A new genus and three new species of miniaturized microhylid frogs from Indochina (Amphibia: Anura: Microhylidae: Asterophryinae)

We report on the discovery of a new genus of microhylid subfamily Asterophryinae from northern and eastern Indochina, containing three new species. Vietnamophryne Gen. nov. are secretive miniaturized frogs (SVL<21 mm) with a mostly semi-fossorial lifestyle. To assess phylogenetic relationships, we studied 12S rRNA – 16S rRNA mtDNA fragments with a final alignment of 2 591 bp for 53 microhylid species. Morphological and osteological characters were analyzed using micro-CT scanning and used to describe the new genus. Results of phylogenetic analyses assigned the new genus into the mainly Australasian subfamily Asterophryinae as a sister taxon to the genus Siamophryne from southern Indochina. The three specimens collected from Gia Lai Province in central Vietnam, Cao Bang Province in northern Vietnam, and Chiang Rai Province in northern Thailand proved to be separate species, different both in morphology and genetics (genetic divergence 3.1%≤P≤5.1%). Our work provides further evidence for the “out of Indo-Eurasia” scenario for Asterophryinae, indicating that the initial cladogenesis and differentiation of this group of frogs occurred in the Indochina Peninsula. To date, each of the three new species of Vietnamophryne Gen. nov. is known only from a single specimen; thus, their distribution, life history, and conservation status require further study.


INTRODUCTION
Frogs of the family Microhylidae form one of the most speciose groups of amphibians with pantropical distribution. Currently, some 642 species are recognized, inhabiting areas from the tropics and subtropics of Africa, Madagascar, Southern and Northern America, and South, East, and Southeast Asia to the islands of the Australasian archipelago and northernmost Australia (Frost, 2018). The basal split within Microhylidae is estimated to have occurred ∼65 Ma, coinciding with the Cretaceous-Paleogene boundary (Feng et al., 2017). The family Microhylidae is assumed to be of Gondwanan origin and is currently divided in 13 subfamilies, each of which are associated with a certain landmass derived from the breakup of Gondwana (De Sá et al., 2012;Kurabayashi et al., 2011;Peloso et al., 2016). Despite significant progress in understanding the evolutionary relationships within the family, the level of congruence between morphology-based and molecular phylogenetic hypotheses is still low and further changes in family-and genus-level taxonomy are required (De Sá et al., 2012;Kurabayashi et al., 2011;Matsui et al., 2011;Peloso et al., 2016;Pyron & Wiens, 2011;Rivera et al., 2017).
The subfamily Asterophryinae is the most speciose group within Microhylidae, currently consisting of 327 species inhabiting the tropical forests of northern Australia, New Guinea, and adjacent Australasian islands westwards to Sulawesi, southern Philippines, and crossing the Wallace line in Bali (Frost, 2018).
The original biogeographic hypothesis for this subfamily suggested that the common ancestor of Asterophryinae dispersed to Australia via an Antarctic land bridge (Hill, 2009;Savage, 1973), where it diversified and subsequently dispersed to New Guinea and adjacent Australasian islands.
However, based on multilocus phylogenetic analyses, Kurabayashi et al. (2011) demonstrated that the enigmatic genus Gastrophrynoides from Sundaland (Borneo and Malay Peninsula) belongs to the subfamily Asterophryinae as a sister-lineage with respect to all Australasian taxa, suggesting that the basal split of the subfamily may not have occurred in Gondwana, but instead on the Eurasian mainland. Thus, Kurabayashi et al. (2011) proposed an "out of Indo-Eurasia" biogeographic scenario for Asterophryinae, suggesting that its colonization route was from Asia to Australia, and not via Antarctica as suggested earlier.
In their work, Kurabayashi et al. (2011:9) predicted, that the "biogeographic findings on Gastrophrynoides imply the possible occurrence of further microhylid taxa with unexpected evolutionary backgrounds and give a basis for future paleontological and biogeographic studies of Asian anurans". Our more recent work (Suwannapoom et al., 2018) reported on the unexpected discovery of Siamophrynea striking troglophilous microhylid frog found in a limestone cave in Tenasserim (southern Thailand) -with phylogenetic analyses placing it as a sister lineage of Gastrophrynoides, further suggesting that mainland Southeast Asia likely served as a cradle of initial divergence and radiation of asterophryine frogs.
In 2016 and 2017, during field surveys in northern and eastern Indochina, we encountered three specimens of miniaturized frogs. Although these frogs were found in different localities in central and northern Vietnam and northern Thailand ( Figure  1), all three specimens were superficially very similar to each other and found in similar microhabitats -soil or leaf litter under large tree logs or among plant roots. They were assigned to Microhylidae due to the presence of morphological characters diagnostic for the family: namely, lack of mandibular teeth, lack of parotoid glands, firmisternal pectoral girdle with non-overlapping epicoracoids, well-developed coracoids reaching midline of girdle and scapulae, large, cartilaginous sternum, and absence of clavicles and omosternum. Further morphological, osteological, and molecular analyses demonstrated that each of the three specimens represented a new species of a previously unknown lineage of frogs, assigned to the subfamily Asterophryinae and sister taxon to Siamophryne. We describe this new genus and three new species herein. ). All fieldwork and collection permits are listed in the Acknowledgements. Geographic coordinates and elevation were obtained using a Garmin GPSMAP 60CSx (USA) and recorded in WGS84 datum. In total, three adult specimens (all males) were collected from three surveyed localities. The specimens were photographed in life and then euthanized using 20% benzocaine prior to fixation in 96% ethanol and subsequent storage in 70% ethanol. Tissue samples for genetic analysis were taken prior to preservation and stored in 95% ethanol. Specimens and tissues were subsequently deposited in the herpetological collections of the Zoological Museum of Moscow University (ZMMU, Moscow, Russia) and School of Agriculture and Natural Resources, University of Phayao (AUP, Phayao, Thailand).

