We report the first record of Limnonectes nguyenorum McLeod, Kurlbaum & Hoang, 2015 outside of Vietnam, namely from China, based on five specimens collected from Daweishan Nature Reserve, southern Yunnan, China. Morphologically, the records from China agree with those of L. nguyenorum from Vietnam, and they also phylogenetically clustered with strong support. In addition, based on the new records from China and the previous descriptions of L. nguyenorum from Vietnam, we provide an extended diagnosis of this species.

16S rRNA, Daweishan Nature Reserve, Honghe Prefecture, morphology, southern Yunnan

The fanged frog genus Limnonectes Fitzinger, 1843 is the most species rich genus of Asian frogs of the family Dicroglossidae which presently comprises 78 species distributed throughout East and Southeast Asia (Frost 2021). To date, six species have been reported from China (Wang et al. 2020; AmphibiaChina 2021), namely L. fragilis (Liu & Hu, 1973); L. liui (Yang, 1983); L. fujianensis Ye & Fei, 1994; L. bannaensis Ye, Fei & Jiang, 2007; L. taylori Matsui, Panha, Khonsue & Kuraishi, 2010; and L. longchuanensis Suwannapoom, Yuan, Chen, Sullivan & McLeod, 2016.

Limnonectes nguyenorum McLeod, Kurlbaum & Hoang, 2015 is a species which was described from Ha Giang Province, Vietnam, and then was recorded from Vinh Phuc (Ziegler et al. 2015), Son La (Pham et al. 2016), Lao Cai (Nguyen et al. 2019), Yen Bai (Le and Do 2019), and Tuyen Quang (Pham et al. 2019) provinces. At the time, this species was still believed to be endemic to northern Vietnam.

During our field survey in southern Yunnan, China, in 2019, some specimens of Limnonectes were collected from Daweishan Nature Reserve. Detailed morphological comparisons and molecular analyses indicated these specimens to be L. nguyenorum. Herein, we provide the first country record for China.

The field survey was conducted in Daweishan Nature Reserve under the permit of Honghe Prefecture Forestry and Grassland Bureau of Yunnan Province. Specimens were euthanized and then fixed in 75% ethanol for storage after taking photographs. Liver tissue samples were preserved in 99% ethanol for molecular analysis. All specimens were deposited at Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ).

Genomic DNA was extracted from liver tissues preserved in 99% ethanol using the standard phenol-chloroform extraction protocol (Sambrook et al. 1989). Partial fragments of the mitochondrial 16S rRNA were amplified for all samples via the polymerase chain reaction (PCR) using the primers 16SAR: 5’-CGCCTGTTTAYCAAAAACAT-3’ and 16SBR: 5’-CCGGTYTGAACTCAGATCAYGT-3’ (Palumbi et al. 1991). PCR cycling conditions followed Suwannapoom et al. (2021). The products were purified and sequenced by Tsingke Biotechnology (Beijing) Co., Ltd., using the same primers as in PCR. All new sequences were deposited in GenBank. Fejervarya limnocharis (Gravenhorst, 1829) and F. iskandari Veith, Kosuch, Ohler & Dubois, 2001 were chosen as outgroups according to Suwannapoom et al. (2021). Homologous and outgroup sequences were obtained from GenBank (Table 1). The technical computation methods for the sequences alignment, best substitution model selection, Bayesian inference and Maximum likelihood phylogenetic analyses were the same as those in Liu et al. (2021).

Measurements were taken with a digital caliper to the nearest 0.1 mm. Morphological terminology followed McLeod et al. (2015). Abbreviations of the morphometric traits are as follows: ED, eye diameter; EN, eye–nostril distance; FEL, thigh (femur) length; FOL, foot length; HL, head length; HW, head width; IN, internarial distance; IO, interorbital width; LAL, lower arm length; OH, odontoid height; PAL, palm length; RFL, relative finger length when digits are adpressed; RL, rostrum length, distance from the level of the anterior corner of the eye to the anteriormost point of the head; RTL, relative toe length when digits are adpressed; SVL, snout–vent length; TBL, shank (tibia) length; TD, tympanum diameter; and UEW, upper eyelid width.

