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A new species of Nanorana Günther, 1896 (Anura, Dicroglossidae) from Yunnan, China
expand article infoShuo Liu, Peisong Zhang§, Dingqi Rao|
‡ Kunming Natural History Museum of Zoology, Kunming, China
§ Research Institute of Xishuangbanna National Nature Reserve, Jinghong, China
| Kunming Institute of Zoology, Kunming, China
Open Access

Abstract

A new species of Nanorana Günther, 1896 is described from Yunnan Province, China, based on morphological and molecular evidence. Morphologically, Nanorana xuelinensis sp. nov. is distinguished from its congeners by a combination of the following diagnostic characters: body size large; adult males with keratinized spines on chest, belly, lateral body, posterior dorsum, buttocks, outer side of the fore limbs, the inner metacarpal tubercle, fingers I and II, and upper eyelids; no spines on the inner side of the lower and upper arm; forelimbs strongly hypertrophied in adult males; anterior dorsum skin smooth; dorsolateral folds absent; finger I longer than finger II; webbing deeply incurved between tips of toes; present outer metacarpal tubercle and absent outer metatarsal tubercle. The new species is separated from all other congeners by uncorrected genetic distances ranging from 5.2% to 7.3% based on mitochondrial 16S rRNA gene and ranging from 3.9% to 7.6% based on mitochondrial 12S rRNA gene.

Keywords

12S, 16S, morphology, phylogeny, spiny frog, systematic, taxonomy

Introduction

The tribe Paini is a widespread, complex taxon, and there are many different views on the classification of this taxon. Dubois (1992) first proposed the tribe Paini to include the genera Paa Dubois, 1975 and Chaparana Bourret, 1939. Roelants et al. (2004) suggested that Nanorana Günther, 1896 is imbedded within Paa on the basis of molecular data. Jiang et al. (2005) presented that Quasipaa Dubois, 1992 is a distinct genus in the tribe Paini. Chen et al. (2005) placed Chaparana, Paa, and Nanorana into Nanorana on the basis that Paa is paraphyletic with respect to Nanorana and Chaparana. Ohler and Dubois (2006) described two new genera in the tribe Paini, namely Allopaa Ohler & Dubois, 2006 and Chrysopaa Ohler & Dubois, 2006. Che et al. (2010, 2020) considered that the high elevation species of Nanorana represent dwarfed and degraded ones derived from lower elevation Paa on the basis of evidence of molecular phylogeny. Dubois et al. (2021) presented a different classification of the tribe Paini that included more genera, namely Chaparana, Diplopaa Dubois, Ohler & Pyron, 2021, Feirana Dubois, 1992, Gynandropaa Dubois, 1992, Nanorana, Ombropaa Dubois, Ohler & Pyron, 2021, and Paa.

To reduce confusion, we currently use the classification system on the “Amphibian Species of the World” website (Frost 2021). In this classification system, the genus Nanorana now contains 30 species (Frost 2021), of which 21 species were recorded in China (AmphibiaChina 2021).

During a field survey in Yunnan, China in 2019, some specimens of the genus Nanorana were collected. Morphological and molecular analyses indicated that these frogs were distinctive, differing from all known species of genus Nanorana. Therefore, we described them here as a new species.

Materials and methods

Sample collection

Specimens were collected by hand from Lancang County, Yunnan, China, euthanized, tissue samples taken, then preserved in 75% ethanol. Tissue samples were taken from liver and placed in 99% ethanol and subsequently stored at −80 °C. All specimens were deposited at Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, the Chinese Academy of Sciences (KIZ).

