A new species of the genus Rana sensu lato Linnaeus, 1758 (Anura, Ranidae) from Wuyi Mountain, Fujian Province, China

Abstract A new species of the frog genus Rana sensu lato from Wuyi Mountain, Fujian Province, China is described. Molecular phylogenetic analyses clustered the new species into the R.johnsi group and indicated that it was genetically divergent from its closely related species. The new species could be distinguished from its congeners by a combination of the following characters: body size medium, SVL 41.4–45.6 mm (42.9 ± 1.9 mm, n = 4) in adult males and 47.6–50.3 mm (n = 2) in adult females; adult male with a pair of internal subgular vocal sacs; lateroventral grooves present on tip of toes; webbing on fourth toes reaching the tip of toe; transverse skin ridges distinctly present on the dorsal surface of thigh and tibia, the number large (mean 26.5 ± 2.7, range 22–29, n = 6); breeding males possess creamy white nuptial pad with tiny velvety spines on the dorsal surface of the first finger, divided into three parts.

Recently, in Wuyishan National Park, Wuyishan City, Fujian Province, China, we collected several specimens which can be assigned to Rana sensu lato based on morphology. Molecular phylogenetic analyses and detailed morphological comparisons indicated the specimens represented an undescribed species of the R. johnsi group. Herein we described it as a new species.

Specimens
Twelve unnamed specimens including four adult males, two adult females, and six tadpoles were collected from Wuyishan National Park, Fujian Province, China (Table 1   collected, i.e., six R. zhengi from Gulin County, Sichuan Province, China; five adult males, two adult females and six tadpoles of R. sangzhiensis from its type locality, Sangzhi County, Hunan Province, China; two adult males, one female, and one larval of R. johnsi from northern Vietnam; two larval of R. johnsi from Jinxiu County, Guangxi Province, China; eight adult males and six tadpoles of R. johnsi from Shiwandashan Mountains, Guangxi Province, China; and one adult male of R. weiningensis from its type locality, Weining City, Guizhou Province, China (Table 1, Fig. 1, Suppl. material 1). In the field, the frogs and tadpoles were euthanized using isoflurane, and the specimens were fixed in 75% ethanol. Muscle tissue samples were taken and preserved separately in 95% ethanol prior to fixation. The specimens were deposited in Chengdu Institute of Biology (CIB), Chinese Academy of Sciences, Nanning Normal University (NNU), and Institute of Ecology and Biological Resources (IEBR), Vietnam (for voucher numbers see Table 1 and Suppl. material 1).

Molecular phylogenetic analyses
A total of 40 samples collected in this study was used in molecular analyses, encompassing twelve unnamed specimens from Wuyi Mountain, six R. sangzhiensis, six R. zhengi, 15 R. johnsi, and one R. weiningensis (Table 1). Total DNA was extracted using a standard phenol-chloroform extraction protocol (Sambrook et al. 1989). Three mitochondrial genes (16S rRNA, ND2, and Cyt b) and three nuclear DNA markers   Table 2. Gene fragments were amplified under the following conditions: an initial denaturing step at 95 °C for 4 min; 36 cycles of denaturing at 95 °C for 30 s, 40 s at appropriate annealing temperature (Table 2); and extending at 72 °C for 70 s. PCR products were sequenced with primers same as used in PCR. Sequencing was conducted using an ABI3730 automated DNA sequencer. New sequences were deposited in GenBank (Table 1).
For phylogenetic analyses, the corresponding sequences for congeners especially for the topotypes of species in the subgenus Rana were downloaded from GenBank (Table 1), mainly derived from previous studies (Yuan et al. 2016;Wang et al. 2017;Wan et al. 2020). For phylogenetic analyses, corresponding sequences of one Odorrana versabilis  and one Pelophylax nigromaculatus (Hallowell, 1861) were also downloaded (Table 1), and used as outgroups according to Yuan et al. (2016).
Sequences were assembled and aligned using the ClustalW module in BioEdit v.7.0.9.0 (Hall 1999) with default settings. The protein-coding gene (Cytb, ND2, BDNF, RAG1, and Tyr1) sequences were translated to amino acid sequences in MEGA v. 6.0 (Tamura et al. 2013), adjusted for open reading frames, and checked to ensure absence of premature stop codons. No-sequenced fragments were treated as missing data. For phylogenetic analyses based on mitochondrial DNA, the dataset was concatenated with mitochondrial gene sequences. The best partition scheme and the best evolutionary model for each partition were chosen for the phylogenetic analyses using PARTITION-FINDER v. 1.1.1 (Robert et al. 2012). In this analysis, 16S gene and each codon position of protein-coding mitochondrial gene were defined, and Bayesian Inference Criteria was used. As a result, the analysis suggested that the best partition scheme is 16S gene/ each codon position of protein-coding gene, and selected GTR + G + I model as the best model for each partition. Phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian Inference (BI) methods, implemented in PhyML v. 3.0 (Guindon et al. 2010) and MrBayes v. 3.2 (Ronquist et al. 2012), respectively. For the ML tree, branch supports (bs) were drawn from 10,000 nonparametric bootstrap replicates. In BI, two runs each with four Markov chains were simultaneously run for 50 million generations with sampling every 1,000 generations. The first 25% trees were removed as the "burn-in" stage followed by calculations of Bayesian posterior probabilities (bpp), and the 50% majority-rule consensus of the post burn-in trees were sampled at stationarity. In addition, to access the genetic isolation between the undescribed species and its closely related species on nuclear DNA, one haplotype network for each nuclear gene dataset was constructed, using the maximum parsimony method in TCS v. 1.21 (Clement et al. 2000). Genetic distance of uncorrected-p-distance model on 16S gene sequences between the new species and its closely related species were estimated using MEGA.

