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

Abstract

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. External morphology characters and osteological characteristics analyzed using micro-CT scanning were used for describing 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.

Full text

ZOOLOGICAL RESEARCH
A new genus and three new species of miniaturized
microhylid frogs from Indochina (Amphibia: Anura:
Microhylidae: Asterophryinae)
Nikolay A. Poyarkov, Jr.1,2,*, Chatmongkon Suwannapoom3, Parinya Pawangkhanant3, Akrachai Aksornneam4, Tang Van
Duong1,5, Dmitriy V. Korost6, Jing Che7,8
1Department of Vertebrate Zoology, Biological Faculty, Lomonosov Moscow State University, Moscow 119234, Russia
2Joint Russian-Vietnamese Tropical Research and Technological Center, Nghia Do, Cau Giay, Hanoi, Vietnam
3Division of Fishery, School of Agriculture and Natural Resources, University of Phayao, Phayao 56000, Thailand
4Department of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
5Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, Hanoi, Vietnam
6Petroleum Geology Department, Geological Faculty, Lomonosov Moscow State University, Moscow 119234, Russia
7
State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan
650223, China
8Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences, Yezin Nay Pyi Taw 05282, Myanmar
ABSTRACT
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.
Keywords:
Vietnamophryne
Gen. nov.
;Vietnamophryne
inexpectata
sp. nov.
;Vietnamophryne orlovi
sp. nov.
;Vietnamophryne occidentalis
sp. nov.
;
Siamophryne;Gastrophrynoides; mtDNA; micro-CT
scanning; Vietnam; Thailand; Herpetofauna;
Amphibia; Biogeography; Taxonomy; Indochina
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
Received: 30 January 2018; Accepted: 03 March 2018; Online:
20 April 2018
Foundation items: This work was supported by the programs of the
National Natural Science Foundation of China (31501843, 31622052),
Southeast Asia Biodiversity Research Institute, Chinese Academy of
Sciences (CAS) (Y4ZK111B01: 2017CASSEABRIQG002), Animal
Branch of the Germplasm Bank of Wild Species, CAS (Large
Research Infrastructure Funding), and Thailand Research Fund
(TRF) (DBG6180001). Molecular experiments, phylogenetic analyses,
specimen storage, examination and microCT-analysis were carried out
with the financial support of the Russian Science Foundation (RSF
14-50-00029)
*Corresponding author, E-mail: n.poyarkov@gmail.com
DOI: 10.24272/j.issn.2095-8137.2018.019
130 Science Press Zoological Research 39(3): 130-155, 2018

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 Siamophryne —
a 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.
MATERIALS AND METHODS
Sample collection
Field work was conducted from 23 May to 2 June 2016 in
Kon Chu Rang Nature Reserve, Gia Lai Province, Tay Nguyen
Plateau, central Vietnam (N14.506◦, E108.542◦; elevation 1 000
m a.s.l.); from 8 to 17 June 2017 in Phia Oac-Phia Den
National Park, Cao Bang Province, northern Vietnam (N22.600◦,
E105.884◦; elevation 1 200 m a.s.l.) and from 5 to 15 February
and 4 to 8 April 2017 in the environs of Doi Tung Mt., Pong Ngam
District, Chaing Rai Province, northern Thailand (N20.344◦,
E99.830◦; elevations from 900 to 1 050 m a.s.l.). 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-chloroform-
proteinase K (final concentration 1 mg/mL) extraction with
subsequent isopropanol precipitation (as per Hillis et al., 1996
and Sambrook & 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 tRNAVal 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 MgCl2, Taq
PCR buffer (10 mmol/L of Tris-HCl, pH 8.3, 50 mmol/L of KCl,
1.1 mmol/L of MgCl2and 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
Zoological Research 39(3): 130-155, 2018 131

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).
Figure 1 Known distribution of main Asterophryinae lineages and new genus Vietnamophryne Gen. nov. (yellow stars)
Biogeographic borders: A: Isthmus of Kra line, approximate biogeographic border between Sundaland and Indochina; B-1: Wallace line (after Huxley
(1868)); B-2: Wallace line (after Mayr, 1944); C: Weber line; D: Lyddeker line. Most Asterophryinae genera inhabit Australasia, east of the Wallace line
(red), and Bali; Gastrophrynoides is confined to Sundaland (Borneo and Malaysian Peninsula; blue circles); Siamophryne is known from a single locality in
Tenasserim, southern Thailand (green diamond); Vietnamophryne Gen. nov. occurs in northern and eastern Indochina in Thailand and Vietnam. Localities:
1: Vietnamophryne inexpectata sp. nov.: Kon Chu Rang N.R., Gia Lai Province, Vietnam; 2: Vietnamophryne orlovi sp. nov.: Phia Oac-Phia Den N.P., Cao
Bang Province, Vietnam; 3: Vietnamophryne occidentalis sp. nov.: Doi Tung Mt., Chiang Rai Province, Thailand.
Phylogenetic analyses
For phylogenetic analysis we used the 12S rRNA and 16S
rRNA Microhylidae dataset of Suwannapoom et al. (2018) with
the addition of the newly obtained sequences of Microhylidae
Gen. spp. from Vietnam and Thailand. Data on sequences and
specimens used in molecular analyses are summarized in Table
1. In total, sequences of the 12S rRNA and 16S rRNA mtDNA
fragments of 53 microhylid representatives were included in the
final analysis: including three samples of Microhylidae Gen.
spp. from central and northern Vietnam and northern Thailand;
27 samples of the subfamily Asterophryinae (25 specimens
of Australasian asterophryine genera and two specimens
of Gastrophrynoides and Siamophryne from Sundaland and
Tenasserim, respectively); 18 samples of Asian microhylids
representing all major lineages of the family inhabiting this
region (including subfamilies Microhylinae, Kalophryninae,
Melanobatrachinae, and Chaperininae); and five outgroup
sequences of non-Asian Microhylidae, including subfamilies
Dyscophinae (Madagascar), Gastrophryninae (North America),
Phrynomerinae (Africa), and Scaphiophryninae (Madagascar).
An mtDNA sequence of Rhacophorus schlegelii (Günther)
(Rhacophoridae) was used as a non-microhylid outgroup.
