Molecular, morphological and acoustic assessment of the genus Ophryophryne (Anura, Megophryidae) from Langbian Plateau, southern Vietnam, with description of a new species

Abstract Asian Mountain Toads (Ophryophryne) are a poorly known genus of mostly small-sized anurans from southeastern China and Indochina. To shed light on the systematics within this group, the most comprehensive mitochondrial DNA phylogeny for the genus to date is presented, and the taxonomy and biogeography of this group is discussed. Complimented with extensive morphological data (including associated statistical analyses), molecular data indicates that the Langbian Plateau, in the southern Annamite Mountains, Vietnam, is one of the diversity centres of this genus where three often sympatric species of Ophryophryne are found, O. gerti, O. synoria and an undescribed species. To help resolve outstanding taxonomic confusion evident in literature (reviewed herein), an expanded redescription of O. gerti is provided based on the examination of type material, and the distributions of both O. gerti and O. synoria are considerably revised based on new locality records. We provide the first descriptions of male mating calls for all three species, permitting a detailed bioacoustics comparison of the species. We describe the new species from highlands of the northern and eastern Langbian Plateau, and distinguish it from its congeners by a combination of morphological, molecular and acoustic characters. The new species represents one of the smallest known members of the genus Ophryophryne. At present, the new species is known from montane evergreen forest between 700–2200 m a.s.l. We suggest the species should be considered Data Deficient following IUCN’s Red List categories.


Introduction
Asian Mountain toads (Ophryophryne Boulenger, 1903) are a small group of frogs from southeast Asia with a rather limited distribution mostly in mountains of eastern Indochina and adjacent parts of southern China (Yunnan and Guangxi) and northern Thailand (Inger et al. 1999, Ohler 2003, Orlov and Ananjeva 2007, Yang 2008. The genus Ophryophryne is still poorly studied, to date five (Ohler 2003, Stuart et al. 2006 or six (Stuart et al. 2010) species are recognized, with little consensus on the taxonomic status of several forms. All of the known Ophryophryne species have been reported from the Truong Son or Annamite mountains in Vietnam, which may be considered as an area of highest diversity for this group (Orlov and Ananjeva 2007).
The systematic status of Ophryophryne has long been a source of confusion. Boulenger (1903) described the genus and species O. microstoma Boulenger, 1903, and though he clearly stated that Ophryophryne is closely allied to Megophrys Kuhl & Van Hasselt, 1822, he noted characters also shared by members of the family Bufonidae Gray, 1825 (lacking vomerine and maxillary teeth and presence of horizontal pupil). Subsequently, Noble (1926), mostly based on morphology of the pectoral girdle, clearly demonstrated that the genus Ophryophryne is a member of Pelobatidae (at the time including the subfamily Megophryinae Bonaparte, 1850), and assumed its close affinities to Megophrys. However, due to widespread misinterpretation of Boulengers' original statement, the genus Ophryophryne was nevertheless incorrectly listed as a member of Bufonidae in several classical works on batrachians (Bourret 1937, 1942, Gorham 1974, Guibé 1950, Nguyen and Ho 1996, Taylor 1962. The systematic status of the genus Ophryophryne among the Megophryidae has been discussed in several works. Liu and Hu (1962) provided the first description of the Ophryophryne tadpole which was remarkably similar to those of Megophrys, which led Dubois (1980) to rank Ophryophryne at the level of subgenus within Megophrys. Soon afterwards, Dubois re-evaluated his proposition and elevated Ophryophryne back to the genus-level status (Dubois 1987). Summarizing available cytological, morphological and ecological evidence, Rao and Yang (1997) proposed to split Megophrys s. lato, regarding Ophryophryne as a separate genus, as well as the former Megophrys subgenera, Megophrys s. stricto, Atympanophrys Tian & Hu, 1983, Brachytarsophrys Tian & Hu, 1983and Xenophrys Günther, 1864. Several studies indicated close affinities of Ophryophryne to the genus Xenophrys (Tian and Hu 1983, Frost 1985, Ye et al. 1993, Rao and Yang 1997, Manthey and Grossmann 1997. Delorme et al. (2006) recognized the tribe Xenophryini, containing two genera Ophryophryne and Xenophrys, and most recent faunal reviews have treated Ophryophryne as a valid genus within Megophryidae (Fei et al. 1999, 2005, Orlov and Ananjeva 2007, Nguyen et al. 2009). Recently, Mahony (2011a) suggested that insufficient evidence was available for the morphological distinction of Xenophrys from Megophrys and suggested to retain the historical usage of Megophrys s. lato for species of both genera pending a taxonomic review of the group, however, he did not discussed the status of Ophryophryne.
Though a comprehensive phylogeny of the genus Ophryophryne is still pending, preliminary molecular data were contradictory, suggesting both as sister-clade relationships of Ophryophryne with respect to a monophyletic group composed of Xenophrys, Megophrys, and Brachytarsophrys (Pyron and Wiens 2011), or providing evidence of the paraphyly of Xenophrys with respect to Ophryophryne (e.g. Wang et al. 2012). A recent phylogenetic study on Megophryinae by Chen et al. (2017) provides new insights on evolutionary relationships within this group, indicating contrasting (though poorly supported) phylogenetic positions of Ophryophryne in their multilocus nuclear-gene based phylogeny and matrilineal mtDNA genealogy. Chen et al. (2017) preliminarily recognized Ophryophryne as one of the five monophyletic genera within Megophryinae (Ophryophryne, Brachytarsophrys, Xenophrys, Atympanophrys and Megophrys). However, Mahony et al. (2017) provides an alternative hypothesis based on extensive morphological studies and a larger nuclear gene dataset. They provided compelling evidence (recent diversification, insufficient morphological or biological distinction of major clades) for the consideration of Megophryinae to be treated as a single genus, Megophrys, with seven sub-clades (including Ophryophryne) being treated as subgenus level taxa. Their phylogenetic analyses provided strong support for the sister taxa relationship of Ophryophryne and a clade corresponding to Panophrys (previously considered a synonym or subclade of Xenophrys, e.g., Delorme et al. 2006, Chen et al. 2017. For a long time after its' description, the genus Ophryophryne was thought to include a single species, O. microstoma, described from "Mau Son" in Tonkin (northern Vietnam). Later, Bourret (1937) described a second species, O. poilani Bourret, 1937, based upon a single, badly preserved specimen from "Dong Tam Ve" in Quang Tri Prov. of Annam (central Vietnam). Almost half a century later a third species, O. pachy-proctus Kou, 1985, was described by Kou (1985) from Mengla County in Yunnan Prov. (southern China). Ohler (2003) revised the available material on the genus and, mostly based on samples collected by M. Smith (in southern Vietnam), and I.S. Darevsky and N.L. Orlov (in central Vietnam), described two more species: O. gerti Ohler, 2003 andO. hansi Ohler, 2003, respectively (the type series of O. gerti included specimens from the Langbian Plateau in southern Vietnam, and Laos). Ohler (2003) revised diagnostic characters and provided a key for the genus; she also examined the type specimen of O. poilani and considered it to be a junior synonym of O. microstoma (opinion not shared by Stuart et al. 2010). The last major progress on the taxonomy of Ophryophryne was made by Stuart et al. (2006), who reported the genus for Cambodia and described one more species, O. synoria Stuart, Sok & Neang, 2006 from Mondolkiri Prov. in eastern hilly Cambodia, near the Vietnamese border. A recent review of southern Vietnamese herpetofauna by Vassilieva et al. (2016) based on morphological evidence recorded O. synoria for lowland areas of Dong Nai and Binh Phuoc provinces.
The Langbian (or Da Lat) Plateau forms the southernmost edge of the Annamite Mountains, or Truong Son Range, a mountain chain spanning the breadth of Indochina, including parts of Vietnam, Laos and Cambodia. To date, following the review by Ohler (2003), only Ophryophryne gerti has been recorded from the high elevations (above 1000 m a.s.l.) of the Langbian Plateau (Stuart et al. 2010, Nguyen et al. 2009. However, our recent fieldwork in this area from 2007 until 2016 revealed the presence of at least three morphologically distinct species, often recorded in syntopy (Orlov et al. 2008, Poyarkov andVassilieva 2011). Further investigation of partial 12S rRNA-16S rRNA mtDNA gene sequences, as well as the study of advertisement calls from the Langbian Ophryophryne populations, herein confirm their specific status and reveals that one of the lineages represents a previously undescribed species. We also provide the first preliminary mtDNA phylogeny for the genus Ophryophryne and discuss the biogeography of the genus in Indochina in light of our new data.
London, United Kingdom (NHMUK, formerly BMNH, though the latter acronym is retained for specimen numbers for comparability with older literature), Field Museum of Natural History, Chicago, USA (FMNH), and Yunnan University, Faculty of Biology, Kunming, China (YU).
Morphology. Specimens were photographed in life, and tissue samples for genetic analyses were taken prior to preservation, and stored in 96% ethanol. We recorded morphological data from specimens fixed and stored in 75% ethanol.
