6): 332–340. doi: 10.1016/j.tree.2009.01.009
Drummond, A.J., Suchard, M.A., Xie, D., Rambaut, A. 2012. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular
Biology and Evolution, 29(8): 1969–1973. doi: 10.1093/molbev/mss075
Dubois, A. 1992. Notes sur la classification des Ranidae (Amphibiens, Anoures). Bulletin Mensuel de la Société Linnéenne de Lyon, 61:
305–352. doi: 10.3406/linly.1992.11011

© Zoological Systematics, 47(4): 275–292 A new species of Rana from China 291
Edgar, R.C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32(5): 1792–
1797. doi: 10.1093/nar/gkh340
Fei, L., Ye, C.Y., Huang, Y.Z. 1990. Key to Chinese Amphibians. Chongqing Branch Science Technol Literature Press, Chongqing. 340pp.
Fei, L., Ye, C.Y., Huang, Y.Z., Jiang, J.P., Xie, F. 2005. An Illustrated Key to Chinese Amphibians. Sichuan Publishing House of Science
and Technology,Chengdu. pp. 102–116.
Fei, L., Hu, S.Q., Ye, C.Y., Huang Y.Z. 2009a. Fauna Sinica. Amphibia Vol. 2 Anura. Science Press, Beijing. pp. 33–35.
Fei, L., Hu, S.Q., Ye, C.Y, Huang, Y.Z. 2009b. Fauna Sinica. Amphibia Vol. 3 Anura: Ranidae. Science Press, Beijing. pp. 969–1042.
Fei, L., Ye, C.Y., Jiang, J.P. 2010. Phylogenetic systematics of Ranidae. In: Ji, X. (ed.), Herpetologica Sinica. Southeast University Press,
Nanjing. pp. 1–43.
Fei, L., Ye, C.Y., Jiang, J.P. 2012. Colored Atlas of Chinese Amphibians and Their Distributions. Sichuan Publishing House of Science
and Technology, Chengdu. pp. 78–96.
Frost, D.R. 2022. Amphibian Species of the World: an Online Reference. Version 6.1. American Museum of Natural History, New York,
USA. Available from https://amphibiansoftheworld.amnh.org (accessed 30 September 2022). doi: 10.5531/db.vz.0001
Frost, D.R., Grant, T., Faivovich, J., Bain, R.H., Haas, A., Haddad, C.F.B., de Sá, R.O., Channing, A., Wilkinson, M., Donnellan, S.C.,
Raxworthy, C.J., Campbell, J.A., Blotto, B.L., Moler, P.E., Drewes, R.C., Nussbaum, R.A., Lynch, J.D., Green, D.M., Wheeler, W.C.
2006. The amphibian tree of life. Bulletin of the American Museum of Natural History, 297: 1–370. doi: 10.1206/0003-0090(2006)
297[0001:TATOL]2.0.CO;2
Grummer, J.A., Bryson, Jr,R.W., Reeder, T.W. 2014. Species delimitation using Bayes factors: simulations and application to the
Sceloporus scalaris species group (Squamata: Phrynosomatidae). Systematic Biology, 63(2): 119–133. doi: 10.1093/sysbio/syt069
Günther, A.C.L.G. 1876. Description of a new frog from north-eastern Asia. Annals and Magazine of Natural History, Series, 4, 17(101):
387–387. doi: 10.1080/00222937608681973
Hoang, D.T., Chernomor, O., von Haeseler, A., Minh, B.Q., Vinh, L.S. 2018. UFBoot2: improving the ultrafast bootstrap approximation.
Molecular Biology and Evolution, 35(2): 518–522. doi: 10.1093/molbev/msx281
Hu, S.Q. 1978. Three new amphibian species in China. In: Hu, S.Q., Fei, L., Ye, C.Y. (eds), Materials for Herpetological Research, 4.
Chengdu Institute of Biology, Chengdu. p. 20.
Jiang, J.P., Xie, F., Li, C., Wang, B. 2020. Species Catalogue of China. Vol. 2. Animals, Vertebrates (IV), Amphibians. Science Press,
Beijing. 129pp.
