A New Species of Nanorana (Anura: Dicroglossidae) from Northwestern Yunnan, China, with Comments on the Taxonomy of Nanorana arunachalensis and Allopaa

Simple Summary Currently, the genus Nanorana contains thirty-two species, and four of them belong to the subgenus Nanorana, namely N. bangdaensis, N. parkeri, N. pleskei, and N. ventripunctata. In this study, on the basis of molecular and morphological evidence, we described a new species of Nanorana (Nanorana) from northwestern Yunnan, China, where only one member (N. ventripunctata) of Nanorana (Nanorana) has been reported. Additionally, the taxonomic status of Nanorana arunachalensis and Allopaa hazarensis were discussed, and subgeneric allocations of Nanorana species were suggested. The findings in this study bring the number of Nanorana species to 33 and improve our understanding on the taxonomy of genus Nanorana and the species diversity of Nanorana (Nanorana), an alpine group widely distributed in the southern and southeastern QTP. Abstract The genus Nanorana contains three subgenera, namely Nanorana, Paa, and Chaparana, and currently, there are four species known to science in Nanorana (Nanorana). In this study, we describe a new species belonging to the subgenus Nanorana from northwestern Yunnan, China. Phylogenetically, the new species, Nanorana laojunshanensis sp. nov., is the sister to the clade of N. pleskei and N. ventripunctata. Morphologically, the new species can be distinguished from known congeners by the combination of following characters: present tympanum, equal fingers I and II, small body size, yellow ventral surface of limbs, distinct vomerine teeth, indistinct subarticular tubercles, head width greater than head length, slender supratympanic fold, absent dorsolateral fold, nuptial spines present on fingers I and II in adult males, absent vocal sac, and paired brown spines on the chest. Moreover, we suggest moving the genus Allopaa into Nanorana (Chaparana) and consider that N. arunachalensis is neither an Odorrana species nor a member of the subfamily Dicroglossinae (therefore Nanorana), but probably represents a distinct genus closely related to Ingerana or belongs to Ingerana, pending more data. Additionally, we consider that Nanorana minica deserves the rank of an independent subgenus, and we suggest assigning N. arnoldi, N. blanfordii, N. ercepeae, N. polunini, N. rarica, N. rostandi, N. vicina, N. xuelinensis, and N. zhaoermii into the subgenus Paa and placing N. kangxianensis, N. phrynoides, and N. sichuanensis in the subgenus Chaparana.