Measurements to the nearest 0.1 mm were taken using either a digital caliper, or a dissecting microscope; morphometrics of adult frogs and character terminology follows Mahony (2011a) and Mahony et al. (2013). Morphometric abbreviations are as follows: Additionally, for the description of the type series we measured the distance between anterior orbital borders (IFE); distance between posterior orbital borders (IBE); first toe length (TIL); second toe length (TIIL); third toe length (TIIIL); fourth toe length (TIVL); fifth toe length (TVL). All measurements were taken on the right side of the specimen, except when a character was damaged, in which case the measurement was taken on the left side. Entire skin surface of all specimens were examined by microscope for the presence of dermal microstructures. Sex was determined by direct observation of calling in life and/or gonadal inspection by dissection.
Morphological description of larval stages included the following 15 measurements: total length (TOL); body length (BL); tail length (TAL); maximum body width (BW); maximum body height (BH); maximum tail height (TH); snout to vent length (SVL); snout to spiracle distance (SSp); maximum upper tail fin height (UF); maximum lower tail fin height (LF); internarial distance (IN); interpupilar distance (IP); rostro-narial distance (RN); naro-pupilar distance (NP); eye diameter (ED). The oral disk width and the labial tooth row formula were not recorded since in Ophryophryne the oral disk is modified to an extensive funnel which is closed when fixed in preservative, and oral disk structures typical for most other anurans are absent. Tadpoles were staged after Gosner (1960); morphometrics followed Grosjean (2001Grosjean ( , 2003 and Poyarkov et al. (2015b).
All statistical analyses were performed with Statistica 6.0 (StatSoft, Inc. 2001). Morphometric characters were used for univariate analyses, corrected by body size. Sexes were separated for subsequent comparisons among the samples. One-way ANO-VA and Duncan's post hoc test were used for morphometric comparisons. Multivariate statistical analyses were conducted for examination of overall morphological variation among studied populations. If some characters showed high correlation between each other, all but one of them were omitted in order to exclude the overweighting effect of these characters on the analyses. After metric values were log e-transformed, a principal component analysis (PCA) was conducted. The additional specimens of the undescribed Ophryophryne species, measured by LTN, were not included in the PCA to avoid potential error due to inter-observer variation of measurement techniques. A significance level of 95% was used in all statistical tests.
Comparative morphological data were obtained from museum specimens of Ophryophryne and (when available) photographs of these specimens in life (see Appendix 1). Data on morphology and taxonomy of Ophryophryne are also available from the following literature: O. microstoma (Bourret 1942, Liu and Hu 1962, Yang 1991, Ye et al. 1993, Fei et al. 1999, Zhang and Wen 2000, Ohler 2003, Bain et al. 2007, Yang 2008, O. pachyproctus (Kou 1985, Yang 1991, Ye et al. 1993, Fei et al. 1999, O. poilani (Bourret 1937, 1942, Ohler 2003. However, due to the considerable undiagnosed diversity within Megophryidae (Chen et al. 2017, Mahony et al. 2017, where available, we relied on the examination of type specimens, topotypic material and/or original species descriptions. Only characters verified on all specimens in the type series and referred specimens are used to represent the new species in the comparison and diagnosis sections. Specimens of O. cf. poilani listed in Appendix 1 were not used in the comparison of the undescribed species with O. poilani. DNA isolation and sequencing. For molecular analysis, total genomic DNA was extracted from ethanol-preserved muscle or liver tissues using either standard phenolchloroform extraction procedures (Hillis et al. 1996) followed with isopropanol precipitation (at Moscow State University: hereafter MSU), or a Qiagen DNeasy® Blood & Tissue Kit primarily following manufacturers' instructions, with the exception of an extended (10 minute) soaking step prior to the elution of extracted DNA from the column, and additional final elution step using 40 μl H 2 O (at University College Dub-lin: hereafter UCD). The isolated total genomic DNA was visualized using agarose gel electrophoresis in the presence of ethidium bromide (MSU), or SafeView TM (Applied Biological Materials Inc. -at UCD). The concentration of total DNA was measured using NanoDrop 2000 (MSU) or NanoDrop 1000 (Thermo Scientific) (UCD), and consequently either adjusted to ca. 100 ng DNA/μl (MSU), or 10 ng DNA/μl (UCD).
We amplified sequences of a continuous fragment including partial sequences of 12S rRNA and 16S rRNA genes and complete t-val gene sequence, to obtain a fragment of up to 2077 bp (base pairs) of mtDNA. For some adult specimens and larvae a partial ca. 460-500 bp fragment of the 16S rRNA gene was sequenced for molecular identification purposes. 16S rRNA is a molecular marker widely applied for biodiversity surveys in amphibians (Vences et al. 2005a, 2005b, Vieites et al. 2009), and has proven to be particularly useful in studies of megophryid diversity (Matsui et al. 2010, Ohler et al. 2011, Stuart et al. 2011, Hamidy et al. 2012, Rowley et al. 2010a, 2011a, Jiang et al. 2013, Poyarkov et al. 2015a and references therein). Amplification was performed in 25 μl reactions using either ca. 50 ng genomic DNA, 10 nmol of each primer, 15 nmol of each dNTP, 50 nmol additional MgCl 2 , Taq PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.1 mM MgCl 2 and 0.01% gelatine) and 1 U of Taq DNA polymerase (MSU), or 2.0 μl of genomic DNA (10 ng/μl), 2.5 μl Sigma 10x PCR buffer (excluding MgCL 2 ), 0.5 μl MgCL 2 , 0.5 μl dNTP mix, 0.5 μl forward and reverse primer (10 ng/μl), 0.2 μl Platinum® Taq DNA Polymerase (Invitrogen) and 18.3 μl PCR grade H 2 O (UCD). Primers used in PCR and sequencing were as follows: forward primers: 12SAL (AAACTGGGATTAGATACCCCACTAT; Zhang et al. 2008 Wilkinson et al. 2002). Tadpoles were assigned to species based on short 16S rRNA sequences obtained using the primer pair 16L-1 (see above) and 16H-1 (CTC-CGGTCTGAACTCAGATCACGTAGG; Hedges 1994). Two Touch-Down (TD) PCR reaction protocols (Murphy and O'Brien 2007) were used: TD 63-57 for 12SA and 16SBr primers and TD 55 for all other primer pairs. Slight differences in reaction protocol were used between MSU, and UCD reactions (in parentheses). TD 55 included an initial denaturation step of 5 (2) min. at 94°C and followed with 10 cycles of denaturation for 30 (45) sec. at 96°C, primer annealing for 30 (40) sec. with annealing temperature decreasing by 1°C per cycle from 65°C to 55°C and extension step for 1 min. at 72°C, followed with 35 cycles of 30 (45) sec. at 96°C, 30 (40) sec. at 55°C and 4 (1) min. at 60°C (72°C), with the final extension step for 10 min. at 72°C. TD 63-57 consisted of 2 min. at 95°C, 6 cycles of 45 sec. at 95°C, 40 sec. at 63°C with a reduction of 1°C each cycle, 1 min. at 72°C, followed by 35 cycles of 45 sec. at 95°C, 40 sec. at 57°C and 1 min at 72°C, and a final step of 10 min. at 72°C. PCR products were loaded onto 1% agarose gels, stained with either GelStar gel stain (Cambrex: at MSU) or SafeView TM (at UCD), and visualized in a Dark reader transilluminator (Clare Chemical). If distinct bands were produced, PCR products were purified either using 2 μl, from a 1:4 dilution of ExoSapIt (Amersham), per 5 μl of PCR product prior to cycle sequencing (MSU), or using TSAP (Promega) following manufacturers' instructions (UCD). At MSU, a 10 μl sequencing reaction included 2 μL of template, 2.5 μl of sequencing buffer, 0.8 μl of 10 pmol primer, 0.4 μl of BigDye Terminator version 3.1 Sequencing Standard (Applied Biosystems) and 4.2 μl of water. The cyclesequencing reaction was 35 cycles of 10 sec. at 96°C, 10 sec. at 50°C and 4 min. at 60°C. Cycle sequencing products were purified by ethanol precipitation. Sequence data collection and visualization were performed on an ABI 3730xl automated sequencer (Applied Biosystems). At UCD, purified PCR products were Sanger sequenced in both directions by Macrogen (Europe). The forward and reverse sequences were checked visually either in Chromas Pro software (Technelysium Pty Ltd., Tewntin, Australia: at MSU) and a consensus sequence was compiled with BioEdit 5.0.9 (Hall 1999: at MSU), or using CodonCodeAligner 3.7.1 (CodonCode Corporation, Dedham, Massachusetts: at UCD). Sequences were submitted to a BLAST search in GenBank to confirm that the intended sequences had been amplified. The obtained sequences are deposited in GenBank under the accession numbers KY425352-KY425411 and KY515232-KY515233 (see Table 1).
Phylogenetic analyses. Sequences coding for the 12S rRNA-16S rRNA mtDNA genes of 66 megophryid specimens: 53 Ophryophryne, representing all currently recognized species, and outgroup sequences of two Brachytarsophrys species, eight Megophrys s. lato species (including seven Xenophrys and one Megophrys s. stricto species), two Leptobrachium Tschudi, 1838, and one Leptolalax Dubois, 1980 species (Table 1), were included in the final alignment and subjected to phylogenetic analyses. Nucleotide sequences were initially aligned using ClustalX 1.81 (Thompson et al. 1997) with default parameters, and then optimized manually in BioEdit 7.0.5.2 (Hall 1999) and MEGA 6.0 (Tamura et al. 2013). Mean uncorrected genetic distances (p-distances) between sequences were determined with MEGA 6.0 (Tamura et al. 2013); the existence of "barcode gap" was estimated using the online version of ABGD (Puillandre et al. 2012). MODELTEST v.3.06 (Posada and Crandall 1998) was used to estimate the optimal evolutionary models to be used for the data set analysis. The best-fitting model as suggested by the Akaike Information Criterion (AIC) was the general time-reversible (GTR) model of DNA evolution with a gamma shape parameter (G).