Laboratory methods

Genomic DNA extracted from 99% ethanol-preserved liver tissues, using DNA extraction kit from Beijing Dingguo Changsheng Biotechnology Co. Ltd. Two mitochondrial genes, 12S and 16S, were amplified. Primers used for 12S were FS01: 5'-AACGCTAAGATGAACCCTAAAAAGTTCT-3' and R16: 5'-ATAGTGGGGTATCTAATCCCAGTTTGTTTT-3' (Qi et al. 2019) and for 16S were 16Sar: 5' -CGCCTGTTTACCAAAAACAT-3' and 16Sbr: 5'-CCGGTYTGAACTCAGATCAYGT-3' (Palumbi et al. 1991). PCR conditions followed Qi et al. (2019). Amplifications were processed with the cycling conditions that initial denaturing step at 94 °C for 5 min, 35 cycles of denaturing at 94 °C for 30 sec, annealing at 55 °C for 30 sec and extending at 72 °C for 1 min, and final extending step at 72 °C for 5 min. PCR products were isolated through electrophoresis using 1% agarose gels, and further purified using Millipore Microcon Kits. Purified PCR products were sequenced by Davis Sequencing using BigDye terminator 3.1 and sequences were edited and manually managed using SeqMan in Lasergene 7.1 (DNASTAR Inc., Madison, WI, USA) and MEGA X (Kumar et al. 2018). All sequences were deposited in GenBank (Table 1).

Table 1.

Information of samples used in molecular analysis.

Species name Locality Specimen voucher 12S 16S Rag1 Rhod Tyr
Nanorana aenea Sa Pa, Lao Cai, Vietnam ROM37984 EU979693 EU979830 HM163609 EU979895 EU979986
Nanorana aenea Sa Pa, Lao Cai, Vietnam MNHN 1999.5818 AY880456 AY880443
Nanorana blanfordii Yatung, Tibet, China SYNU-1507011 MH315954 MH315963
Nanorana chayuensis Zayü, Tibet, China SYNU-XZ64 EU979709 DQ118509 EU979853 EU979944
Nanorana chayuensis Zayü, Tibet, China SYNU-XZ67 EU979708 DQ118510 EU979852 EU979943
Nanorana conaensis Cona, Tibet, China KIZ-YP152 EU979703 EU979834 EU979874 EU979965
Nanorana liebigii Janakpur, Nepal A17_12_NME MN011989 MN012104 MN032528 MN012368 MN012518
Nanorana liebigii Janakpur, Nepal R18_12_NME MN012105 MN032529 MN012369 MN012519
Nanorana maculosa Jingdong, Yunnan, China YNU-HU2002308 EU979706 EU979835 EU979859 EU979950
Nanorana maculosa Jingdong, Yunnan, China YNU-HU2002322 EU979707 DQ118512 EU979860 EU979951
Nanorana medogensis Medôg, Tibet, China SYNU-XZ35 EU979705 DQ118506 EU979862 EU979953
Nanorana medogensis Medôg, Tibet, China SYNU-XZ75 EU979704 DQ118507 EU979861 EU979952
Nanorana parkeri N7_06_NME MN012006 MN012126 MN032549 MN012391 MN012540
Nanorana parkeri Dangxiong, Tibet, China KP317482 KP317482
Nanorana phrynoides Yimen, Yunnan, China YNU-HU20024012 EU979686 EU979825 EU979877 EU979968
Nanorana pleskei KQ47_14_NME MN012019 MN012156 MN032562 MN012422 MN012570
Nanorana pleskei Shiqu, Sichuan, China CIB20080515-1 HQ324232 HQ324232
Nanorana polunini Pangum, Nepal K1553 KR827957
Nanorana quadranus An, Sichuan, China SCUM20030031GP EU979694 EU979831 EU979886 EU979977
Nanorana quadranus Maowen, Sichuan, China SCUM20045195CJ EU979695 DQ118514 EU979887 EU979978
Nanorana rostandi Kyirong, Tibet, China SYNU-1507058 MH315955 MH315964
Nanorana sichuanensis Huili, Sichuan, China SCUM20030091GP EU979685 EU979824 EU979880 EU979971
Nanorana taihangnica Jiyuan, Henan, China KIZ-HN0709001 EU979724 EU979842 EU979893 EU979984
Nanorana taihangnica Jiyuan, Henan, China KIZ-HN0709002 EU979725 EU979843 EU979894 EU979985
Nanorana unculuanus Jingdong, Yunnan, China YNU-HU2002502601 EU979699 DQ118490 DQ458262 DQ458277
Nanorana unculuanus Jingdong, Yunnan, China YNU-HU2002502702 EU979700 DQ118491 HM163585 EU979865 EU979956
Nanorana ventripunctata SH050538_NME MN012066 MN012208 MN032610 MN012478 MN012626
Nanorana ventripunctata Xianggelila, Yunnan, China SCUM045887WD EU979717 DQ118501 HM163585 EU979868 EU979959
Nanorana yunnanensis Yongde, Yunnan, China YNU-HU20011102 EU979691 EU979829 EU979884 EU979975
Nanorana zhaoermii Lhünzê, Tibet, China SYNU-1706049 MH315947 MH315956
Nanorana zhaoermii Lhünzê, Tibet, China SYNU-1706058 MH315948 MH315957
Nanorana xuelinensis sp. nov. Lancang, Yunnan, China KIZL2019012 MZ410625 MZ410628
Nanorana xuelinensis sp. nov. Lancang, Yunnan, China KIZL2019013 MZ410624 MZ410627
Nanorana xuelinensis sp. nov. Lancang, Yunnan, China KIZL2019014 MZ410623 MZ410626
Limnonectes fragilis Hainan, China ZNAC11006 AY899241 AY899241
Quasipaa boulengeri Yichang, Hubei, China KIZ-HUB292 KX645665 KX645665

