We collected six specimens (three adult males and three adult females) of Pelophylax chosenicus from multiple localities of Seoul, South Korea and Shenyang, Dandong, China, six specimens (three adult males and three adult females) of P. plancyi were collected from four localities in China (Fig. 1) and six specimens (three adult males and three adult females) of P. hubeiensis were collected from Lichuan and Wuhan, China, including specimens from the vicinity of the type locality of the three species (detailed collection information presented in the Taxonomic account below). The specimens collected from South Korea were released after morphological data measuring and Non-Disruptive DNA Sampling. The DNA- sampling came from oral throat swab sampling. The specimens collected from China were fixed in 10% formalin and then stored in 75% ethanol. Liver tissue samples were collected from individual specimens before formalin fixation and preserved in 95% ethanol for subsequent molecular analyses. Specimens and tissue samples are preserved at Shenyang Agricultural University (SYAU).

Linear measurements were performed on all the adult specimens, using a Vernier caliper with a precision of 0.1 mm, with the following abbreviations: SVL (snout-vent length, from the tip of snout to vent); HL (head length, from posterior corner of the mandible to tip of snout); HW (head width, the greatest cranial width); SL (snout length, from the tip of snout to the anterior corner of the eye); INS (internasal space, the distance between the two nostrils); IOS (interorbital distance, the minimal distance between upper eyelids); ED (horizontal eye diameter); LAHL (length of lower arm and hand, from the tip of finger III to the elbow joint); TD (horizontal tympanic diameter); LAD (diameter of the lower arm); HLL (hind limb length, from the tip of toe IV to groin); TL (tibia length); TW (tibia width, the greatest width of tibia); FL (foot length, from the proximal end of the inner metatarsal tubercle to the tip of toe IV); and IMT (internal metatarsal tubercle, the length of internal metatarsal tubercle). The morphology comparisons are according to Zhou et al. (2022) for Pelophylax mongolius (Schmidt 1925) and Fei et al. (2009) for P. nigromaculatus (Hallowell, 1861), P. fukienensis (Pope, 1929) and P. terentievi (Mezhzherin, 1992). The toe webbing formula followed the protocol described by Savage (1975).

Measurement data were used for principal component analysis (PCA) on the morphometric differences amongst P. plancyi, P. chosenicus and P. hubeiensis. Statistical analyses were carried out by using the “prcomp” package in R 4.1.1 (Zhang et al. 2022).

Genomic DNA was extracted by Qiaprep Spin Miniprep kits (QiaGen) and a 508 bp mitochondrial genome fragment of 16S ribosomal RNA (16S) using primers L3975 and H4551 (Simon et al. 1994) was specifically amplified for this study. The standard PCR protocol was performed in a 20 µl reaction with at least 20 ng of template DNA and 10 pmol of primers. PCR conditions consisted of an initial denaturation for 5 min at 95 °C, followed by 25 cycles: denaturation at 95 °C for 30 sec, annealing at 54 °C for 60 sec, elongation at 72 °C for 90 sec and then finalised with an extension step of 10 min at 72 °C. Sequencing was conducted by Nanchang Kechang Biotech Co., Ltd. New sequences are uploaded to GenBank (Table 1).

For phylogenetic comparisons, corresponding sequences of 11 recognised species of the genus Pelophylax and one outgroup (Rana taihangensis Chen, 2022) were obtained from GenBank (Table 1). Sequences were assembled and aligned using MEGA7 (Tamura et al. 2013) with default settings and were further revised manually when necessary. Phylogenetic trees were estimated with Maximum Likelihood (ML) using RAxML NG v.0.9.0. (Kozlov et al. 2019) and Bayesian Inference (BI) using MrBayes 3.1.2 (Ronquist et al. 2012). We used JModelTest 2 (Guindon and Gascuel 2003; Darriba et al. 2012) to infer the model of sequence evolution that best fits our 16S sequences. The model HKY + I + G was selected as the best-fitted model for BI testing and HKY + F + R2 was selected as the best-fitted model for ML. All searches consisted of three heated chains and a single cold chain. Three independent iterations, each comprising two runs of 100,000,000 generations, were performed, sampling every 10,000 generations and parameter estimates were plotted against generation. The first 25% of the samples were discarded as burn-in, resulting in a potential scale reduction factor (PSRF) of < 0.005. Maximum likelihood analysis is completed through 1000 rapid guided repetitions. Pairwise distances (p-distance) were calculated in MEGA 6 using the uncorrected p-distance model.