132 www.zoores.ac.cn

Table 1 Specimens and sequences of three new species of Vietnamophryne Gen. nov. from Indochina and outgroup
representatives of Microhylidae and Rhacophoridae used in molecular analyses
Group GenBank accession No. Species Specimen ID Reference
Asterophryinae DQ283195 Aphantophryne pansa ABTC 49605 Frost et al., 2006
Asterophryinae FR832625; FR832642 Asterophrys (Asterophrys) turpicola ZMB 70537 Günther et al., 2010
Asterophryinae KM509160 Asterophrys (Metamagnusia) slateri PT-507 Peloso et al., 2016
Asterophryinae FR832653; FR832636 Asterophrys (Pseudocallulops) eurydactyla ZMB 70534 Günther et al., 2010
Asterophryinae JN048979; JN049004 Austrochaperina guttata LSUMZ 95008 Rittmeyer et al., 2012
Asterophryinae KC822485 Austrochaperina sp. BSFS 11377 Blackburn et al., 2013
Asterophryinae EU100119; EU100235 Barygenys exsul BPBM 20128 Köhler & Günther, 2008
Asterophryinae KM509105 Callulops robustus PT-506 Peloso et al., 2016
Asterophryinae DQ283207 Choerophryne sp. ABTC 47720 Frost et al., 2006
Asterophryinae DQ283206 Cophixalus sphagnicola ABTC 47881 Frost et al., 2006
Asterophryinae DQ283208 Copiula sp. AMS R124417 Frost et al., 2006
Asterophryinae AB634647; AB634705 Gastrophrynoides immaculatus UKMHC 279 Matsui et al., 2011
Asterophryinae JX119248; JX119392 Hylophorbus rufescens LSUMZ 94943 Oliver et al., 2013
Asterophryinae JN048989; JN049014 Mantophryne lateralis LSUMZ 92102 Rittmeyer et al., 2012
Asterophryinae MH004403 Vietnamophryne inexpectata Gen. et sp. nov. ZMMU A-5820 This work
Asterophryinae MH004404 Vietnamophryne orlovi Gen. et sp. nov. ZMMU A-5821 This work
Asterophryinae MH004406 Vietnamophryne occidentalis Gen. et sp. nov. ZMMU A-5822 This work
Asterophryinae MG682553 Siamophryne troglodytes ZMMU NAP-06651 Suwannapoom et al., 2018
Asterophryinae FR832634; FR832635 Oninia senglaubi ZMB 74608 Günther et al., 2010
Asterophryinae KC822488 Oreophryne anulata PNMCMNHH 1366 Blackburn et al., 2013
Asterophryinae DQ283194 Oreophryne brachypus ABTC 50081 Frost et al., 2006
Asterophryinae AB634651; AB634709 Oreophryne monticola MZBAmp 16265 Matsui et al., 2011
Asterophryinae KC822489 Oreophryne variabilis TNHC 58922 Blackburn et al., 2013
Asterophryinae JN048996; JN049021 Paedophryne amauensis BPBM 31882 Rittmeyer et al., 2012
Asterophryinae JX119386; JX119242 Sphenophryne (Sphenophryne) cornuta LSUMZ 94793 Oliver et al., 2013
Asterophryinae DQ283209 Sphenophryne (Genyophryne) thomsoni ABTC 49624 Frost et al., 2006
Asterophryinae DQ283199 Sphenophryne (Liophryne) rhododactyla ABTC 49566 Frost et al., 2006
Asterophryinae EU100323; EU100207 Sphenophryne (Oxydactyla) crassa BPBM 17061 Köhler & Günther, 2008
Asterophryinae FR832655; FR832638 Xenorhina cf. oxycephala ZMB 74628 Günther et al., 2010
Asterophryinae KM509212 Xenorhina obesa PT-529 Peloso et al., 2016
Chaperininae AB598318; AB598342 Chaperina fusca BORN 8478 Matsui et al., 2011
Dyscophinae AB634648; AB634706 Dyscophus guineti KUHE 33150 Matsui et al., 2011
Dyscophinae AB634649; AB634707 Dyscophus insularis KUHE 35001 Matsui et al., 2011
Gastrophryninae AB634650; AB634708 Gastrophryne olivacea KUHE 33224 Matsui et al., 2011
Kalophryninae AB634642; AB634700 Kalophrynus pleurostigma MZBAmp 15295 Matsui et al., 2011
Kalophryninae AB634645; AB634703 Kalophrynus subterrestris KUHE 53145 Matsui et al., 2011
Melanobatrachinae KM509159 Melanobatrachus indicus IND-18 Peloso et al., 2016
Microhylinae AB201182; AB201193 Glyphoglossus molossus KUHE 35182 Matsui et al., 2011
Microhylinae AB634626; AB634684 Glyphoglossus yunnanensis KUHE 44148 Matsui et al., 2011
Microhylinae KP682314 Kaloula rugifera – Deng et al., 2016
Microhylinae AB634634; AB634692 Metaphrynella pollicaris KUZ-21655 Matsui et al., 2011
Microhylinae AB634600; AB634658 Microhyla annectens – Matsui et al., 2011
Microhylinae DQ512876 Microhyla fissipes – unpublished
Microhylinae NC006406 Microhyla heymonsi – Zhang et al., 2005
Microhylinae AB303950 Microhyla okinavensis – Igawa et al., 2008
Microhylinae AB634616; AB634674 Microhyla petrigena – Matsui et al., 2011
Microhylinae NC024547 Microhyla pulchra – Wu et al., 2016
Microhylinae AB598317; AB598341 Micryletta inor nata KUHE 20497 Matsui et al., 2011
Microhylinae AB634638; AB634696 Micryletta steinegeri KUHE 35937 Matsui et al., 2011
Microhylinae AB634636; AB634694 Phrynella pulchra UKMHC 820 Matsui et al., 2011
Microhylinae AB634633; AB634691 Uperodon taprobanicus KUHE 37252 Matsui et al., 2011
Phrynomerinae AB634652; AB634710 Phrynomantis bifasciatus KUHE 33277 Matsui et al., 2011
Scaphiophryninae AB634653; AB634711 Scaphiophryne gottlebei KUHE 34977 Matsui et al., 2011
Rhacophoridae AB202078 Rhacophorus schlegelii - Sano et al., 2005
–: Not available.
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
Zoological Research 39(3): 130-155, 2018 133

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).