Phylogenetic analyses

Total genomic DNA was isolated from the tissue samples of three individuals. Quasipaa boulengeri (Günther, 1889) and Limnonectes fragilis (Liu & Hu, 1973) were used as outgroups according to Qi et al. (2019). The mitochondrial genes 12S ribosomal RNA (12S) and 16S ribosomal RNA (16S), and the nuclear genes recombination activating protein 1 (Rag1), rhodopsin (Rhod), and tyrosinase (Tyr) of 19 known Nanorana species and two outgroup species were obtained from GenBank. Detail information of these materials are given in Table 1.

Sequences were aligned using ClustalW (Thompson et al. 1994) integrated in MEGA X (Kumar et al. 2018) with default parameters. The genetic divergences (uncorrected p-distance) were calculated in MEGA X (Kumar et al. 2018). 12S, 16S, Rag1, Rhod, and Tyr gene segments were concatenated seriatim into a single partition. Bayesian inference (BI) was performed in MrBayes 3.2.7 (Ronquist et al. 2012) and used the Akaike information criterion (AIC) in ModelFinder (Kalyaanamoorthy et al. 2017) to calculate that GTR+F+I+G4 was the best-fit model of evolution for 12S and 16S; HKY+F+I was the best-fit model of evolution for Rag1, Rhod, and Tyr. Two runs were performed simultaneously with four Markov chains starting from a random tree. The chains were run for 1,000,000 generations and sampled every 100 generations. The first 25% of the sampled trees was discarded as burn-in after the standard deviation of split frequencies of the two runs was less than a value of 0.01, and then the remaining trees were used to create a 50% majority-rule consensus tree and to estimate Bayesian posterior probabilities. Maximum likelihood (ML) analysis was performed in IQ-TREE (Nguyen et al. 2015) and used the Akaike information criterion (AIC) in ModelFinder (Kalyaanamoorthy et al. 2017) to calculate that GTR+F+R3 was the best-fit model of evolution for 12S and 16S, and that TPM3+F+I was the best-fit model for Rag1, Rhod, and Tyr. 1000 bootstrap pseudoreplicates via the ultrafast bootstrap (UFB; Hoang et al. 2018) approximation algorithm were used to construct a final consensus tree.

Morphology

All measurements were taken with digital calipers to the nearest 0.1 mm. Morphological characters used and their measurement methods followed Qi et al. (2019). The morphometrics and character terminology include:

AG axilla to groin, distance from posterior base of forelimb at its emergence from body to anterior base of hindlimb at its emergence from body;

EHD eye horizontal diameter;

END eye to nostril distance, distance from anterior corner of eye to nostril;

FL foot length, from proximal end of inner metatarsal tubercle to tip of toe IV;

FML femur length;

HAL hand length, from proximal end of outer metacarpal tubercle to tip of the finger III;

HH head height, greatest height of head;

HL head length, from posterior corner of mandible to tip of snout;

HW head width, at the greatest cranial width;

ID internasal distance, distance between nostrils;

IOD interorbital distance, least distance between upper eyelids;

LAD diameter of lower arm;

LAL length of lower arm, from proximal end of outer metacarpal tubercle to elbow joint;

SL snout length, from tip of snout to the anterior corner of eye distance;

SND snout to nostril distance, distance from tip of snout to nostril;

SVL Snout–vent length, from tip of snout to vent;

TDH horizontal diameter of tympanum;

TDV vertical diameter of tympanum;

TFL length of tarsus and foot, from proximal end of tarsus to tip of the toe IV;

TIL tibia length;

UEW upper eyelid width, maximum width of upper eyelid.