Laboratory methods
Total genomic DNA was extracted from ethanol-preserved femoral muscle tissue using standard phenol-chloroformproteinase K (final concentration 1 mg/mL) extraction with subsequent isopropanol precipitation (as per Hillis et al., 1996 andSambrook &Russell, 2001). The isolated DNA was visualized using agarose electrophoresis in the presence of ethidium bromide. The resulting DNA concentration in 1 µL was measured using a NanoDrop 2000 (Thermo Scientific, USA) and consequently adjusted to 100 ng DNA/µL. We amplified mtDNA fragments, covering partial sequences of the 12S rRNA and 16S rRNA mtDNA genes and complete sequence of the tRNA Val mtDNA gene to obtain a 2 591-bp long continuous fragment of mtDNA. These mtDNA markers have been used for comprehensive phylogenetic studies on Microhylidae frogs (De Sá et al., 2012;Matsui et al., 2011;Peloso et al., 2016;Pyron & Wiens, 2011;Van Der Meijden et al., 2007; and references therein), including molecular taxonomic research on the subfamily Asterophryinae (Blackburn et al., 2013;Frost et al., 2006;Günther et al., 2010;Köhler & Günther, 2008;Kurabayashi et al., 2011;Oliver et al., 2013;Rittmeyer et al., 2012;Suwannapoom et al., 2018). PCR was performed in 20 µL reactions using 50 ng of genomic DNA, 10 nmol of each primer, 15 nmol of each dNTP, 50 nmol of additional MgCl 2 , Taq PCR buffer (10 mmol/L of Tris-HCl, pH 8.3, 50 mmol/L of KCl, 1.1 mmol/L of MgCl 2 and 0.01% gelatin), and 1 U of Taq DNA polymerase. The PCR conditions as well as primers used for PCR procedures and sequencing followed Suwannapoom et al. (2018).
The PCR products were loaded onto 1.5% agarose gels in the presence of ethidium bromide. Visualization was carried out using agarose electrophoresis. If distinct bands were obtained, products were purified prior to cycle sequencing using 2 µL of ExoSapIt (Amersham, UK), diluted at a 1:4 ratio, per 5 µL of PCR product. The 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 v3.1 Sequencing Standard (Applied Biosystems, USA), and 4.2 µL of water. The cycle sequencing reaction included 35 cycles with the following steps: 10 s at 96 • C, 10 s at 50 • C, and 4 min at 60 • C. Cycle sequencing products were then purified by ethanol precipitation. Sequencing was performed on an ABI 3730xl automated sequencer (Applied Biosystems, USA). The obtained sequences were deposited in GenBank under accession numbers MH004403-MH004406 (Table 1).    (Günther) (Rhacophoridae) was used as a non-microhylid outgroup. Nucleotide sequences were initially aligned using ClustalX 1.81 software (Thompson et al., 1997) with default parameters, and then optimized manually in BioEdit 7.0.5.2 (Hall, 1999) and MEGA 7.0 (Kumar et al., 2016). Mean uncorrected genetic distances (P-distances) between sequences were determined using MEGA 6.0. MODELTEST v3.06 (Posada & Crandall, 1998) was applied to estimate the optimal evolutionary models to be used for dataset analysis. The best-fitting model was the GTR+I+G model of DNA evolution, as suggested by the Akaike Information Criterion (AIC).
Phylogenetic trees were inferred using maximum likelihood (ML) and Bayesian inference (BI). The ML analysis was conducted using Treefinder (Jobb et al., 2004). Confidence in tree topology was tested by non-parametric bootstrap (BS) analysis with 1 000 replicates (Felsenstein, 1985). The BI analysis was conducted using MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001;Ronquist & Huelsenbeck, 2003). Metropolis coupled Markov chain Monte Carlo (MCMCMC) analyses were run with one cold chain and three heated chains for four million generations and were 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 using posterior probability (PP) (Huelsenbeck & Ronquist, 2001). We regarded tree nodes with BS values of 75% or greater and PP values over 0.95 as sufficiently resolved, those with BS values between 75% and 50% (PP between 0.95 and 0.90) as tendencies, and those with BS values below 50% (PP below 0.90) as unresolved (Huelsenbeck & Hillis, 1993).

Osteology
Micro-CT scanning protocols followed Suwannapoom et al. (2018). Micro-CT scanning was conducted at the Petroleum Geology Department, Faculty of Geology, Lomonosov Moscow State University using a SkyScan 1 172 desktop scanner (Bruker micro-CT, Kontich, Belgium) equipped with a Hamamatsu 10 Mp digital camera. Scanning was performed only for ZMMU A-5820. The specimen was mounted on a polystyrene baseplate and placed inside a hermetically sealed polyethylene vessel. Scans were conducted with a resolution of 3.7 µm at 100 keV voltages and a current of 100 mA with a rotation step of 0.2 • in oversize mode in which four blocks of sub-scan data were connected vertically to obtain a general tomogram. Data processing was performed using Skyscan software: NRecon (reconstruction) and CTan/CTVol (3D model producing and imaging). Osteological terminology followed Scherz et al. (2017), Suwannapoom et al. (2018), and Trueb (1968. Micro-CT does not render cartilage, and therefore cartilage structures were omitted from the osteological descriptions.