Adult morphology
Sex of adult individuals was determined using gonadal
dissection. All measurements were taken to the nearest
0.02 mm and subsequently rounded to a 0.1 mm precision
from preserved specimens using a digital caliper under a light
dissecting microscope. Measurements included the following
40 morphometric characters, as per Poyarkov et al. (2014)
and Suwannapoom et al. (2018): (1) snout-vent length (SVL;
length from tip of snout to cloaca); (2) head length (HL; length
from tip of snout to hind border of jaw angle); (3) snout
length (SL; length from anterior corner of eye to tip of snout);
(4) eye length (EL; distance between anterior and posterior
corners of eye); (5) nostril-eye length (N-EL; distance between
anterior corner of eye and nostril center); (6) head width (HW;
maximum width of head at level of mouth angles in ventral
view); (7) internarial distance (IND; distance between central
points of nostrils); (8) interorbital distance (IOD; shortest
distance between medial edges of eyeballs in dorsal view);
(9) upper eyelid width (UEW; maximum distance between
medial edge of eyeball and lateral edge of upper eyelid);
(10) forelimb length (FLL; length of straightened forelimb
from limb base to tip of third finger); (11) lower arm and
hand length (LAL; distance between elbow and tip of third
finger); (12) hand length (HAL; distance between proximal
end of outer palmar (metacarpal) tubercle and tip of third
finger); (13) inner palmar tubercle length (IPTL; maximum
distance between proximal and distal ends of inner palmar
tubercle); (14) outer palmar tubercle length (OPTL; maximum
diameter of outer palmar tubercle); (15) hindlimb length (HLL;
length of straightened hindlimb from groin to tip of fourth toe);
(16) tibia length (TL; distance between knee and tibiotarsal
articulation); (17) foot and tibiotarsus length (FTL; length from
tibiotarsal joint to end of fourth toe); (18) foot length (FL;
distance between distal end of tibia and tip of fourth toe);
(19) inner metatarsal tubercle length (IMTL; maximum length
of inner metatarsal tubercle); (20) outer metatarsal tubercle
length (OMTL; maximum length of outer metatarsal tubercle);
(21) tympanum length, maximum tympanum diameter (TYD);
(22) tympanum-eye distance (TED); (23–26) finger lengths
(1–3FLO, 4FLI; for outer side (O) of first, inner side (I) of fourth,
distance between tip and junction of neighboring finger); (27)
first finger width (1FW), measured at distal phalanx; (28–30)
finger disk diameters (2–4FDW); (31) first toe length (1TOEL),
distance between distal end of inner metatarsal tubercle and
tip of first toe; (32–35) second to fifth toe lengths (outer
lengths for toes II–IV, inner length for toe V); (36–40) toe disk
diameters (1–5TDW).
The morphological characters for comparison and data
on states in other Microhylidae representatives were taken
from: Burton (1986), Chan et al. (2009), Günther & Richards
(2016), Günther (2009, 2017), Günther et al. (2010, 2012a,
2012b, 2014, 2016), Köhler & Günther (2008), Kraus & Allison
(2003), Kraus (2010, 2011, 2013a, 2013b, 2014, 2016, 2017),
Menzies & Tyler (1977), Parker (1934), Richards & Iskandar
(2000), Richards et al. (1992, 1994), Rittmeyer et al. (2012),
Suwannapoom et al. (2018), Zweifel (1972, 2000), and Zweifel
(2003).
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, 1973). Micro-CT does not render cartilage,
and therefore cartilage structures were omitted from the
osteological descriptions.
RESULTS
Sequence variation
Final alignment of the studied 12S rRNA and 16S rRNA
mtDNA fragments consisted of 2 591 sites: 1 059 sites were
conserved and 1 408 sites were variable, of which 1 082
were parsimony-informative. The transition-transversion bias
(R) was to 2.14. Nucleotide frequencies were A=34.21%,
T=22.89%, C=24.95%, and G=17.95% (data given only for
Microhylidae ingroup).
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. General
topology of the phylogenetic relationships of the Microhylidae
frogs was consistent with results reported in a number of
134 www.zoores.ac.cn

recent studies (De Sá et al., 2012; Kurabayashi et al., 2011;
Matsui et al., 2011; Peloso et al., 2016; Pyron & Wiens, 2011;
Rivera et al., 2017; Suwannapoom et al., 2018; Van Der
Meijden et al., 2007).
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
(mainland Southeast Asia and Sundaland) and included
the genus Gastrophrynoides (Malay Peninsula and Borneo;
lineage Asterophryinae 4 in Figure 2; range in Figure 1
marked in blue), recently discovered genus Siamophryne
(Tenasserim in southern Thailand; lineage Asterophryinae 3;
locality in Figure 1 marked in green), and the three newly
discovered microhylids from central and northern Vietnam
and northern Thailand (lineage Asterophryinae 2 on Figure 2;
localities in Figure 1 marked in yellow).
Figure 2 Bayesian inference dendrogram of Asterophryinae derived from analysis of 2 591-bp long 12S rRNA – 16S rRNA mtDNA
gene fragments
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.
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
Zoological Research 39(3): 130-155, 2018 135

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,
2005b; Vieites 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. 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., 2005a, 2005b; Vieites 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.
Subsequent analyses of osteology and external morphology
(see below) strongly suggest that the recently discovered
miniaturized Microhylidae Gen. sp. from northern and eastern
Indochina represent a new previously undescribed genus with
three new species, which we describe herein:
Amphibia Linnaeus, 1758
Anura Fischer von Waldheim, 1813
Microhylidae Günther, 1858
Asterophryinae Günther, 1858
Vietnamophryne Gen. nov.
Diagnosis: Small-sized (14.2 mm<SVL<20.5 mm) member
of the mainly Australasian subfamily Asterophryinae (family
Microhylidae), with the following combination of morphological
attributes: (1) both maxillae and dentaries eleutherognathine,
no maxillary teeth; (2) vertebral column procoelous with
eight presacral vertebrae lacking neural crests; (3) no cranial
sagittal crest; (4) frontoparietals connected by long non-calcified
suture; (5) nasals wide, calcified, not contacting medially; (6)
vomeropalatines and neopalatine not expanded, not calcified
(possibly, cartilaginous), vomerine spikes absent; (7) cultriform
process of parasphenoid broad and short, abruptly obtuse
anteriorly; (8) clavicles absent; (9) omosternum absent;
(10) sternum small, non-calcified, completely cartilaginous,
xiphisternum flat, rounded; (11) distinct dorsal crest present
on urostyle at three-quarters of its length, absent on ilium;
(12) terminal phalanges small, bobbin-shaped; (13) no disks
on digits, digit tips rounded; (14) first finger reduced to nub or
shortened, all phalanges present and ossified; (15) subarticular
tubercles absent; (16) toe webbing absent; (17) tympanum
distinct; (18) single transverse smooth palatal fold; (19) pupil
round; (20) snout rounded, subequal to or shorter than eye
length; (21) skin on dorsum warty to shagreened; and (22)
semi-fossorial (mostly subterranean) lifestyle.