All measurements were taken on the left side of the examined specimen. It should be noted that because the limbs of our specimens cannot be spread, the characters FLL (length of forelimb, from axilla to tip of finger III) and HLL (length of hindlimb, from tip of disk of toe IV to vent) in Qi et al. (2019) are not provided here.

Results

Genealogical relationships

The results of BI and ML phylogenetic trees were constructed based on the concatenated DNA sequences and resulted in approximately identical topologies (Fig. 1). The phylogenetic tree showed that the newly discovered population from Xuelin Township, Lancang County is a member of Nanorana; however, its phylogenetic position in the genus was not clearly resolved. The newly discovered population formed a unique clade sister to the clade consisting of Nanorana aenea (Smith, 1922), N. phrynoides (Boulenger, 1917), N. quadranus (Liu, Hu & Yang, 1960), N. sichuanensis (Dubois, 1987), N. taihangnica (Chen & Jiang, 2002), N. unculuanus (Liu, Hu & Yang, 1960), and N. yunnanensis (Anderson, 1879), but the node supports were very low.

Figure 1. 

Bayesian inference tree of the genus Nanorana based on the sequences of the mitochondrial 12S and 16S, and the nuclear Rag1, Rhod, and Tyr gene. Limnonectes fragilis and Quasipaa boulengeri were included as outgroup. Numbers above branches indicate Bayesian posterior probabilities (≥0.9 retained) and numbers below branches indicate the ML ultrafast bootstrap (UFB) (≥90 retained).

Genetic distances

The uncorrected p-distances calculated from 12S rRNA and 16S rRNA gene fragment sequences of the examined species are shown in Tables 2 and 3, respectively. The observed distances calculated from 12S gene between the sequences of the specimens collected from Xuelin Township, Lancang County and the homologous sequences obtained from GenBank ranged from 3.9% to 7.6%. The observed distances calculated from 16S gene between the sequences of the specimens collected from Xuelin Township, Lancang County and the homologous sequences obtained from GenBank ranged from 5.2% to 7.3%.

Table 2.

Mean uncorrected pairwise genetic distances (%) among the species of Nanorana and outgroups based on partial 12S gene.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1 Nanorana aenea
2 N. blanfordii 7.8
3 N. chayuensis 6.9 5.0
4 N. cuonaensis 7.7 5.3 6.6
5 N. liebigii 6.9 5.8 5.6 6.3
6 N. maculosa 7.2 4.8 2.0 5.8 4.9
7 N. medogensis 6.1 3.9 3.3 4.0 4.5 2.8
8 N. parkeri 6.8 6.9 5.3 7.1 5.9 4.9 4.5
9 N. phrynoides 6.6 7.3 7.1 8.3 7.3 7.1 6.0 8.8
10 N. pleskei 6.0 5.1 5.3 6.7 5.7 5.3 4.8 3.6 7.6
11 N. quadranus 7.5 8.1 7.2 8.1 7.4 7.9 7.0 8.4 7.9 7.6
12 N. rostandi 6.1 6.3 5.5 7.5 5.6 5.8 5.4 5.9 7.3 5.5 7.6
13 N. sichuanensis 6.8 7.5 7.3 9.5 7.3 7.4 6.7 9.1 1.2 7.8 8.1 7.5
14 N. taihangnica 5.2 5.3 5.1 6.3 4.4 4.9 4.0 6.1 5.4 4.6 5.4 5.3 5.6
15 N. unculuanus 5.5 5.0 5.4 7.1 5.4 5.2 4.3 7.3 5.4 5.9 7.4 4.8 6.1 3.7
16 N. ventripundata 8.4 6.3 6.7 8.3 6.4 6.6 6.3 5.4 8.2 3.4 8.6 7.4 8.9 6.7 6.7
17 N. yunnanensis 4.5 6.5 5.2 8.1 6.8 5.7 5.6 6.0 1.9 5.2 7.4 5.7 2.2 4.9 5.4 6.6
18 N. zhaoermii 6.0 3.2 3.2 3.5 4.1 2.1 1.4 5.0 5.9 4.1 6.3 4.4 6.8 3.5 3.5 5.1 5.8
19 Nanorana xuelinensis sp. nov. 6.3 5.6 6.0 6.6 4.0 5.4 4.6 5.9 6.8 6.0 7.1 6.0 7.1 3.9 6.1 7.6 6.5 4.5
20 Quasipaa boulengeri 11.9 12.8 12.5 14.1 11.8 12.4 12.5 12.8 14.7 12.7 13.5 11.9 14.9 12.0 12.9 14.8 13.6 14.0 11.8
21 Limnonectes fragilis 18.1 16.0 17.8 20.0 17.5 18.0 17.8 17.8 18.5 17.0 18.7 18.4 17.5 17.5 18.0 19.1 17.3 18.0 17.4 15.9
Table 3.

