Hiding in Plain Sight: Rain Water Puddles in Nicobar Islands of India Reveal Abundance of a New Frog Species of the Genus Microhyla Tschudi, 1838 (Anura: Microhylidae)

© 2022 Academia Sinica, Taiwan Open Access Hiding in Plain Sight: Rain Water Puddles in Nicobar Islands of India Reveal Abundance of a New Frog Species of the Genus Microhyla Tschudi, 1838 (Anura: Microhylidae) Sonali Garg1, Chandrakasan Sivaperuman2, G. Gokulakrishnan2, S. R. Chandramouli3, and S. D. Biju1,* 1Systematics Lab, Department of Environmental Studies, University of Delhi, Delhi 110007, India. *Correspondence: E-mail: [email protected], [email protected] (Biju). E-mail: [email protected] (Garg) 2Andaman and Nicobar Regional Centre, Zoological Survey of India, Port Blair 744102, Andaman and Nicobar Islands, India. E-mail: [email protected] (Sivaperuman); [email protected] (Gokulakrishnan) 3Department of Ecology and Environmental Sciences, School of Life Sciences, Pondicherry University, Puducherry 605014, India. E-mail: [email protected] (Chandramouli) Received 5 September 2021 / Accepted 19 December 2021 / Published 14 February 2022 Communicated by Jian-Nan Liu Recent studies on frogs of the Microhyla heymonsi species complex have demonstrated that high genetic variation exists among its various known populations from regions across Asia. We assessed the taxonomic identity of the Nicobar population of Microhyla cf. heymonsi and compared it to the typical Microhyla heymonsi from Taiwan and the two recently described species in this complex from Vietnam. Our study demonstrates that the Nicobar population is both genetically and morphologically divergent and warrants recognition as a new species, which we formally describe here as Microhyla nakkavaram sp. nov. The new species is closely related to M. daklakensis, M. heymonsi, and M. ninhthuanensis, but diagnosable from all three species by a suite of morphological characters, such as the presence of two small tubercles at mid-dorsum along with ( )-shaped markings, length of finger I longer than half the length of finger II, presence of three distinct metacarpal tubercles on hand, rudimentary foot webbing, as well as its slender body shape, granular dorsal skin texture, and other colour characters and body markings. Statistical analyses based on multiple morphometric characters also clearly separate our new taxon from M. heymonsi, with which it was previously confused. Our phylogenetic analyses based on the mitochondrial 16S rRNA locus find Microhyla nakkavaram sp. nov. to be nested in the Microhyla achatina species group, where it is delimited as a distinct species. This lineage shows genetic distances of ≥ 3.5% from all the other known congeners. Currently, the known distribution of the new taxon is restricted to the southernmost group of Nicobar Islands—Great Nicobar, Kondul, and Little Nicobar—where it is found in abundance across a wide range of habitats during the monsoon season. Our study underlines the need to reassess the identity of all the known populations of M. ‘heymonsi’ from other regions in Southeast and East Asia. To facilitate future taxonomic work in the light of our and other recent findings, we also provide a detailed redescription and revised diagnosis for M. heymonsi based on morphological examination of its century-old type material originating from Taiwan. Key words: Amphibia, Microhyla heymonsi, Integrative taxonomy, Mitochondrial DNA, Species complex. Citation: Garg S, Sivaperuman C, Gokulakrishnan G, Chandramouli SR, Biju SD. 2022. Hiding in plain sight: rain water puddles in Nicobar Islands of India reveal abundance of a new frog species of the genus Microhyla Tschudi, 1838 (Anura: Microhylidae). Zool Stud 61:2. doi:10.6620/ ZS.2022.61-02. Zoological Studies 61: 2 (2022) doi:10.6620/ZS.2022.61-02 1 © 2022 Academia Sinica, Taiwan BACKGROUND Across South and Southeast Asia, with the onset of the rainy season, a chorus of frogs resounds in urban and rural areas. This is particularly common in cultivated and water-logged areas. The chorus is a series of short calls delivered in rapid succession, similar to the sound of a comb rubbed along its teeth. These tiny frogs are commonly referred to as Narrow-mouthed frogs or Rice frogs and belong to the genus Microhyla Tschudi, 1838. Despite their common occurrence and local abundance, many species in this group are often misidentified, largely due to their small adult size, overall similar body colouration, and overlapping external morphological features (Matsui et al. 2011; Poyarkov et al. 2014; Garg et al. 2019). The genus Microhyla belongs to the family Microhylidae Günther, 1858 (1843) and has been a subject of intensive research in recent years, leading to descriptions of several new species, comprehensive taxonomic revisions, and new phylogenetic and biogeographical insights (e.g., Matsui et al. 2011; Poyarkov et al. 2014 2019; Garg et al. 2019; Gorin et al. 2020). There are currently 47 recognised Microhyla species known from regions across South, Southeast, and East Asia (Hoang et al. 2021; Eprilurahman et al. 2021). Of these, 40 species are either known only from their type localities and vicinities, or have geographical ranges restricted to relatively small regions. On the other hand, there are only a handful of species with wide distribution ranges, such as M. berdmorei, M. butleri, M. fissipes, M. heymonsi, M. mukhlesuri, M. nilphamariensis, and M. pulchra (e.g., Yuan et al. 2016; Garg et al. 2018 2019; Gorin et al. 2020; AmphibiaWeb 2021; Frost 2021). Most of these wide-ranging species are recently shown to represent complexes of multiple species based on molecular evidence (Garg et al. 2019; Gorin et al. 2020), thereby re-emphasising on the existence of many potentially undescribed taxa even among the known populations of Microhyla frogs (e.g., Matsui et al. 2005 2011; Hasan et al. 2012 2014a; Poyarkov et al. 2014; Yuan et al. 2016; Garg et al. 2018 2019; Gorin et al. 2020). The trend in the discovery and description of new species in the genus also specifically suggests a high likelihood that there are many additional new taxa in both unexplored and relatively well explored regions in South Asia (Hasan et al. 2014b; Howlader et al. 2015; Seshadri et al. 2016; Wijayathilaka et al. 2016; Khatiwada et al. 2017; Vineeth et al. 2018; Biju et al. 2019; Garg et al. 2019). One such species is the Heymon’s narrowmouthed frog, Microhyla heymonsi Vogt, 1911, originally described based on nine male specimens (ZMB 54906–54913 and ZMB 21944) collected from “Formosa” (= Taiwan) by Hans Sauter in 1909. Over the years, this species was reported from many parts of East, Southeast, and South Asia, such as Cambodia, Laos, Mainland of China, Malaysia, Myanmar, Thailand, Vietnam, the islands of Hainan, Singapore, and Sumatra, as well as the Andaman and Nicobar Archipelago of India (e.g., Taylor 1962; Berry 1975; Sarkar 1990; Lim