Phylogenetic relationships
Results of the phylogenetic analyses are shown in Figure 2. The BI and MI analyses resulted in essentially similar topologies. Though phylogenetic relationships between the subfamilies of Microhylidae remained essentially unresolved, high resolution was achieved among most major lineages of the subfamily Asterophryinae, with major nodes being sufficiently resolved (1.0/100; hereafter node support values are given for BI PP/ML BS, respectively; Figure 2). However, phylogenetic relationships within the Austro-Papuan radiation of Asterophryinae were poorly resolved with low or insignificant levels of support for major nodes.
The BI tree ( Figure 2) suggested the following genealogical relationships among the representatives of Microhylidae: monophyly of the subfamilies Dyscophinae, Kalophryninae, and Asterophryinae well-supported (1.0/100), monophyly of the subfamily Microhylinae not supported, and phylogenetic relationships among Microhylidae subfamilies unresolved.
The subfamily Asterophryinae consisted of the two major well-supported (1.0/100) reciprocally monophyletic clades: (1) The Asterophryinae 1 or "core" Asterophryinae ( Figure 2, in red) clade included all presently known Australasian genera of the subfamily inhabiting islands east of the Wallace line, tropical areas of northern Australia, and Bali (see line B1 in Figure 1; range of Asterophryinae 1 marked in red).
(2) The second clade included three Asterophryinae lineages inhabiting areas derived from the Eurasian landmass Phylogenetic relationships among genera within the Asterophryinae 1 clade were essentially unresolved ( Figure 2). Cophixalus was suggested as a sister lineage to Choerophryne with moderate support (0.92/72). Monophyly of the clade that included Sphenophryne, Liophryne, and Oxydactyla genera was strongly supported (1.0/96), thus supporting synonymy of the two latter genera with Sphenophryne, as suggested by Rivera et al. (2017). However, Sphenophryne thomsoni (Boulenger), previously assigned to the genus Genyophryne, was placed with significant node support (0.97/71) as a sister lineage to the clade that included Cophixalus and Choerophryne and was distantly related to the clade that included the remaining Sphenophryne s. lato taxa. Our data provided only weak support for monophyly of the genus Oreophryne (0.55/80). Callulops was identified as a sister lineage to Mantophryne and Hylophorbus (0.96/63). The monophyly of the clade that included Asterophrys, Oninia, and the formerly recognized genera Metamagnusia and Pseudocallulops, was strongly supported (1.0/95).
The monophyly of the genus Xenorhina also showed high support (1.0/94).
Phylogenetic relationships among Asterophryinae clades 2-4 were well-resolved ( Figure 2). Monophyly of the lineage Asterophryinae 2, joining three small microhylids from northern and eastern Indochina, was strongly supported (1.0/100); among them, the two Vietnamese samples from Cao Bang and Gia Lai provinces formed a strongly supported monophyletic group (1.0/95). The genus Siamophryne from Tenasserim (southern Thailand) was reconstructed as a sister lineage with respect to Asterophryinae 2 (1.0/100).
The genus Gastrophrynoides from Sundaland was suggested as a sister-clade with respect to Indochinese lineages Siamophryne + Asterophryinae 2 with strong node support (1.0/100).
Our phylogenetic analyses indicated that the three newly discovered Microhylidae Gen. sp. from northern and eastern Indochina formed a monophyletic group, belonging to the mainly Australasian subfamily Asterophryinae s. lato, within which they were placed as a sister lineage to the genus Siamophryne (the only other asterophryine genus known from Indochina) with high levels of node support.

Genetic distances
16S rRNA is a widely known molecular marker applied for biodiversity studies in amphibians (Vences et al., 2005a(Vences et al., , 2005bVieites et al., 2009). The uncorrected genetic P-distances among and within the 12S rRNA -16S rRNA gene fragments of the studied Asterophryinae genera are shown in Table 2. Voucher specimen IDs and GenBank accession numbers are given in Table 1. Sequence of Rhacophorus schlegelii was used as an outgroup. Numbers near branches represent posterior probability (PP) or bootstrap support values (BS, 1 000 replicates) for BI/ML inferences, respectively. Photos by N. A. Poyarkov and Y. Lee. The genetic differentiation between the newly discovered Microhylidae Gen. sp. from northern and eastern Indochina and other Asterophryinae genera varied from 12.6% (between Microhylidae Gen. sp. from Cao Bang Province (Vietnam) and genus Metamagnusia) to 21.4% of substitutions (between Microhylidae Gen. sp. from Gia Lai Province (Vietnam) and genus Callulops). Genetic distances between Microhylidae Gen. sp. and its sister lineage Siamophryne varied from 12.6% to 15.1% of substitutions. These genetic divergences were high and corresponded well to genus level differentiation within Asterophryinae (Table 2). Genetic divergence between the three specimens of Microhylidae Gen. sp. was moderate and varied from 3.1% (between samples from Gia Lai Province of Vietnam and Chiang Rai Province of Thailand) to 5.1% (between Gia Lai and Cao Bang samples) of substitutions, slightly higher than the conventional threshold of species-level divergence in other groups of Anura (3.0% of divergence in the 16S rRNA gene according to Vences et al., 2005aVences et al., , 2005bVieites et al., 2009).