Introduction
The Hengduan Mountains, located at the southeastern edge of the Qinghai-Tibetan Plateau (QTP) and having experienced major uplift between the late Miocene and the late Pliocene [1], are composed of a series of discrete north-to-south mountain ranges with alternating deep river valleys.They are characterized by extremely complex and diverse cli-matic and topographic conditions resulting in altitudinal zonation, which supports and isolates species inhabiting different niches [2] and has greatly contributed to the appearance of many new species [3].As one of the 34 global biodiversity hotspots [4,5], the Hengduan Mountains are distributed with approximately 30% of the amphibian species of China and are particularly unusual in having the richest endemic alpine amphibian fauna-adapted cold conditions [6].Furthermore, it has become a hotspot for the discovery of new species (e.g., [7][8][9]), indicating that the species diversity of amphibians in this area is still highly underestimated, and there are still cryptic amphibian species that have been detected but not described (e.g., [10]).
The genus Nanorana Günther, 1896 [11] is endemic to Asia.It has a wide distribution from the Himalayan region of northern Pakistan; northern India, Nepal; and western China through Myanmar, Thailand, Laos, and northern Vietnam to montane central and southern China [12].Currently, it contains 32 species [12] and can be divided into three subgenera, namely subgenus Nanorana, subgenus Paa, and subgenus Chaparana [13], although some species were not included in the study of Che et al. [13].Originally, the two subgenera Paa and Chaparana were erected as two independent genera by Dubois [14] and Bourret [15], respectively.Roelants et al. [16] revealed that Nanorana is imbedded within Paa on the basis of molecular data, and Jiang et al. [17] also provided molecular evidence for paraphyly of Paa with respect to Nanorana and the polyphyly of Chaparana.Subsequently, Chen et al. [18] placed Chaparana and Paa into Nanorana on the basis of a paraphyletic Paa with respect to Nanorana and Chaparana, and this was followed by Frost et al. [19], who placed Chaparana and Paa into the synonymy of Nanorana to resolve the paraphyly of Paa with respect to Nanorana (sensu stricto).Although Ohler and Dubois [20] presented a taxonomy of the tribe Paini in which Chaparana and Nanorana were treated as two independent genera and Paa was treated as a subgenus of Chaparana, it is difficult to address because they did not take into account previous molecular results [19] and recognized paraphyletic (genus Chaparana and its subgenus Paa) and polyphyletic (subgenus Chaparana) taxa [12].Therefore, the placement of Chaparana and Paa into synonymy of Nanorana [18,19] was followed by most recent studies [13,[21][22][23][24][25][26][27][28][29], although recently, Dubois et al. [30] presented a new classification of the tribe Paini in which Nanorana, Paa, Chaparana, Feirana Dubois, 1992 [31], and Gynandropaa Dubois, 1992 [31] remained independent, while two new genera (Diplopaa Dubois, Ohler, and Pyron, 2021 [30] and Ombropaa Dubois, Ohler, and Pyron, 2021 [30]) were erected.Nanorana (Nanorana), which is widely distributed in the southern and southeastern QTP, is an alpine group within the family Dicroglossidae.Currently, it is composed of four species, namely N. pleskei Günther, 1896 [11], N. parkeri (Stejnger, 1927) [32], N. ventripunctata Fei and Huang, 1985 [33], and N. bangdaensis Rao, Hui, Zhu, and Ma, 2022"2020" [9].In Yunnan, Nanorana (Nanorana) is known in three counties (Zhongdian, Deqing, and Weixi) located in the Three Parallel Rivers region, and only N. ventripunctata, a species occurring in lentic environments such as marshes, pools, and ponds at elevations ranging from 3120 to 4100 m, is recorded [34,35].
During field surveys in the sky-island mountains of the Three Parallel Rivers region, we collected some specimens belonging to Nanorana (Nanorana) from Mt. Laojun, Lijiang, northwestern Yunnan, China.Morphological comparisons and molecular phylogenetic analyses supported that these specimens are distinct from the four known species of Nanorana (Nanorana) and other members of genus Nanorana.Herein, we describe them as a new species of the genus Nanorana.

Sampling
The classification of Frost [12] was followed for convenience.Specimens were collected at Mt. Laojun, Lijiang, Yunnan, China (Figure 1) by Guohua Yu in July 2019.Specimens were euthanized with ethyl acetate in a closed vessel and fixed and then stored in 75% ethanol.Liver tissues were preserved in 99.9% ethanol.All specimens were deposited at Guangxi Normal University (GXNU; Table 1).
The classification of Frost [12] was followed for convenience.Specimens were collected at Mt. Laojun, Lijiang, Yunnan, China (Figure 1) by Guohua Yu in July 2019.Specimens were euthanized with ethyl acetate in a closed vessel and fixed and then stored in 75% ethanol.Liver tissues were preserved in 99.9% ethanol.All specimens were deposited at Guangxi Normal University (GXNU; Table 1).
Multivariate principal component analyses (PCAs) were conducted using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) based on a correlation matrix of measurements.For these analyses, the measurements were corrected for size, and males and females were considered.Scatter plots of the first two PCA factors were used to examine the differentiation between the new species and its closely related relatives in the subgenus Nanorana.

