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Article

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

by
Shangjing Tang
1,2,
Shuo Liu
3,* and
Guohua Yu
1,2,*
1
Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection (Guangxi Normal University), Ministry of Education, Guilin 541004, China
2
Guangxi Key Laboratory of Rare and Endangered Animal Ecology, College of Life Science, Guangxi Normal University, Guilin 541004, China
3
Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China
*
Authors to whom correspondence should be addressed.
Animals 2023, 13(21), 3427; https://doi.org/10.3390/ani13213427
Submission received: 15 August 2023 / Revised: 28 October 2023 / Accepted: 4 November 2023 / Published: 6 November 2023
(This article belongs to the Section Wildlife)
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Abstract

:

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.

1. 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 climatic 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.

2. Materials and Methods

2.1. 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).

2.2. Morphology

Morphometric data were taken using electronic digital calipers to the nearest 0.1 mm. The morphological terminology followed Fei et al. [36]. Measurements included the following: snout–vent length (SVL); head length (HL); head width (HW); snout length (SL); internarial distance (IND); interorbital distance (IOD); upper eyelid width (UEW); eye diameter (ED); nostril–eye distance (DNE); tympanum diameter (TD); forearm and hand length (FHL); tibia length (TL); foot length (FL); and length of foot and tarsus (TFL). Besides the specimens of the new species, eight specimens of N. pleskei and eight specimens of N. ventripunctata were also measured because phylogenetically, the new species is closer to these two species (see below). Comparative morphological data of congeners were taken from their original descriptions or re-descriptions [9,11,14,26,32,35,37].
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.

2.3. Molecular Phylogenetic Analyses

Genomic DNA was extracted from liver tissue fixed in 99.9% ethanol using a standard phenol/chloroform protocol. We amplified and sequenced three mitochondrial genes (16S rRNA, COI, and cytb) and one nuclear gene (RAG-1). Primers used for PCR amplification and sequencing were obtained from previous studies [38,39,40] or designed by this study (see Table 2). PCR amplifications were performed in 25 µL reactions using the following cycling conditions: an initial denaturing step at 94 °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 1). Sequences of known Nanorana species were obtained from GenBank. Chrysopaa sternosignata (Murray, 1885) [41], Quasipaa boulengeri (Günther, 1889) [42], Allopaa hazarensis (Dubois and Khan, 1979) [43], Occidozyga lima (Gravenhorst, 1829) [44], Occidozyga lingnanica Lyu and Wang, 2022 [45], Occidozyga myanhessei (Koehler, Vargas, Than, and Thammachoti, 2021) [46], Ingerana borealis (Annandale, 1912) [47], Ingerana tenasserimensis (Sclater, 1892) [48], Hoplobatrachus chinensis (Osbeck, 1765) [49], Fejervarya cancrivora (Gravenhorst, 1829) [44], Sphaerotheca breviceps (Schneider, 1799) [50], Amolops mengdingensis Yu, Wu, and Yang, 2019 [51], and Odorrana hosii (Boulenger, 1891) [52] were included as hierarchical outgroups.
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.

3. Results

3.1. 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 (Figure 2 and Figure 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).

3.2. 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.