Mean uncorrected pairwise genetic distances (%) among the species of Nanorana and outgroups based on partial 16S gene.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
1 Nanorana aenea
2 N. blanfordii 5.1
3 N. chayuensis 5.2 2.4
4 N. cuonaensis 5.5 2.8 3.6
5 N. liebigii 6.2 4.1 4.1 5.3
6 N. maculosa 5.4 2.5 0.9 4.0 3.8
7 N. medogensis 5.9 3.0 2.5 4.4 4.0 2.2
8 N. parkeri 5.5 2.9 3.3 3.5 4.4 3.2 3.8
9 N. phrynoides 4.7 4.5 5.3 4.0 4.6 5.3 5.4 4.4
10 N. pleskei 5.2 2.9 4.1 4.4 4.3 3.6 4.3 3.1 4.7
11 N. polunini 6.0 3.6 4.1 4.4 6.3 4.1 3.8 4.1 6.4 5.2
12 N. quadranus 6.1 4.2 5.8 5.7 6.8 5.8 5.3 5.5 5.1 6.1 6.2
13 N. rostandi 6.6 3.4 4.5 4.1 5.3 4.3 4.3 5.1 5.8 5.3 2.3 5.5
14 N. sichuanensis 5.2 4.4 5.5 4.2 5.3 5.5 5.6 4.5 1.1 4.2 6.6 4.5 5.6
15 N. taihangnica 4.6 3.2 4.5 3.6 4.4 4.2 4.2 3.0 3.8 3.1 5.1 6.3 4.9 4.0
16 N. unculuanus 4.6 5.3 6.1 6.6 6.4 6.2 6.1 5.9 6.0 5.7 8.3 6.9 6.8 5.8 5.4
17 N. ventripundata 4.9 2.5 3.4 3.9 3.9 3.3 3.9 2.3 3.7 2.0 4.9 5.0 4.5 3.5 3.9 5.5
18 N. yunnanensis 5.3 4.5 5.4 4.4 5.3 5.5 5.6 4.5 2.2 4.8 6.1 5.1 5.4 2.2 4.2 6.0 4.3
19 N. zhaoermii 5.5 2.4 2.1 3.1 4.3 2.2 2.6 2.9 4.6 3.0 3.2 4.0 4.7 4.0 3.6 6.0 2.9 4.4
20 Nanorana xuelinensis sp. nov. 6.7 5.8 5.8 5.6 6.5 5.4 5.2 6.0 5.6 6.0 6.6 6.6 5.9 6.2 5.3 7.3 5.7 6.2 5.3
21 Quasipaa boulengeri 8.4 7.8 7.5 6.7 8.0 7.2 7.4 6.5 7.6 7.3 8.8 8.7 9.4 8.0 7.1 9.2 7.7 7.8 6.1 8.3
22 Limnonectes fragilis 12.0 11.8 12.0 11.7 12.2 11.6 11.8 11.1 12.4 12.2 16.2 13.3 14.3 12.6 11.5 12.2 12.5 12.8 11.4 12.3 10.3

Systematics

Nanorana xuelinensis sp. nov.