The morphological comparisons of Pelophylax plancyi and P. chosenicus revealed similarities, our newly-collected specimens of P. plancyi (P) and P. chosenicus (C) both had large body sizes (SVL: 40.7–56.1 mm, n = 6 (P) vs. 41.1–60.1 mm, n = 6 (C)), head length slightly larger than head width (HL/HW: 1.00–1.03, n = 6 (P) vs. 1.01–1.04, n = 6 (C)), relatively short snouts (SL/SVL: 0.14–0.16, n = 6 (P) vs. 0.14–0.16, n = 6 (C)), tympanum diameter slightly smaller than the eye diameter (ED/TD: 1.02–1.09, n = 6 (P) vs. 1.00–1.15, n = 6 (C)), internal subgular vocal sacs and dorsolateral fold slightly thick. Furthermore, all of the newly-collected P. hubeiensis specimens can be distinguished from P. plancyi and P. chosenicus by the combination of the following characteristics: (1) head width slightly larger than head length, HL/HW 0.93–0.97 (n = 6) (vs. head length slightly larger than head width in P. plancyi and P. chosenicus, HL/HW 1.00–1.04, n = 12); (2) tympanum diameter slightly larger than the eye diameter ED/TD 0.86–0.97 (n = 6) (vs. tympanum diameter slightly smaller than the eye diameter, ED/TD 1.00–1.15, n = 12); (3) foot length shorter, FL/SVL 0.49–0.58 (n = 6) (vs. FL/SVL 0.55–0.78, n = 12); (4) silent sac (vs. internal subgular vocal sacs) (Table 2). The differences between P. plancyi, P. chosenicus, P. hubeiensis and other congeneric species distributed in China are summarised in Table 3.

Moreover, the PCA result indicated that the Pelophylax hubeiensis could be distinctly separated from P. plancyi and P. chosenicus, but overlapped between P. plancyi and P. chosenicus (Fig. 2). The first two extracted principal component axes accounted for 74.11% and 15.74% of the variation (Table 4). PC1 separated the sexes and the Interorbital distance, Horizontal tympanic diameter and Foot length are those loading strongly on PC2, were the most important morphological indexes affecting the morphological differences amongst the three species in PCA.

Figure 2. 

Plots of the first principal component (PC1) versus the second (PC2). Pelophylax chosenicus (red), P. plancyi (blue) and P. hubeiensis (green). M: Male; F: Female.

In this study, the topological structures of the Maximum Likelihood (ML) and Bayesian Inference (BI) trees are generally consistent (Fig. 3; Table 5). The genus Pelophylax forms a monophyletic group (Clade A), which is divided into two groups (Clades A1 and A2). In the gene tree, samples of Pelophylax plancyi and P. chosenicus (including topotype) form a supported monophyletic group (Clade B; Bayesian posterior probabilities 100, bootstrap supports 1.00) and small divergences (p-distance 0.0–0.8), which is further close to the P. nigromaculatus with significant support (Bayesian posterior probabilities 96, bootstrap supports 0.98). The samples of P. hubeiensis (including a topotype) also form a strongly-supported monophyletic group (Clade C; Bayesian posterior probabilities 100, bootstrap supports 0.99) far from clade B and with moderate divergence (p-distance 1.4–2.0).

Figure 3. 

Bayesian-inferenced topology of Pelophylax species, based on 16s rRNA sequences. BPP and BS values, respectively, occur at the nodes.

The combined evidence from morphology and molecular phylogeny suggested the specific distinction of Pelophylax hubeiensis that is distant from P. plancyi, while indicating the homogeneity between P. plancyi and P. chosenicus. Thus, we suggest that P. chosenicus is a junior synonym of P. plancyi and P. hubeiensis should be treated as a distinct species and we provide descriptions of these two separate species.

 Pelophylax plancyi (Lataste, 1880)

Rana plancyi – Lataste 1880; Boulenger (1920).

Rana nigromaculata coreana – Okada (1927, 1928).

Rana nigromaculata chosenica – Okada (1931).

Rana chosenica – Kuramoto (1983).

Rana plancyi plancyi – Kuramoto (1983).

Rana (Rana) plancyi – Dubois (1986).

Rana (Rana) chosenica – Dubois (1986).

Rana (Pelophylax) plancyi – Dubois (1992).

Rana (Pelophyxlax) chosenica – Dubois (1992).

Rana (Pelophylax) plancyi chosenica – Dubois and Ohler (1994).

Hylarana plancyi – Chen et al. (2005).