3.3. 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.
http://zoobank.org/urn:lsid:zoobank.org:act:D44F3AC5-A50D-4908-A424-F4EFA512D209 (accessed on 17 August 2023).
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Figure 5. Holotype of Nanorana laojunshanensis sp. nov. in life (A,B) and in preservative (C,D).
Figure 5. Holotype of Nanorana laojunshanensis sp. nov. in life (A,B) and in preservative (C,D).
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Figure 6. Views of tympanum (A), vomerine teeth (B), hand (C), nuptial pad (D), and foot (E) of the holotype of Nanorana laojunshanensis sp. nov.
Figure 6. Views of tympanum (A), vomerine teeth (B), hand (C), nuptial pad (D), and foot (E) of the holotype of Nanorana laojunshanensis sp. nov.
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Figure 7. Dorsal and ventral views of the paratype of Nanorana laojunshanensis sp. nov. (GXNU YU090312) in preservative.
Figure 7. Dorsal and ventral views of the paratype of Nanorana laojunshanensis sp. nov. (GXNU YU090312) in preservative.
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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.
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) finger I equal to finger II; (8) absence of dark blotches on ventral surface and yolk-yellow 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.
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 yolk-yellow 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.
Comparison: Nanorana (Nanorana) contains four species, namely N. pleskei, N. parkeri, N. ventripunctata, and N. bangdaensis. The new species can be distinguished from N. parkeri by having a tympanum (vs. absent; Figure 8), equal fingers I and II (vs. finger I longer than finger II), smaller body size (male SVL 33.3–38.5 mm and female SVL 42.9 in N. laojunshanensis sp. nov. vs. males 40–51 mm and females 50.1–51.5 mm in N. parkeri), yellow ventral surface of limbs (vs. greyish white; Figure 8), and distinct vomerine teeth (vs. absent or extremely weak). It can be distinguished from N. pleskei by its greyish brown dorsal surface (vs. olive green, yellowish green, or dark green), equal fingers I and II (vs. finger II longer than finger I), distinct vomerine teeth (vs. absent or extremely weak), and narrower internarial distance, narrower upper eyelid, and longer limbs (Table 6 and Figure 4). It can be distinguished from N. ventripunctata by indistinct subarticular tubercles (vs. distinct; Figure 9), a greyish white throat and belly with no dark patches and yolk-yellow ventral surface of limbs (vs. greyish white ventral surface scattered with dark blotches on the throat, belly, and/or ventral surface of limbs; Figure 9), and a narrower upper eyelid, larger eye, and greater nostril–eye distance (Table 6 and Figure 4). It can be distinguished from N. bangdaensis by a present tympanum (vs. absent; Figure 8), nuptial pads present on fingers I and II (vs. only finger I), a head width greater than head length (vs. head width equal to head length), a very rough dorsal surface with a dense longitudinal skin ridge on the dorsum and many tubercles on both the dorsum and the dorsal surface of the hindlimbs (vs. a smooth dorsal surface with only a few skin ridges on the dorsum), equal fingers I and II (vs. finger I longer than finger II), large black blotches on the flank (vs. many small spots on the flank), dark bands on the limbs (vs. absent), a white venter and yolk-yellow ventral surface of limbs (vs. beige), and a slender supratympanic fold (vs. thick).
The new species can be distinguished from N. aenea (Smith, 1922) [59], N. annandalii (Boulenger, 1920) [60], N. gammii (Anderson, 1871) [61], N. liebigii (Günther, 1860) [62], N. minica (Dubois, 1975) [14], N. polunini (Smith, 1951) [63], N. rarica (Dubois, Matsui, and Ohler, 2001) [64], N. rostandi (Dubois, 1974) [65], N. unculuanus (Liu, Hu, and Yang, 1960) [66], and N. vicina (Stoliczka, 1872) [67] by the absent dorsolateral fold (vs. present); from N. arnoldi (Dubois, 1975) [14], N. maculosa (Liu, Hu, and Yang, 1960) [66], N. yunnanensis (Anderson, 1879) [68], and N. zhaoermii Qi, Zhou, Lu, and Li, 2019 [22] by nuptial spines which are present only on fingers I and II in adult males (vs. present on fingers I–III); from N. arunachalensis by the greyish brown dorsal surface (vs. green) and small body size (female SVL 42.9 mm vs. female SVL 81.33 mm in N. arunachalensis); from N. blanfordii (Boulenger, 1882) [69], N. chayuensis (Ye, 1977) [70], N. conaensis (Fei and Huang, 1981) [71], N. feae (Boulenger, 1887) [72], N. medogensis (Fei and Ye, 1999) [73], N. mokokchungensis (Das and Chanda, 2000) [74], N. phrynoides (Boulenger, 1917) [75], and N. sichuanensis (Dubois, 1987) [76] by the absent vocal sac (vs. present); from N. kangxianensis (Yang, Wang, Hu, and Jiang, 2011) [77], N. quadranus (Liu, Hu, and Yang, 1960) [78], and N. taihangnica (Chen and Jiang, 2002) [79] by nuptial spines which are present on fingers I and II (vs. nuptial spines which are only present on finger I in N. kangxianensis and nuptial spines which are absent in N. quadranus and N. taihangnica); and from N. xuelinensis Liu, Zhang, and Rao, 2021 [26] by having paired brown spines on the chest (vs. black spines which are present on the chest, belly, lateral body, etc).