and Lim 1992; Fei 1999; Ohler et al. 2002; Nguyen et al. 2005; Stuart 2005; Teynié et al. 2010; Sheridan et al. 2010; Jang-Liaw and Chou 2015; Mulcahy et al. 2018; Harikrishnan and Vasudevan 2018; Garg et al. 2019; Gorin et al. 2020; AmphibiaWeb 2021; Frost 2021). Based on molecular evidence, Garg et al. (2019) revealed the presence of six distinct lineages within the Microhyla ‘heymonsi’ complex and subsequently Gorin et al. (2020) suggested up to eight genetic lineages. Recently, Hoang et al. (2021) recognised and formally described two lineages of this complex from Vietnam as distinct species (M. daklakensis and M. ninhthuanensis). However, the identities of the remaining known populations of the M. ‘heymonsi’ complex remain uninvestigated. The Indian records of Microhyla ‘heymonsi’ are from the Nicobar Islands of the Andaman and Nicobar Archipelago (e.g., Mehta and Rao 1987; Sarkar 1990; Das 1994; Harikrishnan and Vasudevan 2018; Rangasamy et al. 2018). Some authors also reported this taxon from Assam and Manipur in Northeast India (Grosselt et al. 2005; Mathew and Sen 2010), which were doubted by Garg et al. (2019). This recent study also demonstrated that the Nicobar populations (erroneously stated to be from “Andaman”, Sonali Garg and S. D. Biju, pers. comm.) are genetically and perhaps morphologically divergent from the typical and geographically distant M. heymonsi populations of Taiwan (Huang and Hou 2004). Based on these findings, Garg et al. (2019) provisionally referred to the Nicobar populations as M. cf. heymonsi, deferring the confirmation of its species status for future studies. This was further reiterated through the phylogenetic findings of Gorin et al. (2020). In this backdrop, the present study was undertaken to assess and clarify the identity of M. cf. heymonsi from Nicobar Islands. MATERIALS AND METHODS Study area The Andaman and Nicobar Archipelago is formed by a nearly 780 km long chain of 572 islands, islets, and rocks, located in the Bay of Bengal and separated from Southeast Asia by the Andaman Sea (Census of India 2011). The northernmost point of the Andaman Islands page 2 of 23Zoological Studies 61: 2 (2022) © 2022 Academia Sinica, Taiwan (Landfall Island) lies less than 200 km away from the nearest point in Myanmar, while the southernmost Indira Point of the Nicobar Islands (Great Nicobar Island) is less than 150 km from the northernmost point on Rondo Island of Sumatra, Indonesia (Census of India 2011). Both the group of islands are part of two globally recognised biodiversity hotspots: Andaman Islands are part of the Indo-Burma hotspot, whereas Nicobar Islands are part of the Sundaland hotspot (Myers et al. 2000). The Nicobar group lies south of the Ten-degree Channel and consists of 23 islands divided into North Nicobar, Central Nicobar, and South Nicobar groups. Field Surveys and Sampling During fieldwork in the southern group of Nicobar Islands between 2016 and 2021, we sampled several populations of Microhyla frogs previously identified as Microhyla heymonsi (e.g., Mehta and Rao 1987; Sarkar 1990; Das 1994; Harikrishnan and Vasudevan 2018; Rangasamy et al. 2018) or M. cf. heymonsi (Garg et al. 2019). Surveys and sampling were carried out in the day and night time, largely during the monsoon season (June to September). The sampled individuals were photographed, euthanised using Tricaine methanesulphonate (MS-222) solution, fixed in 4% formalin, and finally washed and transferred to 70% ethanol for preservation. Prior to fixation, tissue samples were obtained from the thigh muscle, preserved in absolute ethanol, and later stored at -20°C for molecular studies. Some referred specimens were directly fixed and preserved in absolute or 70% ethanol. Geographical coordinates and elevation at the sampling sites were recorded using a Garmin 64S or eTrex 30x GPS with the WGS84 datum system. Distribution maps were prepared in QGIS version 2.6.1 (http://www.qgis. org). Type specimens are deposited in the amphibian collection of the Zoological Survey of India, Andaman and Nicobar Regional Centre, Port Blair (ZSI/ANRC). Referred specimens are available at Systematics Lab, University of Delhi (SDBDU); and the Department of Ocean studies and Marine Biology, Pondicherry University, Port Blair (DOSMB). Molecular study Genomic DNA was extracted from a newly sampled individual of Microhyla cf. heymonsi from Campbell Bay, Great Nicobar Island, using the DNeasy blood and tissue kit (Qiagen, Valencia, CA, USA). A fragment of the mitochondrial 16S ribosomal RNA gene (~540 bp) was PCR-amplified using the previously published primer set 16Sar and 16Sbr (Simon et al. 1994) and standard protocols. Sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit on ABI 3730 automated DNA sequencer (Applied Biosystems). The forward and reverse strands were checked and assembled in ChromasPro v1.34 (Technelysium Pty Ltd.). The assembled sequence was deposited in the GenBank under accession number OL828246. Previously published homologous 16S rRNA sequences representing 44 out of 46 currently recognised Microhyla species—genetic data were not available for the other two species (M. darevskii and M. maculifera)—were retrieved from the GenBank. A dataset of 77 taxa including five outgroups (Table S1) was aligned using MUSCLE in MEGA 7.0 (Kumar et al. 2016) and the alignment was manually optimised. The resultant alignment of 532 base pairs containing 201 distinct patterns, 149 parsimonyinformative sites, 33 singleton sites, and 350 constant sites was subjected to phylogenetic analyses using the Maximum Likelihood (ML) and Bayesian Inference (BI) approaches. The IQ-Tree Web Server (Trifinopoulos et al. 2016, available at http://iqtree.cibiv.univie.ac.at) was used to perform an ML analysis with 10,000 ultrafast bootstrap (UFB) replicates and the ‘auto’ criterion to select an appropriate model of DNA evolution. The general time-reversible (GTR) model, with empirical base frequencies (+F), proportion of invariant sites (+I), and discrete gamma shape parameter with four rate categories (+G4), was suggested as the best-fit model according to the Bayesian information criterion (BIC), Akaike Information Criterion (AIC), as well as the Corrected Akaike Information Criterion (AICc). The BI analysis was performed in Mr. Bayes v3.2.1 (Ronquist et al. 2012) using the GTR+I+G model selected through PartitionFinder v.2.1.1 (Lanfear et al. 2016) based on AIC. Two parallel runs of four Metropolis-coupled Markov chain Monte Carlo (MCMCMC) chains were executed for 30 million generations with trees sampled after every 1,000 generations. Convergence of the runs was evaluated by split frequencies of < 0.01 standard deviations and potential scale reduction factors of ~1.0. Tracer v. 1.7 (Rambaut et al. 2018) was used to view trace plots and confirm the effective sample size (ESS) values of > 200 for all the parameters. The Bayesian Posterior