Kumar, S., Stecher, G., Tamura, K. 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular
Biology and Evolution, 33(7): 1870–1874. doi: 10.1093/molbev/msw054
Lanfear, R., Frandsen, P.B., Wright, A.M., Senfeld, T., Calcott, B. 2017. PartitionFinder 2: new methods for selecting partitioned models
of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution, 34(3); 772–773. doi:
10.1093/molbev/msw260
Leigh, J.W., Bryant, D. 2015. POPART: full-feature software for haplotype network construction. Methods in Ecology and Evolution,
6(9): 1110–1116. doi: 10.1111/2041-210X.12410
Li, P.P., Lu, Y.Y., Li, A. 2008. A new species of brown frog from Bohai, China. Asiatic Herpetological Research, 11: 62–70.
Liu, C.C. 1946. A new woodfrog Rana chaochiaoensis with a discussion of its allied species, from West China. Journal of the West China
Border Research Society, Series B, 16: 7–14.
Liu, C.C. 1950. Amphibians of western China. Fieldiana. Zoology Memoires, 2: 1–397. doi: 10.5962/bhl.part.4737
Liu, C.C., Hu, S.Q. 1962. A herpetological report of Kwangsi. Acta Zoologica Sinica, 14(sup): 73–104.
Lu, Y.Y., Li, P.P., Jiang, D.B. 2007. A new species of Rana (Anura, Ranidae) from China. Acta Zootaxonomica Sinica, 32(4): 792–801.
Nguyen, L.T., Schmidt, H.A., von Haeseler, A., Minh, B.Q. 2015. IQ-TREE: a fast and effective stochastic algorithm for estimating
maximum-likelihood phylogenies. Molecular Biology and Evolution, 32(1): 268–274. doi: 10.1093/molbev/msu300
Nikolskii, A.M. 1918. Fauna of Russia and Adjacent Countries. Amphibia. Russian Academy of Sciences, Petrograd. 284pp.
Nilsson, S. 1842. Skandanavisk Herpetologi eller Beskrifning öfver de Sköldpaddor, Odlor, Ormar och Grodor, som Förekomma I Sverige
Och Norrige, Hemte Deras Lefnadssätt, Födöamnen, Nytta och skada m.m. Tryckt uti Borlingska Boktryckeriet, Lund. 140pp. doi:
10.5962/bhl.title.43795
Okada, Y. 1928. Frogs in Korea. Chosen Natural History Society Journal, 6: 15–46.
Pyron, R.A., Wiens, J.J. 2011. A large-scale phylogeny of Amphibia including over 2800 species, and a revised classification of advanced
frogs, salamanders, and caecilians. Molecular Phylogenetics and Evolution, 61: 543–583. doi: 10.1016/j.ympev.2011.06.012
Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A., Huelsenbeck, J.P.
2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology,
61(3): 539–542. doi: 10.1093/sysbio/sys029
Shen, Y.H. 1986. A new ranid species (Rana sangzhiensis) from Hunan. Acta Herpetologica Sinica, New Series 5(4): 290–294.
Shen, Y.H., Jiang, J.P., Yang. D.D. 2007. A new species of the genus Rana—Rana hanluica sp. nov. from Hunan Province, China (Anura:
Ranidae). Acta Zoologica Sinica, 53(3): 481–488.
Shen, H.J., Xu, M.Y., Yang, X.Y., Chen, Z., Xiao, N.W., Chen, X.H. 2022. A new brown frog of the genus Rana (Anura, Ranidae) from
north China, with a taxonomic revision of the R. chensinensis species group. Asian Herpetological Research, 13(3): 145–158. doi:
10.16373/j.cnki.ahr.210062

292 Yan et al.
Smith, M.A. 1921. New or little-known reptiles and batrachians from southern Annam (IndoChina). Proceedings of the Zoological Society
of London, 1921: 423–440. doi: 10.1111/j.1096-3642.1921.tb03271.x
Stejneger, L. 1898. On a collection of batrachians and reptiles from Formosa & adjacent islands. Journal of the College of Science,
Imperial University, Japan, 12: 215–225.
Wu, L., Dong, Q., Xu, R.H. 1986. Amphibians of Guizhou Province. Guizhou People Press, Guiyang. pp. 108–112.