The obtained sequence alignment is 900 bp in length. Bayesian Inference and Maximum Likelihood analyses showed consistent topology. The specimens collected from Daweishan Nature Reserve, southern Yunnan, China, clustered with the specimens (including type specimens) of Limnonectes nguyenorum from Vietnam with strong support by both Bayesian Inference and Maximum Likelihood analysis (Fig. 1). The genetic distance (uncorrected p-distance) between the specimens from China and the specimens (including type specimens) of L. nguyenorum from Vietnam was 1.1% (Table 2). Although morphological characters of the specimens from China showed slight variations (smaller body size and nuptial pad present only on first finger) from the original description (McLeod et al. 2015) of L. nguyenorum, they agree with the subsequent descriptions (Pham et al. 2016; Le and Do 2019; Nguyen et al. 2019; Pham et al. 2019) of L. nguyenorum from Vietnam. Therefore, we consider that the specimens from Daweishan Nature Reserve, southern Yunnan, China, belong to L. nguyenorum.

Figure 1. 

Bayesian Inference tree based on 900 bp 16S rRNA sequences. Numbers before slashes indicate Bayesian posterior probabilities (> 0.9 remain) and numbers after slashes indicate ultrafast bootstrap support for Maximum Likelihood analyses (> 90 remain).

Taxonomic account

 Limnonectes nguyenorum McLeod, Kurlbaum & Hoang, 2015

Figs 2, 3

Specimens examined

KIZ20193243–KIZ20193245, three adult males, and KIZ20193246–KIZ20193247, two adult females, all collected on 24 March 2019 from Daweishan Nature Reserve, Manhao Town, Gejiu City, Honghe Prefecture, Yunnan Province, China (23°1'21"N, 103°23'52"E; at an elevation of 500 m).

Morphological description

Morphometric and meristic data are presented in Table 3. Body size small (SVL 36.5–38.5 mm in males, 35.4–35.5 mm in females); head large (HL 42–44% SVL in males, 36–39% in females); head slightly wider than long in males (HL 92–94% HW), head length almost equal to width in females (HL 97–101% HW); canthus rostralis indistinct and rounded, lores flat; supratympanic fold distinct; tympanum not visible in males and indistinct in females; odontoid processes in males robust with rounded tips, angled posteriorly, odontoid processes present but very short in females.

Table 3.

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Measurements (in mm) and proportions of the specimens of Limnonectes nguyenorum from Yunnan, China. For abbreviations see Materials and methods.

	KIZ20193243	KIZ20193244	KIZ20193245	KIZ20193246	KIZ20193247
	male	male	male	female	female
SVL	37.1	36.5	38.5	35.5	35.4
ED	4.7	4.5	5.0	4.4	4.7
EN	3.4	3.4	3.2	2.9	3.0
RL	6.3	6.2	6.7	5.3	5.7
FEL	18.3	18.5	19.2	16.9	17.9
FOL	25.6	24.9	26.3	23.2	24.9
HL	15.6	15.5	17.1	12.9	13.8
HW	16.8	16.7	18.1	13.3	13.7
IN	3.6	3.8	3.9	3.2	3.1
IO	3.3	2.9	3.5	2.6	2.6
LAL	7.1	7.3	7.9	6.2	7.2
PAL	9.2	9.3	10.4	8.3	8.8
RFL	III>IV>II>I	III>IV>II>I	III>IV>II>I	III>IV>II>I	III>IV>I>II
RTL	IV>III>V>II>I	IV>III>V>II>I	IV>III>V>II>I	IV>III>V>II>I	IV>III>V>II>I
TBL	17.1	16.8	18.1	15.1	16.9
TD	2.2	2.3	2.4	2.3	2.3
UEW	2.9	2.5	2.8	2.8	2.8
OH	2.7	2.2	2.8	1.4	1.5
PAL/LAL	1.26	1.22	1.28	1.30	1.19
PAL/SVL	0.25	0.25	0.27	0.23	0.25
ED/HL	0.30	0.29	0.29	0.34	0.34
HL/HW	0.92	0.93	0.94	0.97	1.01
HL/SVL	0.42	0.42	0.44	0.36	0.39
TBL/SVL	0.46	0.46	0.47	0.43	0.48
FEL/SVL	0.49	0.51	0.50	0.48	0.51
LAL/SVL	0.19	0.20	0.21	0.17	0.20
FOL/SVL	0.69	0.68	0.68	0.65	0.70

Limbs robust, relatively short. Finger tips rounded, relative length of fingers usually III>IV>II>I, rarely III>IV>I>II; no webbing on fingers; nuptial pad on finger I present in males. Toe tips rounded, relative length of toes IV>III>V>II>I. Tibia short (TBL 43–48% SVL), slightly shorter than femur. Full webbing between toes, inner metatarsal tubercle oval, a skin ridge from inter metatarsal to tarsus present.