Probabilities (BPP) were summarized after discarding the first 25% trees as burn-in. Nodes with UFB support values ≥ 95% for ML (Minh et al. 2013) and BPP ≥ 0.95 (Leaché and Reeder 2002) were considered strongly supported. Using the Bayesian consensus phylogram, a species delimitation analysis was performed by Bayesian implementation of the Poisson Tree Processor (PTP) method (Zhang et al. 2013) with default parameters on the bPTP Web Server (available at page 3 of 23Zoological Studies 61: 2 (2022) © 2022 Academia Sinica, Taiwan https://species.h-its.org). Uncorrected pairwise genetic distances (p-distances) among 79 samples of the Microhyla achatina species group were computed in PAUP* (Swofford 2002). Morphological study The newly sampled populations from Nicobar were morphologically studied and compared with other congeners for accurate identification. Sex and maturity of the specimens were determined by examining the gonads through a small lateral or ventral incision, or by the presence of external secondary sexual characters such as a calling patch in males. Only sexually mature (adult) individuals were used for the morphological study. Following the terminologies of Biju et al. (2019) and Garg et al. (2019), body measurements were taken to the nearest 0.1 mm with the aid of a Nikon SMZ 1500 stereomicroscope using digital slide-callipers. The following characters were measured: snout-vent length (SVL). Head characters: Head length (HL), from rear of mandible to tip of snout; head width (HW), at the angle of the jaws; snout length (SL), from tip of snout to anterior orbital border; eye length (EL), horizontal distance between bony orbital borders; inter upper eyelid width (IUE), the shortest distance between the upper eyelids; maximum upper eyelid width (UEW); internal front of the eyes (IFE), shortest distance between the anterior orbital borders; internal back of the eyes (IBE), shortest distance between the posterior orbital borders; distance from the nostril to the tip of the snout (NS); distance from the front of the eye to the nostril (EN); internarial distance (IN), distance between the nares; greatest tympanum diameter (TYD); distance from the tympanum to the back of the eye (TYE). Forelimb characters: Forearm length (FAL), from flexed elbow to base of outer palmar tubercle; hand length (HAL), from base of outer palmar tubercle to tip of third finger; finger length (FL); maximum disc width of finger (FD); width of finger (FW), measured at the base of the disc; inner palmar tubercle length (IPTL); outer palmar tubercle length (OPTL). Hindlimb characters: Thigh length (TL), from vent to knee; shank length (SHL), from knee to heel; foot length (FOL), from base of inner metatarsal tubercle to the tip of fourth toe; total foot length (TFOL), from heel to the tip of fourth toe; inner toe length (ITL); maximum disc width of toe (TD); width of toe (TW), measured at the base of the disc; length of inner metatarsal tubercle (IMTL); length of outer metatarsal tubercle (OMTL). Digit number is represented by roman numerals I–V in subscript. All morphometric measurements discussed in the text are in millimetres. The webbing formulae follow Savage and Heyer (1967) as modified by Myers and Duellman (1982). The amount of webbing relative to subarticular tubercles is described by numbering the tubercles 1–3, starting from the toe discs, following Biju et al. (2014). For the convenience of discussion, the adult snout-vent length size and webbing categories follow Garg et al. (2019). Twenty-two morphometric measurements from seven male specimens each of M. heymonsi and the putative new taxon were subjected to statistical analyses. To reduce the impact of allometry, the values were size-corrected (ratio of each measurement to SVL). A multivariate Principal Component Analysis (PCA) was performed using 21 characters (except SVL) to assess the degree of morphological differentiation and spatial distribution of the two taxa. A Welch’s twosample t-test was used to determine the significance of differences in all the morphometric characters between the two taxa, with the significance level set at 0.05. The PCA and Welch’s t-test were carried out in the software R (R Development Core Team 2008) using the FactorMineR and factoextra packages from Kassambara (2017). Box plots of the most significant characters determined in the Welch’s t-test were prepared in PAST v. 2.17 (Hammer et al. 2001). RESULTS Phylogenetic relationships and genetic divergence The topologies recovered in our ML and BI trees were largely similar (Fig. 1) and in agreement with recently published phylogenies of Microhyla frogs (e.g., Biju et al. 2019; Garg et al. 2019; Gorin et al. 2020 2021; Poyarkov et al. 2020; Eprilurahman et al. 2021; Hoang et al. 2021). Our putative new species nested in the Microhyla achatina species group, wherein it formed a distinct and well-supported lineage (UFB 100, BPP 1.0) closely related to members of the Microhyla heymonsi subgroup (M. daklakensis, M. heymonsi, and M. ninhthuanensis). Within this subclade, a sistergroup relationship between the new taxon and M. ninhthuanensis was highly supported (UFB 98, BPP 1.0). Together, the two taxa showed a sister-group relationship with M. daklakensis, albeit with moderate to low support (UFB 88, BPP 0.60). The clade representing M. heymonsi sensu stricto was recovered as the basal lineage with which the remaining three members of the M. heymonsi subgroup shared a common ancestor. Our results support the findings of Garg et al. (2019) and Gorin et al. (2020) and once again indicate that several formerly misidentified populations of M. ‘heymonsi’ represent multiple distinct species. page 4 of 23Zoological Studies 61: 2 (2022) © 2022 Academia Sinica, Taiwan Fig. 1. Maximum Likelihood tree for the genus Microhyla showing the phylogenetic position and relationship of the new species based on 532 bp of the mitochondrial 16S rRNA gene. Values above and below the branches indicate Ultrafast Bootstrap Support (UFB) of > 50% and Bayesian Posterior Probabilities (BPP) > 0.50, respectively. Circles at internal nodes denote species groups. Putative species in the Microhyla achatina species group based on bPTP species delimitation analysis are denoted with dark grey vertical bars (support > 0.90), light grey vertical bars (support > 0.70), or open vertical bars (support < 0.70); black bars represent the recognized species. GenBank accession numbers and other sample details are referenced in table 1. Photographs of frogs in life represent species of the Microhyla heymonsi subgroup. Photos: M. heymonsi and M. daklakensis by Jianping Jiang; M. achatina, M. ninhthuanensis and M. nakkavaram sp. nov. by S. D. Biju. page 5 of 23Zoological Studies 61: 2 (2022) © 2022 Academia Sinica, Taiwan Our putative new taxon was also recovered as a distinct lineage with high support (> 0.95) in the bPTP species