Morphological comparisons
All six adult specimens of the undescribed species were measured (Suppl. material 1). For comparisons, five adult male specimens of R. sangzhiensis, eleven adult male specimens of R. johnsi, and 22 adult male specimens of R. zhengi used in Jiang et al. (1997) were also measured (Suppl. material 1). The terminology and methods followed Fei et al. (2009). Measurements were taken with a dial caliper to 0.1 mm. Twenty-two morphometric characters of adult specimens were measured:

ED
eye diameter (distance from the anterior corner to the posterior corner of the eye); FIIIL third finger length (distance from base to tip of finger III); FIIL second finger length (distance from base to tip of finger II); FIL first finger length (distance from base to tip of finger I); FIVL fourth finger length (distance from base to tip of finger IV); FL foot length (distance from tarsus to the tip of fourth toe); HAL hand length (distance from tip of third digit to proximal edge of inner palmar tubercle); HDL head length (distance from the tip of the snout to the articulation of jaw); HDW maximum head width (greatest width between the left and right articulations of jaw); IND internasal distance (minimum distance between the inner margins of the external nares); IOD interorbital distance (minimum distance between the inner edges of the upper eyelids); LAL length of lower arm and hand (distance from the elbow to the distal end of the Finger IV); LW lower arm width (maximum width of the lower arm); SL snout length (distance from the tip of the snout to the anterior corner of the eye); SNT distance between the nasal the posterior edge of the vent; SVL snout-vent length (distance from the tip of the snout to the posterior edge of the vent); TFL length of foot and tarsus (distance from the tibiotarsal articulation to the distal end of the Toe IV); THL thigh length (distance from vent to knee); TL tibia length (distance from knee to tarsus); TW maximal tibia width; TYD maximal tympanum diameter; UEW upper eyelid width (greatest width of the upper eyelid margins measured perpendicular to the anterior-posterior axis).
To reduce the impact of allometry in adults, the correct value from the ratio of each character to SVL was calculated, and then was log-transformed for the following morphometric analyses. One-way ANOVA tests were conducted to test the significance of differences on morphometric characters between the undescribed species and its closely related species. The significance level was set at 0.05.