4. 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), N. bangdaensis differs from N. parkeri by the nuptial pad present on the base of finger I (vs. present on both fingers I and II), and N. laojunshanensis sp. nov. can be distinguished from N. ventripunctata by the yolk-yellow ventral surface of limbs and the indistinct subarticular tubercles (Figure 9). Furthermore, the distributions of these five species are nearly discrete (Figure 1), and it has been revealed that gene flow between N. parkeri and N. bangdaensis is highly restricted [81], as mentioned above. Therefore, we consider that the five species of subgenus Nanorana are all valid. Generally, genetic distance between recently diverged species is relatively small (e.g., species of the Amolops mantzorum group; [83]). This is the same with the subgenus Nanorana, which started to diverge into different lineages ca. 3.7 Mya according to Hofmann et al. [23].
Additionally, the taxonomic status of N. arunachalensis needs further examination. It was originally placed in Odorrana by Saikia et al. [58] and was later transferred to Nanorana by Qi et al. [22] based on morphological characters. Most recently, Hofmann et al. [84] suggested excluding N. arunachalensis from Nanorana and reassigning it to the genus Odorrana based on a 16S phylogeny and genetic distances. However, this taxonomic change is unreliable in that the 16S phylogeny in Hofmann et al. [84] only contained members of Dicroglossinae and actually did not show any phylogenetic evidence that N. arunachalensis belongs to Odorrana. The two vouchers of nominal N. arunachalensis (ZSIS-M37 and ZSIS-M40) were sequenced by Saikia and colleagues and came from the type locality. In this study, our phylogenetic analyses revealed that the voucher of N. arunachalensis is nested in Occidozyginae and is closer to Ingerana with strong supports. Morphologically, N. arunachalensis is similar to Ingerana in having small finger and toe discs, a prominent supra-tympanic fold, an indistinct or hidden tympanum, and a dark inter-orbital band, according to Saikia et al. [58], Fei et al. [34], and Zug [85]. Therefore, assuming that these two vouchers of N. arunachalensis (ZSIS-M37 and ZSIS-M40) were identified correctly by Saikia and his colleagues, the phylogenetic analyses in this study indicate that N. arunachalensis probably belongs to Ingerana or represents a new genus closely related to Ingerana rather than Odorrana or Nanorana. Recently, Wangyal et al. [86] reported the first record of N. arunachalensis from Bhutan based solely on photographs. However, this record should be treated with caution and needs further investigation because it obviously differs from the types of N. arunachalensis by its brown dorsal surface (vs. green) and the absence of dark bands on the dorsal surface of the limbs. Thus, more studies employing molecular and morphological data are necessary to solve the taxonomy of N. arunachalensis.
Che et al. [13] divided the genus Nanorana into three subgenera, but some members of this genus were not included and therefore were not assigned to a subgenus by them. In this study, most known species of the genus were included, and phylogenetic analyses based on the combined data revealed that Nanorana contains four major lineages, three of which correspond to the three subgenera (Figure 3). Nanorana arnoldi, N. blanfordii, N. ercepeae, N. polunini, N. rarica, N. rostandi, N. vicina, N. xuelinensis, and N. zhaoermii were nested in the clade containing known members of the subgenus Paa (N. liebigii, N. conaensis, N. medogensis, N. maculosa, and N. chayuensis; Che et al. [13]). So, we suggested placing these nine species into the subgenus Paa. Nanorana kangxianensis, N. sichuanensis, and N. phrynoides form a clade with known members of the subgenus Chaparana (N. quadranus, N. taihangnica, N. aenea, N. unculuanus, and N. yunnanensis; Che et al. [13]), suggesting that these three species belong to the subgenus Chaparana (Table 7). Nanorana minica was once placed in same subgenus with members of Paa by Ohler and Dubois [20]. However, molecular phylogenetic analyses in the present study revealed with strong support that it forms a distinct clade with the voucher (RAS VV11.1) of an unnamed species and that it is closer to subgenera Paa and Nanorana, although the phylogenetic relationships between these three clades were not resolved in this study. This finding is consistent with the work of Hofmann et al. [84], which revealed a distinct clade that contains three vouchers (RAS VV5.1, RAS VV8.1, and RAS VV11.1) and is the sister to the clade composed of subgenera Nanorana and Paa with strong support [84]. Therefore, we consider that this clade deserves the rank of independent subgenus in the genus Nanorana and suggest the subgenus name Minipaa 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).