delimitation analysis (Fig. 1). Specifically within the Microhyla achatina species group, one additional candidate species was observed among the studied samples of M. achatina, M. gadjahmadai, and M. orientalis, while two additional candidates were recovered for M. malang. All other recognised congeners (except M. borneeensis and M. nepenthicola) were delimited as distinct species, albeit with variable degrees of support. For the studied 16S gene fragment, the new species diverged from its closest relatives, i.e., members of the Microhyla heymonsi species subgroup, by 3.5–3.7% from M. ninhthuanensis, 4.3–4.4% from M. heymonsi, and 4.8–5.2% from M. daklakensis. These observed interspecific p-distances are comparable to species-level divergences between other recognised congeners (e.g., Garg et al. 2019; Gorin et al. 2020). Detailed p-distance comparisons of the putative new species with other members of the Microhyla achatina species group are provided in table 1. Morphological affinity Our putative new species is morphologically assignable to the Microhyla achatina species group based on the following suite of characters: small to medium adult size (SVL 16–21 mm); nostrils positioned laterally on the snout; fingers with small discs; finger and toe discs with dorso-terminal grooves, cover bifurcate distally, except in M. fodiens; terminal phalanges of toes T-shaped; and rudimentary webbing between toes (Garg et al. 2019). Within the Microhyla achatina group, our new collections are closely related to members of the Microhyla heymonsi subgroup (M. daklakensis, M. heymonsi, and M. ninhthuanensis) in possessing the following combination of characters: presence of ( )-shaped dark marking at the centre of the dorsum; presence of a narrow mid-dorsal skin fold or line, extending from the tip of snout up to the vent; a prominent blackish brown lateral marking starting from the tip of snout and approaching the groin; finger and toe discs with prominent dorsolateral grooves, cover bifurcate distally; and webbing between toes rudimentary or absent. A further morphological comparison with the three closest congeners showed that the populations from Nicobar differed with respect to a combination of characters (see ‘Morphological comparison’ under the new species description) and cannot be assigned to any other available name, thereby warranting a distinct species status. Based on concordance in the morphological and molecular evidence, the Nicobar populations are therefore assigned to a new species, formally described below in the taxonomy section. TAXONOMY Microhyla heymonsi Vogt, 1911 Heymon’s narrow-mouthed frog (Figs. 1, 2; Table 1; Tables S1–S2) Taxonomic note: This taxon was originally described based on nine male specimens (ZMB 54906–54913 and ZMB 21944) collected by Hans Sauter in 1909 from “Formosa” (= Taiwan). The original description is relatively brief and provides a comparison with two other microhylids found in the region (Microhyla okinavensis and Micryletta steinegeri). However, Vogt (1911) clearly defined some of the key morphological characteristics of the species that are useful for differentiating this taxon from other currently recognised congeners, such as: “Die Haut ist vollkommen glatt” [The skin is perfectly smooth], “Eine helle Linie reicht von der Schnauzenspitze bis zum After. In der Mitte des Rückens teilt die Linie einen fast kreisrunden tiefschwarzen Fleck von ungefähr 1,5 mm Durchmesser. Dieser Fleck ist für die Art sehr charakteristisch und bei allen Exemplaren deutlich hervortretend.” [A bright line extends from the tip of the face to the anus. In the middle of the back, the line divides an almost circular, deep black spot about 1.5 mm in diameter. This spot is very characteristic of the species and is clearly visible in all specimens], and “Die Seiten sind tiefdunkel und scharf gegen den Rücken abgesetzt. Auch der After ist dunkel, ebenfalls die Innenseite des Metatarsus und die Unterseite des Fußes” [The sides are deeply dark and set off sharply against the back. The anus is also dark, as is the inside of the metatarsus and the underside of the foot]. Due to the presence of some of these characters, subsequent authors reported M. heymonsi from several regions across mainland Asia (see Introduction). However, in the recent years many of these populations are demonstrated to be genetically and morphologically divergent (e.g., Garg et al. 2019; Gorin et al. 2020; Hoang et al. 2021). While Hoang et al. (2021) recently described two populations from Vietnam previously referred to M. heymonsi as new species, Garg et al. (2019) demonstrated that the population from Nicobar Islands of India potentially represents a new candidate species. We studied the type series of M. heymonsi available at ZMB, Berlin and found it comparable to the original description with respect to all the described page 6 of 23Zoological Studies 61: 2 (2022) © 2022 Academia Sinica, Taiwan Table 1. The uncorrected pairwise genetic distances (in percent) among all the recognised members of the Microhyla achatina species group, based on the studied fragment of the mitochondrial 16S rRNA. Comparisons with Microhyla nakkavaram sp. nov. are indicated in bold. The diagonal indicates intraspecific distances. Distances are reported as mean ± standard deviation (minimum–maximum). Values in parentheses following the species names represent the number of samples A: Microhyla heymonsi species subgroup Species 1 2 3 4 1M. nakkavaram sp. nov. (2) 0 2M. daklakensis (6) 5.0 ± 0.1 (4.8–5.2) 0.2 ± 0.2 (0–0.6) 3M. heymonsi (3) 4.3 ± 0.1 (4.3–4.4) 3.7 ± 0.2 (3.5–4.2) 0.1 ± 0.1 (0–0.2) 4M. ninhthuanensis (4) 3.5 ± 0.1 (3.5–3.7) 4.0 ± 0.2 (3.9–4.4) 3.5 ± 0.1 (3.5–3.8) 0.5 ± 0.5 (0–1.0) B: Microhyla achatina species subgroup Species 1 5 6 7 8 9 10 11 12 13 14 15 5M. achatina (3) 8.7 ± 0.3 (8.3–9.0) 1.9 ± 1.2 (0.6–2.8) 6M. borneensis (1) 7.2 (7.2–7.2) 6.3 ± 0.3 (6.0–6.6) – 7M. gadjahmadai (2) 9.5 ± 0.3 (9.3–9.8) 5.0 ± 0.5 (4.4–5.8) 6.4 ± 0 (6.4) 2.6 8M. irrawaddy (2) 8.0 ± 0 (8.0–8.0) 6.4 ± 0.4 (6.0–6.8) 7.4 ± 0 (7.4) 6.7 ± 0.1 (6.6–6.8) 0 9M. kodial (2) 7.4 ± 0.2 (7.2–7.6) 6.7 ± 0.2 (6.4–7.0) 5.9 ± 0.1 (5.8–6.0) 7.2 ± 0.1 (7.0–7.4) 5.0 ± 0 (5.0) 0 10 M. malang (3) 8.4 ± 0.3 (8.0–8.6) 7.2 ± 0.4 (6.6–8.0) 2.6 ± 0.7 (2.0–3.4) 6.8 ± 0.4 (6.2–7.2) 7.6 ± 0.2 (7.4–7.8) 7.5 ± 0.5 (7.0–8.2) 3.0 ± 0.6 (2.4–3.4) 11 M. mantheyi (3) 8.0 ± 0.2 (7.8–8.2) 5.9 ± 0.4 (5.4–6.4) 5.1 ± 0.2 (5.0–5.3) 5.5 ± 0.3 (5.2–6.0) 7.2 ± 0.1 (7.0–7.2) 5.9 ± 0.2 (5.6–6.2) 6.2 ± 0.2 (6.0–6.6) 0.9 ± 0.1 (0.8–1.0) 12 M. minuta (2) 8.0 ± 0 (8.0–8.0) 6.9 ± 0.4 (6.6–7.4) 5.6 ± 0 (5.6) 6.0 ± 0.7 (5.4–6.6) 7.6 ± 0 (7.6) 7.1 ± 0.1 (7.0–7.2) 5.3 ± 0.5 (5.0–6.0) 6.0 ± 0.3 (5.8–6.4) 0 13 M. nepenthicola (3) 8.0 ± 0.1 (8.0–8.2) 6.2 ± 0.2 (5.8–6.6) 0.9 ± 0.1 (0.8–1.0) 6.1 ± 0.2 (5.8–6.3) 7.2 ± 0.2 (7.0–7.4) 6.3 ± 0.2 (6.0–6.6) 2.7 ± 0.4 (2.2–3.2) 5.2 ± 0.2 (5.0–5.6) 4.8 ± 0.1 (4.8–5.0) 0.3 ± 0.1 (0.2–0.4) 14 M. orientalis (2) 7.8 ± 0.3 (7.5–8.0) 5.7 ± 0.3 (5.2–6.2) 4.5 ± 0.7 (4.0–5.0) 6.1 ± 0.2 (6.0–6.4) 7.3 ± 0.1 (7.2–7.4) 6.7 ± 0.5 (6.2–7.2) 5.4 ± 0.8 (4.6–6.6) 5.2 ± 0.2 (5.0–5.4) 5.7 ± 0.1 (5.6–5.8) 4.6 ± 0.4 (4.2–5.2) 1.2 15 M. sriwijaya (2) 9.6 ± 0.3 (9.4–9.8) 8.6 ± 0.5 (7.8–9.4) 5.7 ± 0.3 (5.5–5.9) 8.2 ± 0.3 (7.9–8.5) 7.9 ± 0.3 (7.7–8.1) 8.9 ± 0.2 (8.7–9.2) 6.7 ± 0.5 (5.9–7.2) 6.7 ± 0.4 (6.1–7.2) 7.2 ± 0.3 (7.0–7.4) 5.8 ± 0.3 (5.5–6.1) 5.0 ± 0.3 (4.8–5.2) 0.4 C: Microhyla pineticola species subgroup Species 1 16 17 16 M. neglecta (2) 10.8 ± 0.2 (10.6–11.0) 0.4 17 M. pineticola (2) 8.3 ± 0.1 (8.2–8.4) 5.9 ± 0.3 (5.9–6.2) 0.2 D: ungrouped Species 1 18 18 M. fodiens (2) 10.4 ± 0.2 (10.3–10.6) 0 page 7 of 23Zoological Studies 61: 2 (2022) © 2022 Academia Sinica, Taiwan characters. Since the type series contains multiple specimens, below we provide a detailed redescription of ZMB 54906, along with photographs of various aspects (Fig. 2), and the body measurements for the syntypes (Table S2), with the aim of facilitating future studies. Furthermore, since recent studies suggest that M. heymonsi is likely to be restricted to Taiwan and neighbouring regions, we also provide below a revised diagnosis for the species based on examination of multiple specimens from the original type series as well as additional museum specimens for circumscription of the taxa and to facilitate comparison with other newly described species of the M. heymonsi species subgroup from Vietnam (Hoang et al. 2021) and India (present study). Material examined: Syntypes: (Nine males, from “Formosa” (= Taiwan): ZMB 54906 (SVL 22. 3 mm), ZMB 54907 (SVL 20.4 mm), ZMB 54908 (SVL 22.3 mm), ZMB 54909 (SVL 22.8 mm), ZMB 54910 (SVL 23.1 mm), ZMB 54911 (SVL 23.5 mm), ZMB 54912 (SVL 21.7 mm), ZMB 54913 (SVL 22.6 mm), and ZMB 21944 (SVL 20.1 mm). Other museum specimens: Four males: CIB 65580 (SVL 21.8 mm), CIB 65583 (SVL 22.4 mm), CIB 65579 (SVL 19.5 mm), CIB 65582 (SVL 22.1 mm), and three females: CIB 65581 (SVL 24.8 mm), CIB 65585 (SVL 26.5 mm), and CIB 65586 (SVL 25.6 mm), from Taiwan Province, China. Revised diagnosis: Microhyla heymonsi can be diagnosed from other congeners by a combination of following morphological traits: body stocky, small in size (male SVL 19–24 mm; female SVL 24–27 mm); snout rounded in dorsal and ventral view, obtusely pointed in lateral view; finger I short, FI ≤ ½ FII; tips of fingers dilated, forming discs, with a dorsal-terminal groove, cover bifurcate distally; metacarpal tubercles two, rounded; tips of all toes dilated into discs, with a dorso-terminal groove, cover bifurcate distally; foot webbing rudimentary, formula: I2––2½II2––3–III3––4– IV4––3–V; lateral surfaces deeply dark, from tip of the snout to groin, clearly demarcated from the light brown dorsum; a narrow, light coloured, mid-dorsal line and skin fold extending from the snout tip to the vent, with a dark brown ( )-shaped mark at the centre of the dorsum, along the mid-dorsal line (Fig. 2) (Vogt 1911; Garg et al. 2019; Hoang et al. 2021; present study). Redescription of syntype, ZMB 54906 (measurements in mm) (Fig. 2): A small-sized adult male (SVL 22.3), rather stocky. Head characters: head wider than long (HW 6.3, HL 5.3); snout rounded in dorsal and ventral view, obtusely pointed in lateral view, its length (SL 2.9) longer than horizontal diameter of eye (EL 2.0); loreal region acute; interorbital space flat; inter upper eyelid distance wider (IUE 2.8) than upper eyelid width (UEW 1.2) and internarial distance (IN 1.8); nostrils oval, placed more towards lateral side of snout, closer to snout (NS 1.3) than eye (EN 1.6); tympanum hidden; supratympanic fold extending from posterior corner of eye to the shoulder, well-developed; vomerine ridge present; tongue small, oval, without papillae. Forelimb characters: Arms short, forearm length (FAL 4.3) shorter than hand length (HAL 5.7); relative length of fingers I<II=IV<III (FLI 1.1, FLII 2.3, FLIII 3.8, FLIV 2.3); tips of all fingers with median dorso-terminal grooves, cover bifurcate distally, finger tips slightly wider compared to finger width (FDI 0.5; FWI 0.3, FDII 0.5; FWII 0.3, FDIII 0.6; FWIII 0.5, FDIV 0.4, FWIV 0.4); dermal fringes absent; webbing absent between fingers; subarticular tubercles prominent, all present; inner and outer metacarpal tubercles well developed, oval; supernumerary tubercles absent; nuptial pad absent. Hindlimb characters: Hind limbs short, thigh (TL 10.8) shorter than shank (SHL 12.2) and foot (FOL 12.9); distance from base of tarsus to tip of toe IV (TFOL 17.1); relative length of toes I<II<V<III<IV; toe tips rounded, slightly enlarged into discs (TDI 0.5; TWI 0.4, TDII 0.6; TWII 0.4, TDIII 0.6; TWIII 0.4, TDIV 0.7, TWIV 0.5,TDV 0.6, TWV 0.5); toe discs with prominent dorso-terminal grooves, cover bifurcate distally; dermal fringes absent; foot webbing rudimentary: I2––2½II2––3–III3––4–IV4––3–V; subarticular tubercles prominent, all present, circular; a weakly developed light grey cutaneous fold on inner side of toe I continuous up to inner metatarsal tubercle; another weakly developed light grey cutaneous fold along toe V from tip of the toe to nearly the weakly developed outer metatarsal tubercle; inner metatarsal tubercle prominent (IMTL 0.8), oval-shaped; outer metatarsal tubercle, small and weakly developed (OMTL 0.5), rounded; supernumerary tubercles absent. Skin: Skin of snout and upper eyelids shagreened; anterior and posterior parts of dorsum smooth; upper and lower parts of flank smooth; dorsal surfaces of forelimb shagreened, thigh and shank shagreened with scattered flattened tubercles; posterior parts of thigh and cloacal region shagreened to sparsely granular. Ventral surfaces of throat, chest, belly, and limbs smooth (Fig. 2). Colour in preservation: Dorsum greyish-brown with a feeble brownish ‘teddy-bear’ shaped pattern; a well-developed, thin, light-coloured mid-dorsal line from the tip of the snout to vent; a single small tubercle at mid-dorsum along with a ( )-shaped marking; a prominent darker brown lateral band extending from the lateral surfaces of the head to nearly the groin; a prominent brown streak above the cloacal opening. Throat blackish-brown; chest and lateral surfaces of the belly greyish-brown without markings; ventral surfaces page 8 of 23Zoological Studies 61: 2 (2022) © 2022 Academia Sinica, Taiwan Fig. 2. A–G: Syntype (ZMB 54906) of Microhyla heymonsi: A: dorsal view; B: ventral view; C: lateral view of head; D: ventral view of hand; E: illustration of the third finger tip morphology in dorsal view; F: ventral view of foot; G: illustration of the fourth toe tip morphology in dorsal view. H– M: Diagnostic morphological characters for members of the Microhyla heymonsi species subgroup. Photos and illustrations: S. D. Biju. page 9 of 23Zoological Studies 61: 2 (2022) © 2022 Academia Sinica, Taiwan triangular); length of finger I longer