﻿A new species of Bush frog (Anura, Rhacophoridae, Raorchestes) from southeastern Yunnan, China

﻿Abstract In this study, based on morphological and molecular data, a new bush frog species is described from Yunnan, China. Eleven samples of Raorchestesmalipoensissp. nov. were collected from Malipo County, southeastern Yunnan. This species can be distinguished from other congeners by a combination of 13 morphological characters. Phylogenetic analyses based on the 16S rRNA gene indicate that these individuals form a monophyletic group, and genetic divergence between this clade and its closest relatives is higher than 3.1%, which is comparable to the divergence between recognized Raorchestes species. The discovery of this new species suggests that additional extensive surveys in the southeastern Yunnan would yield more amphibian lineages yet unknown to science.


Introduction
The genus Raorchestes Biju, Shouche, Dubois, Dutta & Bossuyt, 2010 belongs to the family Rhacophoridae Hoffman, 1932. It includes bush frogs with adult size ranging from 10.0 mm to 50.5 mm (Priti et al. 2016). They are distinguished by the presence of a transparent/translucent vocal sac, the absence of vomerine teeth, and direct development without free swimming tadpoles (Seshadri et al. 2012). The genus Raorchestes currently contains 74 species, ranging from the southern tip of the Indian Peninsula to northeastern India, Indo-China, and southwestern China (Frost 2021): most are from south and Southeast Asia including southern India to Nepal, Myanmar, Thailand, Laos, southern China, Vietnam, and West Malaysia. Of the 74 recognized species, seven species have been originally described from China: Raorchestes longchuanensis (Yang & Li, 1978), R. menglaensis (Kou, 1990), R. andersoni (Anderson, 1927), R. cangyuanensis (Wu et al., 2019), R. dulongensis (Wu et al., 2021), R. hillisi , and R. huanglianshan . Detailed ecological data is not available for the species reported in China except for R. longchuanensis, for which Yan et al. (2021) reported the breeding mode.
Many Raorchestes species from the region were described with few diagnostic characters and limited morphological data, which hampers the identification of these smallsized bush frogs ). In addition, the taxonomy of Raorchestes gryllus is under dispute. It was originally described as Philautus gryllus Smith, 1924, from Langbian Peaks, southern Vietnam. Biju et al. (2010) classified this species into Raorchestes according to the 16S sequences from Pac Ban, Tuyen Quang, northern Vietnam, and recently Poyarkov et al. (2021) suggested a transfer to Kurixalus based on morphological and molecular data of specimens from the type locality (Langbian, southern Vietnam).
In this work we studied specimens allocated to Raorchestes from Malipo County. This county is located in the southeast of Yunnan Province, and lies on the China-Vietnam border where few herpetological investigations have been conducted. During the fieldwork, we collected 11 specimens of a small-sized bush frog that could be assigned to the genus Raorchestes based on morphological and molecular evidence. Phylogenetically, these specimens were grouped together with a misidentified "R. gryllus" from Pac Ban, Tuyen Quang, northern Vietnam. However, considering that the type locality of Philautus gryllus, Langbian Plateau, is 1200 km far from the China-Vietnam border and that obvious morphological differences exist between Philautus gryllus and the lineage consisting of individuals from China-Vietnam border region, we consider that these specimens represent a new species that we formally describe here.

Morphology and morphometrics
All the measurements were made with slide calipers to the nearest 0.1 mm. Morphological terminology and measurement methods followed Fei et al. (2009). The morphological characters include: snout-vent length (SVL); head length (HL); head width (HW); snout length (SL); internarial distance (INS); interorbital distance (IOS); eye horizontal diameter (EHD); maximum width of upper eyelid (UEW); tympanum diameter (TD); forelimb and hand length (FAHL); width of lower arm (LAW); hand length (HAL); femur length (FML); tibia length (TBL); length of tarsus and foot (TFL); foot length (FOL); tibia width (TBW); and femur width (FMW). Morphological measurements of the specimens are given in Table 1. Males and females (breeding individuals) were identified based on the presence or absence of an external single subgular vocal sac. Comparative morphological data of congeneric species were taken from previous studies and are presented in Table 2.