Wan, H., Lyu, Z.T., Qi, S., Zhao, J., Li, P.P., Wang, Y.Y. 2020. A new species of the Rana japonica group (Anura, Ranidae, Rana) from
China, with a taxonomic proposal for the R. johnsi group. ZooKeys, 942: 141–158. doi: 10.3897/zookeys.942.46928
Wang, C., Qian, L., Zhang, C., Guo, W., Pan, T., Wu, J., Wang, H., Zhang, B.W. 2017. A new species of Rana from the Dabie Mountains
in eastern China (Anura, Ranidae). ZooKeys, 724: 135–153. doi: 10.3897/zookeys.724.19383
Wang, K., Ren, J.L., Chen, H.M., Lyu, Z.T., Guo, X.G., Jiang, K., Chen, J.M., Li, J.T., Guo, P., Wang, Y.Y., Che, J. 2020. The updated
checklists of amphibians and reptiles of China. Biodiversity Science, 28(2): 189–218. doi: 10.17520/biods.2019238
Wu, Y., Shi, S., Zhang, H., Chen, W., Cai, B., Hoang, V.C., Wu, J., Wang, B. 2021. A new species of the genus Rana sensu lato Linnaeus,
1758 (Anura, Ranidae) from Wuyi Mountain, Fujian Province, China. ZooKeys, 1065: 101–124. doi: 10.3897/zookeys.1065.67005
Xiao, N., Luo, Q.H., Luo, T., Wei, F., Zeng, Y.J. 2019. A new record species of Rana longicrus species group in Guizhou, Rana culaiensis.
Sichuan Journal of Zoology, 38(6): 616–622.
Xie, W., Lewis, P.O., Fan, Y., Kuo, L., Chen, M.H. 2011. Improving marginal likelihood estimation for Bayesian phylogenetic model
selection. Systematic Biology, 60(2): 150–160. doi: 10.1093/sysbio/syq085
Yan, F., Jiang, K., Chen, H., Fang, P., Jin, J., Li, Y., Wang, S.N., Murphy, R.W., Che, J., Zhang, Y. 2011. Matrilineal history of the Rana
longicrus species group (Rana, Ranidae, Anura) and the description of a new species from Hunan, southern China. Asian
Herpetological Research, 2(2): 61–71. doi: 10.3724/SP.J.1245.2011.00061
Yang, B.T., Zhou, Y., Min, M.S., Matsui, M., Dong, B.J., Li, P.P., Fong, J.J. 2017. Diversity and phylogeography of Northeast Asian
brown frogs allied to Rana dybowskii (Anura, Ranidae). Molecular Phylogenetics and Evolution, 112: 148–157. doi: 10.1016/j.ympev.
2017.04.026
Ye, C.Y., Fei, L., Hu, S.Q. 1993. Rare and Economic Amphibians of China. Sichuan Publishing House of Science and Technology,
Chengdu. 412pp.
Ye, C.Y., Fei, L., Matsui, M. 1995. Taxonomic studies of Chinese Rana japonica Guenther. In: Amphibian Reptile Branch of Chinese
Zoology Society & Zunyi Medical University (eds), Acta Herpetologica Sinica, 4/5. Guizhou Science and Technology Publishing
House Co., Ltd., Guiyang. pp. 82–87.
Yuan, Z.Y., Zhou, W.W., Chen, X., Poyarkov, Jr.N.A., Chen, H.M., Jang-Liaw, N.H., Chou, W.H., Matzke, N.J., Iizuka, K., Min, M.S.,
Kuzmin, S.L., Zhang, Y.P., Cannatella, D.C., Hillis, D.M., Che, J. 2016. Spatiotemporal diversification of the true frogs (genus Rana):
a historical framework for a widely studied group of model organisms. Systematic Biology, 65(5): 824–842. doi: 10.1093/sysbio/
syw055
Zhang, L., Ran, H., Sheng, Z.X., Liang, L. 2010. A new record of amphibian Rana zhenhaiensis in Guizhou Province. Journal of Anhui
Agricultural Sciences 38(26): 14442. doi: 10.13989/j.cnki.0517-6611.2010.26.167
Zhang, J., Kapli, P., Pavlidis, P., Stamatakis, A. 2013. A general species delimitation method with applications to phylogenetic placements.
Bioinformatics, 29(22): 2869–2876. doi: 10.1093/bioinformatics/btt499
Zhao, H., Yang, J., Wang, C., Li, P., Murphy, R.W., Che, J., Yuan, Z. 2017. A new species of the genus Rana from Henan, central China
(Anura, Ranidae). ZooKeys, 694: 95–108. doi: 10.3897/zookeys.694.12513