than half the length of finger II (vs. shorter); presence of three metacarpal tubercles, inner oval, middle and outer rounded (vs. two); absence of dark brownish lines parallel to the middorsal line (vs. present); absence of light brown stripe extending from posterior corner of eye to axilla (vs. present); ventral surfaces blackish-brown to off-white (vs. purplish-grey with indistinct whitish mottling); and foot webbing rudimentary: I2–2½II2–3III3–4IV4–3V (vs. relatively more: I1½–2½II1¾–3III2¾–3¾IV4– 2½V). Microhyla nakkavaram sp. nov. differs from M. neglecta by its snout rounded in lateral view (vs. sharply acuminate); comparatively shorter hind limbs, with tibiotarsal articulation of straightened limb projecting up to the eye (vs. longer, projecting well beyond the snout); dorsal skin granular (vs. smooth with small, flat, irregularly placed tubercles); absence of a distinct cream-white stripe from posterior corner of eye to axilla (vs. present); and ventral surfaces blackish-brown to off-white (vs. yellowish belly with indistinct greyish marbling laterally). Microhyla nakkavaram sp. nov. differs from all other members of the Microhyla achatina species group by its prominently dark blackish-brown lateral surfaces from the snout tip to nearly the groin (vs. dark lateral colouration absent or partial); and presence of ( )-shaped markings on anterior part of the dorsum, along the middorsal line (vs. absent). Specifically, it also differs from M. achatina by its slender body (vs. stout); relatively smaller adult male size, SVL 16–19 mm (vs. larger, SVL 21–24 mm); snout rounded in lateral view (vs. obtusely pointed); length of finger I longer than half the length of finger II: FLI > ½FLII (vs. shorter: FLI < ½FLII); and dorsal skin granular (vs. smooth to shagreened). Microhyla nakkavaram sp. nov. further differs from M. borneensis, M. malang, and M. mantheyi by its snout rounded in lateral view (vs. obtusely pointed in all three species); and foot webbing rudimentary, below the first subarticular tubercles on toes II to V: I2–2½II2– 3III3–4IV4–3V (vs. above the second subarticular Fig. 5. Statistical analyses showing the morphometric distinctness of Microhyla nakkavaram sp. nov. from M. heymonsi with which it was previously confused. A: Scatter plot of the first two principal components recovered from a principal component analysis of morphometric data from seven males of each species. The sampled individuals are enclosed in 95% confidence ellipses; B–E: total contributions of the first four principal components. Dashed line on the histograms indicates the expected average contribution: B: PC1 (34.8% variance); C: PC2 (22.1% variance); D: PC3 (14.8% variance); E: PC4 (10.9% variance); F–J: Box plots depicting differences between the two species with respect to the five most significant diagnostic characters (p < 0.001) recovered in the Welch’s Two Sample t-test; F: SVL (p = 0.000001019); G: HW/SVL (p = 0.000000007186); H: TL/SVL (p = 0.000001057); I: IN/SVL (p = 0.00001086); J: HL/SVL (p = 0.00006885). page 16 of 23Zoological Studies 61: 2 (2022) © 2022 Academia Sinica, Taiwan tubercles on either side of toe IV in all the three species: I1–2II1–3III2½–3⅓IV3½–2V, I1–2II1–2⅔III1⅔– 3IV3–1V, and I1–2II1–2III2–3IV3–1½V, respectively). It specifically also differs from M. borneensis by the well-developed first finger, length of finger I longer than half the length of finger II: FLI > ½FLII (vs. greatly reduced to a nub); and further differs from M. malang and M. mantheyi by comparatively shorter hind limbs, with tibiotarsal articulation of straightened limb projecting up to the eye (vs. longer, projecting well beyond the snout); and presence of a mid-dorsal line (vs. absent). Microhyla nakkavaram sp. nov. differs from M. fodiens by its slender body (vs. stout); relatively smaller adult male size, SVL 16–19 mm (vs. larger, SVL 20–30 mm); presence of a mid-dorsal line (vs. absent); and outer metatarsal tubercle small and not shovel-shaped (vs. large and shovel-shaped). It differs from M. gadjahmadai in having a slender body (vs. stout); relatively smaller adult male size, SVL 16–19 mm (vs. larger, SVL 18–22 mm); absence of a light-coloured stripe from posterior corner of eye to axilla (vs. present); and comparatively shorter hind limbs, with tibiotarsal articulation of straightened limb projecting up to the eye (vs. longer, projecting well beyond the snout). It differs from M. irrawaddy by the presence of a mid-dorsal line (vs. absent); absence of a light-coloured stripe from posterior corner of eye to axilla (vs. present); and finger and toe discs with dorsoterminal grooves, cover bifurcate distally (vs. absent). It differs from M. kodial by the presence of a mid-dorsal line (vs. absent); prominently dark blackish-brown lateral surfaces (vs. absent); and absence of a light coloured stripe from posterior corner of eye to axilla (vs. present). It differs from M. minuta by the length of finger I longer than half the length of finger II (vs. finger I almost equal or slightly shorter than half the length of finger II); absence of a light-coloured stripe from posterior corner of eye to axilla (vs. present); presence of mid-dorsal line (vs. absent); and absence of crescentshaped bright yellow lines on dorsum (vs. presence of two crescent-shaped bright yellow lines on dorsum). It differs from M. nepenthicola by relatively larger adult male size, SVL 16–19 mm (vs. SVL 10–13 mm); snout rounded in lateral view (vs. obtusely pointed); relatively well-developed first finger (vs. greatly reduced to a nub); and foot webbing rudimentary: up to the first subarticular tubercles on toes II–V: I2–2½II2–3III3– 4IV4–3V (vs. relatively more, up to or slightly below the second subarticular tubercle on either side of toe IV). It differs from M. orientalis by relatively larger adult male size, SVL 16–19 mm (vs. smaller, SVL 15–18 mm); prominently dark blackish-brown lateral surfaces (vs. absent); absence of a light-coloured stripe from posterior corner of eye to axilla (vs. present); and length of finger I longer than half the length of finger II (vs. shorter). It differs from M. sriwijaya by relatively larger adult male size, SVL 16–19 mm (vs. smaller, SVL 12–15 mm); prominently dark blackishbrown lateral surfaces (vs. absent); absence of a lightcoloured stripe from posterior corner of eye to axilla (vs. present); presence of a mid-dorsal line or skin fold (vs. absent); and length of finger I longer than half the length of finger II (vs. shorter). Comparison with Indian species outside the Microhyla achatina group: The new species cannot be confused with any of the other 11 known Microhyla species from India, outside the Microhyla achatina species group (M. berdmorei, M. chakrapanii, M. darreli, M. eos, M. laterite, M. mukhlesuri, M. mymensinghensis, M. nilphamariensis, M. ornata, M. rubra, M. sholigari, and M. taraiensis) due to a suite of