﻿A new species of Raorchestes (Anura, Rhacophoridae) from Yunnan Province, China

﻿Abstract A new bush frog species is described from Yunnan, China, based on phylogenetic analyses, species delimitation analyses, and morphological comparisons. Raorchesteshekouensissp. nov. is distinguished from all other congeners by a combination of 11 morphological characters. The new species brings the current number of Raorchestes species in China to ten, nine of which are distributed in Yunnan. Molecular analyses supported an unnamed lineage previously recorded as “Raorchestesgryllus” in northern Vietnam. Further studies including additional samples are necessary to clarify the species diversity and boundaries of Raorchestes in China and Indochina.


Introduction
The genus Raorchestes Biju, Shouche, Dubois, Dutta & Bossuyt, 2010, which currently contains 76 species (Frost 2023), is one of the most speciose genera within the family Rhacophoridae.Members of Raorchestes are characterized by a small body size (15-45 mm), lack of vomerine teeth, transparent/translucent vocal sac when calling, and direct development (Biju et al. 2010;Vijayakumar et al. 2014).Raorchestes is widely distributed in South and Southeast Asia, from India to Nepal, Myanmar, Thailand, and Laos to southwestern China, Vietnam, Cambodia, and West Malaysia (Frost 2023).
Most Raorchestes species were initially assigned to the genus Philautus Gistel, 1848 (Bossuyt and Dubois 2001); however, Yu et al. (2009) and Li et al. (2009) revealed that frogs traditionally classified in Philautus consisted of two groups rather than being a monophylum, and Li et al. (2009) proposed the name Pseudophilautus Laurent, 1943 for the group primarily distributed on the Indian subcontinent, which itself consists of two reciprocally monophyletic groups, i.e., a radiation with notably large diversity in the Western Ghats of India and a radiation with large diversity in Sri Lanka.Biju et al. (2010) later erected the genus Raorchestes for the clade with substantial diversity in the ZooKeys 1192: 213-235 (2024), DOI: 10.3897/zookeys.1192.106013Lingyun Du et al.: ShortTitle Western Ghats to distinguish it from Pseudophilautus sensu stricto, a clade of 80 species largely restricted to Sri Lanka (Biju et al. 2010;Meegaskumbura et al. 2019).Based on phylogenetic analysis, Li et al. (2013) suggested that Raorchestes and Pseudophilautus formed a sister group of Kurixalus Ye, Fei & Dubois, 1999; however, more recent studies based on wider genus-level sampling suggested Raorchestes is sister to Pseudophilautus (Vijayakumar et al. 2014;Chan et al. 2018;Garg et al. 2021) or the clade composed of Raorchestes and Pseudophilautus is sister to Mercurana Abraham, Pyron, Ansil, Zachariah & Zachariah, 2013(Meegaskumbura et al. 2019).
As one of the most diverse groups in the Rhacophoridae family, Raorchestes frogs form a distinct radiation with more than 80% of the known species distributed in South Asia, especially in India.As such, most research attention has been paid to the taxonomy and evolution of Indian Raorchestes.For examples, Vijayakumar et al. (2014) reported on Raorchestes relationships within the Western Ghats, naming nine species and recognizing 15 clades within the Western Ghats complex; Vijayakumar et al. (2016) revealed that geological processes, Quaternary glaciations, and ecological gradients drove diversification of Raorchestes frogs in the Western Ghats; and Garg et al. (2021) named five species in the Western Ghats and delimited Raorchestes into 16 species groups.
Six Raorchestes species are known from Southeast Asia, i.e., R. parvulus (Boulenger, 1893), R. gryllus, R. menglaensis, R. huanglianshan, R. longchuanensis, and R. malipoensis (Frost 2023).However, the taxonomic status of R. gryllus is problematic.This species was originally described from Langbian Plateau in Lam Dong Province, southern Vietnam, and has been widely reported in Vietnam (Lam Dong, Dak Lak, Gia Lai, and Kon Tum, Lao Cai, Cao Bang, Vinh Phu, and Bac Thai) and Laos (Sepian, Boloven Highlands, Champasak Province) (Bourret 1937(Bourret , 1939(Bourret , 1942;;Orlov et al. 2002Orlov et al. , 2012;;Teynié et al. 2004;Nguyen et al. 2009).Biju et al. (2010) confirmed the affiliation of R. gryllus with the genus Raorchestes based on molecular data from Li et al. (2009).However, those specimens used in Li et al. (2009) were collected from Pac Ban, Tuyen Quang, northern Vietnam, and Orlov et al. (2012) considered records of R. gryllus in this region to be highly improbable.Furthermore, the species contains a series of tubercles along the outer side of the forearm and foot, and a dermal projection on the snout (Smith 1924), very similar to members of Kurixalus, and differing in egg capsule appearance from other Raorchestes species, with thick and semi-transparent eggs in R. gryllus compared to transparent eggs in other Raorchestes species (Orlov et al. 2012).
Recently, Poyarkov et al. (2021) suggested the transfer of R. gryllus to Kurixalus based on unpublished molecular data of specimens from the type locality and unpublished morphological data from type material, implying that the socalled "R.gryllus" specimens from northern Vietnam used in previous phylogenetic analyses (e.g., Li et al. 2009Li et al. , 2013;;Nguyen et al. 2014;Wu et al. 2019) are not actually true K. gryllus, but represent an unnamed species.Moreover, Huang et al. (2023) considered that the specimen of R. UI ROM30288 from Pac Ban, Tuyen Quang, northern Vietnam was misidentified and revised it to R. malipoensis.This suggests that other records of the species from Vietnam and Laos need further examination.
Yunnan Province harbors the highest amphibian species diversity in China (AmphibiaChina, 2022), with many new species described in recent years (e.g., Gan et al. 2020;Liu et al. 2021;Wang et al. 2022).During recent field surveys in Hekou, Yunnan, China, we collected eight specimens of Raorchestes.Morphological comparison and phylogenetic analysis indicated that these specimens could be distinguished from all other members of the genus Raorchestes, except for the R. UI ROM 38828 from northern Vietnam in molecular analysis, indicating that the eight specimens from Hekou and the ROM38828 specimen from northern Vietnam represent a new species.