Locus
Primer Name Primer Sequence Source DNA sequences were aligned using the MUSCLE option in MEGA version 7.0 [53] with the default parameters.Phylogenetic analyses were conducted for both 16S rRNA sequences and combined data of the four genes.For the analysis of combined data, the four gene alignments were defined using genes and codon positions, and then the best partitioning scheme and evolutionary models were estimated (Table 3) with PartitionFinder v.2.1.1 [54] using the "greedy" algorithm [55] for subsequent phylogenetic analyses.Bayesian phylogenetic analyses were performed in MrBayes v. 3.2.[56].Two runs were performed simultaneously with four Markov chains starting from a random tree.The chains were run for 3,000,000 generations and sampled every 100 generations.The first 25% of the sampled trees were 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 consensus tree and to estimate Bayesian posterior probabilities (BPPs).In addition, maximum likelihood (ML) analyses were conducted in raxmlGUI 2.0 [57] with 1000 rapid bootstrap replicates.

Molecular Phylogeny
The aligned sequences of 16S rRNA, COI, cytb, and RAG-1 were 575 bp, 674 bp, 965 bp, and 1189 bp, respectively.Both phylogenetic analyses for 16S rRNA sequences and phylogenetic analyses for the combined data based on the best partitioning scheme and models revealed that Nanorana (Nanorana) from Yunnan consists of two distinct lineages (Figures 2 and 3), one containing individuals from Zhongdian Plateau, known as N. ventripunctata (Clade I), and one containing samples from Mt. Laojun, Lijiang (Clade II).Nanorana ventripunctata (Clade I) was recovered as the sister to N. pleskei, and Clade II was recovered as the sister to the clade consisting of N. ventripunctata and N. pleskei.Genetic distances (p-distance) between the new lineage and known species of Nanorana (Nanorana) ranged from 1.6% (vs.N. ventripunctata) to 2.0% (vs.N. bangdaensis) in 16S, which is roughly equal to the distances between other species of subgenus Nanorana, and from 7.4% (vs.N. ventripunctata) to 10.6% (N.bangdaensis) in COI, which is greater than the distance between N. bangdaensis and N. parkeri (Table 4).
The specimen under the name Nanorana arunachalensis (Saikia, Sinha, and Kharkongor, 2017) [58] in GenBank did not cluster together with the subfamily Dicroglossinae (therefore Nanorana) but was nested in the subfamily Occidozyginae and was closer to Ingerana with strong supports (Figure 2).The sample of A. hazarensis was nested in the genus Nanorana and grouped together with members of the subgenus Chaparana with strong supports (Figure 3).

Morphometric Analyses
Morphological measurements of the new species are presented in Table 5, and the measurements of N. pleskei and N. ventripunctata examined in this study are presented in Tables S1 and S2.For the PCA analysis on the new species and N. ventripunctata, the first two principal components accounted for 58.68% of the total variance (Table 6), the loadings for PC2 were heavily loaded on UEW, ED, and NED (loading factor > 0.7), and differentiation between the new species and N. ventripunctata was found along the PC2 axis (Figure 4a), indicating that the new species differs from N. ventripunctata by its narrower upper eyelid, larger eye, and greater nostril-eye distance.For the PCA analysis on the new species and N. pleskei, the first principal component (PC1) accounted for 48.24% of the total variance, the loadings for PC1 were heavily loaded on IND, UEW, FHL, TL, TFL, and FL, and obvious differentiation was found along the PC1 axis (Figure 4b), indicating that the new species is different from N. pleskei by its narrower internarial distance, narrower upper eyelid, and longer limbs.

Morphometric Analyses
Morphological measurements of the new species are presented in Table 5, and the measurements of N. pleskei and N. ventripunctata examined in this study are presented in Tables S1 and S2.For the PCA analysis on the new species and N. ventripunctata, the first two principal components accounted for 58.68% of the total variance (Table 6), the loadings for PC2 were heavily loaded on UEW, ED, and NED (loading factor > 0.7), and differentiation between the new species and N. ventripunctata was found along the PC2 axis (Figure 4a), indicating that the new species differs from N. ventripunctata by its narrower upper eyelid, larger eye, and greater nostril-eye distance.For the PCA analysis on the new

Taxonomic Account
The results of molecular phylogenetic and morphological analyses indicated that the specimens from Mt. Laojun, Lijiang, represent a distinct lineage, and it can be distinguished from its congeners by body size and the combination of texture and coloration pattern.Therefore, we describe it here as a new species.Nanorana (Nanorana) laojunshanensis sp.nov.(Figures 5-7; Table 5) http://zoobank.org/urn:lsid:zoobank.org:act:D44F3AC5-A50D-4908-A424-F4EFA512D209 (accessed on 17 August 2023).