Taxonomy
Based on our phylogenetic analyses, the newly discovered miniaturized microhylid frogs from northern and eastern Indochina formed a monophyletic group, clearly distinct from all other members of Microhylidae for which comparable genetic data were available.
This group was placed in the radiation of the subfamily Asterophryinae with strong support. Though the 12S rRNA -16S rRNA mtDNA fragment sequences did not achieve full phylogenetic resolution for all lineages of the subfamily Asterophryinae, the phylogenetic relationships within our focal group, Asterophryinae lineages 2-4, were well-resolved. Our data strongly suggest that the three main lineages of Asterophryinae inhabiting Indochina and Sundaland were monophyletic, whereas the miniaturized Microhylidae Gen.
sp. from northern Indochina were suggested as the sister-lineage of the genus Siamophryne from southern Indochina. Table 2 Uncorrected P-distances (percentages) between 12S rRNA -16S rRNA sequences of Vietnamophryne Gen. nov. and other Asterophryinae genera included in phylogenetic analyses (below diagonal line) and standard error estimates (above diagonal line) Vietnamophryne Gen. nov.
Distribution: To date, Vietnamophryne Gen. nov. is known only from three localities in northern and eastern Indochina: two localities in Vietnam (Gia Lai Province, Tay Nguyen Plateau of the central Annamite (Truong Son) Mountains and mountainous area in Cao Bang Province, northern Vietnam) and one locality in northern Thailand (limestone mountainous area in northern Chiang Rai Province) (Figure 1). This distribution pattern, joining the north-eastern part of Vietnam (Dong Bac), central Annamites (Tay Nguyen), and northern Thailand, suggests that members of the new genus may be found in other areas of northern and eastern Indochina, and its occurrence in adjacent regions of Laos and central-northern Vietnam is strongly anticipated.
well-developed neural crests on presacral vertebrae). Vietnamophryne Gen. nov. can be further distinguished from Aphantophryne by its eight presacral vertebrae (vs. seven). Vietnamophryne Gen. nov. can be distinguished from members of the genus Sphenophryne s. lato (including Liophryne and Oxydactyla) and Austrochaperina by absence of clavicles (vs. well-developed long and slender clavicles). Vietnamophryne Gen. nov. can be further distinguished from Sphenophryne s. lato by its lack of vomeropalatines (vs. broad vomeropalatines contacting each other medially, with post-choanal portion overlying palatine region). Vietnamophryne Gen. nov. can be diagnosed from Sphenophryne s. stricto (S. cornuta Peters & Doria) by smooth upper eyelid and semi-fossorial lifestyle (vs. spine-like projection on upper eyelid and arboreal lifestyle in S. cornuta). Vietnamophryne Gen. nov. can be further distinguished from the genus Liophryne (considered as a synonym of Sphenophryne by Rivera et al., 2017) by absence of finger disks (vs.
nov. can be further distinguished from Austrochaperina by lack of vomeropalatines (vs. vomeropalatines expanded). Vietnamophryne Gen. nov. differs from the genus Paedophryne by having all digit phalanges ossified (vs. cartilaginous phalanges in first digit), and eight presacral vertebrae (vs. seven). Vietnamophryne Gen. nov. can be diagnosed from the genus Choerophryne by lack of vomeropalatines (vs. palatine portions of vomeropalatines fused with broad sphenethmoids). Vietnamophryne Gen. nov. can be distinguished from the genus Copiula by lack of disks on fingers, but tiny disks on toes (vs. well-developed disks on fingers and toes) and absence of conspicuous rostral dermal gland (vs. rostral gland present). Semi-fossorial Vietnamophryne Gen. nov. can be easily distinguished from the mostly arboreal or terrestrial genus Oreophryne by its lack of toe webbing (vs. distinct toe webbing) and absence of vomeropalatines (vs. vomeropalatines expanded). Vietnamophryne Gen. nov. can be distinguished from Hylophorbus by comparatively better developed nasals (vs. poorly developed nasals), comparatively broad cultriform process of parasphenoid (vs. narrow cultriform process of parasphenoid), and F1 very small or reduced to nub, 1FL 1 /22FL (vs. F1 well-developed, 1FL≥ 1 /22FL).
Finally, the 12S-16S rRNA mtDNA fragment sequences for the new genus were markedly distinct from all sequences for Asterophryinae members for which homologous sequences were available (Figure 2, Table 2).
Comparisons with other Microhylidae genera inhabiting mainland Southeast Asia: From other genera of Microhylidae inhabiting mainland Southeast Asia, all members of the genus Vietnamophryne Gen.
nov. can be distinguished by a combination of the following characters: small body size (SVL≤21.0 mm); stout body habitus; externally distinct tympanum (vs. hidden tympanum in Glyphoglossus, Microhyla, Micryletta, Kaloula, Phrynella, Metaphrynella, and Gastrophrynoides); absence of subarticular tubercles (vs. subarticular tubercles of fingers greatly enlarged in Phrynella and Metaphrynella), absence of toe webbing or fringing on digits (vs. webbing or digit fringes present in Microhyla, Phrynella, and Metaphrynella); absence of tibiotarsal projection (vs. bony tibiotarsal projection present in Chaperina); lack of bony ridge along posterior border of each choana (vs. present in Kaloula); short rounded or obtuse snout (vs. long pointed snout 2.6-3.0 times eye diameter in Gastrophrynoides); and absence of disks on digits (vs. long limbs with digits bearing large disks, with those on fingers up to 2.5 times wider than penultimate phalanges in Siamophryne).

Etymology:
The generic nomen Vietnamophryne is derived from "Vietnam", the name of the country where the representatives of this genus were first recorded and where two of the three known species of the genus occur; and Greek noun "phryne" (ϕρύνη; feminine gender), meaning "toad" in English; this root is often used in generic names in Asterophryinae frogs. Gender of the new genus is feminine.

Vietnamophryne inexpectata sp. nov.
Description of holotype: Measurements of holotype are given in Table 3. Holotype in life is shown in Figure 5A and Figure 6. Body miniaturized, with SVL 14.2 (hereafter all measurements in mm), in good state of preservation; ventral surface of left thigh dissected 1.5 mm and partial femoral muscles removed. Body habitus stout ( Figure 5A), head as long as wide (HL/HW 101.1%); snout short, obtuse in dorsal view ( Figure 6A), rounded in profile ( Figure 6C), subequal to eye diameter (SL/EL 96.8%); eyes medium-sized (EL/SVL 13.0%), slightly protuberant in dorsal and lateral views ( Figure  6A, C), pupil round, horizontal ( Figure 6C); dorsal surface of head slightly convex, canthus rostralis distinct, rounded; loreal region weakly concave; nostril rounded, lateral, located almost same distance from tip of snout and eye; tympanum well discerned, circular, comparatively large (TL/SVL ratio 7.9%), located distantly from eye (TED/SVL ratio 3.6%), tympanic rim not elevated above skin of temporal area, supratympanic fold present, glandular; vomerine teeth and spikes absent, single transverse palatal fold with smooth edge present across palate anteriorly to pharynx, tongue spatulate and free behind, lacking papillae, and vocal sac opening not discernable.
Skin on anterior dorsal and dorsolateral surfaces shagreened with numerous small flat tubercles ( Figure 6A); tubercles larger and more prominent on posterior parts of dorsum, sacral area, and dorsal surfaces of hindlimbs; dorsal surface of forelimbs smooth with few small tubercles on forearm; upper eyelids and supratympanic folds with rows of enlarged tubercles forming flat glandular ridge; ventral sides of trunk, head, and limbs completely smooth ( Figure 6B); weak indistinct dermal ridge present on midline of dorsal surface, running from tip of snout to scapular area ( Figure 5A; Figure 6A).