Hylarana chosenica – Chen et al. (2005).

Pelophylax chosenicus – Frost et al. (2006); Che et al. (2007); Zhou et al. (2022).

Type series

Syntypes : Not traced, two specimens presumably originally in Lataste’s personal collection and which were deposited ultimately in the BMNH; BMNH 1920.1.20.1020 is by museum records a syntype (Lataste 1880; Frost 2022). Neotype: CIB 79I1764, adult male, collected from Yongfeng, Beijing, China (Fei et al. 2009).

Specimens examined

Twelve newly-collected specimens (six adult males and six adult females): adult males SYAU BAA000035, SYAU BAA000072 and adult female SYAU BAA000073, from Nanshan Park, Jiujiang, Jiangxi, China (29.6698°N, 116.0057°E, 28 m a.s.l.) (type locality of Pelophylax plancyi); adult male HGSE 01 and adult female HGSE 02 from Liulin Wetland, Seoul, South Korea (37.5663°N, 126.9779°E, 45 m a.s.l.) (type locality of “P. chosenicus”); adult male SYAU BAA000046 and adult female SYAU BAA000016 from Puhe Park, Shenyang, Liaoning, China (41.5114°N, 122.7579°E, 30 m a.s.l.); adult male SYAU BAA000061 and adult female SYAU BAA000062 from Donggang District, Dandong, Liaoning, China (39.8260°N, 124.0526°E, 60 m a.s.l.); adult female SYAU BAA000074 from Yuzui Park, Nanjing, Jiangsu, China (31.9801°N, 118.6723°E, 26 m a.s.l.); adult female SYAU BAA000075 from Bailuhu Park, Binzhou, Shandong, China (37.4209°N, 118.1575°E, 55 m a.s.l.); adult male SYAU BAA000077 from Si River, Jining, Shandong, China (35.5338°N, 116.8349°E, 45 m a.s.l.).

Diagnosis

(1) large body size, SVL 40.7–59.6 mm in adult males (n = 16), SVL 55.2–70.5 mm in adult females (n = 16) (Fei et al. 2009); (2) head length slightly larger than head width; (3) maxillary gland pronounced; (4) tympanum diameter large, slightly smaller than the eye diameter; (5) loreal region oblique, concave; (6) nostril located dorsally, between the eye and the tip of snout; (7) vomerine teeth two small masses; (8) tongue pyriform, free-margin notched; (9) fingers with rudimentary webs; toes almost full webs, webbing formula I 0 -0- II 0-⅓ III 0 – ⅓ IV I ⅓ – 0 V; (10) heels only meeting.

Common names

“Green Pond Frog” in English / “金线侧褶蛙 (jīn xiàn cè zhě wā)” in Chinese.

Comparison

Pelophylax plancyi can be differentiated from other species in the Pelophylax nigromaculatus species group (P. nigromaculatus, P. terentievi and P. mongolius), based on the internal subgular vocal sacs in males (Fei et al. 2009). Pelophylax plancyi additionally differs from P. hubeiensis, based on its head slightly longer than wide, its tympanum slightly smaller than the eye diameter and longer foot (see Morphological Comparisons in the Results above). Pelophylax plancyi further differs from P. fukienensis, based on the dorsolateral fold wider and heels meeting (vs. dorsolateral fold narrower and heels meeting in P. fukienensis).

Colouration

The living specimens exhibit varying degrees of green, olive green or emerald green body colour variation, without spots on their backs, a tympanic membrane that was golden yellow or brown with a green margin and yellow eyelids. The dorsolateral fold ranged from green to golden yellow or light brown, with yellow at the ends and some minuscule irregular yellow spots. The legs were slightly lighter than the body, with brownish-yellow transverse stripes. The throat, chest and belly were light yellow. The backs of the elbows were yellow, with light brown cloudy spots behind the thighs. The ventral surface of the forelimbs and hind limbs were yellow. The nuptial pads were light grey (Fig. 4A, B).

Figure 4. 

Colouration of Pelophylax plancyi A. ♀ SYAU BAA000073; B. ♂ SYAU BAA000061 and P. hubeiensis; C. ♂ SYAU BAA000052; D. ♀ SYAU BAA000051.

In preservative, the dorsal surface turned dark grey without patches, while the dorsolateral fold and backline were light grey, the limbs were brown with black stripes, the ventral surface was flesh-coloured, the ventral surface of the limb was beige and the hand and toe webs were dark grey (Fig. 5).

Figure 5. 