Sampling
Field surveys were conducted in March 2019 and April 2023 at Liangzi village, Hekou, Yunnan, China (Fig. 1).Specimens were euthanized, fixed, and preserved in 75% ethanol.Liver tissues were taken and preserved in 99% ethanol.Voucher specimens and tissue samples were deposited at Guangxi Normal University (GXNU), China.
step at 95 °C for 4 min, 35 cycles of denaturing at 94 °C for 1 min, annealing at 51 °C for 1 min, and extension at 72 °C for 1 min, with a final extension step of 72 °C for 10 min.Sequencing was conducted using the corresponding PCR primers.All new sequences were deposited in GenBank under accession numbers ON986419-ON986422, OQ029526, OQ859106 and OQ859107 (Table 2).

Phylogenetic analysis and species delimitation
To examine the phylogenetic position of the specimens collected from Hekou, Yunnan, China, we reconstructed phylogenetic trees of the genus Raorchestes based on sequences of the 16S rRNA (16S) genes.Furthermore, 35 homologous sequences of other Raorchestes species were obtained from GenBank (Table 2).Pseudophilautus kani (Biju & Bossuyt, 2009) and Pseudophilautus amboli (Biju & Bossuyt, 2009) were selected as outgroups based on Wu et al. (2021).All sequences were aligned in MEGA v. 7.0 (Kumar et al. 2016) using the ClustalW tool and both ends of the sequence were trimmed to minimize missing characters.
Phylogenetic relationships were inferred based on maximum likelihood (ML) and Bayesian inference (BI) analyses.BI analysis was conducted in MrBayes v. 3.2.6 (Ronquist et al. 2012).The best-fitting model (GTR + I + G) was chosen using the Akaike Information Criterion (AIC) in JModelTest v. 2.1.10( Darriba et al. 2012).Four Monte Carlo Markov chains were started from a random tree.The chains were run for three million generations and sampled every 100 generations, with the first 25% of sampled trees discarded as burn-in.The remaining trees were used to create a consensus tree and to estimate Bayesian posterior probabilities (BPP).ML analysis was performed using RAxML v. 8.2.10 (Stamatakis 2014) under the GTR + I + G model.Tree searches were performed 100 times with 1000 bootstrap (BS) replicates to assess node support.Nodes with BPP ≥ 0.95 and BS ≥ 70 were considered well supported.Additionally, uncorrected pairwise genetic distances (p-distances) between species in 16S rRNA sequences were calculated using MEGA v. 7.0.(Kumar et al. 2016).We used two approaches, i.e., the Bayesian Poisson Tree Processes (bPTP; Zhang et al. 2013) and Assemble Species by Automatic Partitioning (ASAP; Puillandre et al. 2021), to delimit species boundaries.The bPTP method was run on the bPTP server (http://species.h-its.org/)using the tree generated by Bayesian phylogenetic analysis and default parameters.For the ASAP method, the simple distance (p-distance) model was used and the partitioning with the lowest ASAP score was selected as the best, as per Puillandre et al. (2021).