Coloration of holotype in life:
Dorsum grayish-brown, anteriorly light brown, posteriorly darker, with small reddish speckling anteriorly ( Figure 6A); tubercles on sacral area, posterior parts of dorsum, and dorsal surfaces of hindlimbs dark gray with whitish pustules in middle; upper eyelids with tiny reddish speckles, two dorsolateral rows of darker tubercles running from scapular area toward vent; dorsal surfaces of forearms dark brown with red-brown blotches; dorsal surfaces of hindlimbs dark brown with rare reddish spots and dark gray to whitish tubercles and pustules; lateral sides of head dark brown with beige mottling present in tympanic area and mouth corners ( Figure 6C); canthus rostralis ventrally dark brown, dorsally reddish-brown; supratympanic fold with whitish glandular tubercles; ventrally gray-beige with weak gray marbling, more scarce on belly, denser on chest, throat, and ventral surfaces of limbs ( Figure 6B); fingers and toes dorsally dark brown with indistinct dark brown or reddish blotches, ventrally uniform gray ( Figure 6D, E). Pupil round, black, iris uniform black ( Figure 6C).

Coloration of holotype in preservative:
Coloration pattern unchanged after preservation in ethanol for two years; however, dorsal coloration changed to grayish-brown and ventral surface of chest, belly, and limbs turned light gray.
Osteological characteristics: Osteological description is based on microtomographic data from male holotype. Main skeletal features are shown in Figure 3. Details of skull morphology are presented in Figure 4.

Natural history notes:
Our knowledge on the biology of Vietnamophryne inexpectata sp. nov. is scarce. The single new species specimen was recorded in primary polydominant tropical montane evergreen forests of Tay Nguyen Plateau at an elevation of ca. 1 000 m a.s.l.. It was found during heavy rain at 2100 h in wet soil at the bottom of a 20-cm deep hollow formed after a large 2-m long rotten tree log was turned over. The new species location was situated approximately 7 m from a small cascading stream (Figure 7). The frog was hiding among soil and leaf litter, suggesting that the new species has a semi-fossorial (subterranean) lifestyle or at least spends a considerable portion of its life hiding in leaf litter and under logs. The forest where the new species was recorded has a multi-layered canopy and heavy undergrowth,  Despite intensive fieldwork, no additional specimens of the new species were encountered either on the ground or in leaf litter over a 7-d period, suggesting a secretive biology for this frog. Diet and reproductive biology of the new species remain unknown. No calling activity was recorded during the survey. The male specimen was active at an air temperature of 21°C with 100% humidity. The male possessed a pair of well-developed testes.

Distribution and biogeography:
At present, Vietnamophryne inexpectata sp. nov. is known only from its type locality in montane tropical forest in Kon Chu Rang Nature Reserve, Gia Lai Province, central Vietnam at an elevation of ca. 1 000 m a.s.l.. The discovery of this secretive species in montane forests of other parts of Tay Nguyen Plateau at similar elevations in central Vietnam (Kon Tum, Quang Nam, Quang Ngai and Thua Thien-Hue provinces) and possibly in adjacent Laos is highly anticipated.

Conservation status:
To date, the new species is known only from a single specimen, likely due to its secretive biology. The range and population status of Vietnamophryne inexpectata sp. nov. are unknown and further survey efforts in other parts of Tay Nguyen Plateau are required to understand its distribution and life history. Given the available information, we suggest Vietnamophryne inexpectata sp. nov. be considered as a Data Deficient (DD) species following IUCN's Red List categories (IUCN Standards and Petitions Subcommittee, 2016).

Etymology:
The specific name "inexpectata" is a Latin adjective in the nominative singular meaning "unexpected"; referring to the surprising discovery of this frog species in 2016, which belongs to the mainly Australasian subfamily Asterophryinae; until recently (Suwannapoom et al., 2018) members of Asterophryinae were not recorded from mainland Southeast Asia or eastern Indochina.

Description of holotype:
Measurements of holotype are given in Table 3. Holotype in life is shown in Figure 5B and Figure 8. Body miniaturized, SVL 15.4, in good state of preservation; ventral surface of left thigh dissected 1.6 mm and partial femoral muscles removed. Body habitus stout ( Figure 5B), head notably longer than wide (HL/HW 86.5%); snout comparatively long, rounded in dorsal view ( Figure 8A), truncate in lateral view ( Figure 8C), snout length greater than eye length (SL/EL ratio 141.3%); eyes medium-sized (EL/SVL ratio 11.6%); eye to nostril distance 12.7% of SVL; eyes slightly protuberant in dorsal and lateral views ( Figure 8A, C), pupil round, horizontal ( Figure 8C); dorsal surface of head slightly convex, canthus rostralis distinct, rounded; loreal region concave; nostril rounded, lateral, located closer to tip of snout than to eye; tympanum well discernable, circular, comparatively small (TL/SVL ratio 5.5%), located distantly from eye (TED/SVL ratio 4.2%), tympanic rim not elevated above skin of temporal area, supratympanic fold present, distinct, glandular; vomerine teeth and spikes absent, single transverse palatal fold with smooth edge present across palate anteriorly to pharynx, tongue spatulate and free behind, papillae on tongue absent, vocal sac opening absent.
Forelimbs comparatively short, around one-third of hindlimb length (FLL/HLL 37.2%); hand shorter than lower arm, almost one-third of forelimb length (HAL/FLL 38.7%); fingers short, round in cross-section, first finger well developed, half of length of second finger (1FL/2FL 47.9%); relative finger lengths: I<IV<II<III ( Figure 8D). Finger webbing and dermal fringes on fingers absent. First finger tip rounded, first finger well developed.
Skin on anterior dorsal and dorsolateral surfaces smooth, shagreened on posterior dorsum and dorsal surfaces of hindlimbs; small flat tubercles loosely scattered on sacral area and dorsal surfaces of limbs ( Figure 8A); dorsal surface of forelimbs smooth; upper eyelids smooth, supratympanic folds with low glandular ridges; ventral sides of trunk, head and limbs completely smooth ( Figure 8B); well-developed distinct dermal ridge present on midline of head dorsal surface, running from tip of snout to sacral area ( Figure 5B; Figure 8A). Coloration of holotype in life: Dorsum reddish-brown, anteriorly orange-brown, numerous small red speckles densely scattered on dorsal surfaces of head, body, and limbs ( Figure  8A); posterior parts of dorsum and dorsal surfaces of hindlimbs with tiny whitish pustules; upper eyelids and canthus rostralis with narrow whitish stripe formed by tiny flat tubercles: stripe from snout tip toward eye along canthus rostralis, continuing to superciliary area and indistinct on supratympanic fold; dorsal surfaces of forearms brick-red; dorsal surfaces of hindlimbs reddish-brown with numerous reddish spots and rare whitish tubercles and pustules; lateral sides of head dark brown with whitish mottling on upper jaw and mouth corners ( Figure 8C); canthus rostralis ventrally dark brown, dorsally with whitish stripe continuing to upper eyelid; supratympanic fold with reddish glandular tubercles lacking white stripe; ventrally bright lemon-yellow with weak dark brown marbling, marbling more scarce on ventral part of thighs and vent area, denser anteriorly toward chest and throat area ( Figure 8B); fingers and toes dorsally gray-brown with indistinct reddish blotches, ventrally gray-brown with irregular beige or yellowish blotches ( Figure  8D, E). Pupil round, black, iris uniform dark brown ( Figure 8C).