5. 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.
  • A key to members of Nanorana (Nanorana)
1
Tympanum absent………………………………………………………………………….2
–
Tympanum present…………………………………………………………………………3
2
Nuptial pad present on base of finger I……………………………………N. bangdaensis
–
Nuptial pad present on both fingers I and II……………………………………N. parkeri
3
Finger I shorter than finger II………………………………………………………N. pleskei
–
Finger I equal to finger II……………………………………………………………………4
4
Subarticular tubercles distinct; ventral surface grayish white scattered with dark blotches………………………………………………………………….…N. ventripunctata
–
Subarticular tubercles indistinct; lacking dark blotches on ventral surface and ventral surface of limbs yolk yellow………………………………….N. laojunshanensis sp. nov.
  • Minipaa subgen. nov.
http://zoobank.org/urn:lsid:zoobank.org:act:F6036C62-88DD-4435-BDBC-CD9A2592E588 (accessed on 19 October 2023).
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.
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.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ani13213427/s1, Table S1: Measurements (in mm) of N. pleskei collected from Xinduqiao, Sichuan, China; Table S2: Measurements (in mm) of N. ventripunctata.

Author Contributions

Conceptualization, G.Y. and S.L.; data curation, S.T.; formal analysis, S.T.; methodology, S.T.; visualization, S.T.; writing—original draft, S.T.; writing—review and editing, G.Y. and S.L.; supervision, G.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (Nos. 31872212 and 32060114), Guangxi Natural Science Foundation Project (2022GXNSFAA035526), Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection (Guangxi Normal University), Ministry of Education (ERESEP2022Z04), and Guangxi Key Laboratory of Rare and Endangered Animal Ecology, Guangxi Normal University (19-A-01-06).

Institutional Review Board Statement

The study was approved by the Ethics Committee of Guangxi Normal University (protocol code: GXNU-202308-010; date of approval: 22 August 2023).

Informed Consent Statement

Not applicable.

Data Availability Statement

Sequence data used in this study are deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) (accessed on 17 October 2023).

Acknowledgments

Thanks to Shuwei Liu, Chi Ma, and Zhengshun Mao for their help with the field surveys.

Conflicts of Interest

The authors declare no conflict of interest.