DNA sequencing and analyses of sequences
Total DNA was extracted using a commercial tissue DNA isolation kit (Chenlu Biotech, China). For seven specimens in this study, the mitochondrial gene 16S ribosomal RNA (16S rRNA) gene was sequenced. The fragments of 16S rRNA were amplified using primers 16Sar-L (5'-CGCCTGTTTATCAAAAACAT-3') and 16Sbr-H (5'-CCGGTCTGAACTCAGATCACGT-3') (Palumbi et al. 1991). Polymerase chain reactions (PCR) amplifications were performed in a 25 μl reaction volume with an initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 51 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 10 min. The PCR products were sequenced using an ABI 3730 automated sequencer. To study the phylogenetic relationships among Raorchestes species, matrilineal genealogies were reconstructed based on the 16S fragment. Fifty-two sequences of Raorchestes and representative outgroups  were downloaded from GenBank (Table 3). The dataset was checked by eye and manually adjusted using MEGA 6.0 with default settings (Tamura et al. 2013), and the alignment was checked by eye and adjusted manually. JMODELTEST v. 2.1.7 (Darriba et al. 2012) was used to select an appropriate nucleotide substitution model for Bayesian Inference (BI). The GTR+G+I model was chosen as the best-fit model following the Bayesian information criterion (BIC; Posada 2008). Bayesian analysis was performed using MrBayes 3.2 (Ronquist et al. 2012). For BI analyses, the Monte Carlo Markov chain length was run for 120,000,000 genera-  Raorchestes longchuanensis (Yang & Li, 1978) Al-Razi et al. 2020 b; Yang and Li 1978 7 Raorchestes menglaensis (Kou, 1990 (Stamatakis, 2014). Mean genetic distances (uncorrected p-distance) between and within species were calculated in MEGA v. 6.0.6 (Tamura et al. 2013) based on 16S sequences.

Results
The final DNA sequence dataset is consisted of 59 sequences and the length of the sequence alignment is 542 base pairs (bp) (Table 3), of which 194 sites are variable and 135 are parsimony informative. The BI and ML trees had almost identical topologies (Fig. 3). The samples from Malipo County, Yunnan Province form a monophyletic group and the sample from Pac Ban, Tuyen Quang (northern Vietnam) previously identified as R. gryllus was also nested in the clade with strong support (Fig. 3). Genetic distances between the samples from Malipo County and the other species of Raorchestes varied from 3.1% (R. longchuanensis) to 6.0% (R. huanglianshan) ( Table 4).   Diagnosis. The genus Raorchestes is a group of small frogs, diagnosed primarily on the basis of an adult snout-vent length between 15 and 45 mm; vomerine teeth absent; large gular pouch transparent while calling; nocturnally active; direct development without free-swimming tadpoles in all species for which the development is known (Biju et al. 2010). Although the mode of development in the new species remains unknown, R. malipoensis sp. nov. is placed in the genus Raorchestes due to the combination of following characters: small body size, vomerine teeth absent, single translucent external subgular vocal sac present, and tips of all fingers and toes expanded into discs with circum-marginal grooves. The new species is distinguished from geographically and molecularly relevant congeners by the following combination of characters: (1) very small body size (males SVL 14.6-17.7 mm, n = 7; females SVL 18.3-19.3 mm, n = 4); (2) head wider than long; (3) tympanum small, supratympanic fold distinct; (4) tips of all fingers and toes yellow; (5) webbing formula (I 2 -2 II 2 -2 III 2 -3 IV 3 -2 V); (6) inner and outer metacarpal tubercle indistinct; (7) heels not meeting when limbs held at right angles to body; (8) tibiotarsal articulation reaching anterior border of eye when hindlimb is stretched alongside of body; (9) iris golden brown; (10) nuptial pad small and milky white; (11) inner metatarsal tubercle rounded, outer metatarsal tubercle absent; (12) fingers and toes having lateral dermal fringe; and (13) interorbital distance larger than eye horizontal diameter.
Description of the holotype. Adult male (Fig. 4), body size small (SVL 17.7 mm); head wider than long (HL 6.4 mm; HW 6.8 mm); top of head relatively flat; snout rounded in profile, projecting beyond lower jaw; snout length almost equal to interorbital distance at narrowest point (SL 2.6 mm; IOS 2.6 mm); the canthus rostralis rounded, loreal region slightly concave; tympanum small (TD 1.5 mm); internarial distance wider than maximum width of upper eyelid (INS 2.1 mm; UEW 1.3 mm); nostril slightly closer to tip of snout than to anterior corner of eyes; tongue pyriform, with a deep notch at posterior tip; vomerine teeth absent; pineal ocellus absent; eyes moderately large (EHD 2.6 mm) and protruding, pupil horizontal; supratympanic fold distinct, from posterior corner of eye to above insertion of arm.
Forelimbs fairly robust (FAHL 8.2 mm); relative finger lengths: I < II < IV < III, tips of all four fingers expanded into discs with circum-marginal grooves; all fingers with lateral dermal fringes on both sides; subarticular tubercles distinct, rounded; supernumerary tubercles absent; no webbing between fingers; inner and outer metacarpal tubercle indistinct; nuptial pad is small and milky white on dorsal surface of the first finger.
Dorsal surfaces of head, body, forelimbs, thighs, and tibia rough with small granules; upper eyelid with several small granules; throat, chest, and ventral surfaces of forelimbs smooth; abdomen, ventral side of thigh, and area around vent with granules; dorsolateral folds absent.