Figures 2, 3, 4, 5, 6

Holotype

KIZL2019016, adult male, collected on 13 July 2019 by Shuo Liu from Xuelin Township, Lancang County, Puer City, Yunnan Province, China (23°2'38"N, 99°32'35"E; at an elevation of 1840 m asl).

Paratypes

KIZL2019012 and KIZL2019015, two subadult males; KIZL2019013 and KIZL2019014, two subadult females; and KIZL2019017, adult female. All with same collection information as for the holotype.

Figure 2. 

Dorsal and ventral views of the specimens of the type series of Nanorana xuelinensis sp. nov. in preservative.

Diagnosis

Large body size, SVL 101.7–107.3 mm in adults; adult males with keratinized spines on chest, belly, lateral body, posterior dorsum, buttocks, outer side of the fore limbs, the inner metacarpal tubercle, fingers I and II, and upper eyelids; no spines on the inner side of the lower and upper arm; forelimbs strongly hypertrophied in adult males; tympanum big but indistinct, ca 2/3 of eye diameter; anterior dorsum skin smooth; dorsolateral folds absent; finger I longer than finger II; webbing deeply incurved between tips of toes; no tarsal fold; present outer metacarpal tubercle and absent outer metatarsal tubercle; vomerine teeth distinct.

Figure 3. 

Various views of the male holotype (KIZL2019016) of Nanorana xuelinensis sp. nov. in preservative.

The living specimens were yellowish brown with distinct or indistinct black spots on the dorsum and sides of the body and the dorsal side of limbs; no band on arms and legs. Ventral surface white with no spots, throat yellow in adult males.

Figure 4. 

Various views of the female paratype (KIZL2019017) of Nanorana xuelinensis sp. nov. in preservative.

Description of holotype

Adult male, habitus very stout, SVL 107.3 mm, large size in genus Nanorana; head flat and broader than long (HL/HW 0.85, HH/HL 0.53); snout blunt and rounded in both dorsal and lateral views; canthus rostralis obtuse; tympanum large and very indistinct (TDH/EHD 0.76); supratympanic fold extending from eye over tympanum to shoulder; transversal fold behind eyes; eye relatively large (EHD/HL 0.26), pupil slightly rhombic; vomerine teeth distinct; tongue large and cordiform, deeply notched posteriorly.

Figure 5. 

Different views of the male holotype (KIZL2019016) of Nanorana xuelinensis sp. nov. in life.

Forelimbs short and strongly hypertrophied (LAD 18.8 mm); relative finger length: II < I < IV < III; inner metacarpal tubercle enlarged, dorsal surface of inner metacarpal tubercle, fingers I, and finger II with black keratinized nuptial spines, no spine on inner side of fore limbs, and a few spines on outer side of fore limbs; finger tips rounded but not dilated, fingers free, without webbing, no circum-marginal groove or lateroventral groove; subarticular tubercles distinct, outer metacarpal tubercle indistinct.

Figure 6. 

Different views of the female paratype (KIZL2019017) of Nanorana xuelinensis sp. nov. in life.

Hindlimbs rather long and stout; relative toe length: I < II < V < III < IV; tips of toes rounded but not dilated; subarticular tubercles oval and distinct, formula is 1, 1, 2, 3, 2; inner metatarsal tubercles elongated and pronounced; outer metatarsal tubercle absent; webbing deeply incurved between tips of toes, formula I 0-0- II 0-0- III 0--0- IV 0--0 V; lateral fringe on the outer side of toe V developed; no circum-marginal groove or lateroventral groove; tarsal fold absent.

Figure 7. 

Habitat of Nanorana xuelinensis sp. nov. at the type locality.

Anterior dorsum skin smooth; keratinized spines present on chest, belly, lateral body, posterior dorsum, buttocks, and upper eyelids; spines most dense on axilla and each side of chest.

Coloration of holotype in life

The coloration of dorsum is yellowish brown with very indistinct black spots in dorsum, and no band on arms and legs. Ventral surface white with no spots. The throat is yellow. The pupil is black, and the iris is light yellow with many black radial strips around the pupil.