Coloration of holotype in preservative:
Coloration pattern unchanged after preservation in ethanol for one year; however, dorsal coloration changed to dark gray yellow tint on ventral surfaces of body and limbs faded to gray-beige.

Natural history notes:
The biology of Vietnamophryne orlovi sp. nov. is unknown. The only encountered specimen of the new species was discovered at 2300 h under heavy rain in soil around the roots of cf. Dicranopteris sp. ferns (Gleicheniaceae, Gleicheniales), approximately 10 cm underground; the frog burrow was located on a steep slope of Phia Oac Mt. ( Figure  9A), ca. 20 m from a small cascading stream ( Figure 9B) at an elevation of ca. 1 200 m a.s.l. and air temperature of 17°C. Thus, this species may exhibit a semi-fossorial lifestyle. Despite thorough search efforts, no additional individuals were recorded during a 10-d field survey in Phia Oac-Phia Den National Park, possibly due to the secretive biology of this frog. Diet and reproductive biology of Vietnamophryne orlovi sp. nov. remain unknown. No calling activity was recorded during the survey. The male possessed a pair of well-developed testes.
In Phia Oac, under the influence of the monsoon tropical climate of northeast Vietnam with cold winters and summer rains, the mean annual temperature, precipitation, and humidity are 20.6°C, 1 718 mm, and 83.4%, respectively (Averyanov et al., 2003;Le, 2005). Unusually for northern Vietnam, the temperature can fall below freezing and snow is not rare in December and January. The dry season extends from November to April, with a mean precipitation of 295 mm (17.2% of total annual rainfall); the rainy season runs from May to November, with peak rainfall in July and August and mean rainfall of 1 423 (82.8% of total annual rainfall; Le, 2005). These conditions support a variety of forest types, particularly low to high montane broadleaf evergreen forests (Tran et al., 2014). Currently, vegetation covers approximately 84% of the total area of Phia Oac, though mostly consists of secondary forests or plantations. Mature (primary) and undisturbed forests are found only above 1 000 m a.s.l. (Tran et al., 2014).
Comparisons: For comparisons with other members of the family Microhylidae occurring in Indochina, see "Comparisons with other Microhylidae genera inhabiting mainland Southeast Asia" above. For comparisons with Vietnamophryne inexpectata sp. nov. see the "Comparisons" section above.

Distribution and biogeography:
Description of holotype: Measurements of holotype are given in Table 3. Holotype in life is shown in Figure 5C and Figure  10. Body size small, SVL 20.5, in poor state of preservation (specimen was partially decayed prior to preservation, soft tissues absent from distal part of left hindlimb and middle part of belly); ventral surface of left thigh dissected 2.0 mm and partial femoral muscles removed. Body habitus stout ( Figure  5C), head width equal to head length (HL/HW 99.0%); snout very short, truncate in dorsal view, rounded in lateral view ( Figure 10A), snout length much shorter than eye length (SL/EL ratio 85.5%); eyes medium-sized (EL/SVL ratio 12.1%); eye to nostril distance 6.7% of SVL; eyes slightly protuberant in dorsal and lateral views ( Figure 5C; Figure 10A, B), pupil round, horizontal; dorsal surface of head rather flat, canthus rostralis distinct, rounded; loreal region vertical; nostril rounded, lateral, located closer to tip of snout than to eye; tympanum well discernable, circular, comparatively small (TL/SVL ratio 5.0%), located very close to eye (TED/SVL ratio 1.8%); tympanic rim not elevated above skin of temporal area, supratympanic fold present, distinct and thick, rounded, glandular; vomerine teeth and spikes absent, single transverse palatal fold with smooth edge present across palate anteriorly to pharynx, tongue spatulate and free behind, lacking papillae, vocal sac opening absent.
Skin on dorsal and dorsolateral surfaces smooth; rare small flat tubercles present on dorsal surfaces of hindlimbs and posterior dorsum ( Figure 5C); dorsal surface of forelimbs smooth; upper eyelids smooth, supratympanic folds with low thick glandular ridges; ventral sides of trunk, head, and limbs completely smooth ( Figure 10B); well-developed distinct dermal ridge present on midline of dorsal surface, running from tip of snout to cloacal area ( Figure 5C; Figure 10A).