Skin on top of the head and venter smooth, skin on dorsum, flanks, and dorsal surface of forelimbs with sparse tubercles; precloacal area and dorsal surface of hindlimbs covered with heterogeneous dense tubercles.

Coloration

Head yellow–brown with brownish black bar on posterior interorbital region, yellowish white bar extending from nares to insertion of arm, upper lip with distinct white spots and dark brown bars, dorsum and dorsal surfaces of limbs greyish yellow–brown, supratympanic fold brownish black, lateral sides greyish, ventral surface of throat and chest white with black marbling, ventral surface of limbs and abdomen white, dorsal surface of thighs and tibias with brownish black bars; iris reddish brown in upper half and greyish white in lower half.

Figure 2. 

Specimens of Limnonectes nguyenorum from Daweishan Natural Reserve, southern Yunnan, China, in preservative. A. Dorsal view; B. Ventral view.

Extended diagnosis

Based on the newly collected specimens from China and the descriptions (McLeod et al. 2015; Pham et al. 2016; Le and Do 2019; Nguyen et al. 2019; Pham et al. 2019) of L. nguyenorum from Vietnam, we extend the diagnosis of this species: (1) adult male SVL 36.5–47.3 mm, adult female SVL 35.4–47.1 mm; (2) males with nuptial pads on first finger or first and second fingers; (3) males with slightly enlarged heads (HL 37–46% of SVL in males; 36–43% in females); (4) head slightly longer than wide or slightly wider than long in males(HL 92–112% HW), head slightly longer than wide or length almost equal to width in females (HL 97–116% HW); (5) canthus rostralis indistinct and rounded, lores flat; (6) supratympanic fold distinct or indistinct; (7) tympanum indistinct (or not visible); (8) white or yellow bar extending from nares to insertion of arm, upper lip with distinct white spots and dark brown bars; (9) skin on top of head and venter smooth, skin on dorsum and flanks very feebly crenulate; (10) pericloacal area, and dorsal surfaces of shank and foot covered with heterogeneous tubercles; (11) toes fully webbed; (12) relative finger length when adpressed: III>IV>II>I or III>IV>I>II.

Figure 3. 

Limnonectes nguyenorum from Daweishan Natural Reserve, southern Yunnan, China, in life. A. Dorsal view of the adult male (KIZ20193245); B. Ventral view of the adult male (KIZ20193245); C. Dorsal view of the adult female (KIZ20193246); D. Ventral view of the adult female (KIZ20193246).

Ecological notes

The specimens were found at night in the artificial drainage ditch along a road; there are primary forests on both sides of the road, and there was shallow water flowing in the ditch. No eggs or tadpoles were found.

Limnonectes nguyenorum was known previously only from northern Vietnam (Frost 2021). This is the first record of L. nguyenorum from China and thus from outside of Vietnam. The new locality in China is approximately 150 km away from the type locality in Vietnam (Fig. 4). Our work brings the total species number of the genus Limnonectes in China to seven.

Figure 4. 

Map showing the known distribution of Limnonectes nguyenorum in Vietnam (red dots) and in China (black dot).

Two new species of amphibians and reptiles have been recently described from Daweishan Nature Reserve (Liu et al. 2021; Zhang et al. 2021) and the diversity of amphibians and reptiles in this area is still underestimated. Further study is necessary to document the true amphibian and reptile diversity of Daweishan Nature Reserve.

We would like to thank Decai Ouyang, Dangshou Yang, Fanjing Yang, and Dazhi Dong for assistance in the field. Thanks also to our colleagues for their help and advice. We also thank the reviewers for their valuable comments on the manuscript. This work was supported by Science-Technology Basic Condition Platform from the Ministry of Science and Technology of the People’s Republic of China (Grant No. 2005DKA21402), and the project of Ministry of Ecology and Environment of China: Investigation and assessment of amphibians and reptiles in southern Yunnan.