Sexual dimorphism

The forelimbs of adult males are strongly hypertrophied; in addition, adult males have keratinized spines on chest, belly, lateral body, posterior dorsum, buttocks, outer side of the fore limbs, the inner metacarpal tubercle, fingers I and II, and upper eyelids. The forelimbs of adult females are not hypertrophied, and adult females have distinct black spots on the dorsum, lateral body, and the dorsal side of limbs, no keratinized spines on chest, belly, lateral body, posterior dorsum, buttocks, and upper eyelids, and only some keratinized spines on finger I and a few small spines on finger II.

Figure 8. 

Map showing the type locality of Nanorana xuelinensis sp. nov. (red dot).

Etymology

The name refers to Xuelin Township, the locality where the new species was found. We propose “Xuelin Paa Frog” or “Xuelin Spiny Frog” for the common English name and “雪林棘蛙” (Xuě Lín Jí Wā) for the common Chinese name of the new species.

Table 4.

Morphological measurements (mm) of the type series of Nanorana xuelinensis sp. nov.

KIZL2019016 Holotype Adult male KIZL2019017 Paratype Adult female KIZL2019012 Paratype Subadult male KIZL2019013 Paratype Subadult female KIZL2019014 Paratype Subadult female KIZL2019015 Paratype Subadult male
SVL 107.3 101.7 60.3 79.2 75.1 66.9
AG 36.2 40.6 15.9 29.1 29.7 20.1
HL 35.9 36.4 23.7 27.0 27.7 25.8
HW 42.1 38.1 23.6 28.8 28.9 27.0
HH 19.1 18.9 11.6 14.1 15.4 13.1
SL 16.4 14.4 9.7 11.3 11.6 11.2
ID 7.3 7.4 4.6 5.7 5.6 5.5
IOD 4.1 4.7 2.5 3.3 3.9 3.3
UEW 7.0 7.2 4.3 5.6 5.7 5.1
EHD 9.5 10.8 6.6 8.4 8.3 8.4
TDH 7.2 7.7 4.5 5.6 5.7 4.6
TDV 6.6 4.8 3.5 4.4 4.2 3.6
SND 8.4 7.1 4.5 6.4 5.1 4.9
END 7.9 7.3 4.6 5.6 5.8 5.4
LAl 22.5 19.9 11.7 15.3 13.9 14.1
LAD 18.8 10.3 7.9 8.8 8.2 9.4
HAL 22.7 19.0 14.6 17.8 15.9 15.6
FML 45.4 41.8 26.8 34.6 33.5 29.5
TIL 42.5 39.2 25.7 33.2 31.1 28.9
TFL 65.9 63.1 41.9 54.2 49.9 45.8
FL 44.3 42.7 29.1 36.0 33.6 33.1

Distribution

Nanorana xuelinensis sp. nov. is recorded in Lancang County (Pu’er City), Shuangjiang County (Lincang City), and Jinghong City (Xishuangbanna Prefecture), Yunnan Province, China.

Habitat

The type series was found in a still-water pond. At the type locality we found three other species of amphibians: Chirixalus cf. doriae Boulenger, 1893; Raorchestes hillisi Jiang Ren, Guo, Wang & Li, 2020; Tylototriton verrucosus Anderson, 1871a; and three species of reptiles: Calotes emma Gray, 1845; Pareas xuelinensis Liu & Rao, 2021; and Pseudocalotes microlepis (Boulenger, 1887).

Comparisons

Nanorana xuelinensis sp. nov. differs from N. aenea, N. annandalii (Boulenger, 1920), N. gammii (Anderson, 1871b), N. liebigii (Günther, 1860), N. polunini (Smith, 1951), N. rarica (Dubois, Matsui & Ohler, 2001), N. rostandi (Dubois, 1974), and N. unculuanus by the absence of dorsolateral fold (vs presence).

Nanorana xuelinensis sp. nov. differs from N. arnoldi (Dubois, 1975), N. maculosa (Liu, Hu & Yang, 1960), N. yunnanensis, and N. zhaoermii Qi, Zhou, Lu & Li, 2019 by the spines present only on finger I and finger II in adult males (vs present on finger I–III).

