Rhacophorus tuberculatus and Rhacophorus verrucopus are two morphologically similar species described in 1871 and 1983 respectively. Their taxonomic distinctiveness has been questioned in the past. In the current study, we encountered frogs that we confer to R. tuberculatus based on morphological similarity to a syntype of this species. We redescribe the species based on a re-examination of a syntype, which is designated as a lectotype here, and additional specimens from Garo hills of Meghalaya. We also present molecular data, natural history notes, and report a range extension of this species. Molecular phylogenetic analysis based on the 16S rRNA fragment revealed minimal genetic divergences (0.20–1.74% uncorrected p-distance) between specimens identified as either R. tuberculatus or R. verrucopus from different locations. On the basis of molecular data and morphological characteristics, we conclude that R. verrucopus is a junior synonym of R. tuberculatus.

Darwinian shortfall, Indo-Burma hotspot, range extension, Rhacophoridae, synonymy, systematics

Frogs of the genus Rhacophorus Kuhl & Van Hasselt, 1822 occur across South and Southeast Asia and are represented by 44 extant species (Frost 2023). Despite many species of Rhacophorus being widespread, most of them are poorly studied, and information on their distribution is sparse (Ohler and Delorme 2005). Rhacophorus tuberculatus (Anderson, 1871) is one such poorly known species. It was described in 1871 from “Seebsaugor, Assam” (now Sivasagar 26.98515°N, 94.63878°E) and was subsequently reported from Arunachal Pradesh, Meghalaya, and West Bengal, India (Annandale 1912; Chanda 2002; Sen 2004; Das and Dutta 2007; Ahmed et al. 2009; Mathew and Sen 2010; Roy et al. 2018). Huang (1983) described Rhacophorus verrucopus from “Beibeng, Medo Xian” (Beibeng Township, Motuo County, Xizang Autonomous Region, China 29.23942°N, 95.17644°E), which closely resembles R. tuberculatus and this species was known only from its type locality and northern Myanmar (Fei 1999; Fei et al. 2009, 2010; Li et al. 2010; Fei et al. 2012; Liu et al. 2020; Fei 2020; Zug 2022). By studying the type and near-topotypical specimens of R. verrucopus and comparing them with the descriptions of R. tuberculatus, Che et al. (2020) considered that R. verrucopus and R. tuberculatus could be the same species. However, since Che et al. (2020) did not obtain any molecular data or examined the types of R. tuberculatus from India and only made comparisons of external morphology, Frost (2023) does not currently adopt their view and still treats R. verrucopus as a valid separate species.

We encountered a Rhacophorus species, the identity of which was difficult to determine, during our recent surveys between 2021 and 2022 in the West Garo hills of Meghalaya, India. Herein, we ascertain the identity of that Rhacophorus species as R. tuberculatus based on comparison with the syntypes of R. tuberculatus and provide a redescription of the species after more than 150 years since its original description, provide photographs of live individuals, morphological measurements and genetic divergence of R. tuberculatus with other congeners using 16S rRNA gene and discuss its relationship with R. verrucopus.

The suggested best substitution model for BI was GTR+F+I+G4 and for ML was TIM2+F+I+G4, both analyses showed an essentially consistent topology (Fig. 1). The sequences of the newly collected specimens from Meghalaya, India clustered with the sequences of R. verrucopus from Myanmar with strong supports by both BI and ML (0.97/99), and they together clustered with the sequences of R. verrucopus from China with strong supports by both BI and ML (1/100). The genetic divergence (uncorrected p-distance) between the sequences of the newly collected specimens and the sequences of R. verrucopus from China ranged from 1.50% to 1.74%, the genetic divergence (uncorrected p-distance) between the sequences of the newly collected specimens and the sequence of R. verrucopus from Myanmar ranged from 0.20% to 0.44% (Table 2).

Figure 1. 

Bayesian inference tree of the genus Rhacophorus based on partial 16S rRNA fragments. Numbers before slashes indicate Bayesian posterior probabilities (≥ 0.90 remain) and numbers after slashes indicate ultrafast bootstrap support for ML analyses (≥ 90 remain).

Morphologically, the newly collected specimens (R. tuberculatus) from Meghalaya, India agree with the Syntype (ZSI 10154) and subsequent descriptions and figures by Annandale (1912) and Mathew and Sen (2010) of Rhacophorus tuberculatus in most aspects, especially in having a distinct tympanum, almost half as large as the eye; absence of vomerine teeth; presence of partial, sheath-like webbing on fingers and fully developed webbing on toes; pointed projection at tibio-tarsal articulation; well-developed and expanded discs on toe. Hence, we consider these newly collected specimens to belong to R. tuberculatus. In addition, there were also no obvious morphological differences between the newly collected specimens of R. tuberculatus and R. verrucopus from China and Myanmar (see Table 3).