Coloration of holotype in life:
Dorsally uniform dark brick-brown, continued on dorsal surfaces of limbs; rare small flat tubercles somewhat darker (dark brown) ( Figure 10A); loosely scattered pustules on dorsal surfaces of posterior parts of dorsum and hindlimbs gray; dorsal surfaces of fore-and hindlimbs dark brick-brown; lateral sides of head dark brown (almost black); whitish mottling head sides or jaws absent ( Figure 10A); canthus rostralis and supratympanic fold ventrally dark brown, dorsally brick-brown; ventrally bright orange-red with weak and rare dark brown marbling, denser on throat and ventral surfaces of hindlimbs ( Figure 10B); fingers and toes dorsally dark brown, ventrally gray-brown to gray with occasional reddish blotches ( Figure 10C, D). Pupil round, black, iris uniform dark brown ( Figure 5C; Figure 10A).

Coloration of holotype in preservative:
Coloration pattern unchanged after one year in ethanol; however, dorsal coloration changed to dark brown, reddish tint from dorsum and ventral surfaces faded completely; latter look yellowish-gray.
Natural history notes: The first record of Vietnamophryne occidentalis sp. nov. from Doi Tung Mt. was made by Akrachai Aksornneam on 10 February 2017. The specimen was encountered under a tree log at an elevation of ca. 1 000 m a.s.l. but was not collected. The holotype male specimen of the new species was encountered on 5 April 2017 during the day (1400 h) after heavy rain. The specimen was found at an elevation of ca. 1 050 m a.s.l. in leaf litter near a forest trail ( Figure 11B) on the slope of Doi Tung Mt. with limestone outcrops ( Figure 11A).
The climate of Doi Tung Mountain, Chiang Rai Province, is monsoonal with three distinct seasons: cool-dry from November to February, hot-dry from March to May, and rainy from May-June to November. The average annual rainfall is 2 500 mm at 1 200 m.
As in other species of Vietnamophryne Gen. nov., the biology of Vietnamophryne occidentalis sp.
nov. remains completely unknown. Both known specimens were encountered during the day in soil under a large log or in leaf litter after heavy rain. As in other species of Vietnamophryne Gen. nov., we assume that Vietnamophryne occidentalis sp. nov. has a secretive lifestyle and spends considerable time underground or in leaf litter. Despite intensive search efforts, only two specimens were encountered during two surveys. No calling activity was recorded during either survey, and reproductive biology and diet of Vietnamophryne orlovi sp. nov. remain unknown.  Comparisons: For discrimination from other microhylid frogs occurring in Indochina, see "Comparisons with other Microhylidae genera inhabiting mainland Southeast Asia" above. For comparisons with Vietnamophryne inexpectata sp. nov. and Vietnamophryne orlovi sp. nov. see the "Comparisons" sections above.

Distribution and biogeography:
To date, Vietnamophryne occidentalis sp. nov. is known only from its type locality in montane subtropical forest on limestone outcrops of Doi Tung Mt., Pong Ngam District, Chaing Rai Province, northern Thailand, at an elevation of ca. 1 050 m a.s.l. Mt. Doi Tung belongs to a small mountain ridge located on the border between Chiang Rai Province of Thailand and Shan State of Myanmar; thus, the occurrence of the new species in adjacent parts of Myanmar is highly anticipated.

Conservation status:
To date, Vietnamophryne occidentalis sp. nov. is known from a single locality based on one unvouchered record and the holotype specimen. Similar to other members of the genus Vietnamophryne Gen. nov., it is likely that the new species has a secretive semi-fossorial biology. Additional focused survey efforts in adjacent parts of Thailand and Myanmar are required to clarify the range and population status of Vietnamophryne occidentalis sp. nov. Given the available information, we suggest Vietnamophryne occidentalis sp. nov.sp. nov. be considered as a Data Deficient (DD) species following IUCN's Red List categories (IUCN Standards and Petitions Subcommittee, 2016).

Etymology:
The specific name "occidentalis" is a Latin adjective in the nominative singular meaning "western"; referring to the type locality of the new species in western Indochina (Chiang Rai Province of Thailand) -to date, the westernmost area where members of the subfamily Asterophryinae are recorded.

Suggested common names:
We recommend the following common names for the new species: "Chiang Rai Dwarf Frog" (English) and "Eung Tham Khaera Chiang Rai" (Thai).