Maximum Likelihood (ML) analysis was conducted using Treefinder (Jobb et al. 2004). Transitions and transversions were equally weighted, and gaps were treated as missing data. Confidence in tree topology was tested by non-parametric bootstrap analysis (Felsenstein 1985) with 1000 replicates. Bayesian inference (BI) was conducted using MrBayes 3.1.2 Ronquist 2001, Ronquist andHuelsenbeck 2003); Metropolis-coupled Markov chain Monte Carlo (MCMCMC) analyses were run with one cold chain and three heated chains for four million generations and sampled every 1,000 generations. Five independent MCMCMC runs were performed and 1,000 trees were discarded as burn-in. Confidence in tree topology was assessed by posterior probability (Huelsenbeck and Ronquist 2001). We a priori regarded tree nodes with bootstrap (BS) values 75% or greater and posterior probabilities (BPP) values over 0.95 as sufficiently resolved, BS values between 75% and 50% (BPP between 0.95 and 0.90 ) were regarded as tendencies, and BS values below 50% (BPP below 0.90) were considered to be unresolved (Huelsenbeck and Hillis 1993).
All recordings were standardized by Avisoft SASLab Pro software v. 5.2.05 in mono format with sampling rate at 48 kHz and 16-bit precision, and low-frequency noise was reduced using the low-pass filter (up to 1000 Hz). Calls were analyzed using Avisoft SASLab Pro software v. 5.2.05; all parameters were measured using the reticule and standard cursors in the spectrogram window of Avisoft. Spectrograms for analyses were created using the Hamming window, FFT-length 1024 points, frame 100%, and overlap 87.5%. Figure spectrograms were created using the Hamming window, FFTlength 512 points, frame 100%, and overlap 75%. In total, we measured 1797 calls of the new Ophryophryne sp., 533 calls of O. gerti and 200 calls of O. synoria.
Four temporal parameters were measured: the duration of each call, the interval between successive calls within each series, the duration of series, the interval between successive series, and five frequency parameters: the initial and final fundamental frequency, the minimum and maximum of fundamental frequency and the frequency of maximum amplitude (also "F peak"). Then we calculated the frequency range as the difference between the maximum and minimum of fundamental frequencies and the call repetition rate per recording/series (calls/s) for each recording/series as a ratio of number of all calls within the recording/series (excluding series consisting of just one call) to recording/series duration. All numerical parameters are given as mean ± SE, the minimum and maximum values are given in parentheses (min-max).
To compare acoustic characteristics between three species of Ophryophryne we applied one-way ANOVA with Tukey post hoc for the values of the parameters for which distributions did not differ from normality (p > 0.05, Kolmogorov-Smirnov test). Otherwise we used nonparametric Kruskal-Wallis ANOVA with Mann-Whitney U post hoc test.
The records of advertisement calls were deposited at the Fonoteca Zoologica and are available at the website http://www.fonozoo.com (under the accession numbers 9954-9964).

Sequence data
The final alignment of the studied 12S rRNA-16S rRNA mtDNA gene fragment consisted of 2077 sites: 1439 sites were conserved and 567 sites were variable, of which 465 were found to be parsimony-informative. The transition-transversion bias (R) was estimated as 2.06. Nucleotide frequencies were A = 32.8%, T = 27.6%, C = 21.6%, and G = 17.9% (all data given for ingroup only).

Phylogenetic relationships and geographic distribution of mtDNA haplotypes
We achieved high resolution of phylogenetic relationships among taxa within Ophryophryne, with all major nodes fully resolved (BPP = 1.0; BS = 100%: Fig. 2). Monophyly of species-level groups and species complexes in Ophryophryne were also significantly supported (BS > 90%; BPP ≥ 0.95). However, phylogenetic relationships between the taxa of outgroup Megophryinae are poorly resolved with major nodes in the tree having low or insignificant levels of support (BPP < 0.95; BS < 75%). Bayesian and Maximum Likelihood analyses resulted in essentially similar topologies (see Fig. 2) slightly differing from each other only in associations for several poorly supported outgroup nodes.
Our analyses (Fig. 2) inferred the following set of phylogenetic relationships among studied megophryid taxa:  Our data confirm the monopyly of Megophryinae with respect to outgroup taxa (Leptobrachium and Leptolalax) (1.0/100; hereafter node support values are given for BPP/BS respectively). Within Megophryinae, the sample of Megophrys nasuta (Schlegel, 1858), representing the genus Megophrys s. stricto, forms the most basal split; this lineage is recovered as a sister group with respect to all other Megophryinae. Phylogenetic relationships among other genera of Megophryinae remain essentially unresolved; while monophyly of Brachytarsophrys received high support (0.99/100), species assigned to Xenophrys form two weakly supported groups, paraphyletic with respect to Ophryophryne and Brachytarsophrys.
Monophyly of the genus Ophryophryne is strongly supported by all analyses (1.0/100). General topology of the Bayesian tree suggests that the genus Ophryophryne is divided into two major groups: the first group joins taxa from southern China, northern and central Indochina (Group I, see  (Fig. 2). In the present paper we treat this lineage as the species level clade representing O. gerti s. stricto (see discussion below).
The second species level clade comprises large-sized Ophryophryne from comparatively lowland populations in the western foothills of the Langbian Plateau ( Fig. 1, Locs 1-4, < 1000 m a.s.l.) and large-sized Ophryophryne from medium elevations in the northern parts of the plateau (Fig. 1, Locs 6 and 11; 1000-1500 m a.s.l.). Two reciprocally monophyletic (1.0/100) subclades are revealed in this clade (Fig. 2). Subclade B joins montane populations from 1000-1500 m a.s.l. (Fig. 1, Locs 4, 6 and 11) and a recently discovered lowland (200 m a.s.l.) population from Cat Tien N.P. in Dong Nai Prov. Subclade C joins two populations from the westernmost edge of the Langbian Plateau, which include the holotype of O. synoria from Mondolkiri Prov. in eastern Cambodia ( Fig. 1, Loc. 1; 500 m a.s.l.) and a population from Bu Gia Map N.P. in the adjacent Vietnamese province of Binh Phuoc (Fig. 1, Loc. 2; 400 m a.s.l.). These localities are close to each other and geographically belong to one hilly region on the western edge of the Langbian Plateau. Based on phylogenetic and morphological data we herein regard both subclades B and C as the species level clade representing O. synoria.
The third species level clade forms a sister clade with respect to a clade comprised of O. gerti s. stricto and O. synoria (Fig. 2). It joins small-sized Ophryophryne specimens, all collected from both high elevations ( > 1750 m a.s.l.) in the northern and eastern parts of the Langbian Plateau (see Fig. 1: Bidoup and Hon Giao Mts., Lam Dong Prov., Locs 6-7; and Chu Pan Fan and Chu Yang Sin Mts., Dak Lak Prov., Locs 10-11), and from lower elevations (700-1510 m a.s.l.) on the summits of three mountains representing the easternmost outcrops of the Langbian Plateau (see Fig. 1: Hon Ba Mt., Khanh Hoa Prov., Loc. 8; Nui Chua Mt., Ninh Thuan Prov., Loc. 9; Tay Hoa, Phu Yen Prov., Loc. 12). Among these populations, samples from the summit of Nui Chua Mt. (Fig. 1, Loc. 9) form Subclade D (Fig. 2), forming a sister clade with respect to all other populations ( Fig. 2, Subclade E; monophyly support 0.95/98). This clade of small-sized Ophryophryne from the northern and eastern parts of the Langbian Plateau currently cannot be assigned to any of the recognized species and represents a new species described herein.

Intra-and interspecific differentiation of mtDNA haplotypes
The observed interspecific sequence divergence within the genus Ophryophryne varied from p = 4.1% to p = 13.0% (Table 2). The values of uncorrected genetic p-distances in ingroup and outgroup comparisons slightly overlapped: sequence divergence between Ophryophryne and outgroup taxa varied from p = 8.8% to p = 24.7%. The minimal interspecific p-distance between recognized nominal species in our analysis was found between the sister species O. gerti and O. synoria (p = 4.1%-5.0%). The maximum pdistance for Ophryophryne was observed between O. synoria and O. hansi (p = 12.6%-13.0%) (see Table 2). The ABGD analysis revealed the existence of a "barcoding gap" at genetic distance value p = 4.0% in the 16S rRNA gene.