Taxonomic Account
The results of molecular phylogenetic and morphological analyses indicated that the specimens from Mt. Laojun, Lijiang, represent a distinct lineage, and it can be distinguished from its congeners by body size and the combination of texture and coloration pattern.Therefore, we describe it here as a new species.Holotype: GXNU YU090314, adult male, collected on 24 July 2019 by Guohua Yu at Mt. Laojun, Lijiang, Yunnan, China (26°37' N, 99°42' E, 3982 m a.s.l.).
Paratypes: Four adult males (GXNU YU090313 and 090315-090317) and an adult female (GXNU YU090312) collected from the type locality at the same time by Guohua Yu.
Etymology: The specific epithet is named after the type locality, Mt.Laojun, Lijiang, Yunnan, China.We suggested "laojunshan slow frog" for the common English name and "老君山倭蛙 (Lǎo Jūn Shān Wō Wā)" for the common Chinese name.
Paratypes: Four adult males (GXNU YU090313 and 090315-090317) and an adult female (GXNU YU090312) collected from the type locality at the same time by Guohua Yu.
Etymology: The specific epithet is named after the type locality, Mt.Laojun, Lijiang, Yunnan, China.We suggested "laojunshan slow frog" for the common English name and "老君山倭蛙 (Lǎo Jūn Shān Wō Wā)" for the common Chinese name.
Diagnosis: The new species was assigned to Nanorana (Nanorana) using the following morphological characters: oval tongue, shallowly notched posterior; blunt finger and toe tips; absent webbing between fingers, absent supernumerary tubercle; developed webbing between toes; paired patches of spine on chest [37].The new species can be distinguished from other members of Nanorana by having a combination of the following characters: (1) present tympanum; (2) small body size; (3) distinct vomerine teeth; (4) indistinct subarticular tubercles; (5) head width greater than head length; (6) slender supratympanic fold; (7)  Description of holotypes: Adult male (SVL 36.1 mm; Table 5); head width (HW 11.3 mm) greater than head length (HL 10.7 mm); rounded snout, slightly protruding beyond lower jaw in ventral view; distinct canthus rostralis; sloping loreal region; nostrils are oval, lateral, and nearer to the eye; interorbital distance (IOD 1.9 mm) is smaller than the internarial distance (IND 2.3 mm) and smaller than the upper eyelid width (UEW 2.4 mm); a small and indistinct pineal spot between the eyes; horizontal oval pupil; small, rounded tympanum (TD 1.3 mm), smaller-than-half eye diameter (ED 3.9 mm); slender, distinct supratympanic; distinct vomerine teeth in two short oblique rows between the choanae; oval choanae; oval tongue; shallowly notched posterior; no vocal sac.
Forelimbs are robust; length of fingers I and II is nearly equal, relative length of fingers I ≈ II < IV < III; fingertips are blunt, not expanded; webbing between fingers is absent; subarticular tubercles are indistinct; supernumerary tubercles at base of fingers are small and indistinct; inner metacarpal tubercle is flat, outer metacarpal tubercle is indistinct.
Hindlimbs are robust, with the tibiotarsal articulation reaching the posterior edge of the eye when the hindlimb is stretched alongside the body; the heels meet when the legs are positioned at right angles to the body; the length of toes III and V is nearly equal, relative length of toes I < II < III ≈ V < IV; all toe tips are blunt, not expanded; toes are fully webbed, webbing formula I0-1II0-1III0-1IV1-0V; subarticular tubercles are indistinct, formula 1, 1, 2, 3, 2; inner metatarsal tubercle is oval and small; outer metatarsal tubercle is absent.
Skin is rough with a longitudinal skin ridge on the anterior part of the dorsum and scattered large tubercles on the posterior part of the dorsum; many large tubercles are on the dorsal surface of the hindlimbs; the dorsal surface of the forelimbs is smooth; a few tubercles are on the body flanks.The ventral surface is relatively smooth; many white tubercles are around the vent.
Color of holotype in life: Dorsal surface of body, body flanks, and dorsal surface of limbs are brown mottled with irregular dark patches; side of head is grayish brown, with a dark band on the canthus rostralis from the tip of the snout to the anterior border of the eye and a dark band below the supratympanic fold.Ventral surface is grey white, with yolkyellow patches on the ventral surface of limbs; iris is black with golden brown mottling.
Color of holotype in preservative: dorsal surface is faded to grayish brown, mottled with dark patches, pattern as in life; ventral surface is white.
Sexual dimorphism: Males possess nuptial pads with dense small brown spines on the base of fingers I and II and an extremely developed nuptial pad on the base of finger I (Figure 6D), as well as paired "/ \"-shaped patches of small brown spines on the chest.In addition, males have a M-shaped patch of brown spines on the edge of the lower jaw.
Variations: The female individual (GXNU YU090312) has a mid-dorsal vertebral stripe running from the posterior of the snout to the vent and a more smooth dorsal surface without conical spines (Figure 7).
Distribution and ecology: Currently, the new species is only known from the type locality and inhabits marshes and ponds.Adult males have nuptial pads and nuptial spines, suggesting that the breeding season of the new species is about July and August.No tadpoles were collected for the new species.