Coloration of holotype in life.
For coloration of the holotype in life see Fig. 4. Dorsal surface beige, with pale brown band between eyes; dorsal surface with a dark brown X-shaped marking; pale brown interorbital rectangle between eyes; upper and lower lips with white and black dots; supratympanic fold pale brown; iris golden brown; dorsal parts of arms and legs with dark brown crossbars that align; crotch with a distinct black patch bordering large creamy white plaque below the black patch near the groin; dorsal thigh beige with one brown crossbar when leg is bent in resting position; ventral surface body and beige, and area around vent with small black spots; discs of fingers and toes yellow.
Coloration in alcohol. After preservation in alcohol, the general pattern did not change. Dorsal color changed to grayish brown, the blotches or spots blackish brown, discs on the fingers become pale gray similar to the body color, ventral side become whiter (Fig. 5).
Etymology. The specific epithet is named for the type locality, Malipo County, Yunnan Province, China. We suggest "Malipo Bush Frog" as its English common name, and "Ma Li Po Guan Shu Wa (麻栗坡灌树蛙)" as its Chinese common name. Distribution. Currently known from the type locality, Malipo County (Fig. 1), Yunnan Province, China and Pac Ban, Tuyen Quang, in north of Vietnam.
Variation. The measurements are given in Table 1. GXNU 000338 has large black spots on dorsal side and GXNU000342 has distinctly darker ground color on dorsal side.
Comparisons. Rather than comparing R. malipoensis sp. nov. to all known Raorchestes, we focus on our morphological comparison with phylogenetically closely related taxa and species without genetic data in adjacent countries ( Table 5).
The new species differs from R. dulongensis by (1) head wider than long; (2) interorbital distance larger than eye horizontal diameter; (3) nuptial pad present; (4) yellow disc; and (5) inner and outer metacarpal tubercle indistinct (vs. head smaller than long; interorbital distance smaller than eye horizontal diameter; nuptial pad absent; greyish or orange disc; inner and outer metacarpal tubercle indistinct present).
The new species differs from R. cangyuanensis by (1) interorbital distance larger than eye horizontal diameter; (2) nuptial pad small and milky white; and (3) yellow discs (vs. interorbital distance smaller than eye horizontal diameter; reddish nuptial pad at the base of first finger; orange disc).
The new species differs from R. annandalii by (1) head wider than long; and (2) relative toe lengths: I < II < V < III < IV (vs. head longer than wide; relative toe lengths: I < II < V = III < IV).

