A new species of Nanorana Günther, 1896 (Anura, Dicroglossidae) from Yunnan, China

Abstract A new species of Nanorana Günther, 1896 is described from Yunnan Province, China, based on morphological and molecular evidence. Morphologically, Nanorana xuelinensissp. 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.


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 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, 2006and Chrysopaa Ohler & Dubois, 2006. Che et al. (2010 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 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.

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).

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 bestfit 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. 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.

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.

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%. 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.
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. 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.  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).
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. 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 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.
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.