Intraspecific distances within Ophryophryne species in our analysis varied from p = 0.5% (in O. gerti), to p = 3.3% among two samples of O. cf. poilani from Quang Nam and Thua Thien-Hue provinces of Vietnam, and to p = 3.7% among two samples of O. microstoma from China and Vietnam respectively. The latter two values are higher than usual intraspecific distances in the 16S rRNA gene in Anura (Vences et al. 2005a, 2005b, Vieites et al. 2009); we recognize that identification of these lineages as conspecific is preliminary, based on morphology and topology of the mtDNA tree. Further studies are needed to clarify their taxonomic status. We also found significant genetic differentiation between intraspecific lineages in two of the three Ophryophryne species inhabiting the Langbian Plateau. Sequence divergence between Subclades B and C of O. synoria is p = 2.6%, while the differentiation between the Nui Chua population (Subclade D) and all other populations of the new species (Subclade E) was even greater at p = 3.1%.
The newly discovered lineage of Ophryophryne from highlands of the northern and eastern parts of the Langbian Plateau was found to have the lowest genetic distance with respect to O. gerti (p = 8.2%-9.1%). This value is much higher than the minimum genetic distances observed in intraspecific comparisons between species of Ophryophryne included in this study (Table 2).

Morphological differentiation
Among the three species examined, mean SVL varied significantly, ranging from 26.9 to 53.7 mm in males and from 35.1 to 70.7 mm in females (Table 3). For SVL, post hoc analyses of one-way ANOVA revealed that males were significantly smaller than females in all three species of Langbian Ophryophryne (one-way ANOVA, p < 0.05; Duncan test, p < 0.05). Body size variation among adult males and females of Langbian Ophryophryne is shown in Figure 3. All three species are clearly different in body size, with O. synoria being the largest, and the undescribed Ophryophryne species being the smallest species known for the genus (male SVL values overlap with values for O. pachyproctus, N.A. Poyarkov, pers. observ.). Ophryophryne gerti occupies an intermediate position between these two species, with SVL values of males (31.7-42.2 mm) slightly overlapping both with those for O. synoria (38.2-53.7 mm) and the undescribed Ophryophryne species (26.9-33.9 mm).
The results of the multivariate PCA-analysis of the morphometric data are shown in Fig. 4 (data given for males only). The discriminative power of PCA factors derived from analysis of morphometric characters is shown in Appendix 2. For males, F1 explained 77.80% of the variability and F2 explained 5.99%. The two-dimensional plots of both the first two principal components (Factor 1 and Factor 2; Fig. 4A) and the first vs. the third principal components (Factor 1 and Factor 3; Fig. 4B) for males completely discriminated the following three morpho-groups: (I) O. gerti, (II) O. synoria, and (III) the undescribed Ophryophryne species. Our multivariate analysis included meristic data for the holotypes of two Ophryophryne species: O. synoria (FMNH 262779, male) and O. gerti (BMNH 1921.4.1.324, male). Both holotypes were assigned correctly to the respective groups, representing O. synoria and O. gerti, in full concordance with the results of molecular analyses (Fig. 4).
In summary, both in body size and other meristic characters, all three examined lineages of Langbian Ophryophryne form clearly separated morphological groups, also supported by multivariate statistical analysis. The small-sized population of the undescribed Ophryophryne species appears to be morphologically distinct from sympatric O. gerti and O. synoria, as well as from other congeners (see Comparisons for details).
Advertisement calls of all studied species were similarly uttered in series (Fig. 5). The call repetition rate/recording/series were one of the most significant differentiating parameters between the three species (Table 4). Parameters values varied both between and within recordings for each of the three species. Though some parameters values overlapped between species, the means of most of the parameters differed significantly. The frequency of maximum amplitude always coincided with the fundamental frequency and greatly varied within recordings: from 4030 to 4920 Hz for the undescribed Ophryophryne species, from 4450 to 5100 Hz for O. gerti, and from 3600 to 3890 Hz for O. synoria. The values of the maximum amplitude frequency in O. synoria were the lowest and least variable among the three species. The minimum fundamental frequency always coincided with the initial fundamental frequency whereas the maximum fundamental frequency either could coincide with the final fundamental frequency, or was close to it. Thus, the frequency modulation was expressed either in lift of fundamental frequency during the whole call, or in an unsymmetrical arch with the peak shifted to the end of the frequency band. The form of frequency modulation varied between these two forms for each species but arch-formed calls appeared most of time in O. synoria. The frequency range expressing depth of frequency modulation also varied within each species' calls: from 40 to 840 Hz for the undescribed Ophryophryne species, from 370 to 890 Hz for O. gerti and from 230 to 650 Hz for O. synoria (Table 4).
Number of harmonics varied between/within recordings but this characteristic mostly depended on recording quality (e.g., sensitivity of recording equipment, distance from vocalizing animal, signal volume and background noise). Calls from the highest quality recordings (of the undescribed Ophryophryne species) contained two harmonics but a major portion of other calls contained only one harmonic.

Taxonomic implications
Our study, based on three lines of evidence -phylogenetic analysis and distribution of mtDNA haplotypes (Figs 1, 2), multivariate statistical analysis of 23 standard morphometric traits (Fig. 4), and acoustic analysis of advertisement calls (Fig. 5), strongly indicate the presence of three independent and distinct evolutionary lineages of Ophryophryne on      Smith, presented to BMNH in 1921;BMNH 1972.1524 (see 'Remarks') adult male, "Huey Sapan, Pak Maat (precise location not found), Mekong, Laos", collector M.A. Smith, accessioned in the BMNH from Smith's private collection in 1972, collection date unknown (Ohler 2003; NHMUK specimen catalogue).
Remarks. Ohler (2003) provides the paratype number BMNH 1972.15.2.4 for the Laos paratype specimen, however, the number on the specimen tag and NHMUK specimen catalogue reads BMNH 1972.1524 (Mahony 2011b). Our present data suggests that O. gerti is not found beyond the limits of the Langbian Plateau, thus we are confident that the paratype BMNH 1972.1524 does not represent this biological taxon. Ohler (2003: andby implication Stuart et al. 2006) preliminarily identified two specimens from Ankhe Dist. in northern Gia Lai Prov. as O. gerti. This locality is disconnected from the Langbian highlands by a wide lowland area, indicating that these specimens are biogeographically isolated from O. gerti s. stricto. Further work is necessary to ascertain the taxanomic status of these specimens. Orlov et al. (2002), without providing data on examined specimens, considered that the distribution of O. microstoma extended south in Vietnam as far as the Lam Dong and Dak Lak provinces. Our results suggest that these southern Vietnamese populations most likely represented the superficially similar O. gerti, O. synoria, or possibly the new taxon described below. Stuart (2005) reports O. gerti from Champasak Prov. in southern Laos, based on a single specimen (FMNH 258564: not examined here). This locality is biogeographically not connected to the known range of this species, as redefined here, thus the taxonomic status of this specimen requires further confirmation. Bain et al. (2007) identifies two specimens AMNH A-169287 (Thua Thien-Hue Prov., Vietnam) and AMNH A-163668 (Quang Nam Prov., Vietnam) as O. gerti. We re-examined these specimens and regard them to be morphologically more similar to O. poilani. Furthemore, both specimens were included in our molecular analysis and found to be distantly related to O. gerti s. stricto (Fig. 2). Stuart et al. (2010) provides the SVL range (SVL 37.5-42.5 mm, mean ± SD 40.4 ± 1.6, N = 8) for 'topotype' female specimens of O. "gerti", however these specimens are smaller than females of O. gerti provided herein (SVL 43.1-47.4 mm, mean ± SD 45.07 ± 2.16, N = 3), and larger than the new taxon described below (SVL 35.1-36.5 mm, mean ± SD 35.6 ± 0.5, N = 6) (see Table 3 for details). The taxonomic status of these specimens remains unknown. Vernacular name. English: "Gerti's Mountain Toad"; Vietnamese: "Cóc Núi Got" (Nguyen et al. 2009), "Cóc Núi Goti" .
Redescription of the holotype. Mature male (SVL 35.7 mm), habitus slender (Fig. 7A, B). Specimen in good state of preservation; two incisions are present on trunk, one longitudinally orientated on mid-abdomen, another longitudinally oriented on upper flank on right side; liver and testes observable through incisions, testes enlarged; jaw is dislocated on right allowing visual access to buccal cavity.
Skin texture and skin glands in preservation. Skin of dorsal and lateral surfaces of head, body and limbs smooth with numerous small tubercles finely and relatively evenly scattered on dorsal surfaces of trunk, head and limbs (Fig. 7A); small tubercles present on temporal region, tympanum smooth with borders weakly raised; tubercles arranged in distinct transverse ridges on dorsal surfaces of forearms, shanks and thighs; numerous large tubercles on flanks irregularly scattered from axilla to groin, intermixed with smaller tubercles; central portion of outer edge of upper eyelids slightly thickened, with a single short tubercular spine (Fig. 7C), transverse fold on posterior edges of upper eyelids absent; well-developed glandular supratympanic folds, narrow anteriorly, considerably widening posteriorly, from posterior corner of orbits, extending along upper margin of tympanum, terminating above forelimb insertions (Fig. 7C); dorsolateral glandular ridge well-developed, extending from posterior to supratympanic ridges to ca. 75% of trunk length, on each side; a moderately well-developed " >-< " shaped glandular parietoscapular-sacral ridge present on dorsum (see Fig. 7A); two small tubercles present above vent; gular region, chest, and ventral surfaces of limbs smooth to weakly shagreened, abdomen weakly granular; two nuptial pads per limb, one large on dorsal surface of F1 from base of metacarpal to near distal joint, another small pad on inner dorsal surface of F2 on metacarpal; pectoral glands round, weakly raised, positioned level with axilla; femoral glands slightly raised, average size, on posterior surface of thighs, situated slightly closer to knee than to cloaca; numerous small white asperities present on posterior half of dorsum, sparse anteriorly, increasing in density posteriorly to above cloaca, absent from all remaining surfaces.