Discussion
Recently, Poyarkov et al. (2021) placed Philautus gryllus in the genus Kurixalus based on unpublished molecular evidence and a study of type materials. In this study, the sample previously identified as R. gryllus from northern Vietnam (voucher number: ROM 30288) nests in the clade of R. malipoensis sp. nov. without distinct genetic divergence (Table 4), indicating that they are likely conspecific (Table 4). Morphologically, Raorchestes malipoensis sp. nov. is obviously distinguishable from K. gryllus as described by Smith (1924 ; Table 6) by (1) smaller body size 14.6-19.3 mm, n = 11; (2) tympanum distinct (TD 1.1-1.6 mm, n = 11); (3) webbing formula (I 2 -2 II 2 -2 III 2 -3 IV 3 -2 V); (4) no webbing between fingers; (5) outer metatarsal tubercle absent (vs. 25.0-27.0 mm, n = 3; tympanum distinct; toes a little more than half webbed; fingers free except for a rudiment of a web between the two outer; outer metatarsal tubercle separated for approximately two-thirds of their length). Therefore, we consider that Raorchestes malipoensis sp. nov. is not conspecific with K. gryllus and the record of R. gryllus (ROM 30288) from northern Vietnam should be revised to R. malipoensis sp. nov. We also suggest that the taxonomic status of other records of R. gryllus from Vietnam and Laos need further examinations.
In recent years, many new species have been found along the border between China and Vietnam, such as Odorrana geminata (Bain et al., 2009), Tylototriton ziegleri (Nishikawa et al., 2013), Leptobrachella feii (Chen et al., 2020), Amolops shihaitaoi (Wang et al., 2022), and Theloderma hekouense (Du et al., 2022). Tropical montane forests in the border region between China and Vietnam are known to harbor a high level of species richness and local endemism (Sterling et al. 2006). One of the main reasons assumed to be responsible for this richness is the greater environmental heterogeneity observed in the montane regions as opposed to the lowland regions, allowing for a larger number of habitats to be occupied by species (Keller et al. 2009). It is expected that more new species from this region would be discovered, and further studies are required to accurately determine the species richness of tree frogs in China-Vietnam border region. Due to historical reasons, herpetological surveys of this region had been scarce, but considering the biogeographical interest of the region it is important to facilitate collaborative research to comprehensively understand herpetofaunal diversity, community composition, and species range limits around the region in order to better protect them and their environment in the face of global warming and habitat destruction. Table 6. Morphological comparison between Raorchestes malipoensis sp. nov. and Kurixalus gryllus (Smith, 1924).

Raorchestes malipoensis sp. nov. (n = 11)
Kurixalus gryllus (n = 3) SVL 14.6-19.3 mm 25.0-27.0 mm, HL 5.2-7.9 mm 8.0-9.5 mm HW 5.5-8.2 mm 10.0-11.0 mm EHD 2.1-2.8 mm 3.0-3.5 mm SL 1.8-2.9 mm 4-4.5 mm HAL 4.2-5.6 mm 7.5-8.5 mm TBL 7.5-9.2 mm 12-13 mm TD 1.1-1.6 mm n = 11 tympanum indistinct Tubercles along forearm and foot absent present Web of toes I 2 -2 II 2 -2 III 2 -3 IV 3 -2 V toes a little more than half webbed Web of fingers no webbing between fingers fingers free except for a rudiment of a web between the two outer fingers Metatarsal tubercle inner metatarsal tubercle rounded, outer metatarsal tubercle absent a small inner metatarsal tubercle Coloration dorsal surface beige, with pale brown and dark brown spots, an individual having large black spots on its body surface dorsal color with pale or dark brown, green, yellow, or grey, many individuals had a bright green patch on the snout, and patches of similar color on the knees and round the vent