Discussion
Currently, four species are recognized in the subgenus Nanorana (Nanorana), and only one is recorded in Yunnan, China [35].In this study, morphological comparison and phylogenetic analyses based on mtDNA and nuDNA revealed that the populations from the east side of Jinsha River represent a new species that is the sister to the clade of N. ventripunctata and N. pleskei, bringing the species number of the subgenus Nanorana to five.It is worthwhile to further investigate the distribution boundary and species diversity of the subgenus Nanorana.Nanorana bangdaensis had been confused with N. parkeri.Zhou et al. [80] revealed that N. parkeri consists of two major lineages (lineages E and W) based on COI sequences.Moreover, recent comprehensive analyses based on whole genomic data [81] found substantial genomic isolation between the E and W lineages with highly restricted gene flow in a narrow geographic zone lying between them and suggested that endogenous selection is a dominant factor resulting in speciation between the W and E lineages.The type localities of N. parkeri and N. bangdaensis are Tingri, Tibet, China, and Bangda, Baxoi, Tibet, China, respectively.Therefore, the west lineage (W) refers to N. parkeri because it contains topotypes of N. parkeri, whereas the east lineage (E) represents N. bangdaensis because it contains topotypes of N. bangdaensis (Figure 1).The 16S distances between members of subgenus Nanorana range from 1.0% to 2.8%, smaller than the value of 3% as defined by Vieites et al. [82], but morphologically, they can be easily distinguished from each other.For instance, N. bangdaensis and N. parkeri differ from N. pleskei, N. ventripunctata, and N. laojunshanensis sp.nov.by the absent tympanum (vs.present), paa subgen.nov. to accommodate N. minica, which has a small body size and was once placed in Paa.The 16S distance between N. minica and the three vouchers (RAS VV5.1, RAS VV8.1, and RAS VV11.1) is very small (0.7-0.9%), implying that they are probably conspecific, pending additional morphological data.For N. annandalii, N. feae, N. gammii, and N. mokokchungensis, their phylogenetic placements have never been investigated, and currently there are no sequences from them in GenBank, so more studies are needed to address the sub-generic allocation of these four species.The genus Allopaa was erected by Ohler and Dubois [20], who recovered the type species of Allopaa (A. hazarensis) as the sister-group of other Paini based on morphological data, and currently, it only contains A. hazarensis [12].Most previous phylogenetic analyses involving Paini [13,21,23,26,87] did not address its phylogenetic placement, until recently, Hofmann et al. [24,25,84] and Akram et al. [27] found that A. hazarensis was nested in the genus Nanorana.However, both Hofmann et al. [24,25,84] and Akram et al. [27] did not render the taxonomic remedy.In this study, we also found with strong support that A. hazarensis was nested in Nanorana and that it was closer to Nanorana (Chaparana), rendering currently recognized Nanorana paraphyletic.According to Ohler and Dubois [20], Allopaa can be distinguished from all other genera of Paini in several features, such as a first finger that is longer than the second, blunt tips of fingers and toes, no tarsal fold, complete webbing, a dermal fringe along the fifth toe that does not reach the basis of the metatarsus, and males with an internal vocal sac and black nuptial spines scattered on the dorsal part of the metacarpal tubercle and along finger I.However, these characters are not unique to Allopaa and vary among Nanorana species.Firstly, the first finger is also longer than the second finger in most members of the subgenus Chaparana (N.quadranus, N. taihangnica, N. unculuanus, N. kangxianensis, N. sichuanensis, N. yunnanensis), in some species of subgenus Paa, such as N. liebigii and N. xuelinensis, and in a member of subgenus Nanorana (N.parkeri) [35,88].Secondly, the tips of the fingers and toes are round and swollen in most species of Nanorana, with a few dilated to small disks [35].Thirdly, the absence of the tarsal fold is also not unique to Allopaa, in that many members of the genus Nanorana also lack it (e.g., N. quadranus, N. unculuanus, N. kangxianensis, N. conaensis, and N. liebigii).Fourthly, complete webbing in Nanorna is very common (e.g., N. quadranus, N. taihangnica, N. kangxianensis, N. sichuanensis, N. phrynoides, N. liebigii, and N. ventripunctata).Fifthly, a dermal fringe along the outer edge of toe V that also does not reach the basis of the metatarsus is present in many Nanorana species; for instance, the fringe only reaches the base of the toe in N. quadranus, N. unculuanus, N. yunnanensis, N. liebigii, and N. polunini.Sixthly, an internal vocal sac also presents in some Nanorana species, such as N. sichuanensis, N. yunnanensis, N. phrynoides, N. blanfordii, and N. chayuensis.Finally, the presence of nuptial spines on the fingers also varies among Nanorana species; for instance, N. kangxianensis has nuptial spines on the first finger, N. quadranus, N. taihangnica, and N. unculuanus have no nuptial spines on the fingers, and N. sichuanensis and N. phrynoides have nuptial spines on fingers I and II or fingers I-III.Therefore, based on the present phylogenetic analyses and morphological comparisons, we consider that the genus Allopaa is invalid and suggest moving it into Nanorana (Chaparana).