Integrating the results of morphological data and also considering the shallow genetic divergence that is usually considered as intraspecific variation in the genus Rhacophorus, we second Che et al. (2020) in stating that R. tuberculatus and R. verrucopus are conspecific, and formally place R. verrucopus under the subjective synonymy of R. tuberculatus. Below, we give a formal synonymy list and provide the description of the specimen ZSI 10154 and designate it as the lectotype of Rhacophorus tuberculatus. Additionally, we also provide measurements of the newly collected material for comparison.

“Darwinian shortfall” is a major challenge faced by conservationists today, the lack of availability of molecular data for several extant species is a common phenomenon across taxa, leading to a situation where phylogenetic information is absent for most organisms, thus inhibiting a robust understanding of phylogenetic relationships within a particular group (Diniz-Filho et al. 2013). Rhacophorus tuberculatus is one such species which was described by Anderson (1871), based on three adult specimens. The original description was brief and lacked photographs or diagrams since it was from more than 150 years ago. Although several subsequent studies such as Ahmed et al. (2009) and Roy et al. (2018) reported photographic records of this species, there had been no studies involving a detailed taxonomic assessment of the species to date. As a result, the species had been overlooked in several studies, including the description of Rhacophorus verrucopus by Huang (1983). The validity of this species has been doubted by Che et al. (2020) who suggested that R. verrucopus could be a junior synonym of R. tuberculatus but due to the lack of molecular evidence this was not accepted. In the current study, we provide genetic divergence between R. tuberculatus and other congeners, including ‘R. verrucopus’ using 16s rRNA gene, thus filling an important knowledge gap. Further examination of R. verrucopus from China and Myanmar and R. tuberculatus from India based on re-examination of types and also freshly collected specimens used in this study revealed that there is an extensive overlap in morphology, and shallow molecular divergence (of a level that usually qualifies to be considered an intraspecific variation in the genus) between R. verrucopus and R. tuberculatus. Based on these lines of evidence we endorse the conclusions of Che et al. (2020) and formally place R. verrucopus under the junior synonymy of R. tuberculatus. Examination of the types (BMNH 1940.6.1.30 and BMNH 1974.828-832) of Rhacophorus turpes described from Kachin region of Northern Myanmar (26.24972°N, 97.23878°E) by Smith (1940) revealed that these specimens also closely match the morphological characters of R. tuberculatus except for a slightly truncated snout, a relatively less prominent tibio-tarsal projection, and fewer granulations on the ventral surface (Fig. 3). However, further studies utilizing molecular approaches based on fresh material are necessary to assess the taxonomic status of this species.

Figure 3. 

The ventral and dorsal view of a syntype of Rhacophorus turpes BMNH 1940.6.1.30.

Figure 4. 

Rhacophorous tuberculatus in life from Garo hills, Meghalaya.

The current study also addresses the re-assessment of the threat status of R. tuberculatus as per IUCN Red List criteria of this Data Deficient species. Rhacophorus tuberculatus was known with certainty only from its type locality until now. However, in this study, we were able to resolve the taxonomic confusion with this species thus mapping its actual distribution range for the first time. The new records of this species from West Garo hills of Meghalaya mark the westernmost limit of the distribution of the species (Fig. 5). The up-listing or down-listing of species from one threat category to another of the IUCN Red List requires an assessment against all the five criteria (A–E, with 11 sub-criteria) but only one criterion needs to be fulfilled for designation of threat categories (IUCN 2001). In the case of R. tuberculatus, the information on geographic distribution seems the most accurate and reliable among all other criteria and hence, used for a conservative estimate of the extent of occurrence (Criteria B, B1). The current Extent Of Occurrence for this species estimated based on all known localities is about 1,07,600 km² and hence, with all the updated information presented here, we recommend transferring the species from Data Deficient to Least Concern.

Figure 5. 

Updated distribution map of R. tuberculatus including records from Meghalaya (Current study) in green and black circle, from Myanmar in green and black square, type locality of “R. verrucopus” in red circle and type locality of R. tuberculatus in green circle.

First and foremost, we are grateful to Mr. Lising G. Momin and the villagers of Sasatgre village for their invaluable assistance during the fieldwork. Our sincere thanks are due to the Additional Principal Chief Conservator of Forests (Wildlife) and Chief Wildlife Warden (CWLW), Meghalaya, and officers of the Department of Forest and Environment, Government of Meghalaya, for facilitating permission from the Community Reserve Management Committees to carry out the field study (No. FWC/Research/15/603-04 dated 31^st May 2019). We extend our thanks to the Technology Platform Services, C-CAMP, National Centre for Biological Sciences, for their expertise in sequencing samples used in this study. Additionally, our gratitude goes to Dr. Kaushik Deuti for helping with the examination of specimens from the Zoological Survey of India, and to Dr. David Gower, Dr. Jeff Streicher, Dr. Simon Loader and Dr. Rikki Gumbs for facilitating the examination of specimens from the British Natural History Museum.