Type species: Vietnamophryne inexpectata sp. nov.
Other included species: Vietnamophryne orlovi sp. nov.;
Vietnamophryne occidentalis sp. 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.
136 www.zoores.ac.cn

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)
Taxon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
1Vietnamophryne inexpectata Gen. et sp. nov. — 1.1 1.1 1.9 2.3 2.5 2.0 1.7 2.0 2.2 2.2 2.4 2.1 2.2 2.1 2.1 2.1 2.1 2.3 1.7 2.2 1.9 1.9 2.3 1.9
2Vietnamophryne orlovi Gen. et sp. nov. 5.1 — 1.0 1.9 2.3 2.6 2.1 1.9 2.2 2.2 2.6 2.7 2.3 2.4 2.3 2.1 2.1 2.1 2.5 1.8 2.3 2.1 2.1 2.3 2.1
3Vietnamophryne occidentalis Gen. et sp. nov. 3.1 4.7 — 1.9 2.2 2.2 1.8 1.7 1.9 2.2 2.2 2.5 1.9 2.1 2.1 2.0 2.0 2.0 2.2 1.5 2.2 2.0 1.8 2.2 1.9
4Siamophryne 15.1 12.6 14.8 — 2.1 2.3 2.2 1.7 2.2 2.2 2.3 2.3 2.1 2.1 2.2 2.1 2.3 2.1 2.1 1.9 2.2 1.9 2.3 2.3 2.1
5Gastrophrynoides 17.5 16.5 16.8 17.2 — 2.4 2.4 1.9 2.3 2.6 2.5 2.8 2.4 2.4 2.4 2.2 2.5 2.4 2.4 2.0 2.1 2.0 2.0 2.4 2.3
6Aphantophryne 19.6 20.9 19.9 17.5 17.9 — 2.0 1.6 2.5 2.2 2.2 2.4 2.1 2.2 2.1 2.0 2.4 2.0 2.2 1.8 2.1 2.0 2.1 2.1 2.0
7Asterophrys s.str. 16.8 15.4 15.5 17.9 16.5 15.8 — 1.4 1.9 1.8 2.0 2.2 1.9 2.2 2.0 1.7 2.1 1.3 1.8 1.4 1.9 1.9 1.7 2.0 1.8
8Austrochaperina 17.7 17.7 17.9 17.9 17.0 15.8 12.9 12.7 1.7 1.7 1.7 1.8 1.6 1.9 1.6 1.5 1.6 1.5 1.7 1.2 1.5 1.4 1.3 1.8 1.5
9Barygenys 17.6 17.4 17.2 19.7 18.3 19.7 12.4 14.5 — 2.1 2.1 2.3 2.2 2.0 2.2 1.8 2.0 2.1 2.1 1.8 2.1 2.1 2.0 1.9 2.2
10 Callulops 21.4 19.8 21.4 20.0 17.6 16.6 12.8 15.5 16.3 — 2.0 2.3 2.0 2.3 1.6 2.0 1.7 2.0 2.2 1.7 1.9 2.1 1.8 1.8 2.0
11 Choerophryne 20.0 20.9 20.7 24.8 22.4 19.3 19.3 17.9 20.1 19.7 — 2.2 2.3 2.1 2.2 2.2 2.2 2.2 2.3 1.7 2.2 2.2 2.0 2.2 2.2
12 Cophixalus 19.0 19.0 20.0 20.7 21.0 17.9 16.2 16.9 16.3 17.0 20.4 — 2.1 2.3 2.5 2.1 2.7 2.4 2.5 1.8 2.2 2.3 2.3 2.2 2.3
13 Copiula 16.7 16.4 16.4 17.4 18.1 15.3 14.6 14.1 17.5 17.5 18.9 17.5 — 2.1 2.0 2.0 2.1 1.7 2.0 1.6 2.2 2.0 1.7 2.2 1.9
14 Genyophryne 16.6 15.0 16.2 17.9 17.9 16.2 15.9 16.6 15.2 19.7 18.0 17.3 17.5 — 2.4 1.9 2.4 2.2 2.2 1.8 2.3 2.1 2.0 2.2 2.1
15 Hylophorbus 17.2 15.4 17.2 18.2 18.6 17.2 14.4 14.9 19.0 13.4 17.2 19.0 14.6 18.6 — 2.1 1.6 2.0 2.2 1.6 2.0 2.1 1.9 2.2 1.8
16 Liophryne 17.2 16.9 16.5 15.8 15.8 14.8 12.0 14.4 13.8 12.4 18.6 15.2 13.2 16.2 13.7 — 2.1 1.7 2.0 1.4 1.8 1.9 2.0 1.6 1.8
17 Mantophryne 16.2 13.8 16.2 19.0 17.9 17.2 15.2 13.3 16.6 13.1 18.3 17.6 15.4 16.3 9.0 13.8 — 2.1 2.3 1.8 2.2 2.2 2.0 2.0 2.2
18 Metamagnusia 15.1 13.8 14.1 17.5 15.8 15.5 5.8 12.4 14.8 14.5 18.3 15.2 12.5 15.2 14.4 12.4 12.8 — 1.9 1.4 2.0 1.8 1.6 1.8 1.8
19 Oninia 19.0 17.8 17.6 17.6 18.3 17.6 17.2 17.9 18.3 20.4 21.1 21.8 17.9 19.7 17.2 15.5 18.0 15.9 — 1.7 1.9 2.1 2.1 2.1 1.9
20 Oreophryne 17.9 18.7 17.6 19.8 18.4 16.9 13.8 15.3 16.8 17.6 19.6 17.7 15.7 18.4 17.3 14.1 17.7 13.5 18.8 15.1 1.5 1.5 1.4 1.6 1.7
21 Oxydactyla 17.5 17.7 17.5 19.6 14.8 18.9 12.4 13.1 15.5 13.4 19.0 13.8 17.1 18.3 14.8 10.7 14.1 12.4 17.6 15.6 — 2.0 1.8 1.9 1.9
22 Paedophryne 18.6 18.6 18.6 19.3 16.9 17.6 14.8 15.2 17.6 18.3 21.8 18.3 18.2 17.3 19.0 15.5 17.3 12.8 20.4 16.7 14.8 — 2.0 2.1 2.0
23 Pseudocallulops 17.2 15.7 17.2 19.2 17.5 15.8 12.0 13.4 17.2 14.1 15.9 17.2 14.2 14.8 13.7 15.1 13.1 10.7 18.3 15.7 14.8 15.9 — 1.9 1.8
24 Sphenophryne s.str. 18.6 17.3 17.5 18.2 17.9 16.2 12.4 14.4 15.2 13.8 18.3 16.2 14.6 15.9 15.1 9.6 12.8 11.3 16.9 15.3 13.4 15.9 16.5 — 2.0
25 Xenorhina 17.5 17.3 17.7 18.6 18.2 15.6 14.1 14.2 16.6 14.5 19.7 17.5 16.3 18.7 16.7 15.1 16.5 13.2 19.6 17.0 16.6 17.9 13.8 16.2 14.0
Mean uncorrected intrageneric P-distances for the ingroup are shown in the diagonal in bold.