Phylogenetic analysis and genetic divergence
The obtained sequence alignment was 552 bp long and included 211 variable sites and 152 parsimony informative sites.Phylogenetic analysis (Fig. 2) revealed that the specimens from Hekou, Yunnan, China, R. malipoensis and R. UI from northern Vietnam formed a monophyletic group, which itself contained three distinct branches, one consisting of the specimens from Hekou and a specimen of R. UI from Pac Ban, Tuyen Quang, Vietnam (ROM 38828) with strong support (BPP = 100, BS = 100) and short internal branch lengths, one consisting only of R. UI from Tam Dao, Vinh Phuc, Vietnam (ROM 30298), and one consisting of the recently named bush frog species R. malipoensis, which included a specimen previously mistaken of "R.gryllus" from Pac Ban, Tuyen Quang, Vietnam (ROM 30288).The clade containing specimens from Hekou was recovered as the sister to R. malipoensis with strong support.The bPTP analysis delimited the three lineages into three candidate species (Fig. 3).The ASAP analysis identified 10 partitions (Fig. 3) and the best partition (score = 2.5) also grouped the three lineages into three candidate species.The 16S p-distances between the clade consisting of Hekou specimens and the other Raorchestes lineages included in this study ranged from 2.5% (R. malipoensis) to 12.9% (R. archeos), greater than the divergence between R. hillisi and R. yadongensis (2.0%; Table 3).
Etymology.The specific epithet hekouensis is named after the type locality, Hekou County, Yunnan, China.We suggest "Hekou bush frog" as its English common name, and "Hé Kǒu Guàn Shù Wā (河口灌树蛙)" as its Chinese common name.
Description of holotype.GXNU YU000159, adult male, body size small (SVL 17.5 mm); head wider than long (HW = 6.9 mm, HL = 6.1 mm); snout rounded in profile, projecting beyond lower jaw, snout length almost equal to diameter of eye (SL = 2.4 mm; ED = 2.5 mm); canthus rostralis rounded, loreal region slightly concave; internarial distance slightly less than interorbital distance, and wider than maximum width of upper eyelid (INS = 2.2 mm; IOS = 2.4 mm; UEW = 1.9 mm); tympanum distinct (TD = 1.3 mm); tongue pyriform, with deep notch at posterior tip; vomerine teeth absent; temporal fold distinct; dorsolateral fold absent.Length of forelimb and hand slightly shorter than half of snout-vent length (LAHL = 8.5 mm, SVL = 17.5); relative fingers lengths: I < II < IV < III; tips of all four fingers expanded into discs with circummarginal grooves; lateral dermal fringes on all fingers; subarticular tubercles distinct, rounded; supernumerary tubercles absent; no webbing between fingers; inner metacarpal tubercle present, outer metacarpal tubercle indistinct; nuptial pads present on first and second fingers in male.Hindlimbs relatively slender, thigh length (TIL = 9.2) shorter than tibia length (TL = 11.4), but greater than foot length (FL = 6.6); tibiotarsal articulation reaching anterior of eye when hindlimb stretched alongside body; heels meeting when limbs held at right angles to body; relative toe lengths: I < II < III < V < IV; tips of toes with well-developed discs with circummarginal grooves; all toes with lateral dermal fringes; subarticular tubercles distinct, rounded; supernumerary tubercles absent; rudimentary webbing between toes; inner metatarsal tubercle rounded, outer metatarsal tubercle absent.Dorsal surfaces rough, dorsum, dorsal surface of limbs, snout, between eyes, and upper eyelid shagreened with numerous tubercles; flank of body, dorsal part of forelimbs, thighs, and tibia relatively smooth, scattered with sparse granules; throat, chest, and ventral surfaces of forelimbs smooth; abdomen, underside of thigh, and around vent with granules; dorsolateral folds absent; dorsal, dorsal surface of limbs and around vent with several beige patches.