characters: presence of mid-dorsal line, presence of ( )-shaped markings at mid-dorsum, and rudimentary foot webbing. Specifically, Microhyla nakkavaram sp. nov. also differs from M. berdmorei by smaller adult male size, SVL 16–19 mm (vs. larger, SVL 33–36 mm); presence of supratympanic fold (vs. absent); and foot webbing rudimentary: I2–2½II2–3III3–4IV4–3V (vs. more, extending well beyond the third subarticular tubercle on either side of toe IV and up to discs on the remaining toes: I1–1II1–1+III1–1½IV1½–1V). It further differs from M. chakrapanii, M. mukhlesuri, and M. mymensinghensis by the presence of prominent dorsoterminal grooves and cover bifurcate distally on finger and toe discs (vs. without grooves); and absence of a light coloured streak from posterior corner of eye to axilla (vs. present). It also differs from M. darreli, M. eos, M. laterite, and M. sholigari by the placement of nostrils towards lateral side of snout (vs. towards dorsal side); and foot webbing rudimentary, up to or below the first subarticular tubercles on toes II to V: I2– 2½II2–3III3–4IV4–3V (vs. well above, up to the second subarticular tubercle on either side of toe IV). It differs from M. nilphamariensis, M. ornata, and M. taraiensis by the presence of prominent dorso-terminal grooves and cover bifurcate distally on finger and toe discs (vs. without grooves). Distribution and natural history: Microhyla nakkavaram sp. nov. is currently known only from the southern group of Nicobar Islands: Great Nicobar Island, Kondul Island, and Little Nicobar Island (Fig. 6). The type series originates from an area surrounding human habitations at Campbell Bay on the east coast of Great Nicobar Island. This species is otherwise widely distributed across a range of habitats, from evergreen, semi evergreen, moist and dry deciduous, littoral and beach forests, to temporary rain water puddles in agricultural fields and human habitations, at page 17 of 23Zoological Studies 61: 2 (2022) © 2022 Academia Sinica, Taiwan Fig. 6. A: Type localities of the four currently recognised species of the Microhyla heymonsi species subgroup. B: The geographical location of Andaman and Nicobar Islands; C: Enlarged view of the Nicobar group of islands; D: The sampling localities of Microhyla nakkavaram sp. nov. in the southern group of Nicobar Islands. Red circle indicates the type locality. page 18 of 23 Zoological Studies 61: 2 (2022) © 2022 Academia Sinica, Taiwan multiple localities within its reported range (Table S6). It is locally common and fairly abundant during the monsoon season (June–September). Calling activity has been observed between 18:00–22:00 hours, especially on rainy nights with the onset of southwest monsoon. DISCUSSION The early documented amphibian surveys on the islands of Andaman and Nicobar during the 19th century (Fitzinger 1860; Stoliczka 1870) and 20th century (e.g., Annandale 1917; Cherchi 1954; Pillai 1977 1991; Mansukhani and Sarkar 1980; Mehta and Rao 1987; Sarkar 1990; Das 1994 1996 1998; Daniels and David 1996) largely recorded the occurrence of species found in the neighbouring biogeographical regions, with sporadic descriptions of new taxa. The past nearly two decades have seen progress in description of new species and genera, new family records, and taxonomic clarifications, particularly with the aid of molecular techniques (e.g., Chandramouli et al. 2016 2020a b; Harikrishnan and Vasudevan 2018; Garg et al. 2019; Biju et al. 2020). The recent surveys on these islands suggest the occurrence of 20 species representing 15 genera and five families (e.g., Harikrishnan and Vasudevan 2018; Rangasamy et al. 2018; Garg et al. 2019; Biju et al. 2020; Chandramouli et al. 2020a). Of these, 12 species are found in the Andaman group of islands, with five species and one genus known to be endemic. On the other hand, the Nicobar group of islands is home to nine species of which three species, including the newly described Microhyla nakkavaram sp. nov., are endemic (Das 1995; Chandramouli and Prasad 2020; present study). However, a proper inventory of the region’s amphibian fauna remains obscure. Even the information on distribution, ecology, and natural history of most species is scant. A major contributing factor is perhaps the general assumption that these islands have faunal affinities with the neighbouring regions—the Andamans with Indo-Burma, while the Nicobar Islands with Sundaland (Mani 1974; Das 1999). Although this holds true to a large extent, it has long deterred a close examination and proper assessment of the taxonomic identities of amphibian species in the region. A classic example is that of our new species Microhyla nakkavaram sp. nov., which was long misidentified as M. heymonsi, a taxon presumed to occur in Nicobar due to its widely reported distribution across Southeast Asia and superficial similarities but without any detailed studies. In other words, an entirely new species was hiding in plain sight under a wrong scientific name, despite being locally common and fairly abundant. Hence, our study emphasises on the primary need to accurately identify the amphibians of Andaman and Nicobar Archipelago, which holds the potential to reveal new or previously unidentified species with much narrower distribution ranges. Such taxonomic studies will also pave way for further scientific inquiries and meaningful conservation assessments of species inhabiting this unique biogeographical region. Based on our extensive surveys, the new species is currently restricted to three islands in the southern Nicobar. However, M. nakkavaram sp. nov. could be found at several additional localities apart from those reported in our study (Table S6) and is also likely to be more widely distributed within the southern group of islands. Our surveys in the central and northern Nicobar over the past six years have not yielded any specimens of this species. However, considering the close proximity of the southernmost population of M. nakkavaram sp. nov. in Great Nicobar to northern Sumatra of Indonesia, the possibility of finding this species in Sumatra cannot be ruled out. Future surveys are required in these regions to ascertain whether the geographical range of M. nakkavaram sp. nov. extends outside of Nicobar. It is also noteworthy that this species is known to occupy a wide range of habitats. Future studies could focus on its ecological preferences, breeding requirements, and adaptability, especially in the light of increasing rate of development and infrastructure on these islands. Hence, potential threats to M. nakkavaram sp. nov. and its habitats will need to be evaluated, all the more because of its relatively narrow geographical range. The family Microhylidae represents the second highest number of species on the Andaman and Nicobar Islands. The relatively high diversity combined with the unique biogeography of the region indicates the research