Zoological Research 39(3): 130-155, 2018 137

Comparisons with other Asterophryinae genera:
Information on character states for other Asterophryinae genera
is based on Parker (1934), Zweifel (1972, 2000), Menzies
& Tyler (1977), Burton (1986), Zweifel (2003), Günther et
al. (2010), Kraus (2010, 2017), Suwannapoom et al. (2018),
and references therein. Vietnamophryne Gen. nov. can be
distinguished from Asterophrys (including recently synonymized
Pseudocallulops and Metamagnusia; Rivera et al., 2017),
Callulops,Mantophryne,Oninia, and Xenorhina (including
recently synonymized Xenobatrachus Peters & Doria) by
eleutherognathine maxillae and dentaries (vs. symphignathine
maxillae and dentaries in all these Asterophryinae genera),
and from Barygenys (vs. symphignathine dentaries and
eleutherognathine maxillae). Vietnamophryne Gen. nov. can
be differentiated from genera Aphantophryne and Cophixalus
by lack of distinct neural crests on presacral vertebrae
(vs. 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. small finger disks present).
Vietnamophryne Gen. nov. can be further diagnosed from
the genus Oxydactyla (coined as a synonym of Sphenophryne
by Rivera et al., 2017) by F1 small or greatly reduced to
nub (1FL1
/22FL) (vs. F1 well-developed, 1FL≥1
/22FL).
Vietnamophryne Gen. nov. can be distinguished from the
genus Genyophryne (coined as a synonym of Sphenophryne by
Rivera et al., 2017) by absence of clavicles (vs. small clavicles
present), lack of vomeropalatines and vomerine spikes (vs.
expanded vomeropalatines with vomerine spikes), and F1 very
small or reduced to nub, 1FL<1
/22FL (vs. F1 well-developed,
1FL≥1
/22FL). Vietnamophryne Gen. 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).
Among the Asterophryinae lineages inhabiting areas derived
from the Eurasian landmass, Vietnamophryne Gen. nov.
can be easily distinguished from the genus Gastrophrynoides
(Malay Peninsula and Borneo) by snout rounded, length equal
to or slightly more than eye length (vs. snout pointed, 2.5 times
longer than eye; Figure 2), distinct tympanum (vs. tympanum
obscured by skin; Figure 2), F1 very small or reduced to nub,
1FL1
/22FL (vs. F1 well-developed, 1FL≥1
/22FL), generally
smaller body size, SVL≤20.5 mm (vs. SVL>20.0 mm), distinct
crest on urostyle (vs. no crest on urostyle), bobbin-shaped
terminal phalanges (vs. T-shaped terminal phalanges), single
smooth palatal fold (vs. two palatal folds), comparatively broad
cultriform process of parasphenoid (vs. narrow cultriform
process of parasphenoid), and shagreened to warty skin (vs.
completely smooth skin).
Vietnamophryne Gen. nov. can be easily distinguished
from its sister genus Siamophryne (Tenasserim, south-western
Thailand) by absence of finger disks (vs. large and wide
finger disks; Figure 2), stout body habitus and generally
smaller body size, SVL≤20.5 mm (vs. slender body habitus,
SVL>20.0 mm), F1 very small or reduced to nub, 1FL1
/22FL
(vs. F1 well-developed, 1FL≥1
/22FL), distinct crest on
urostyle (vs. weak crest on urostyle), lack of clavicles
(vs. small clavicles present), sternum fully cartilaginous (vs.
anterior portion of sternum containing calcified cartilage),
bobbin-shaped terminal phalanges (vs. large T-shaped
terminal phalanges), single smooth palatal fold (vs. two palatal
folds), comparatively broad cultriform process of parasphenoid
(vs. narrow cultriform process of parasphenoid narrow), lack
of vomeropalatines (vs. reduced but present), and shagreened
to warty skin (vs. completely smooth skin).
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).
138 www.zoores.ac.cn

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” (ϕρ´υνη; feminine gender), meaning
“toad” in English; this root is often used in generic names in
Asterophryinae frogs. Gender of the new genus is feminine.
Suggested common names: We suggest the name
“Indochinese Dwarf Frogs” as a common name of the new
genus in English, “Nhái Lùn” as a common name of the new
genus in Vietnamese, and “Eung Tham Khaera” as a common
name of the new genus in Thai.
Vietnamophryne inexpectata sp. nov.
Figure 3, Figure 4, Figure 5A, Figure 6; Table 3.
Holotype: ZMMU A-5820, adult male in good state of
preservation, from a primary montane tropical forest in Kon
Chu Rang Nature Reserve, Gia Lai Province, Tay Nguyen
Plateau, central Vietnam (N14.506°, E108.542°; elevation
1 000 m a.s.l.); collected on 31 May 2016 by Nikolay A.
Poyarkov at 2100 h from soil under a large ca. 2-m long
rotten log approximately 7 m from a small cascading stream
(Figure 7).
Diagnosis: Assigned to the genus Vietnamophryne Gen.
nov. based on morphological characteristics and phylogenetic
position (see Diagnosis of the new genus and Results).