Conclusions
We described a new species of Nanorana, Nanorana laojunshanensis sp.nov.from Mt. Laojun in northwestern Yunnan, China, based on molecular and morphological evidence.The new species belongs to the subgenus Nanorana and is the sister to the clade of N. pleskei and N. ventripunctata.Additionally, we revealed that N. arunachalensis probably does not belong to the subfamily Dicroglossinae (therefore Nanorana) but maybe represents a distinct genus closely related to Ingerana or belongs to Ingerana, pending more data.We suggested placing Allopaa into the synonymy of Nanorana and moving it into the subgenus Chaparana.We considered that N. minica deserves the rank of an independent subgenus in genus Nanorana, and allocations of subgenus were suggested for other species.Type species: Nanorana minica (Dubois, 1975) Type locality: "Dial Bajar, au sud de Chainpur, sur la riviére Seti, Ouest-Népal".Diagnosis: This subgenus can be distinguished from all three other subgenera of the genus Nanorana by the following combination of characters according to Dubois [14] and Ohler and Dubois [20]: (1) body size small (SVL of male adults 28.5-33 mm and SVL of female adults 30.5-41 mm); (2) tips of toes obviously enlarged, twice or more than twice of the diameter of phalanges; (3) webbing very incurved between extremities of adjacent toes; (4) vocal sacs present; (5) nuptial spines large, distinct, countable, and translucent or creamy, present on fingers I and II and chest; and (6) eggs entirely whitish or creamy, without colored animal pole.