Phylogenetic analyses
ML and BI trees of the mitochondrial DNA dataset presented almost consistent topology ( Fig. 2A, B). In mitochondrial DNA trees, all samples of the undescribed species were strongly nested into one clade (all supports = 100; Fig. 2B). The R. johnsi group was strongly supported as a monophyletic group containing all samples of R. johnsi, R. sangzhiensis, R. zhengi, and the undescribed species (all supports = 100; Fig. 2B). The R. johnsi group was clustered into the clade corresponding to the subgenus Rana ( Fig. 2A). The R. johnsi group contained two clades. In the first clade, samples of R. sangzhiensis and R. zhengi were nested into a clade (all supports = 100), which was weakly clustered as the sister of the undescribed species clade (bs = 52; bpp = 0.80; Fig. 2B). In the second clade, three R. johnsi samples from Vietnam were clustered into one clade, which was sister to the clade containing samples of R. johnsi from two localities of Guangxi Province, China (Figs 1, 2B; Table 1). In addition, the topotype specimen of R. weiningensis was phylogenetically far from the R. johnsi group, and clustered as the basal clade of the genus Rana ( Fig. 2A). Haplotype networks based on three nuclear genes all indicated that the undescribed species did not share haplotype with its closely related species R. johnsi, R. sangzhiensis, and R. zhengi (Fig. 2C-E), further indicating the genetic divergence between the undescribed species and its closely related species. As note, on each gene, samples of R. sangzhiensis and R. zhengi massively shared common haplotypes (Fig. 2C-E), indicating their very shallow genetic divergence. The genetic distance on 16S between all samples of undescribed species is less than 0.2% (range 0.0%-0.2%). The genetic distance between the species and its closely related species were as following: vs. R. johnsi from Vietnam 1.3% (range 1.1%-1.7%), vs. R. johnsi from Guangxi, China 0.8% (range 0.8%-0.9%), vs. R. zhengi 1.0% (range 0.9%-1.1%), and vs. R. sangzhiensis 0.9% (range 0.8%-1.1%), being similar to that between the latter four groups (range 0.8%-1.4%). As note, the genetic distance between R. zhengi and R. sangzhiensis was 0.2% (range 0.0%-0.4%), and that between R. johnsi from Vietnam and R. johnsi from Guangxi, China was 0.5% (range 0.4%-1.1%).

Morphological comparisons
The R. johnsi group is phylogenetically clustered into the subgenus Rana, but this group could be identified from other species of the subgenus Rana by the tip of toes with lateroventral grooves (vs. absent in other species of subgenus Rana). The undescribed  Table 1 C-E haplotype networks constructed based on sequences of nuclear genes BDNF, RAG1, and Tyr, respectively. Different species of the R. johnsi group were denoted as different colors. species could be assigned to this species group by a series of morphological characters: tip of toes flat with lateroventral grooves; body size medium, SVL 41.4-45.6 mm (42.9 ± 1.9 mm, n = 4) in adult males and 47.6-50.3 mm (n = 2) mm in adult females; dorsolateral fold distinct and thin, extending straight from posterior margin of upper eyelid to above groin; tympanum distinct, oval; tibio-tarsal articulation reaching forward beyond tip of snout when leg starched forward; skin ridges distinctly arranged on the dorsal surface of thighs and tibias; adult males with a pair of internal subgular vocal sacs; breeding males possess creamy white nuptial pad with tiny hoar velvety spines on the dorsal surface of the first finger, divided into three parts.
Although the R. johnsi group and R. weiningensis both have lateroventral grooves on the tip of toes, the undescribed species in the R. johnsi group could be easily distinguished from R. weiningensis by the following characters: males with internal subgular vocal sacs (vs. absent in the latter); males with lager body size (41.4-45.6 mm, n = 4 vs. 32.8-37.4 mm, n = 3 in the latter); and more developed webbing between toes (webbing on fourth toes reaching tip of toe vs. reaching distal subarticular tubercle in the latter).
On morphometric characters, the results of One-way ANOVA showed that in male, the undescribed species was significantly different from R. sangzhiensis on SVL,  Table 4).
In total, molecular phylogenetic analyses and morphological comparisons indicated that our specimens from Wuyi Mountain, Fujian Province, China should be classified into the R. johnsi group, and are significantly divergent from its closely related species. The specimens should represent a new species which is described as following section. Table 4. Morphometric comparisons between the adult male specimens of Rana wuyiensis sp. nov. and its closely related species. Units given in mm. See abbreviations for the morphological characters in Materials and methods section. P-value was resulted from One-way ANOVA test. Significant level at 0.05. P-value < 0.05 denoted as bold.  Table 3. Comparisons on number of skin ridges on thighs and tibias between Rana wuyiensis sp. nov. and its closely related species. P-value was resulted from Oneway ANOVA test. Significant level at 0.05. Five adult specimens from the same place as holotype collected by Yanqing Wu. One female CIB WYS20200829001 and two males CIB WYS20200829002 and CIB WY20200829003 were collected on 29 August 2020. One female CIB WY20200913002 and one male CIB WY20200913001 were collected on 13 September 2020.