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Animals 13 03427 g001
Figure 1. Map showing the type locality of Nanorana laojunshanensis sp. nov. in northwestern Yunnan, China (a) and known distribution localities of N. parkeri (blue), N. pleskei (red), N. ventripunctata (yellow), N. bangdaensis (green), and the new species (black) in China (b).
Figure 1. Map showing the type locality of Nanorana laojunshanensis sp. nov. in northwestern Yunnan, China (a) and known distribution localities of N. parkeri (blue), N. pleskei (red), N. ventripunctata (yellow), N. bangdaensis (green), and the new species (black) in China (b).
Animals 13 03427 g001
Animals 13 03427 g002
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. 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).
Animals 13 03427 g002
Animals 13 03427 g003
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. 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).
Animals 13 03427 g003
Animals 13 03427 g004
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. 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).
Animals 13 03427 g004
Animals 13 03427 g008
Figure 8. Tympanum region and ventral surface of N. laojunshanensis sp. nov. (A1,A2), N. bangdaensis (B1,B2), and N. parkeri (C1,C2). Tympanum of the new species is highlighted with arrow.
Figure 8. Tympanum region and ventral surface of N. laojunshanensis sp. nov. (A1,A2), N. bangdaensis (B1,B2), and N. parkeri (C1,C2). Tympanum of the new species is highlighted with arrow.
Animals 13 03427 g008
Animals 13 03427 g009
Figure 9. Hand and ventral surface of N. laojunshanensis sp. nov. (A1,A2) and N. ventripunctata (B1,B2).
Figure 9. Hand and ventral surface of N. laojunshanensis sp. nov. (A1,A2) and N. ventripunctata (B1,B2).
Animals 13 03427 g009
Table 1. Species used in phylogenetic analyses of this study.
Table 1. Species used in phylogenetic analyses of this study.
SpeciesVoucher no.Locality16SCOICYTBRAG-1
Odorrana hosiiUSNM:Herp:586991Yeybu Village, Tanintharyi, MyanmarMG935960MG935666--
Amolops mengdingensisKIZ20160265Mengding, Yunnan, ChinaMK501808MK501811--
Occidozyga limaUSNM:Herp:520376Chatthin, Sagaing, MyanmarMG935924MG935630--
Occidozyga lingnanicaSYS a005585Shenzhen, Guangdong, ChinaON615075ON615615--
Occidozyga myanhesseiUSNM:Herp:587105Dawei, Bago, MyanmarMG935916MG935622--
Ingerana tenasserimensisCAS 205064MyanmarAY322302--
Ingerana borealisMZMU1644Tibet, ChinaMT799709--KU243100
Fejervarya cancrivoraHW6Guangxi, ChinaEU652694EU652694EU652694HM163581
Hoplobatrachus chinensisTHW1Jinhua, Zhejiang, ChinaJX181763JX181763JX181763-
Sphaerotheca brevicepsUSNM:Herp:537466Sagaing, MyanmarMG935993MG935699--
Limnonectes bannaensisZNAC 21020Yunnan, ChinaAY899242AY899242AY899242
Quasipaa boulengeriXM3632Sichuan, ChinaKX645665KX645665KX645665-
Chrysopaa sternosignataUSNM:Herp:589844Parvan, AfghanistanMG700155MG699938--
Allopaa hazarensis9386PakistanMW598397MW603006-MW598465
Nanorana arunachalensisZSIS-M40Cona, Tibet, ChinaMN636773---
Nanorana (Chaparana) aenea2001.0277Yunnan, ChinaKR827954KR087830-HM163609
Nanorana (Chaparana) quadranusCIB 20060644Sichuan, ChinaGQ225907OL762449-HM163591
Nanorana (Chaparana) taihangnicaLGW-LC-001Henan, ChinaKF199146KF199146KF199146-
Nanorana (Chaparana) yunnanensisSTJW-LP-001Yunnan, ChinaKF199150KF199150KF199150HM163592
Nanorana (Paa) chayuensisMNU20190419Chayu, ChinaMN411630MN411630MN411630HM163587
Nanorana (Paa) liebigiiSH080524-NMENepalMN012113MN012241-MN032536
Nanorana (Paa) medogensisSYNU-XZ35Motuo, ChinaMH315960--HM163590
Nanorana raricaA1961-13-NMENepalMN012202MN012322
Nanorana (Paa) rostandiSYNU-1507058Tibet, ChinaMH315964--MW111374
Nanorana ercepeaeA2017-13-NMENepalMN012076MN012212 MN032500
Nanorana zhaoermiiSYNU-1706063Tibet, ChinaMH315958---
Nanorana xuelinensisKIZL2019013Lancang, Yunnan, ChnaMZ416027---
Nanorana vicinaWLM:NV289171Murree, PakistanMW898174---
Nanorana unculuanusKizYP010Yunnan, ChinaDQ118491--HM163595
Nanorana sichuanensisCIBYY20080693Sichuan, ChinaKU140064---
Nanorana polunini2003.3085Pangum, NepalKR827957 MW111375
Nanorana phrynoidesCIBYN090223Yunnan, ChinaKU140002---