DISCUSSION
In this work, we report on the discovery of a new lineage of Asterophryinae microhylid frogs from Indochina. Vietnamophryne is a genus of small miniaturized frogs. Although the specimens were mostly recorded in soil or under large tree-trunks, suggesting a semi-fossorial lifestyle, they lack obvious adaptations for digging. Due to their secretive underground biology, they have been encountered by herpetologists only rarely and have remained almost unnoticed despite 200 years of herpetological studies in Indochina. Even with our intensive effort, we were unable to collect additional specimens of the three new species from the three localities in Vietnam and Thailand. It is anticipated, however, that members of the genus Vietnamophryne will be discovered in other parts of Indochina, including central and northern Vietnam, Laos, and northern Myanmar. Our work calls for intensification of focused herpetological surveys combined with molecular analyses to further our understanding of amphibian biodiversity in Indochina. Intensive examination of museum herpetological collections also might result in the discovery of Asterophryinae specimens, as these frogs may have been misidentified as juveniles of other microhylid species in previous work.
As predicted by Kurabayashi et al. (2011), Vietnamophryne represents an ancient lineage of Asterophryinae differentiation distributed deep in mainland Southeast Asia (northern Indochina). Here, Vietnamophryne was reconstructed as a sister lineage to Siamophryne from southern Indochina (north of Isthmus of Kra, Figure 1; Suwannapoom et al., 2018), and the clade joining the two latter genera was determined to be a sister clade to Gastrophrynoides from Sundaland (south of Isthmus of Kra, Figure 1). Thus, our discovery of the genus Vietnamophryne and three constituent species brings the number of Asterophryinae species reported for Indochina to five, and illustrates that the basal cladogenetic events within the subfamily most likely occurred on the Eurasian landmass, followed by subsequent radiation. This further supports the "out of Indo-Eurasia" scenario of Kurabayashi et al. (2011): according to their divergence estimates, the common ancestor of Asterophryinae diverged from other Microhylidae lineages during the late Cretaceous (possibly on the Indian subcontinent), and the basal split within the subfamily occurred during the Eocene (∼48 Ma, Kurabayashi et al., 2011). Our data suggest that this split, separating the ancestor of Gastrophrynoides+Siamophryne+Vietnamophryne from the ancestor of all other "core" Australasian Asterophryinae, most likely took place in Indochina.
While the "core" Asterophryinae ancestors dispersed further eastwards, crossed the Wallace line, colonized the Australasian landmass, and diversified during the late Oligocene (∼25 Ma, Rivera et al., 2017), the cladogenesis within the Eurasian Asterophryinae was less intensive. Divergence within the genus Vietnamophryne was, most likely, a comparatively recent event due to the small genetic distances observed among species. A similar biogeographic "out of Indochina to Australasia" pattern has been reported in several other taxonomically diverse groups of amphibians and reptiles. For example, Yan et al. (2016) demonstrated that the speciose frog family Ceratobatrachidae (Natatanura) originated in the eastern Himalayas and Tibet, from where it colonized and subsequently radiated to the islands of the Australasian archipelago. Wood et al. (2012) reported a generally similar biogeographic pattern for the most diverse genus of geckoes (Cyrtodactylus), suggesting that the genus formed in the eastern Tibet-Himalayan region, from where it colonized the tropical areas of South and Southeast Asia. According to this scenario, Indochina served as a local diversification center of Cyrtodactylus, with several waves of dispersal allowing this genus to colonize Sundaland, Lesser Sunda Islands, the Philippines, Papua New Guinea, and adjacent Australasian islands and northern Australia (Wood et al., 2012). Hence, the biogeographic scenarios for at least two of most speciose Australasian frog families and the most speciose gecko genus argue an initial origination and cladogenesis in mainland Southeast Asia followed by dispersal into the Australasian archipelago and subsequent radiation. Our study further suggests that the Indochinese Peninsula played a key role in the formation of the herpetofauna of Southeast Asia and Australasia.
Our dataset on the "core" Asterophryinae was based on sequences obtained from earlier studies (see Table 1 for details), and our results on phylogenetic relationships among members of the Asterophryinae 1 clade were generally in accordance with previously published data. This speciose group underwent adaptive radiation in the Australo-Papuan region, with members of the Asterophryinae 1 clade demonstrating various lifestyles, including arboreal, scansorial, terrestrial, burrowing (fossorial), and semi-aquatic (Rivera et al., 2017). This adaptive radiation has led to numerous homoplasies and reversal shifts in the evolution of morphological characteristics, thus hampering the progress of generic taxonomy based solely on morphological evidence (Burton, 1986;Köhler & Günther, 2008;Menzies, 2006;Rivera et al., 2017;Zweifel, 1972).
The multilocus analysis of phylogenetic relationships following wide sampling of New Guinean asterophryines by Rivera et al. (2017) showed that basal radiation of Asterophryinae occurred in a narrow timeframe between 20-27 Ma and was accompanied by numerous ecomorphological shifts. Rivera et al. (2017) pointed out 11 asterophryine genera as paraphyletic, suggesting that in most cases they can be brought into monophyly by collapsing genera (Albericus is synonymized with Choerophryne; Oreophryne clade 3 is synonymized with Aphantophryne; Genyophryne, Oxydactyla and Liophryne are synonymized with Sphenophryne). However, some of the groups in Rivera et al. (2017) tree got low or no node support, thus hampering further taxonomic decisions (e.g., Austrochaperina and Copiula). Though based on limited taxon and molecular sampling, our analysis indicated that Sphenophryne thomsoni, previously assigned to the genus Genyophryne, was a sister lineage to the clade that united Cophixalus and Choerophryne, thus suggesting that the synonymization of Genyophryne with Sphenophryne may be premature.
It is obvious that generic taxonomy of Asterophryinae is still in a state of flux and further molecular and morphological research is needed to achieve a better taxonomic hypothesis for this group.
Due to the paucity of observations and very limited sampling, the natural history of Vietnamophryne remains almost completely unknown. Our data suggest that the new genus prefers undisturbed evergreen forests with a secretive, possibly semi-fossorial, lifestyle, and spends substantial time sheltering in leaf litter and soil. We have no information on diet, enemies, reproduction, or life cycle of the new genus. All members of the "core" Asterophryinae clade inhabiting Australasia, for which breeding has been observed, are known to have direct development -i.e., a life cycle with metamorphosis taking place within the egg (Günther et al., 2012b;Menzies, 2006). However, the recently discovered Siamophryne has a peculiar tadpole, which is, to date, the only record of the existence of a larval stage for Asterophryinae (Suwannapoom et al., 2018). The reproductive biology and development of Gastrophrynoides also remain unknown (Chan et al., 2009;Parker, 1934). Superficially, the miniaturized Vietnamophryne resembles some small ground-dwelling genera of the "core" Asterophryinae, which exhibit direct development. However, as all collected specimens of Vietnamophryne were males, we cannot speculate on the possible reproduction mode of the new species. Due to the ancient divergence and phylogenetic history, morphological differences, and peculiarities of life cycle (e.g., larval development in Siamophryne), we cannot exclude that the taxonomic status of the Eurasian Asterophryinae lineage might be reconsidered in the future.
Our work adds a new genus and three new species of frogs to the batrachofauna of Indochina. The real extent of distribution of the species described herein is unknown and requires further study. Undisturbed montane forests of eastern and northern Indochina cradle one of the richest herpetofaunas in the world (Poyarkov et al., unpublished data). However, deforestation is a growing threat in Indochina, especially in Vietnam (Meyfroidt & Lambin, 2008), and habitat loss and modification are widely recognized as major threats to amphibians in Southeast Asia. Forest specialist species restricted to primary undisturbed broadleaf evergreen montane forests would be especially vulnerable to changes in their environment.
Further field survey efforts and molecular taxonomic studies are essential for the effective estimation and conservation management of amphibian biodiversity in Indochina.