Color of holotype in preservative (Figure 7). Dorsal surfaces of head, body, forearms and hindlimbs mid to light brown, slightly lighter on flanks and dorsal surface of upper arms; a distinct darker brown "V"-shaped marking on dorsal surface of head; no distinct "X"-shaped or hourglass marking on mid dorsum; most flank tubercles are bordered below by a small dark brown spot anteriorly, increasing in size posteriorly towards groin; a broad brown stripe extends around lateral surfaces of snout, from anterior borders of orbits between canthus rostralis and the level of lower border of nostrils; two broad darker brown vertical stripes below orbits, one at level of anterior orbital border, and a second extends from central lower border of orbits to edge of jaw, a faint darker brown stripe extends from posterior border of orbits to cover tympanum; color of supratympanic folds same as surrounding sufaces, but lower border dark brown; edge of lower eyelid dark brown; dorsal surfaces of forearms each with two dark brown transverse blotches, and thighs and shanks with faint darker brown transverse stripes; ventral surfaces of throat, chest and anterior half of abdomen, and ventral surface of hands primarily plain light brown, fading to a mottled brownish beige with small dark brown blotches on posterior half of abdomen, and ventral surfaces of forelimbs, thighs and shanks, and dorsal surfaces of tarsi and feet; ventral surface of tarsi and feet dark brown fading distally on toes to a mid-brown; area surrounding cloaca dark brown, fading distally on lateral surfaces of thighs.
Distribution. Ophryophryne gerti is herein confirmed from three localities on the Langbian Plateau in southern Vietnam, between 700-2000 m a.s.l. (Fig. 1) (Orlov et al. 2008; this study). Additional localities reported in literature require confirmation pending further study of voucher material (see Remarks section above).  1, Fig. 1). Subsequently, during field surveys in 2009-2011, the species was reported in southern Vietnam from Bu Gia Map N.P., Binh Phuoc Prov. (Loc. 2, Fig. 1) and Cat Tien N.P. in Dong Nai Prov. (Loc. 3, Fig. 1) based on morphological evidence (Vassilieva et al. 2016). Herein, we confirm the identity of these specimens based on morphological and molecular genetic evidence, and further expand its distribution in southern Vietnam to include medium and low elevation localities in the central and western parts of the Langbian Plateau (Dak Lak, Lam Dong, Dong Nai and Binh Phuoc provinces between 200 and 1500 m a.s.l.; its presence in Dak Nong Prov. is anticipated). We also identify two mtDNA lineages within O. synoria with a moderate level of sequence divergence (p = 2.6%: Table 2 Fig. 1) whereas Subclade C is only found in Mondolkiri Prov. of Cambodia and adjacent Binh Phuoc Prov. of Vietnam (Fig. 1, Locs 1-2) and corresponds to O. synoria s. stricto.

New records and range extension for
Variation. The studied specimens of O. synoria showed substantial variation in morphological characters, including SVL (Fig. 3) and other morphometric characters (Fig. 4), coloration, and degree of development of palpebral projection (Fig. 6D, E and F). Overall morphology, coloration, and skin glands of the newly discovered populations of O. synoria are in general agreement with the description of the holotype by Stuart et al. (2006). Young specimens from Bidoup-Nui Ba N.P. in life often have reddish or orange coloration of thighs and groin, which was not observed in the type specimens from Cambodia (Stuart et al. 2006), nor in the Bu Gia Map population. The degree of development of short dorsolateral glandular folds varied among specimens, but they were always distinct (Fig. 6D, E and F). The holotype and the Bu Gia Map population (Subclade C) have the finger length formula F1 = F2 < F4 < F3, while in populations from Bidoup-Nui Ba N.P. and Chu Yang Sin N.P. (Subclade B), the finger length formula is F1 = F4 < F2 < F3. Subclade B populations also tend to have a slightly larger tympanum (TYD/ED 68.4%-80.1%; TYE/TD 73.3%-80.6%) than the nominative O. synoria (TYD/ED 62.0%-71.9%; TYE/TD 83.9%-103.3%). Though the taxonomic status of the two revealed lineages is not completely clear, herein we tentatively regard them as deep intraspecific mtDNA lineages based on observed genetic differentiation and overall morphological similarity. Vernacular name. English: "O'Reang Mountain Toad" (this paper); "O'Reang horned frog" (Vassilieva et al. 2016); Vietnamese: "Cóc Núi O-Reng" (Vassilieva et al. 2016).

Description of a new species of Ophryophryne
Based upon several lines of evidence, including the analyses of diagnostic morphological characters, acoustic analyses of advertisement calls and phylogenetic analyses of mtDNA sequences for the 12S rRNA-16S rRNA genes, the new species of Ophryophryne from mid to high elevations of the western Langbian Plateau represents a highly divergent mtDNA lineage, clearly distinct from all other Ophryophryne species. These results support our hypothesis that this recently discovered lineage of Ophryophryne represents an undescribed species, described below:  (Figs 8 and 9).
Paratypes. ZMMU A-5691 (field numbers ABV-00580; ABV-00581), two juveniles from the north-western slope of Chu Yang Sin Mountain, Chu Yang Sin N.P., Hoa Le Commune, Krong Bong Dist., Dak Lak Prov., Vietnam (12°24'47.70 Etymology. The specific epithet is an adjective (in agreement with the genus name in feminine gender), derived from "elf", the English spelling of "alfus" in Latin, referring to usually forest-dwelling supernatural mythological creatures in Germanic mythology and folklore; the name is given in reference both to the funny appearance and small size of the new species, as well as to the their endangered habitat, restricted to wet evergreen montane forests at high elevations of the Langbian Plateau; such forests are often called "elfin forests".
Recommended vernacular name. The recommended common name in English is "Elfin Mountain Toad". The recommended common name in Vietnamese is "Cóc Núi Tiểu Yêu Tinh".
Diagnosis. The species is allocated to Ophryophryne based on its obvious similarities with its sister taxa, its molecular phylogenetic affinities, and the absence of maxillary teeth considered diagnostic for the genus (previous authors, e.g. Ohler 2003, Delorme et al. 2006and Fei et al. 2009 also indicated a horizontal pupil and the absence of vomerine teeth as diagnostic for Ophryophryne, this is reconsidered by Mahony et al. 2017). Ophryophryne elfina sp. n. is distinguished from its congeners by a combination of the following morphological attributes: (1) small adult body size, male SVL 26.9-33.9 mm (N = 29), female SVL 35.1-36.5 mm (N = 6); (2) snout sharply protruding in profile; (3) tympanum diameter approximately half of eye diameter; tympanum to eye distance approximately 70-90% of tympanum diameter; (4) finger length formula: F1 < F4 ≤ F2 < F3, or F1 ≤ F2 < F4 < F3; toe webbing rudimentary, toe length formula: T1 < T5 < T2 < T3 < T4; (5) short dorsolateral glandular ridge present above shoulder; (6) palpebral projection present as a small single tubercle to moderately developed single projection; (7) dermal cloacal protuberance and dermal flaps above cloacal opening absent; (8) skin of dorsal and lateral surfaces of head, body and limbs shagreened with numerous small tubercles, large warts on the flanks; (9) skin on dorsal and lateral surfaces of body, head and limbs with numerous bright orange-red (in life) asperities; (10) males with a redorange (in life) nuptial pad on F1; (11) dorsal coloration light yellow-brown with dark hourglass-shaped marking on dorsum usually edged with white or beige (in life); (12) posterior suborbital light bar well-defined, usually clearly separated from dark-brown temporal triangular spot, uniformly covering temporal area and tympanum.
The new species is also markedly distinct from all congeners for which comparable sequences are available (16S rRNA mitochondrial gene; uncorrected genetic distance > 8.2%). The advertisement call of the new species consists of whistling notes uttered in series: average 12.84 ± 0.41 calls per series, with an average dominant frequency of 4645.94 ± 4.39 Hz, repetition rate per recording/series 1.18 ± 0.2 calls/s and 3.87 ± 0.07 calls/s, respectively, with average call duration 73 ± 0.23 ms and inter-call interval 207 ± 2.06 ms, also distinguishes the new species from Ophryophryne species for which calls are known, including the two species found in sympatry.