Nanorana maculosaYNU-HU2002308Yunnan, ChinaEU979835--HM163588
Nanorana kangxianensisCIB1247625Gansu, ChinaMZ895123MZ895123MZ895123-
Nanorana conaensisKizYP152Tibet, ChinaDQ118513--HM163589
Nanorana blanfordiiSYNU-1507011Tibet, ChinaMH315963--MW111370
Nanorana arnoldiYNU-HU200109006Yunnan, ChinaEU979837---
Nanorana minicaWT008Himachal Pradesh, IndiaOQ079488---
Nanorana sp.VV11.1-RAS-OP173783OP174426-OP204887
Nanorana (Nanorana) bangdaensisN7_06_NMENimu, Lasa, Tibet, ChinaMN012126MN012249-MN032549
Nanorana (Nanorana) bangdaensisCAS802LDangxiong, Lasa, Tibet, ChinaMN012138MN012261-MN032555
Nanorana (Nanorana) bangdaensisKIZ014955Bangda, Baxoi, Tibet, China-KJ811447--
Nanorana (Nanorana) bangdaensisYPX32289Daritang, Uzi, Tibet-KJ811502--
Nanorana (Nanorana) bangdaensisYPX18274Lulang, Nyingchi, Tibet-KJ811344--
Nanorana (Nanorana) bangdaensisYPX15383Lhunze, Tibet-KJ811253--
Nanorana (Nanorana) bangdaensisN3_06_NMESeni, Naqu, Tibet, ChinaMN012150MN012272-MN032561
Nanorana (Nanorana) bangdaensisKP317482Dangxiong, Lasa, Tibet, ChinaKP317482KP317482KP317482-
Nanorana (Nanorana) bangdaensisKIZ20181001Bangda, Baxoi, TibetOR678588OR671665-OR678566
Nanorana (Nanorana) bangdaensisKIZ20181002Bangda, Baxoi, TibetOR678589OR671666-OR678567
Nanorana (Nanorana) bangdaensisKIZ20181003Bangda, Baxoi, TibetOR678590OR671667-OR678568
Nanorana (Nanorana) bangdaensisKIZ20181004Bangda, Baxoi, TibetOR678591OR671668-OR678569
Nanorana (Nanorana) bangdaensisKIZ20181005Bangda, Baxoi, TibetOR678592OR671669-OR678570
Nanorana (Nanorana) parkeriKIZ20181101Jilong, Rikaze, Tibet, ChinaOR678593OR671670-OR678571
Nanorana (Nanorana) parkeriKIZ20181102Jilong, Rikaze, Tibet, ChinaOR678594OR671671-OR678572
Nanorana (Nanorana) parkeriKIZ20181103Jilong, Rikaze, Tibet, ChinaOR678595OR671672-OR678573
Nanorana (Nanorana) parkeriKIZ20181104Jilong, Rikaze, Tibet, ChinaOR678596OR671673-OR678574
Nanorana (Nanorana) parkeriKIZ20181105Jilong, Rikaze, Tibet, ChinaOR678597OR671674-OR678575
Nanorana (Nanorana) parkeriKIZ08139Gangga, Tingri, Tibet, China-KJ811162--
Nanorana (Nanorana) parkeriYPX14998Nierixiong, Rikaze, Tibet-KJ811364--
Nanorana (Nanorana) parkeriYPX15060Saga, Tibet-KJ811396--
Nanorana (Nanorana) parkeriYPX14646Nierixiong, Rikaze, Tibet, China-KY172326KY172509-
Nanorana (Nanorana) pleskeiYPX32958Maqu, Ganan, Gansu, China-KY172146KY172329KY172539
Nanorana (Nanorana) pleskeiYPX25853Banma, Guoluo, Qinghai, China-KY172155KY172338KY172541
Nanorana (Nanorana) pleskeiKIZ014906Jiangda, Changdu, Tibet-KY172183KY172366KY172552
Nanorana (Nanorana) pleskeiYPX32403Kangding, Sichuan, China-KY172218KY172401KY172605
Nanorana (Nanorana) pleskeiKQ13_14_NMEGanzi, Sichuan, ChinaMN012160MN012282-MN032566
Nanorana (Nanorana) pleskeiKQ47_14_NMEKangding, Ganzi, Sichuan, ChinaMN012156MN012278-MN032562
Nanorana (Nanorana) pleskeiCIB20080515-1Shiqu, Sichuan, ChinaHQ324232HQ324232HQ324232-
Nanorana (Nanorana) ventripunctataGXNU YU130021Xiaozhongdian, Zhongdian, Yunnan, ChinaOR678598OR671675OR678554OR678576
Nanorana (Nanorana) ventripunctataGXNU YU130022Xiaozhongdian, Zhongdian, Yunnan, ChinaOR678599OR671676OR678555OR678577
Nanorana (Nanorana) ventripunctataGXNU YU090163Bitahai, Zhongdian, Yunnan, ChinaOR678600OR671677OR678556OR678578
Nanorana (Nanorana) ventripunctataGXNU YU000498Bitahai, Zhongdian, Yunnan, ChinaOR678601OR671678OR678557OR678579
Nanorana (Nanorana) ventripunctataGXNU YU000502Bitahai, Zhongdian, Yunnan, ChinaOR678602OR671679OR678558OR678580
Nanorana (Nanorana) ventripunctataGXNU YU000503Bitahai, Zhongdian, Yunnan, ChinaOR678603OR671680OR678559OR678581
Nanorana (Nanorana) laojunshanensis sp. nov.GXNU YU090312Mt. Laojun, Lijiang, Yunnan, ChinaOR678604OR671681OR678560OR678582
Nanorana (Nanorana) laojunshanensis sp. nov.GXNU YU090313Mt. Laojun, Lijiang, Yunnan, ChinaOR678605OR671682OR678561OR678583
Nanorana (Nanorana) laojunshanensis sp. nov.GXNU YU090314Mt. Laojun, Lijiang, Yunnan, ChinaOR678606OR671683OR678562OR678584
Nanorana (Nanorana) laojunshanensis sp. nov.GXNU YU090315Mt. Laojun, Lijiang, Yunnan, ChinaOR678607OR671684OR678563OR678585