Rana wuyiensis
Other material examined. Six tadpoles collected from the same place as holotype (Table 1) by Yanqing Wu on 01 November 2020.
Diagnosis. Rana wuyiensis sp. nov. is distinguished by a combination of the following morphological characters: body size medium, SVL 41.4-45.6 mm (42.9 ± 1.9 mm, n = 4) in adult males, and 47.6-50.3 mm (n = 2) in adult females; lateroventral  grooves present on tip of toes; transverse skin ridges distinctly present on the dorsal surface of thighs and tibias, the number large (mean 26.5 ± 2.7, range 22-29, n = 6); adult male with a pair of internal subgular vocal sacs; webbing on fourth toes reaching the tip of toe; breeding males possess creamy white nuptial pad with tiny hoar velvety spines on the dorsal surface of the first finger, divided into three parts.
Etymology. The specific name wuyiensis is in reference to the type locality, Wuyi Mountain, Fujian Province, China.
Suggested common name. Wuyi Brown Frog (in English), Wuyi Lin Wa (in Chinese; 武夷林蛙).  (Figs 4, 5). Adult male; SVL 41.4 mm. Head significantly longer than wide (HDW/HDL ratio = 0.85); snout pointed and projecting over lower lip; nostril closer to tip of snout than eye; canthus rostralis distinct; internasal distance distinctly wider than interorbital distance (IOD/IND ratio = 0.81); loreal region slightly oblique and concave; upper eyelids narrower than interorbital distance; tympanum rounded, diameter three quarters of eye (TD/ED ratio = 0.75), and separated from eye by a short distance about one quarter of tympanum diameter; tympanic rim feebly elevated; pupil oval and horizontal, notched at middle lower margin; a skin fold present posterior to tympanum, disconnected with dorsolateral fold, swollen near shoulder; vomerine teeth in two short row, four or five for each, oblique and separated by a distance about one row of teeth; tongue deeply notched posteriorly, depth about one sixth of entire tongue length; a pair of internal subgular vocal sacs present, openings slit like, small, length as wide as finger tips, positioned at on inner mandible near the corners of mouth.