From other congeners Vietnamophryne inexpectata sp.
nov. can be distinguished by the following combination of
morphological characters: (1) miniaturized body size, SVL
of single male 14.2 mm; (2) body habitus stout, FLL/SVL
and HLL/SVL ratios 51.8% and 151.8%, respectively; (3)
head as long as wide, HW/HL ratio 101.1%; (4) snout short,
obtuse in dorsal view, rounded in lateral view, subequal to
eye length (96.8% of eye length); (5) eye medium-sized, eye
length/snout-vent length ratio 13%; eye to nostril distance
6.3% of SVL; (6) tympanum comparatively large and rounded,
7.9% of SVL; well separated from eye (TED/SVL ratio 3.6%);
(7) tips of all digits rounded, not expanded in F1–F4, T1,
T2, and T5, weakly expanded in T3 and T4; (8) first
finger (F1) reduced to nub, less than one-third of F2 length
(1FL/2FL ratio 29.2%); terminal phalanx of F1 reduced to
tiny rounded ossification, relative finger lengths: I<II<IV<III,
relative toe lengths: I<II<V<III<IV; (9) subarticular tubercles
under fingers and toes weak, indistinct; (10) outer metatarsal
tubercle absent, inner metatarsal tubercle small, rounded
(3.5% of SVL); (11) skin of ventral surface completely smooth,
skin of dorsal and lateral surfaces shagreened anteriorly,
distinctly warty posteriorly with large flat tubercles or pustules
finely scattered on posterior dorsum and dorsal surface of
hindlimbs; (12) dorsomedial vertebral skin ridge indistinct,
discernable only on dorsal surface of head; (13) dorsally
grayish-brown with small reddish speckles anteriorly, darker
tubercles posteriorly; lateral sides of head dark brown with
beige mottling; ventrally gray-beige with weak gray marbling.
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.
Forelimbs comparatively short, about one-third of hindlimb
length (FLL/HLL 34.3%); hand shorter than lower arm,
almost one-third of forelimb length (HAL/FLL 34.3%); fingers
short, slender, round in cross-section, first finger reduced
to nub, length comprising less than one-third of second
finger (1FL/2FL 29.6%); relative finger lengths: I<II<IV<III
(Figure 6D). Finger webbing and dermal fringes on fingers
absent. First finger tip rudimentary, slightly protuberant as nub.
Tips of three outer fingers II–IV rounded, not dilated, finger
disks absent, terminal grooves absent; longitudinal furrow on
dorsal surface of fingers absent; subarticular tubercles under
fingers indistinct; nuptial pad absent; two palmar (metacarpal)
tubercles: inner palmar tubercle small, rounded; outer palmar
tubercle rounded with indistinct borders, slightly shorter than
inner palmar tubercle (IPTL/OPTL 120.0%); palmar surface
smooth, supernumerary palmar tubercles absent.
Hindlimbs comparatively short and thick, tibia length half
of snout-vent length (TL/SVL 50.1%); tibiotarsal articulation of
adpressed limb reaching eye level; foot slightly shorter than
tibia (FL/TL 90.9%); relative toe lengths: I<II<V<III<IV; tarsus
smooth, tarsal fold absent; tips of toes rounded, tips of toes
III and IV slightly dilated (Figure 6E), terminal grooves on toes
absent; toes rounded in cross-section, dermal fringes on toes
absent; toe webbing absent between all toes; subarticular
tubercles under toes indistinct; single metatarsal tubercle: inner
metatarsal tubercle rounded, flattened (IMTL/SVL ratio 3.5%).
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
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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).
Figure 3 Osteology of Vietnamophryne inexpectata sp. nov. (male holotype, ZMMU A-5820), showing full skeleton in dorsal (A)
and ventral views(B); right forelimb in dorsal (C) and palmar aspects (D); and right foot in thenar (E) and dorsal aspects (F)
Digits numbered I–V. Abbreviations: antbr.: os antebrachii (radius+ulna); car p.d.II-IV: carpale distale F2-F4; centr.: centrale; cor.: coracoid bone; crur.: os
cruris (tibia+fibula); fem.: femoral bone; fib.: fibulare; hm.: humeral bone; il.: ilium; mtc.I-IV: metacarpalia F1-F4; mtt.I-V: metatarsalia T1-T5; ph.d.I-IV: finger
pahlanges F1-F4; ph.d.I-V: toe pahlanges T1-T5; pr.p.-m.: processus postero-medialis; prsac.v.: presacral vertebrae; rad.: radiale; sac.v.: sacral vertebra;
sc.: scapula; tar.d.II-III: tarsale distale T2-T3; tib.: tibiale; uln.: ulnare; ur.: urostyle.
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Figure 4 Osteology of Vietnamophryne inexpectata sp. nov. (male holotype, ZMMU A-5820), showing skull in dorsal (A); ventral
(B); lateral (C); and frontal views (D)
Abbreviations: angspl.: angulosplenial; col.: columella; cond.oc.: occipital condylus; dent.: dentary bone; exoc.: exoccipital; fpar.: frontoparietal bone; max.:
maxilla; mmk.: mentomeckelian bone; nas.: nasal bone; pmax.: premaxilla; proot.: prootic; psph.: parasphenoid; pter.: pterygoid; qj.: quadratojugal; smax.:
septomaxilla; spheth.: sphenethmoid; sq.: squamosal. Scale bar: 1 mm.
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.
Skull clearly wider than long (Figure 3). Frontoparietals
separate along entire length, longer than broad, narrower
anteriorly than posteriorly, connected medially with long
non-calcified suture, lacking sagittal crest, clearly separated
from exoccipital by distinct suture posteriorly (Figure 4A).
Exoccipitals separate, not contacting medially, sculptured
laterally. Nasals large, not meeting at midline, lacking
posterior ramus, with gently rounded ventrolateral processes,
chondrified peripherally, separated from sphenethmoid (Figure
4A). Sphenethmoid poorly ossified only laterally, chondrified
anteriorly, ventrally, and dorsally (Figure 4B). Prootics
partially chondrified, with distinct dorsal crest (Figure
4C). Squamosal boomerang-shaped, well ossified, distally
chondrified, articulating on lateral surface of prootic (Figure
4C). Columella large, centrally ossified (Figure 4C), distally
chondrified, bent and barely pointing to otic area medially;
tympanic annulus completely chondrified. Premaxilla with
slender, well-ossified dorsal process not reaching nasal; labial
process of premaxilla well ossified (Figure 4D). Maxilla largely
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chondrified, ossified in central and anterior parts. Upper jaw
with eleutherognathine condition: anterior ends of maxillaries
not reaching labial portions of well-developed premaxillaries
(Figure 4D). Quadratojugal mostly cartilaginous, ossified only
in posterior portion. Vomers possibly completely chondrified
plates, lacking teeth or lateral processes; septomaxilla well
ossified (Figure 4B). Mentomeckelians ossified, connected to
dentaries and each other by strips of cartilage (Figure 4B).