Skin texture and skin glands. Skin of dorsal and lateral surfaces of head, body and limbs shagreened, with numerous small skin asperities present on anterior two thirds of dorsum, sparse posteriorly, increasing in density along dermal ridges, densely covering dorsal and lateral surfaces of head, upper eyelids, and dorsal surfaces of thighs, shanks, upper forelimbs, forearms, hands, feet and digits, and absent from all remaining surfaces. Small tubercles finely and relatively evenly scattered on dorsal surfaces of trunk, head and limbs, including maxilla, mandible, eyelids and dorsal surfaces of head, forelimbs and hindlimbs (Figs 8A and 9); small tubercles present on temporal region, tympanum smooth, tympanic rim distinct but not elevated relative to skin of temporal region; on dorsal surfaces tubercles arranged in distinct longitudinal ridges on upper forelimbs, forearms, shanks and thighs, becoming less distinct on dorsal surfaces of hands, feet and digits (Fig. 9); six large tubercles on left flank and seven large tubercles on right flank irregularly scattered from axilla to groin, intermixed with smaller tubercles; central portion of outer edge of upper eyelid slightly thickened, with a distinct small single tubercle on a thickened ridge (Figs 8C,9); distinct thick glandular supratympanic fold, narrow anteriorly, considerably widening posteriorly, extending from posterior corner of eye gently sloping down towards dorsal margin of tympanum (but not concealing it), where it broadly curves down, terminating above axilla (Figs 8C,9); short dorsolateral glandular ridge present above shoulders, on anterior part of dorsum, its length comparable with eye diameter (Fig. 9); a weak " >-< "-shaped glandular dermal parietoscapular-sacral ridge present on dorsum (Figs 8A,9); transverse fold at head basis absent; dermal projection above cloaca absent; gular region, chest, abdomen and ventral surfaces of limbs smooth to weakly shagreened (especially on the posterior surface of abdomen); nuptial pad present, single, covered with microgranules, covering entire dorsal metacarpal of first finger extending distally to ca. 3/4 basal phalange length; pectoral glands round, flat, of medium size, positioned level with axilla; femoral gland flat, indistinct, on posterior surface of thighs.
Color of holotype in life. Entire dorsum light olive-brown to yellow-brown with large irregular brownish grey spots; dorsal surfaces of head yellowish brown from tip of snout to eyes; small oval-shaped spot with irregular borders on dorsal surface of snout between anterior canthi; a small dark dot on dorsal surface of snout tip; similar single dark dots on anterior parts of upper eyelids; brown "V"-shaped marking on crown between supraorbital horns with apex pointing posteriorly, outlined with thin light-beige edging; round brownish spot at head basis; " >-< "-shaped marking surrounded with dark olive-brown, outlined with thin light-beige edging forming a hourglass-shaped dorsal marking (Fig. 8A); two small roundish brown spots at sacrum (Fig. 8A); supratympanic fold dorsally light yellowish brown, ventrally dark-brown; front and lateral surfaces of snout and lateral canthus rostralis dark reddish brown; lateral surfaces of maxilla dark brown with four distinct orange-brown to yellowish beige bars extending from orbits towards edge of maxilla: smallest anteriormost light band borders nostril ventrally, with two posterior light bands extend from posterior corner of eye towards angle of mouth (Fig. 9); axilla purplish brown; tympanum uniform purplish brown; temporal region uniform dark purplish brown, clearly defined from light beige area on posterior part of maxilla; pupil black, outlined in copper-gold; iris golden dorsally and ventrally, copper-orange at medial part, with tiny dark reticulations spreading from pupil; sclera lemon-yellow; upper surface of limbs yellowish brown with irregular darkbrown spots on forearms and transverse spots forming dark-brown and greyish bands across shanks, thighs and tibio-tarsus (three complete transverse bands on left leg, two complete [both on shank and thigh] and one incomplete [on shank only] transverse bands on right leg: Figs 8 and 9), knee joint dark brown; sides beige-yellow with indistinct greyish white flecking and large black spots with irregular borders: marking location of large warts on each side of body, four smaller spots located dorsally, and two large brown-black spots located ventrally, top of larger flank tubercles brownish cream (Fig. 9); throat brownish to purplish grey with greyish white flecking and irregular dark-brown spots; chest and anterior half of abdomen purplish grey with whitish flecking and grey-brown blotches; posterior half of abdomen lighter greyish pink with irregular dark blotches; lower surface of limbs purplish grey with white and beige flecking; area surrounding vent and posterior surface of thighs dark black-brown with whitish spots, posterior surface of thighs near tibio-tarsal articulation black-brown with sparse whitish dots; dorsal surface of feet and shanks yellowish beige with brown flecking; ventral surface of feet and shanks brown-black. Pectoral and femoral glands creamy white. Nuptial pad, outer metacarpal (palmar) and metatarsal tubercles pink-red to orange-red. Asperities covering dorsal surfaces of body, head, limbs and digits, lateral sides of head and anterior part of chest in life bright orange-red, forming reddish rows and ridges on dorsal surfaces of limbs as well as on edge of upper eyelid, palpebral projection also with orange-red asperities.
Color of holotype in preservative. In preservative coloration faded to light greybrown on dorsum and flanks, with slightly paler limbs and greyish beige to whitish on venter; reddish and orange tints, as well as iris coloration, faded completely; dark markings on dorsum, sides and venter and other features remain without significant change (Fig. 8). Banding on limbs is less pronounced. Chest, abdomen, throat, interior portions of forelimbs and thighs are pale greyish brown; formerly brightly colored dorsal asperities and nuptial pads, palmar and metatarsal tubercles turned transparent or creamy white (Fig. 8C, E).
Variation. Morphometric variation within the type series and other referred specimens of the new species is shown in Table 5. Individuals of the type series are similar in morphology and body proportions (Figs 9, 10). There is a clear and significant difference in body size between males and females (Fig. 3): females (SVL 35.1-36.5 mm, N = 6) are significantly bigger than males (SVL 26.9-33.9 mm, N = 29) (Duncan test, p < 0.05); sexual differences were not significant for other mensural characters possibly due to the small sample size of females. Certain variation is observed in finger lengths: most of the examined specimens have the finger length formula F1 < F4 < F2 < F3 (N = 14), in some specimens, including the holotype, the second and the fourth fingers are of equal length (F1 < F4 = F2 < F3; N = 5), or the fourth finger is longer than the second (F1 < F2 < F4 < F3; N = 5); rarely the second finger is as long as the first finger (F1 = F2 < F4 < F3; N = 2). Specimens vary in the number and size of black spots and blotches on flanks (Fig. 10A,  B). In life, both sexes of the new species have lighter dorsum and belly coloration when nocturnally active. Other in-life variation was observed for throat coloration: throat can be dark brownish with clear dark-grey blotches (Fig. 10C) to almost uniform brown-violet to purple with dark blotches not discernable (Fig. 10D). There is significant variation in dorsal pattern: in some specimens the dorsum looks almost uniform yellowish brown with an indistinct hourglass-shaped figure (Figs 6G, 10A, B, 14 [right]) whereas in other specimens the hourglass-shaped figure is distinct, dark brown and edged with light beige (Figs 6H, 9). There is some variation in the length of palpebral projections, from a small almost indistinct tubercle (Figs 6H, 13C) to a moderately well-developed projection (Figs 6G, 14). Coloration of the lateral surfaces of the head vary, but on all specimens two light suborbital bars are distinct, clearly separated from the uniform dark-brown coloration of the tympanal area. Iris coloration shows insignificant variation: Nui Chua Mt. population appear to have copper-red coloration of the entire iris " (Fig. 14B), somewhat different from the coloration of the holotype (Fig. 9). Recently metamorphosed and juvenile specimens have numerous bright red-orange tubercles (Fig. 10E, F) which are more conspicuous than in adults. Excluding the presence of nuptial pads on males, the new species shows no significant variation in dermal characters among sexes (Fig. 10); in preservative smaller tubercles become flattened and less distinct.
Tadpole description. Tadpoles were allocated to Ophryophryne elfina sp. n. based on the following evidence: (1) morphological features characteristic for megophryine larvae in general; Ophryophryne or Megophrys s. lato in particular (elliptical shaped body with long muscular tail, oral disk forms a dorsally oriented funnel); (2) collected in the stream where calling males of the new species were recorded; (3) species identification confirmed by mtDNA sequences of short 16S rRNA gene fragment (up to 500 bp) (GenBank Acession numbers: KY515232-KY515233, see Table 1).