Description of holotype
Forearms moderate, width 0.09 ratio of SVL; hand 0.27 ratio of SVL; fingers elongated, with narrow lateral fringes, rudimentary webbed, webbing formula I 3⅔ -2⅔ II 2½ -3½ III 3½-3 IV; tips of fingers rounded, not swollen, without lateroventral groove; finger II distinctly shorter than I, relative finger lengths II < I < IV < III; subarticular tubercles prominent, rounded; supernumerary tubercles indistinct, oval, present on bases of all fingers; inner metacarpal tubercle distinct, near oval, positioned near inner surface of base of finger I, inner side partially covered with nuptial pad; two outer metacarpal tubercles partially separated near the joint of metacarpals of fingers III and IV, the inner oval and larger, the outer elongated and smaller; nuptial pad present on inner and dorsal surface of finger I, covered with velvety spines, partially divided into three parts, the basal part on inner side of inner metacarpal tubercle, the middle part largest, on third phalanx, the distal part smallest, on first and second phalanxes.
Hindlimbs long, tibia 0.64 ratio of SVL and length of foot and tarsus 0.84 ratio of SVL; thigh shorter than tibia, heels overlap when hindlimbs flexed at right angles to axis of body; tibio-tarsal articulation reaching far beyond snout when hindlimb stretched forward along body; toes entirely webbed, inner edge of toe I and outer edge of toe V with narrow lateral fringe, relative toe lengths I < II < III < V < IV, toes webbing formula: 1⅓ -2 II 1⅓ -2⅓ III 1½ -2⅔ IV 3 -1⅓ V; tip of toes somewhat flat, lateroventral grooves present on all tip of toes and disconnected at middle of front edge; subarticular tubercles prominent and oval; supernumerary tubercles absent; inner metatarsal tubercle oval and prominent, outer metatarsal tubercle rounded, indistinct.
Dorsal skin smooth, supratympanic fold absent; dorsolateral folds distinct, narrow, extending from edge of upper eyelid to hip, not curve above tympanum. Ventral skin smooth, skins around cloaca with numerous flat tubercles. Skin on hindlimbs with transvers paralleled ridges, eight on both thighs, six and seven on left and right tibias, four and two on left and right tarsal. Tarsal fold present.
Coloration in life (Fig. 4). Dorsal surface basically medium brown, scattered with dense dark brown pigments all over; dorsolateral skin folds and skin ridges on dorsal limbs yellow brown with deep drown fringes; five ambiguous deep brown cross bands present on dorsal forelimbs; irregular black patches present on inner surface of forearm near wrist, anterior knee and lateral tibia; lower edge of canthus rostralis dark brown; skins on tympanum and anterior to the fold behind tympanum deep brown; ventral skin basically cream white on body and arm; lips light brown with cream white patches; throat, chest, and upper abdomen with irregular light orangish short bars; ventral hindlimbs mostly flesh colored, with a small region near base of tinged yellowish white; ventral hand flesh-colored with brown pigments; ventral feet covered with dense brown pigments. Nuptial pad hoar. Iris mostly copper with dark cracks, regions anterior and posterior to pupil deeper. Coloration in preserve (Fig. 5). Body coloration lighter than in life, dark brown pigments more prominent. Skins between upper eyelids with an ambiguous brown pattern. Ventral body mostly white, with brown pattern; ventral limbs yellowish. Ventral hand and feet greyish. Skins on temporal region with prominent dark patches. Lateral head before eyes blackish. Iris dark with metal luster.
Secondary sexual characters. Breeding males with nuptial pad on dorsal surface of finger I, covered with velvety spines, divided into three parts. Male with a pair of internal subgular vocal sacs.
Variations. For measurements of type series specimens see Tables 4, Suppl. material 1. Coloration of the two females lighter (Fig. 6A), basically yellowish brown. Black edges of dorsolateral fold absent on CIB WY20200913002 (Fig. 6C) and indistinct on CIB WYS20200829001. The number of skin ridges on dorsal thigh range from five to eight. The skin ridges on tibia range from four to eight.
Distribution and ecology. Currently, Rana wuyiensis sp. nov. is known from Wuyishan National Park, Wuyishan City, Fujian Province, China. In our surveys from 2017 to 2021, the species was found only at one site. All individuals of the new species used in this work were collected from a stream and nearby grassland under the evergreen broad-leaf forest (Fig. 7). Six adult individuals and some very small tadpoles at early stages of development were found in the late August and early September. Only relative larger and middle-staged tadpoles were collected in the early November. This suggests that the breeding season of this species may begin in July or early August.

Discussion
Our results based on mitochondrial DNA and nuclear DNA of several populations of R. zhengi and R. sangzhiensis indicated that the two groups have very low genetic divergence. This is identical to the results of previous molecular phylogenetic analyses in Wan et al. (2020). In addition, we did not find morphological characters for separating the two groups, being consistent with the results of Jiang et al. (1997) and Fei et al. (2009). Based on this evidence, we support the proposal that R. zhengi should be synonymized with R. sangzhiensis. Accordingly, R. sangzhiensis is at least distributed from southwestern part of Sichuan to western Hunan provinces, China. On the contrary, Rana wuyiensis sp. nov. differs from its closely related species not only on morphology but also on molecular data, supporting the separation of the new species.
Moreover, the divergence between Rana wuyiensis sp. nov. and its closely related species in the R. johnsi group is likely corresponding to their separated distributional ranges (Fig. 1). Wuyi Mountain is located at the southeastern edge of the mainland China, far from the "west" distributional ranges of R. johnsi and R. sangzhiensis in southwestern China (at least > 400 km in a straight line between them; Fig. 1), and the distribution ranges belong to different biota (e.g., Zhang 2009; Fei et al. 2010). This indicates that vicariance might be the primary factor for the speciation of the species. Whatever, the discovery of the new species greatly expanded the distributional range of the R. johnsi group to the southeastern China and would promote exploring the biogeographical patterns in the frog group.
However, to date, Rana wuyiensis sp. nov. was found only at one site in Wuyi Mountain, and it probably has a low population size according to our eleven-times surveys which included forty sites every time in April, June, and August from 2018 to 2021. Although this site is in the central part of the Wuyishan National Park, the breeding habitat is vulnerable due to local human activities especially tea plantation (Fig. 7A) and/or local nature disaster (for example, the novel rainstorm in 2020 in Wuyi Mountain; our unpublished data). Therefore, we need to understand its population status and major threats, and then take appropriate actions to prepare strategies for its conservation.