A key to members of
Included species: Nanorana minica (Dubois, 1975).Distribution: Nepal, India, Bhutan.Etymology: From the Latin mini, "small", and from the generic name Paa, Dubois, 1975 (from the Tamang name paa, "frog"), in which this species was originally placed.This name refers to the small body size of this taxon.
Note: We describe it as a new subgenus because N. minica was recovered as the sister taxon to the clade composed of subgenus Paa and subgenus Nanorana, meaning that it deserves a rank of subgenus based on the present taxonomic framework.Additionally, according to Ohler and Dubois [20], this monotypic subgenus displays two unique characters: translucent or creamy nuptial spines and entirely whitish or creamy eggs, without colored animal pole.

Figure 2 .
Figure 2. Bayesian phylogram of the genus Nanorana inferred from 16S rRNA sequences.Numbers above and below branches are Bayesian posterior probabilities and ML bootstrap values, respectively (only values above 50% are shown).

Figure 2 .
Figure 2. Bayesian phylogram of the genus Nanorana inferred from 16S rRNA sequences.Numbers above and below branches are Bayesian posterior probabilities and ML bootstrap values, respectively (only values above 50% are shown).

Figure 3 .
Figure 3. Bayesian phylogram of the genus Nanorana inferred from combination of 16S rRNA, COI, cytb, and Rag-1 sequences.Numbers above and below branches are Bayesian posterior probabilities and ML bootstrap values, respectively (only values above 50% are shown).

Figure 3 .
Figure 3. Bayesian phylogram of the genus Nanorana inferred from combination of 16S rRNA, COI, cytb, and Rag-1 sequences.Numbers above and below branches are Bayesian posterior probabilities and ML bootstrap values, respectively (only values above 50% are shown).

Figure 4 .
Figure 4. Scatterplots of principal components 1 and 2 of morphometric data of N. laojunshanensis sp.nov.and its two relatives, N. ventripunctata (a) and N. pleskei (b).

Figure 4 .
Figure 4. Scatterplots of principal components 1 and 2 of morphometric data of N. laojunshanensis sp.nov.and its two relatives, N. ventripunctata (a) and N. pleskei (b).
. The new species can be distinguished from other members of Nanorana by having a combination of the following characters: (1) present tympanum; (2) small body size; (3) distinct vomerine teeth; (4) indistinct subarticular tubercles; (5) head width greater than head length; (6) slender supratympanic fold; (7) finger I equal to finger II; (8) absence of dark blotches on ventral surface and yolk-

Table 1 .
Species used in phylogenetic analyses of this study.

Table 1 .
Species used in phylogenetic analyses of this study.
• C for 3 min; 35 cycles of denaturing at 94 • C for 60 s, annealing at 48-54 • C (51 • C for 16S, 48 • C for COI, 50 • C for cytb, and 55 • C for RAG-1), and extending at 72 • C for 60 s; and a final extending step of 72 • C for 10 min.The DNA sequences of both strands were obtained using the BigDye Terminator v.3.1 on an ABI PRISM 3730 following the manufacturer's instructions.All new sequences have been deposited in GenBank under Accession Nos.OR671665-OR671686 and OR678554-OR678609 (Table

Table 2 .
Primers used for PCR amplification and sequencing in this study.

Table 3 .
Best partition scheme and models estimated by the partition finder analysis for the combined data.

Table 6 .
Factor loading of first two principal components of 13 size-adjusted morphometric characteristics of N. laojunshanensis sp.nov./N.ventripunctata and of N. laojunshanensis sp.nov./N.pleskei.N. laojunshanensis sp.nov.and N. ventripunctata N. laojunshanensis sp.nov.and N. pleskei finger I equal to finger II; (8) absence of dark blotches on ventral surface and yolkyellow ventral surface of limbs; (9) absent dorsolateral fold; (10) nuptial spines present only on fingers I and II in adult males; (11) absent vocal sac; and (12) paired brown spines on chest.

Table 7 .
Suggested assignment of subgenus for Nanorana species."Yes" means that the species belongs to the corresponding subgenus.