The following description is based on five tadpoles at stage 25 (ZMMU A-5679, field number NAP-01169). In lateral view (Fig. 11A), body slightly compressed dorsoventrally (BH/BW 83.5 ± 4.09%), especially anteriorly, convex both dorsally and ventrally. Body elliptical in dorsal view (Fig. 11B), with maximum width at middle of body (BW/BL 51.0 ± 4.2%); snout short, rounded, blunt. Eyes of moderate size (ED/BL 9.8 ± 0.3%), not bulging, separated by a distance which equals approximately 1.1 times internarial distance (IP/IN 110.2 ± 6.8%), directed and positioned dorsolaterally, not visible in ventral view; pupils oriented dorsolaterally. Nares tubular, positioned dorsally (near anterior edge of eye), much closer to pupils than to tip of snout; directed laterally. Spiracle sinistral, conical, very short, opening at half of distance from snout tip to vent (SSp/SVL 52.2 ± 3.4%); spiracle attached to body wall for most of its length, extremity is free, positioned at the level of longitudinal axis, oriented dorsoposteriorly, opening varies from rounded to oval. Tail long, more than two times longer than body (TAL/BL 231.3 ± 11.0%), lanceolate; almost equal in height along its length (point of maximum height of tail located just anterior to midlength of tail); tail tip bluntly rounded, without terminal filament; tail musculature strong, gradually tapering, almost reaching tail tip. Tail fins shallow, moderately well developed, not extending onto body: dorsal fin originating almost at body-tail junction, much shorter than lower fin proximally and nearly equal in height to it on middle of tail; dorsal fin slightly higher than ventral fin on distal half of tail (LF/UF 77.1 ± 4.9%); free margin of dorsal fin horizontal and shallow on anterior half of tail; free margin of ventral fin parallel to tail musculature. Vent opening medial, tubular, directed posteriorly, not linked to ventral tail fin. Neither skin glands nor neuromasts visible in preservative, but neuromasts of the lateral line system are distinct in life (Fig. 12A) forming two curved lines running from snout towards orbits and along orbital margins ventrally. Subterminal oral disk with lips expanded vertically forming a dorsally oriented funnel (Fig. 11B); lateral corners of funnel distinct; upper lip notably smaller than lower; lips lack keratodonts, but bear short, low ridges, more densely arranged on upper than on lower labium, arranged in 18-24 (mean = 22) longitudinal rows and from 2-4 (mean = 3) transverse rows on upper labium to 4-6 (mean = 5) transverse rows on lower labium. Marginal papillae absent. Width of expanded funnel comprises over 75% of body length (Fig. 12A) and just 30% when folded in preservation (Fig. 12B). Table 5. Measurements of the Ophryophryne elfina sp. n. specimens. For museum accession numbers relative to specimen collection numbers (NAP, ABV, HB, ROM, CYS) see Appendix 1. For locality details relative to population number see Table 1 and Fig. 1. For abbreviations see Material and methods. All measurements are given in mm. In life tadpoles have dorsal side of body and upper flanks uniform brownish red or brownish orange (Fig. 12A, B). Lower flanks weakly mottled with dark brown, few round blackish spots on tail and dorsum; with orange neuromasts visible on dorsal surface (Fig. 12A). Abdomen light brownish orange, intestine not visible through body wall. Caudal muscles pale; tail fins translucent with a few darker spots (more on upper than on lower fin); dorsally tail with indistinct middorsal orange line (Fig. 12A). Eyes golden with black reticulations. Oral funnel pinkish orange with brownish red papillae (Fig. 12B). In preservative tadpole coloration gets much duller, but the general coloration pattern is still visible after 7 years in ethanol.
Advertisement call characteristics. Refer to the Acoustic differentiation section, Table 4  Position in mtDNA phylogeny and sequence divergence. The new species is reconstructed as a member of the Ophryophryne Group II (Fig. 2), forming a sister group with respect to the clade joining O. gerti and O. synoria (see Fig. 2). Uncorrected genetic p-distances between Ophryophryne elfina sp. n. partial sequences for the 16S rRNA gene and all homologous sequences available on GenBank included in the analysis (see Table 1) varied from 8.2% (with O. gerti s. stricto, clade A) to 10.0% (with O. hansi and O. synoria, clade C) (see Table 2). This degree of pairwise divergence in the 16S rRNA gene is greater than that usually representing differentiation at the species level in anura (Vences et al. 2005a, 2005b, Vieites et al. 2009, Poyarkov et al. 2015a, 2015b. Intraspecific variation in this gene fragment for Ophryophryne elfina sp. n. is significant -maximum sequence divergence between Nui Chua Mt. population  (Fig. 2, clade E) is p = 3.1%. Intraspecific variation in 16S rRNA gene fragment within one geographic population was lower, and ranged from 0.0% to 0.8% of substitutions.
Distribution. Ophryophryne elfina sp. n. is found to be endemic to five provinces in (Lam Dong, Dak Lak, Khanh Hoa, Ninh Thuan and Phu Yen) in the northern and eastern part of the Langbian Plateau and its foothills in southern Vietnam (localities 6-12, Fig. 1). The new species is restricted to wet evergreen montane tropical and elfin forests, receiving high precipitation from the sea. Such wet forests are found only on high elevations in the central parts of the Langbian Plateau (e.g. 1900-2100 m a.s.l. on Bidoup Mt., Lam Dong Prov., Fig. 1, Loc. 6) or peripheral mountains remote from the sea (e.g. 1900-2300 m a.s.l. on Chu Pan Fan and Chu Yang Sin Mts., Dak Lak Prov., Fig. 1, Locs 10 and 11), but on the eastern foothills of the plateau which receive more precipitation, the new species is found at lower elevation (from 950 to 1510 m a.s.l. on Hon Ba Mt., Khanh Hoa Prov., Fig. 1, Loc. 8; 780 m a.s.l. on Nui Chua Mt., Ninh Thuan Prov., Fig. 1, Loc. 9; and 700 m in Phu Yen Prov., Fig. 1, Loc. 12).
Ecology. All specimens were collected at night after heavy rains along montane cascade rocky streams, along small waterfalls, or intermittent rocky brooks; or found during the day time under tree-logs and within leaf litter in the limited fragments of primary montane wet polydominant evergreen tropical forests, with a high abundance of large rocks and fallen trees covered with a thick layer of mosses. This including high montane forests that are composed of the specific floral community known as "elfin" forests, with miniature trees (up to 10 m tall). These areas always have high precipitation and have much milder climate than other tropical forests in southern Vietnam: active breeding of the new species was recorded in February with temperatures of ca. 11-12°C.
Reproductively active males were found while calling along streams, usually sitting on leaves of ferns or on the stone banks, rarely on rocks or large stones (see Fig. 14B, C). Some specimens were collected hiding amongst fern stems and were difficult to locate. Females were found hiding under tree logs or in the forest litter.
The ovaries of females contained well-developed unpigmented eggs with a diameter of approximately 2.2-2.8 mm (N = 15; measured from ZMMU ABV-00455, gravid female). On Hon Ba Mt., calling males were observed between 22 to 24 December, 22 to 28 March and 15 to 18 October. On Bidoup, Hon Giao and Chu Yang Sin Mts., reproductive activity and calling males were recorded from 10 February until mid-July. Tadpoles were found from April until July in the same streams where calling males were recorded; during the day time tadpoles hide under flat stones or dead leaves on the stream bed, but come out and can be visible in the shallow sandy parts of the stream at night. Metamorphosed individuals were observed in Chu Yang Sin N.P. in May. Comparisons. Ophryophryne elfina sp. n. is one of the smallest species of its genus, with adult male size (SVL 26.9-33.9 mm) similar to that of O. pachyproctus (adult male SVL 28.0-30.0 mm).
Ophryophryne elfina sp. n. differs from sympatric O. synoria (found at lower elevation from 200 to 1500 m a.s.l. in the foothills of the Langbian Plateau in southern Vietnam and adjacent easternmost hilly Cambodia) by much smaller adults body size: Ophryophryne elfina sp. n. male SVL 26.9-33.9 mm, N = 29, female SVL 35.1-36.5 mm, N = 6 (vs. O. synoria male SVL 38.2-53.7 mm, N = 14, female SVL 51.4-70.7 mm, N = 3; our data; Fig. 3), red-orange nuptial pad (in life) on first finger only (vs. two nuptial pads, covered in brown microgranules, large on first finger, covering entire dorsal metacarpal extending to 3/4 basal phalange length, on second finger medium sized on metacarpal extending to mid basal phalange on inner dorsal side), numerous bright red-orange asperities (in life) on dorsal and lateral surfaces of body, head and dorsal surfaces of limbs (vs. black and white asperities, small sized, spinular, moderately dense in narrow band along lower jaw, and on posterior upper jaw, few on tympanic region [exlcuding tympanum], along supratemporal folds and on posterior upper eyelids; some on anterior dorsum, becoming moderately dense posteriorly, above and surrounding cloaca, few on dorsal shanks, and absent on remaining surfaces on holotype of O. synoria), and smaller tympanum/eye diameter ratio, TYD/ED 48.9%-62.6%, N=29 (vs. TYD/ED 64.8%-85.2%, N=14).
Ophryophryne elfina sp. n. differs from sympatric O. gerti (found at mid-elevations from 700 to 2000 m a.s.l. in the central and northern parts of the Langbian Plateau in southern Vietnam) by typically smaller adults body size: Ophryophryne elfina sp. n. male SVL 26.9-33.9 mm, N = 29, female SVL 35.1-36.5 mm, N = 6 (vs. O. gerti male SVL 31.7-42.2 mm, N = 15, female SVL 43.1-47.4 mm, N = 3; our data; Fig. 3), bright red-orange nuptial pads on males in life (vs. grey or black-brown nuptial pads on males in life), short dorsolateral glandular ridge above each shoulder, not connected to posterior tips of " >-< "-shaped parietoscapular-sacral glandular ridge, see Figs 6G-H, and 8 (vs. strong dorsolateral glandular ridge from above each shoulder to approximately 4/5 distance between axilla and groin, connecting with posterior tips of " >-< "-shaped parietoscapular-sacral glandular ridge; see Figs 6A-C, 7), skin on dor-sal and lateral surfaces of body shagreened with numerous small tubercles (vs. skin on dorsum and sides of body granular, with numerous small and medium-sized tubercles and larger warts, see Fig. 6A-C), dark hourglass-shaped markings on dorsum normally edged with white, see Figs 6H and 8 (vs. dark hourglass-shape on dorsum indistinct or, if present, unclear and not edged with white, Fig. 6A-C), and throat, chest and abdomen having generally lighter coloration than in O. gerti.