Specimen (SYAU BAA000073) of Pelophylax plancyi. A. Dorsal view; B. Ventral view; C. Lateral view; D. Ventral view of the hand; E. Ventral view of the foot.

Sexual dimorphism

Male with a pair of internal subgular vocal sacs; in the breeding season, a single, light grey nuptial pad on the dorsal surface of finger I. Males slightly smaller than females with linea masculina.

Distribution and ecology

At present, specimens of Pelophylax plancyi have been identified in eastern China (except Hunan, Guangdong, Jilin and Heilongjiang) and the Korean Peninsula (Fei et al. 2009; Zhou et al. 2022). Specimens were collected from ponds full of aquatic plants (Fig. 7A, B), where they perch along the river bank during the night-time, although, when startled, they jump into the water.

 Pelophylax hubeiensis (Fei & Ye, 1982)

Rana hubeiensis – Fei and Ye (1982).

Hylarana hubeiensis – Chen et al. (2005).

Pelophylax plancyi – Dufresnes and Litvinchuk (2022).

Type series

Holotype : CIB 74I0570, adult male, SVL 43.7 mm, collected from Lichuan, Hubei, China (Fei and Ye 1982).

Specimens examined

Six newly-collected specimens (three adult males and three adult females): adult male SYAU BAA000052 and adult female SYAU BAA000053, from Qing river, Lichuan, Hubei, China (30.3465°N, 108.9988°E, 780 m a.s.l.) (type locality of Pelophylax hubeiensis); adult males SYAU BAA000049, SYAU BAA000050 and adult females SYAU BAA000048, SYAU BAA000051 from Jiefang Paek, Wuhan, Hubei, China (30.6141°N, 114.3045°E, 40 m a.s.l.).

Diagnosis

(1) large body size, males slightly smaller SVL 38.5–47.1 mm in adult males (n = 23), SVL 41.1–61.9 mm in adult females (n = 23) (Fei et al. 2009); (2) head width slightly larger than head length; (3) maxillary gland pronounced; (4) tympanum diameter large, slightly larger than the eye diameter; (5) loreal region oblique, concave; (6) nostril located dorsally, between the eye and the tip of snout; (7) vomerine teeth two small masses; (8) tongue long pyriform, free-margin notched; (9) fingers with rudimentary webs; toes almost full webs, webbing formula I 0- – ⅓ II 0- -⅓ III ⅓ – I⁺ IV I⁺ – 0- V; (10) heels not meeting.

Common names

“Hubei Gold-striped Pond Frog” in English / “湖北侧褶蛙 (hú běi cè zhě wā)” in Chinese.

Comparison

Pelophylax hubeiensis can be differentiated from all species in the Pelophylax genus, based on the silent sac in males (Fei et al. 2009).

Colouration

The living specimens exhibit green, olive green or light brown body colour variation, with some individuals exhibiting green flecks on their backs that were absent on other individuals, a tympanic membrane that was golden yellow or light brown and yellow eyelids. The dorsolateral fold ranged from yellow to light brown or golden yellow. The legs exhibit yellow, brown or light brown colour variation, with green or olive green markings. The throat, chest and belly were light yellow. The backs of the elbows were dark brown, with brown cloud spots behind the thighs. The ventral surface of the forelimbs and hind limbs were yellow. The nuptial pads were grey (Fig. 4C, D).

In preservative, the dorsal surface turned dark olive green with light black patches, while the dorsolateral fold and backline were brownish-yellow, the limbs were light brown with dark brown patches, the ventral surface was beige with black stripe patterns, the ventral surface of the limb was light yellow and the hand and toe webs were beige (Fig. 6).

Figure 6. 

Specimen (SYAU BAA000053) of Pelophylax hubeiensis. A. Dorsal view; B. Ventral view; C. Lateral view; D. Ventral view of the hand; E. Ventral view of the foot.

Sexual dimorphism

Males with a single, grey nuptial pad on the dorsal surface of finger I in the breeding season. Males slightly smaller than females with linea masculina.

Distribution and ecology

At present, specimens of Pelophylax hubeiensis have been identified in Henan, Hubei, Anhui, Hunan, Chongqing and Jiangxi, China (Fei et al. 2009). Specimens were collected from ponds with lotus flowers or aquatic plants (Fig. 7C, D), where they perch on the leaves of the aquatic plants during the night-time, although, when startled, they plunge underwater.

Figure 7. 

The habitat of Pelophylax plancyi (A) macrohabitats, (B) microhabitats and P. hubeiensis (C, D).