Nanorana (Nanorana) laojunshanensis sp. nov.GXNU YU090316Mt. Laojun, Lijiang, Yunnan, ChinaOR678608OR671685OR678564OR678586
Nanorana (Nanorana) laojunshanensis sp. nov.GXNU YU090317Mt. Laojun, Lijiang, Yunnan, ChinaOR678609OR671686OR678565OR678587
Table 2. Primers used for PCR amplification and sequencing in this study.
Table 2. Primers used for PCR amplification and sequencing in this study.
Locus Primer NamePrimer SequenceSource
16S16Sar5′-CGCCTGTTTATCAAAAACAT-3′[38]
16Sbr5′-CCGGTCTGAACTCAGATCACGT-3′[38]
cytbCytbF5′-ACACCCGCCAATTTGTCTTC-3′This study
CytbR5′-TGAGAGGGGAAGGAGAAGGA-3′This study
COIChmf45′-TYTCWACWAAYCAYAAAGAYATCGG-3′[39]
Chmr45′-ACYTCRGGRTGRCCRAARAATCA-3′[39]
RAG1L-RAG1Ran5′-CTGGTCGTCAGATCTTTCAGC-3′[40]
H-RAG1Ran5′-GCAAAACGTTGAGAGTGATAAC-3′[40]
Table 3. Best partition scheme and models estimated by the partition finder analysis for the combined data.
Table 3. Best partition scheme and models estimated by the partition finder analysis for the combined data.
SubsetPartitionsModel
116S rRNAGTR + I + G
2COI_pos1SYM + I
3COI_pos2HKY + I
4COI_pos3GTR + G
5cytb_pos1SYM + G
6cytb_pos2HKY + I
7cytb_pos3GTR + G
8Rag1_pos1GTR + G
9Rag1_pos2GTR + I
10Rag1_pos3GTR + G
Table 4. Genetic distances (%) between members of the subgenus Nanorana estimated from 16S (lower triangle) and COI sequences (upper triangle).
Table 4. Genetic distances (%) between members of the subgenus Nanorana estimated from 16S (lower triangle) and COI sequences (upper triangle).
Species12345
1N. laojunshanensis sp. nov. 7.48.710.610.0
2N. ventripunctata1.6 8.811.210.9
3N. pleskei1.61.9 12.912.4
4N. bangdaensis2.02.12.8 3.4
5N. parkeri1.61.82.71.0
Table 5. Measurements (in mm) of Nanorana (Nanorana) laojunshanensis sp. nov. from the type locality (M: male; F: female).
Table 5. Measurements (in mm) of Nanorana (Nanorana) laojunshanensis sp. nov. from the type locality (M: male; F: female).
CharacterGXNU YU090314GXNU YU090313GXNU YU090315GXNU YU090316GXNU YU090317GXNU YU090312
SexMMMMMF
SVL (snout–vent length)36.138.535.934.933.342.9
HL (head length)10.711.010.610.210.111.4
HW (head width)11.312.911.911.510.713.6
SL (snout length)4.84.95.04.54.55.0
IND (internarial distance)2.32.42.32.12.12.7
IOD (interorbital distance)1.92.22.11.91.82.3
UEW (upper eyelid width)2.42.42.02.01.92.6
ED (eye diameter)3.94.53.93.83.94.6
TD (tympanum diameter)1.31.81.21.11.21.5
DNE (nostril–eye distance)1.72.11.71.61.62.1
FHL (forearm and hand length)15.115.115.416.213.817.4
TL (tibia length)15.315.815.715.514.216.9
TFL (length of foot and tarsus)26.527.626.227.324.130.7
FL (foot length)18.920.818.920.117.122.4
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.
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
CharacterPC1PC2PC1PC2
Eigenvalue4.3463.2836.2723.403
% variation33.43%25.25%48.24%26.18%
HL0.742−0.123−0.5520.711
HW0.878−0.0150.3830.810
SN0.384−0.574−0.4110.783
IND0.1260.563−0.8400.289
IOD0.677−0.2970.753−0.109
UEW−0.2490.729−0.8000.320
ED0.251−0.755−0.890.698
TD−0.090.162−0.6380.346
NED0.390−0.733−0.6800.224
FHL0.6000.6500.8710.321
TL0.6290.5960.7700.605
TFL0.8380.1910.8540.418
FL0.8180.2300.8840.359
Table 7. Suggested assignment of subgenus for Nanorana species. “Yes” means that the species belongs to the corresponding subgenus.
Table 7. Suggested assignment of subgenus for Nanorana species. “Yes” means that the species belongs to the corresponding subgenus.
SpeciesSuggested Subgenus
Subg. NanoranaSubg. PaaSubg. ChaparanaSubg. MinipaaUnknown
N. aenea yes
N. annandalii yes
N. arnoldi yes
N. bangdaensisyes
N. blanfordii yes
N. chayuensis yes
N. conaensis yes
N. ercepeae yes
N. feae yes
N. gammii yes
N. hazarensis yes
N. kangxianensis yes
N. liebigii yes
N. maculosa yes
N. medogensis yes
N. minica yes
N. mokokchungensis yes
N. parkeriyes
N. phrynoides yes
N. pleskeiyes
N. polunini yes
N. quadranus yes
N. rarica yes
N. rostandi yes
N. sichuanensis yes
N. taihangnica yes
N. unculuanus yes
N. ventripunctatayes
N. vicina yes
N. xuelinensis yes
N. yunnanensis yes
N. zhaoermii yes
N. laojunshanensis sp. nov.yes
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MDPI and ACS Style