Lower jaw with eleutherognathine condition: dentaries not
fused (Figure 4D). Parasphenoid smooth; cultriform process
of parasphenoid rather broad, abruptly terminating at middle
of sphenethmoid with distinct anterior notch (Figure 4B).
Hyoid plate completely cartilaginous; posteromedial processes
strongly ossified, elongated, notably enlarged and widened at
proximal ends, chondrified at distal ends (Figure 3B).
Figure 5 Three male holotypes of Vietnamophryne Gen. nov. species in life
A: Vietnamophryne inexpectata sp. nov. (ZMMU A-5820); B: Vietnamophryne orlovi sp. nov. (ZMMU A-5821); C: Vietnamophryne occidentalis sp. nov.
(ZMMU A-5822). Scale bar: 5 mm. Photos by N.A. Poyarkov (A, B) and P. Pawangkhanant (C).
Eight nonimbricate procoelous presacral vertebrae (PSV),
stout, length approximately one-seventh to one-third of
width; first presacral vertebra longer than posterior vertebrae,
vertebrae width not changing posteriorly; all except first with
wide diapophyses; transverse processes with chondrified tips,
longer anteriorly (3d PSV with longest transverse processes),
decreasing in length progressively to posterior (Figure 3A, B).
Diapophyses of vertebrae PSV2, PSV7, and PSV8 oriented
anteriad, those of PSV6 straight, and those of PSV3 to PSV5
oriented posteriad. Neural crests on PSV absent. Sacrum
with notably expanded diapophyses (diapophyses length ca.
35% of sacrum width). Urostyle with well-pronounced dorsal
crest running about 80% of shaft; ilia smooth, lacking dorsal
crest (Figure 3A).
Coracoids, scapulae, and suprascapulae present; first two
fully ossified; suprascapulae largely chondrified. Coracoids
robust with narrow distal ends oriented anteriad; proximal
ends greatly expanded, centrally notably narrowed (Figure
3B). Omosternum absent and clavicles absent. Sternum
completely cartilaginous.
Hand bones (Figure 3C, D) with three poorly calcified
carpal elements: carpale distale I chondrified, carpale distale
II–IV fused into single large element, partially chondrified;
prepollex chondrified; radiale large, partially calcified; ulnare
rounded, partially calcified. Metacarpals short, distally and
proximally chondrified, medially calcified; hand phalangeal
formula: 2-2-3-3; all phalanges ossified; distal phalanx of
finger I tiny, rudimentary, rounded (Figure 3C, D); terminal
phalanges of fingers II–IV small, bobbin-shaped, notably
narrower than penultimate phalanges (Figure 3C, D). Tarsal
elements of foot mostly chondrified (Figure 3E, F), tiny
ossifications present within generally cartilaginous tarsale
distale II–III and central; prehallux chondrified. Metatarsals
fully ossified medially, partially ossified distally, mostly
chondrified proximally; metatarsals longer and relatively
more massive than metacarpals; foot phalangeal formula:
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2-2-3-4-3; all phalanges ossified medially, chondrified distally
and proximally (Figure 3E, F). Terminal phalanges of all
toes small, bobbin-shaped; notably narrower than penultimate
phalanges on all toes (Figure 3E, F).
Figure 6 Male holotype of Vietnamophryne inexpectata sp. nov. (ZMMU A-5820) in life
A: Dorsal view; B: Ventral view; C: Lateral view of head; D: Palmar view of right hand; E: Thenar view of left foot. Photos by N. A. Poyarkov.
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 2 100 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, predominated
by large trees of the families Podocarpaceae (Dacrydium elatum,
Dacrycarpus imbricatus), Magnoliaceae, Burseraceae (Canarium
sp.), Myrtaceae (Syzygium sp.), Hamamelidaceae (Rhodoleia sp.,
Exbucklandia sp.), Lauraceae (Litsea sp.), Fagaceae (Lithocarpus
sp.), and Sterculiaceae (Scaphium sp.) (Figure 7).
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.
Zoological Research 39(3): 130-155, 2018 143

Table 3 Measurement data for holotypes of three new species of Vietnamophryne Gen. nov. from Indochina
Species V. inexpectata
sp. nov. V. or lovi sp. nov. V. occidentalis
sp. nov. Species V. inexpectata
sp. nov. V. or lovi sp. nov. V. occidentalis
sp. nov.
Specimen ID ZMMU A-5820 ZMMU A-5821 ZMMU A-5822 Specimen ID ZMMU A-5820 ZMMU A-5821 ZMMU A-5822
Holotype Holotype Holotype Holotype Holotype Holotype
Sex Male Male Male Sex Male Male Male
1. SVL 14.2 15.4 20.5 21. TYD 1.1 0.9 1.0
2. HL 5.4 6.7 6.9 22. TED 0.5 0.7 0.4
3. SL 1.8 2.5 2.1 23. 1FL 0.3 0.6 0.8
4. EL 1.9 1.8 2.5 24. 2FL 1.0 1.2 1.9
5. N-EL 0.9 2.0 1.4 25. 3FL 1.7 1.7 3.6
6. HW 5.4 5.8 6.8 26. 4FL 1.1 1.0 2.1
7. IND 1.5 1.8 2.4 27. 1FW 0.2 0.2 0.4
8. IOD 1.6 1.9 2.3 28. 2FDD 0.3 0.3 0.6
9. UEW 0.9 0.8 1.1 29. 3FDD 0.3 0.4 0.7
10. FLL 7.4 8.2 12.9 30. 4FDD 0.3 0.3 0.6
11. LAL 5.9 5.7 9.7 31. 1TOEL 0.4 0.7 1.0
12. HAL 3.2 3.2 5.6 32. 2TOEL 1.4 1.6 2.2
13. IPTL 0.5 0.6 0.7 33. 3TOEL 2.4 3.1 3.8
14. OPTL 0.5 0.7 0.6 34. 4TOEL 3.9 4.1 5.8
15. HLL 21.4 22.1 28.8 35. 5TOEL 1.6 1.8 2.8
16. TL 7.2 7.1 10.0 36. 1TDD 0.3 0.3 0.5
17. FTL 9.6 11.0 14.6 37. 2TDD 0.4 0.5 0.6
18. FL 6.6 7.1 8.2 38. 3TDD 0.5 0.6 0.8
19. IMTL 0.5 0.7 0.9 39. 4TDD 0.6 0.7 0.9
20. OMTL – – – 40. 5TDD 0.4 0.4 0.6
For abbreviations see Materials and Methods. All measurements are in mm. –: Not available.