Coloration of holotype in life.Dorsal surface yellowish brown, with distinct dark brown X-shaped marking on back; blackish line between eyes; tea-brown spots on both sides of lower jaw; dorsal side of limbs with several brown bands; flank near crotch with distinct black region between two creamy white patches, thighs with similar black patch near groin, next to another creamy white patch; ventral surface of throat, chest, ventral side of limbs, and belly opaque creamy white with small black spots and white tubercles; finger and toe discs yellow (Fig. 4).
Coloration of holotype in preservative.Dorsal color changed to grayish brown; forelimbs and hindlimbs with black-brown bands; patches or spots blackish brown; abdomen and ventral sides of limbs still milky white with several small black spots (Fig. 5).
Male secondary sexual characteristics.Adult male with nuptial pads on dorsal surface of first and second fingers and external single subgular vocal sac with slit-like opening at posterior of jaw.White lineae masculinae visible on ventral body.
Variation.Specimen GXNU YU000160 significantly has more black spots on the abdomen and near the cloaca (Fig. 6), specimen GXNU YU000156 differs from the other seven type specimens (GXNU YU000159, GXNU YU000160, GXNU YU000153, GXNU YU000154, GXNU YU000536, GXNU YU000537, and GXNU YU000538) by pale yellow mid-dorsal vertebral stripe from snout to vent, pale yellow stripe along hindlimbs crossing at vent region, mid-ventral stripe from snout to vent and stripe along forelimbs crossing at breast region (Fig. 7), and the specimen GXNU YU000537 has distinctly darker ground color on the dorsal side, especially on the head (Fig. 8).
Distribution.Currently known from the type locality, Hekou County, Yunnan Province, China, and Bac Pan, Tuyen Quang, Vietnam.
Habitat.In Yunnan, Raorchestes hekouensis sp.nov.was found in shrubs and herbs on the edge of a small stream near the road at an elevation of ca 1200 m a.s.l.(Fig. 9) on the nights of 25 March 2019 and 4 April 2023.There were many herbaceous plants near the stream, such as Ageratina adenophora.
No male was heard calling and no eggs were observed during our surveys in late March, but there were males calling during our surveys in April.Therefore, the breeding season for this species starts in April.
Remarks.Raorchestes hekouensis sp.nov. is assigned to the genus Raorchestes based on its molecular phylogenetic position and the following morphological characters: relatively small body size (SVL 15.0-45.0mm); absence of vomerine teeth; large transparent/translucent vocal sac.Due to the  close phylogenetic relationship and distribution (Figs 1, 2), we compared the new species with 16 recognized congeners distributed in Southeast Asia, southwestern China, the Himalayas, and northeastern India, as mentioned above.Raorchestes hekouensis sp.nov. is distinguished from all other 16 congeners by a unique combination of characters.A detailed morphological comparison table of currently known Raorchestes species from China is provided (Table 4).
Raorchestes gryllus is still considered a member of Raorchestes in Frost (2023), although Poyarkov et al. (2021) suggested that it should be transferred to the genus Kurixalus.Raorchestes hekouensis sp.nov.can be distinguished from K. gryllus based on the following characters: no webbing between fingers (vs rudimentary webbing between fingers), rudimentary webbing between toes (vs little more than half webbed), heel with no pointed appendage (vs heel with small, pointed appendage), snout rounded (vs snout pointed with dermal tip), and series of tubercles along outer side of forearm and foot absent (vs present).reaching tip of snout), ventral surface of throat, chest, and belly opaque creamy white, with small black spots (vs chest and belly yellowish, with brown punctuations), and flank near crotch with distinct black region between two creamy white patches (vs irregular large black patch on groin, extending to half of side, with two yellow patches).Raorchestes hekouensis sp.nov.differs from R. yadongensis by lacking webbing between fingers (vs fingers with rudimentary webbing) and tibiotarsal articulation reaching anterior of eye when adpressed (vs tibiotarsal articulation reaching tip of snout when adpressed).