potential of this group to address questions on the patterns of diversification and colonisation of microhylids (Garg and Biju 2019; Gorin et al. 2020), which are one of the most speciose and geographically widespread group of frogs in the world. Our discovery of another new species of Microhyla raises the total recognised species in the genus to 48, of which 14 species are found in India. The finding also shows that the known diversity of this group of microhylid frogs remains underestimated despite active ongoing research (e.g., Matsui et al. 2011; Poyarkov et al. 2014 2019; Garg et al. 2019; Gorin et al. 2020). Our study also highlights the need to reassess the taxonomic identities of all the known M. ‘heymonsi’ populations from regions across Asia (Garg et al. 2019; Gorin et al. 2020). Until now, only four genetically delimited lineages of this complex have been properly identified (Tominaga et al. 2019; Hoang et al. 2021; present study). Since M. heymonsi sensu stricto is page 19 of 23Zoological Studies 61: 2 (2022) © 2022 Academia Sinica, Taiwan possibly restricted to Taiwan and neighbouring regions, the remaining unidentified lineages could either represent other previously available names or reveal additional new species. Further comprehensive and integrative studies are required to fully resolve the taxonomic status of decades-old misidentifications of this taxon. CONCLUSIONS The study describes a new frog species of the genus Microhyla from Nicobar Islands of India. This taxon was previously misidentified as M. heymonsi, a species presumed to be widely distributed across Asia due to a lack of proper taxonomic studies. Based on concordant evidence from multiple analyses using both morphological and phylogenetic data, the new species is formally described as M. nakkavaram sp. nov. and compared with all known congeners, particularly its closely related members in the Microhyla achatina species group. Extended distribution of the new species is reported from three islands of southern Nicobar, where it is currently known to be restricted; however, the possible occurrence of M. nakkavaram sp. nov. in neighbouring Southeast Asian regions requires further investigations. Our study excludes M. heymonsi from the list of Indian amphibians and further highlights the need for proper identification and reassessment of the taxonomic status of several other Asian populations assigned to this species complex over a period of nearly 100 years. The detailed redescription and revised diagnosis of M. heymonsi included in the present work based on the syntype series will also aid future studies on this group of taxonomically challenging frogs. List of abbreviations ZSI/ANRC, Zoological Survey of India, Andaman and Nicobar Regional Centre, Port Blair. SDBDU, Systematics Lab, University of Delhi. DOSMB, Department of Ocean studies and Marine Biology, Pondicherry University, Port Blair. NHM, Natural History Museum, London. ZMB, Zoologisches Museum, Berlin, Germany. CIB, Chengdu Institute of Biology, China. HT, Holotype. PT, Paratype. ST, Syntype. TT, Topotype. RS, Referred Specimen. Acknowledgments: This work and the new species name have been registered with ZooBank under urn:lsid:zoobank.org:pub:EF1F5474-FEAE-43469B17-D5EB0B83A875. We thank the Department of Environment and Forests, Andaman and Nicobar Islands, India for study permissions and logistic support; Shri. Apurba Kumar Das, Research Associate, ZSI/ANRC, Port Blair, and Shri. Jayabal, Driver, Campbell Bay for their assistance and support during the field survey; Mark Oliver Rödel and Frank Tillack (ZMB, Berlin), Jianping Jiang and Jiatang Li (CIB, Chengdu), Barry Clarke and David Gower (NHM, London) for access to specimens under their care and museum support to SDB and SG; Frank Tillack for providing access to original literature; Jianping Jiang for photographs of Microhyla daklakensis and M. heymonsi used in Figure 1; and Naitik Patel for help in statistical analyses. This study was partially supported by a Faculty Research Programme Grant-Institution of Eminence (Ref. No./IoE/2021/12/FRP) from University of Delhi to SDB. Field travel and logistics were partially supported by grants from Ministry of Environment, Forest and Climate Change, Government of India; SERB, Department of Science and Technology, Government of India to CS. SG is supported by the Council for Scientific and Industrial Research [CSIR No. 09/045(1694)/2019-EMR-I], Government of India; and a small research grant (5409-0260) from Re:wild (formerly Global Wildlife Conservation), USA. CS and GG express sincere thanks to the Director, Zoological Survey of India for cooperation and encouragement during the period of the study. SRC thanks K.V. Devi Prasad and P. M. Mohan, the faculty of the Departments of Ecology & Environmental Sciences and Ocean Studies & Marine Biology, Pondicherry University for the facilities and infrastructure provided; and the Mohamed bin Zayed Species Conservation Fund for a grant (#160514249). Authors’ contributions: SDB, SG, and CS conceived and designed the study; GG, CS, SG, SDB, and SRC collected material in the field; SG, SDB, SRC, GG, and CS generated and analysed the data; SG and SDB wrote the manuscript; CS, GG, and SRC reviewed and edited the manuscript drafts; all authors approved the final draft. Competing interests: The authors declare that they have no conflicts of interests. Availability of data and materials: DNA sequence data generated in the study is deposited in the NCBI GenBank. Type specimens are deposited in the Zoological Survey of India - Andaman and Nicobar Regional Centre (ZSI/ANRC), Port Blair, India. page 20 of 23Zoological Studies 61: 2 (2022) © 2022 Academia Sinica, Taiwan Consent for publication: All authors agree to the publication of this work in Zoological Studies. Ethics approval consent to participate: Not applicable. REFERENCES AmphibiaWeb. 2021. Amphibiaweb: information on amphibian biology and conservation. Berkeley: University of California. http://www.amphibiaweb.org. Accessed 10 Aug. 2021. Annandale N. 1917. Zoological results of a tour in the Far East. Batrachia. Mem Asiat Soc Bengal 6:119–155. Atmaja VY, Hamidy A, Arisuryanti T, Matsui M, Smith EN. 2019. A new species of Microhyla (Anura: Microhylidae) from Sumatra, Indonesia. 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Factor loadings, eigenvalues, and percent variance for the first five principal components (PC) with eigenvalues > 1, recovered from principal component analysis of 22 morphometric variables from Microhyla nakkavaram sp. nov. and M. heymonsi. (download) Table S5. Relative significance of morphometric characters for differentiating Microhyla nakkavaram sp. nov. and M. heymonsi based on Welch’s Two Sample t-test. (download) Table S6. Distribution of Microhyla nakkavaram sp. nov. in Nicobar Islands, India. (download) page 23 of 23Zoological Studies 61: 2 (2022)