Nanorana xuelinensis sp. nov. differs from N. arunachalensis (Saikia, Sinha & Kharkongor, 2017), N. blanfordii (Boulenger, 1882), N. chayuensis (Ye, 1977), N. conaensis (Fei & Huang, 1981), N. minica (Dubois, 1975), and N. mokokchungensis (Das & Chanda, 2000) by its larger body size.

Nanorana xuelinensis sp. nov. differs from N. feae (Boulenger, 1887) by the absence of spines on the inner side of the fore limbs in adult males (vs. presence).

Nanorana xuelinensis sp. nov. differs from N. kangxianensis (Yang, Wang, Hu & Jiang, 2011), N. quadranus, N. taihangensis by the strongly hypertrophied forelimbs in adult males (vs not hypertrophied), and by the presence of nuptial spines on the chest and fingers in adult males (vs absence).

Nanorana xuelinensis sp. nov. differs from N. medogensis (Fei & Ye, 1999), N. phrynoides, and N. sichuanensis by smooth anterior dorsum skin (vs many warts present).

Nanorana xuelinensis sp. nov. differs from N. parkeri (Stejneger, 1927), N. pleskei Günther, 1896, and N. ventripunctata Fei & Huang, 1985 by the shape of the nuptial spines (large and conical spines vs tiny and compact spines).

Nanorana xuelinensis sp. nov. differs from N. vicina (Stoliczka, 1872) by its toes ca 2/3 webbed (vs fully webbed) and by the absence of bands on the hind limbs (vs presence).

Discussion

Most species of Nanorana live in running waters, especially in swiftly running waters (Dubois and Ohler 2005; Ohler and Dubois 2006) such as rivers or streams, except for N. parkeri, N. pleskei, and N. ventripunctata, which have produced a series of specialized adaptations to high-altitude habitats (Che et al. 2020). However, the habitat of the Nanorana xuelinensis sp. nov. is distinctive. All specimens of Nanorana xuelinensis sp. nov. were found in still waters in different seasons. Why this species lives in still waters needs further study.

Morphologically, Nanorana xuelinensis sp. nov. is obviously different from all other known species of the genus Nanorana. The skins of most species of Nanorana are rough with more or less tubercles or warts (Che et al. 2020). However, the skin of Nanorana xuelinensis sp. nov. is quite smooth on most areas of the body. Most males of the tribe Paini have spines on the fingers, arms, or breast. The presence of these spines is an adaptation to breeding in swiftly running waters, helping the males grasp of the females (Ohler and Dubois 2006). Although Nanorana xuelinensis sp. nov. does not live in running waters, the males still may need spines to help grasp females due to their smoother skins. But why the males of Nanorana xuelinensis sp. nov. have so many keratinized spines on the other areas of the body except for the fingers and breast we do not yet know, and the reason for this feature also needs further study.

The genus Nanorana contains 30 species, of which 22 species are recorded in China (Frost 2021); however, N. arnoldi is not recorded from China according to AmphibiaChina (2021), which lists only 21 species. This is probably due to an erroneous synonymy: N. chayuensis was placed into the synonymy of N. arnoldi by Dubois (1980), which subsequently was rejected by Hu (1985). In the phylogenetic analyses of Che et al. (2009), the gene sequences of N. arnoldi and N. chayuensis clustered together, but these sequences of N. arnoldi were from Yunnan, China, which were possibly wrongly identified and probably belong to N. chayuensis. We speculate that the true N. arnoldi is distributed in northern Myanmar, and not in China. Because we do not have specimens from northern Myanmar, whether N. chayuensis and N. arnoldi are the same species remains to be solved, but for the time being, we support AmphibiaChina (2021) in treating N. chayuensis as valid and considering that N. arnoldi is not distributed in China. Further collections from both countries will clarify this taxonomic conundrum.

Acknowledgements

We thank Decai Ouyang, Lei Ouyang, and Zhongqiang Yang for assistance in the field. Thanks are also due to our workmates 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 Lancang County, and Investigation and assessment of amphibians and reptiles in Jinghong City, Menghai County, and Mengla County.

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