Discussion
The small body size, morphological conservativeness, and remarkably similar characters in the Raorchestes genus have resulted in ambiguities in taxonomy and distribution (Jiang et al. 2020), necessitating the application of molecular identification (Orlov et al. 2012).Morphologically, the types and topotypes of Kurixalus gryllus are very similar to other members of the genus due to the series of tubercles along the outer side of the forearm and feet, small pointed appendage on the heel, and pointed snout with a dermal tip (Smith 1924;Orlov et al. 2012;Poyarkov et al. 2021), with wide variation in living color patterns of K. gryllus from the type locality shown to be very similar to that seen in Kurixalus motokawai and Kurixalus banaensis (Nguyen, 2015; see Fig. 10).Therefore, we agree with Poyarkov et al. (2021) that "R.gryllus" from the type locality should be reassigned to Kurixalus.We also consider that the samples of so-called "R.gryllus" from northern Vietnam used in the present study (ROM 38828 and ROM 30298) are not conspecific with K. gryllus from the type locality as they are phylogenetically nested within the genus Raorchestes (Fig. 2).
Our results showed that the R. UI ROM 38828 from northern Vietnam clustered with Raorchestes hekouensis sp.nov.with a short branch length, indicating that R. UI ROM 38828 belonged to the new species (Fig. 2), and recently Huang et al. (2023) revised the specimen ROM 30288 from northern Vietnam, which had been recorded as R. gryllus, to R. malipoensis so the taxonomic status of the R. UI specimen from northern Vietnam (ROM 30298) needs further confirmation.The genetic divergences between R. malipoensis, R. UI ROM 30298, and Raorchestes hekouensis sp.nov.were greater than the divergence between R. hillisi and R. yadongensis (Table 3), and species delimitations grouped them into three different candidate species (Fig. 3), indicating that the clade comprised of ROM 30298 likely represented an unnamed species, pending further morphological study.Of note, both the ROM 38828 and ROM 30288 specimens were collected from Pac Ban, Tuyen Quang, Vietnam, suggesting the coexistence of R. malipoensis and the new species Raorchestes hekouensis sp.nov. in that region, which means the records of Raorchestes from that region also need verification.
In this study, we used distance-based (ASAP) and tree-based (bPTP) delimitation methods, and the two different species delimitation methods give the same results.The ASAP analysis divides species based on pairwise genetic distance, but it can provide a score for each partitioning result for users to refer to and select partitioning results.The difference is that bPTP delimits species using non-hypermetric phylogenies, and estimates speciation events in terms of a number of substitutions; therefore, it only requires a standard phylogenetic tree as input.The combination of both methods confirms the species delimitation and helps overcome the constraints of each approach (Carstens et al. 2013).
With the description of the new species, there are now ten Raorchestes species known from China, all of which occur in Yunnan except for R. yadongensis, which is only known from southern Tibet, China (Zhang et al. 2022).Recently Garg et al. (2021) assigned the Raorchestes species into 16 species groups and the clade containing species from Southeast and East Asia (e.g., R. parvulus, R. menglaensis, R. cangyuanensis) was placed in the R. parvulus species group.Based on Garg et al. (2021) and our phylogenetic results, the new species also belongs to the R. parvulus group.The continuous discovery of new Raorchestes species from Yunnan in recent years (Wu et al. 2019(Wu et al. , 2021;;Jiang et al. 2020;Huang et al. 2023;this study) indicates that Raorchestes diversity is seriously underestimated in Yunnan.We expect that more Raorchestes species will be found from southern Yunnan given the unnamed lineage in adjacent northern Vietnam mentioned above, from Tam Dao, Vinh Phuc (ROM 30298).Therefore, further studies employing a wider range of Raorchestes samples across its distribution are necessary to clarify the species boundary in Yunnan.
Due to the placement of "R.gryllus" sensu stricto in Kurixalus, the number of recognized Raorchestes species known from Southeast Asia is decreased to five, including R. parvulus, R. longchuanensis, R. menglaensis, R. malipoensis, and R. huanglianshan based on recent studies (Poyarkov et al. 2021;Jiang et al. 2020;Wu et al. 2022;Huang et al. 2023); our results revealed the existence of an additional but unnamed lineage in northern Vietnam.Previous phylogenetic analyses have also revealed that nominal R. parvulus, which is widely reported across Indochina (Frost 2023), also contains multiple clades that do not form a monophyly (Chan et al. 2018;Wu et al. 2019;Yu et al. 2019;Jiang et al. 2020), indicating that multiple cryptic species may exist within the species.Therefore, Raorchestes species diversity in Southeast Asia may be highly underestimated.

Figure 2 .
Figure 2. Bayesian phylogram of Raorchestes parvulus group estimated from 16S rRNA showing placement of Raorchestes hekouensis sp.nov.Nodal support values are shown above branches as Bayesian posterior probability (BPP) / ML bootstrap support (BS), and the symbol "-" indicates value below 50.

Table 2 .
Information on voucher numbers, localities, and GenBank accession numbers for all specimens used in this study.

Table 3 .
Uncorrected p-distance (%) in 16S rRNA sequences of Raorchestes species used in this study.
Figure 3. ASAP species delimitation within Raorchestes based on 16S sequences.ASAP analysis generated 10 partitions and ranked them using the lowest ASAP score as the best option, and the best partition is highlighted in red.Black and gray vertical bars indicate results of bPTP species delimitation.