Tang, S.; Liu, S.; Yu, G. A New Species of Nanorana (Anura: Dicroglossidae) from Northwestern Yunnan, China, with Comments on the Taxonomy of Nanorana arunachalensis and Allopaa. Animals 2023, 13, 3427. https://doi.org/10.3390/ani13213427

AMA Style

Tang S, Liu S, Yu G. A New Species of Nanorana (Anura: Dicroglossidae) from Northwestern Yunnan, China, with Comments on the Taxonomy of Nanorana arunachalensis and Allopaa. Animals. 2023; 13(21):3427. https://doi.org/10.3390/ani13213427

Chicago/Turabian Style

Tang, Shangjing, Shuo Liu, and Guohua Yu. 2023. "A New Species of Nanorana (Anura: Dicroglossidae) from Northwestern Yunnan, China, with Comments on the Taxonomy of Nanorana arunachalensis and Allopaa" Animals 13, no. 21: 3427. https://doi.org/10.3390/ani13213427

APA Style

Tang, S., Liu, S., & Yu, G. (2023). A New Species of Nanorana (Anura: Dicroglossidae) from Northwestern Yunnan, China, with Comments on the Taxonomy of Nanorana arunachalensis and Allopaa. Animals, 13(21), 3427. https://doi.org/10.3390/ani13213427

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