Though available information on tadpole morphology of Oprhyophryne is very limited (Liu and Hu 1962, Huang et al. 1991, Grosjean 2003, Fei et al. 2009), the tadpoles assigned to the new species based on the analysis of short 16S rRNA gene sequences (Table 1) have certain morphological characteristics that could be useful for distinguishing the larval stage of Ophryophryne elfina sp. n. from other Ophryophryne species. From tadpoles of O. microstoma, described in detail by Grosjean (2003), tadpoles of Ophryophryne elfina sp. n. differ mainly by possessing a longer tail: TOL/BL ratio 231.3 ± 11%, N = 5 (vs. TOL/BL < 210%, N = 52 on O. microstoma), and tail tip rounded (vs. tail tip bluntly pointed), mean = 22 longitudinal rows of papillae and from 2-4 (mean = 3) transverse rows of papillae on the upper labium and 4-6 (mean = 5) transverse rows of papillae on the lower labium, N = 5 (vs. mean = 20 longitudinal rows of papillae, and two upper labium and four lower labium transverse rows of papillae [without clear limits], N = 52); however, some of these differences may relate to the fact that Grosjeans' description was based on later developmental stages (Gosner stage 37) than our sampling (Gosners' stage 25).
DNA-barcoding using short sequences for 16S rRNA (Table 1) also enabled us to identify tadpoles of sympatric Ophryophryne species from the Langbian Plateau, and though our sampling is not big enough to provide detailed morphological descriptions of larval morphology for O. gerti and O. synoria, we found some differences in coloration of tadpoles which may be useful for preliminary diagnostics of the three sympatric Ophryophryne species in the wild. Despite overall morphological similarity, both O. gerti and O. synoria show the presence of light golden to copper blotches on dorsal surfaces of the body and tail, whereas Ophryophryne elfina sp. n. tadpoles always have distinctive uniform brownish coloration with small coppery dots ( Fig. 12A-B).
Finally, the new species is markedly distinct from all other congeners for which comparable sequences are available, including it closest relatives O. gerti and O. synoria, by relatively large genetic distances in 16S rRNA mtDNA gene fragment (p ≥ 8.2%).

Discussion
The data presented here provide the most extensive molecular sampling for the elucidation of phylogentic relationships within the genus Ophryophryne. According to our data, genetic variation within Ophryophryne appears to be strongly geographically structured. Thus, our results indicate the division of the genus Ophryophryne into two major reciprocally monophyletic groups: one corresponding to species found on the Langbian Plateau (Group II, Fig. 2), and another joining species found outside the plateau from central and northern Truong Son and adjacent areas (Group I, Fig. 2). Our data support the hypothesis that eastern Indochina, including the central and southern parts of the Truong Son Mountains (known also as Tay Nguyen Plateau), host the highest diversity of Ophryophryne, and was the center of radiation for this genus Ananjeva 2007, Mahony et al. 2017). Similar patterns of geographic structuring of mtDNA lineages were reported for the genus Leptolalax, another megophryid genus inhabiting the Truong Son Mountains (Poyarkov et al. 2015a. A hidden diversity of Ophryophryne frogs is revealed in the mountains of the Langbian Plateau, where previously only one species, O. gerti, was correctly reported (Ohler 2003, Nguyen et al. 2009, Stuart et al. 2010. In our study it is shown that the previous records of O. cf. gerti from central Vietnam andLaos (Ohler 2003, Bain et al. 2007) actually belong to different species of Ophryophryne and thus we clarify the range of O. gerti showing that this species is likely endemic to the Langbian Plateau. The known distribution of O. synoria is also extended, previously known exclusively from Cambodia (Stuart et al. 2006) and adjacent provinces of Vietnam (Vassilieva et al. 2016), and demonstrate that this species has a considerably wider range encompassing the central, northern and western edges of the Langbian Plateau. Finally, we describe the new spe-cies Ophryophrye elfina sp. n., which is endemic to the northern and eastern edges of the plateau. Thus, the Langbian Plateau was a center of Ophryophryne radiation and cradles three endemic species of these frogs; all of them are sympatric in eastern and northern parts of the plateau and often can be recorded in synbiotopy.
Ophryophrye elfina sp. n. represents one of the smallest known species of the genus Ophryophryne. We found that the three Ophryophryne species of the Langbian Plateau are differentiated in body size with the largest species O. synoria preferring lowland and foothill monsoon forests at elevations from 200 to 1500 m a.s.l., medium-sized O. gerti found in evergreen montane tropical forests at mid-elevations from 700 to 2000 m a.s.l. and the smallest species Ophryophryne elfina sp. n. being restricted to wet montane subtropical forests at elevations from 700 to 2100 m a.s.l., including elfin forests at high elevations. It is probably not surprising that advertisement calls of the three occasionally sympatric Ophryophryne species show significant differences both in call structure and frequency parameters (see Table 4, Fig. 5), and the three studied species are characterized by relatively high values of the frequency parameters (as compared to several other Megophryidae species studied acoustically, especially of the genera Leptolalax, see review in Rowley et al. 2016, and Leptobrachium, see e.g. Stuart et al. 2010). The high frequency parameters may be related with their tendency to vocalize in close proximity to mountain cascade streams, which would create a low-frequency background noise (Preininger et al. 2007). It was shown that low background noise may induce frogs to call at higher frequency rates than expected from their body size, thereby improving the signal-to-noise ratio of their calls (Penna et al. 2005, Wells 2007, Goutte et al. 2016). Our study also recorded that the values of some temporal call parameters of Ophryophryne elfina sp. n. significantly differ between February (average temperature 11.3°C) and April recordings (average temperature 17.5°C; see Appendix 3 for details). Our results correspond with previous reports that intraspecific variation of temporal parameters of anuran calls can depend upon temperature (e.g., Gerhardt and Huber 2002).
The frequency of maximum amplitude coincides with the fundamental frequency for all Ophryophryne species, and have almost equal values for Ophryophryne elfina sp. n. and O. gerti (4645.94 ± 4.39 Hz, N = 1797, and 4845.99 ± 4.22 Hz, N = 533, respectively). The frequency of maximum amplitude of O. synoria is significantly lower (3798.9 ± 4.87 Hz, N = 200; see Table 4 for details), which may be related to the larger body size of the latter species (Stuart et al. 2006, Wells 2007. Further studies on acoustic communication of Langbian Ophryophryne species in areas of allopatry and sympatry would be valuable for better understanding the bioacoustic patterns observed here.
The Langbian Plateau is known for its high herpetofaunal diversity and endemism, a significant portion of which has been discovered only recently (e.g., Orlov et al. 2008, Rowley et al. 2010c, 2011a, 2011b, Stuart et al. 2011 and Vassilieva 2011, Nazarov et al. 2012, Chan et al. 2013, Hartmann et al. 2013, 2015a, 2015b. Despite this increase in species discoveries, many areas of the Annamites have received little scientific attention and are very likely to host further previously unknown diversity. The need for biological exploration in this region is made more urgent given the ongoing loss of natural habitats due to logging, road construction, increasing agricultural pressure and other human activities (Meijer 1973, De Koninck 1999, Laurance 2007, Meyfroidt and Lambin 2008, Kuznetsov and Kuznetsova 2011. Habitat loss is the greatest threat to amphibians in southeast Asia, and the amphibians of the region appear to be particularly vulnerable to habitat alterations (Rowley et al. 2010b). Frogs of the genus Ophryophryne depend on fast-flowing clean mountain streams for reproduction, and appear to be restricted to relatively undisturbed broadleaf evergreen forests: such habitat specialist range-restricted species are likely to be most at risk , Rowley et al. 2010b. Deforestation, habitat loss and modification are continued threats in southern Indochina (Meyfroidt and Lambin 2008), and further studies on herpetofaunal biodiversity in this region are urgently required for elaboration of effective conservation measures.

Addendum (added post manuscript acceptance)
Due to the simultaneous review period of the present paper, and the now recently published Mahony et al. (2017), we chose to preliminarily use Ophryophryne at the genus level (following Chen et al. 2017; published online 1 December 2016), pending the publication of the taxonomic justification by Mahony et al. (2017) which supports a subgenus level classification of Ophryophryne within Megophrys. Mahony et al. (2017) also provided the replacement name Megophrys (Ophryophryne) koui Mahony, Foley, Biju & Teeling, 2017 for Ophryophryne pachyproctus Kou, 1985. We suggest that the new species combination Ophryophryne elfina sp. n. should hereafter be referred to as Megophrys (Ophryophryne) elfina (Poyarkov, Duong, Orlov, Gogoleva, Vassilieva, Nguyen, Nguyen, Nguyen, Che & Mahony) to reflect this revised taxonomy.

Author contributions
NA Poyarkov envisioned the original idea of the manuscript, collected material and data in the field and in the lab, executed this study and wrote the manuscript; TV Duong performed morphometric, molecular and phylogenetic analyses; NL Orlov collected material in the field; SS Gogoleva collected data in the field and performed acoustic analyses and wrote the relevant parts of the manuscript; AB Vassilieva collected material and data in the field; LT Nguyen collected material in the field and assisted with morphological analysis; VDH Nguyen, J Che and SN Nguyen collected material in the field and provided additional molecular data; S Mahony examined type and comparative specimens, performed molecular analysis, provided redescription of types, and edited the manuscript. All authors contributed to this paper sufficiently.

Appendix 2
Factor coordinates of the morphometric characters used in PCA analysis, based on correlations (factors 1 to 3).