Figure 7 Habitat at type locality of Vietnamophryne inexpectata sp. nov. in Kon Chu Rang Nature Reserve, Gia Lai Province,
Vietnam (Photo by A.V. Alexandrova)
Other species of anurans recorded syntopically at the
type locality included Ingerophrynus galeatus (Günther,
1864), Kurixalus banaensis (Bourret, 1939), Rhacophorus
annamensis Smith, 1924, Rhacophorus rhodopus Liu & Hu,
1960, Rh. robertingeri Orlov, Poyarkov, Vassilieva, Ananjeva,
Nguyen, Nguyen & Geissler, 2012, Rana johnsi Smith, 1921,
Microhyla pulverata Bain & Nguyen, 2004, Leptolalax cf.
ardens Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang,
Nguyen & Ziegler, 2016, and Ophryophryne hansi Ohler, 2003.
Comparisons: For discrimination from other Microhylidae
genera occurring in Indochina, see “Comparisons with other
Microhylidae genera inhabiting mainland Southeast Asia” above.
Vietnamophryne inexpectata sp. nov. can be distinguished
from its congeners based on the following morphological
attributes. The new species can be distinguished from
Vietnamophryne orlovi sp. nov. (inhabiting Cao Bang Province,
northern Vietnam, described below) by warty skin on posterior
and shagreened skin on anterior dorsum (vs. mostly smooth
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skin, slightly shagreened posteriorly, lacking enlarge tubercles),
grayish-beige ventral coloration with gray marbling (vs. bright
lemon-yellow belly with dark brown marbling), F1 reduced to
nub, 1FL/2FL 29.6% (vs. F1 well-developed, 1FL/2FL 47.9%),
head length almost equal to head width, HW/HL 101.1% (vs.
head longer than wide, HW/HL 86.5%), snout length subequal
to eye length, SL/EL 96.8% (vs. snout notably longer than
eye length, SL/EL 141.3%), slightly larger tympanum, TYD/EL
60.5% (vs. TYD/EL 47.5%), and eye to nostril distance twice as
short as eye length, N-EL/EL 48.1% (vs. N-EL/EL 109.5%).
Vietnamophryne inexpectata sp. nov. can be discriminated
from Vietnamophryne occidentalis sp. nov. (inhabiting
Chiang Rai Province, northern Thailand, described below)
by the following combination of morphological characters:
smaller body size, SVL 14.2 mm in single male holotype
(vs. larger SVL 20.5 in single male holotype), warty skin on
posterior and shagreened skin on anterior parts of dorsum
(vs. mostly smooth skin with rare flat tubercles), grayish-beige
ventral coloration with gray marbling (vs. bright orange-red
belly with sparse dark brown marbling), F1 reduced to nub,
1FL/2FL 29.6% (vs. F1 well-developed, 1FL/2FL 42.7%),
slightly larger tympanum, TYD/EL 60.5% (vs. TYD/EL
41.5%), and slightly shorter forelimb, FLL/SVL 51.7% (vs.
comparatively longer forelimb, FLL/SVL 62.7%).
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.
Suggested common names. We recommend the following
common names for the new species: “Tay Nguyen Dwarf Frog”
(English) and “Nhái Lùn Tây Nguyên” (Vietnamese).
Vietnamophryne orlovi sp. nov.
Figure 5B, Figure 8, Figure 9; Table 3.
Holotype: ZMMU A-5821, adult male in good state of
preservation, from a primary montane subtropical forest
on the southern slopes of Phia Oac Mt., Phia Oac-Phia
Den National Park, Cao Bang Province, northern Vietnam
(N22.600°, E105.884°; elevation 1 200 m a.s.l.); collected on
9 June 2017 by Nikolay A. Poyarkov at 2300 h from soil in the
roots of a tree fern on a steep mountain slope (Figure 9A), ca.
20 m from a small cascading stream (Figure 9B).
Diagnosis: Assigned to the genus Vietnamophryne Gen.
nov. based on morphological attributes and phylogenetic
position in mtDNA genealogy (see Diagnosis of the new genus
and Results). From other congeners Vietnamophryne orlovi
sp. nov. can be distinguished by the following combination
of morphological traits: (1) miniaturized body size, SVL of
single male 15.4 mm; (2) body habitus stout, FLL/SVL and
HLL/SVL ratios 53.3% and 143.4%, respectively; (3) head
longer than wide, HW/HL ratio 86.5%; (4) snout comparatively
long, rounded in dorsal and lateral views, snout length greater
than eye length (SL/EL ratio 141.3%); (5) eye medium-sized,
eye length/snout-vent length ratio 11.6%; eye to nostril
distance 12.7% of SVL; (6) tympanum comparatively small,
rounded, 5.5% of SVL; well separated from eye (TED/SVL
ratio 4.2%); (7) tips of all digits rounded, not expanded in
F1–F4, T1, T2, and T5, weakly expanded in T3 and T4; (8)
first finger (F1) well developed, half of F2 length (1FL/2FL ratio
47.9%), relative finger lengths: I<IV<II<III, relative toe lengths:
I<II<V<III<IV; (9) subarticular tubercles under fingers and toes
weak, indistinct; (10) outer metatarsal tubercle absent, inner
metatarsal tubercle small, rounded (4.2% of SVL); (11) skin of
ventral surface completely smooth, skin of dorsal and lateral
surfaces smooth anteriorly, somewhat shagreened posteriorly
with small flat pustules loosely scattered on posterior dorsum
and dorsal surface of hindlimbs; (12) dorsomedial vertebral
skin ridge distinct, discernable only on midline of dorsum
and head; (13) dorsally reddish-brown, pustules on posterior
dorsum whitish; lateral sides of head dark brown with whitish
mottling; ventrally lemon-yellow with fine brown marbling.
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.
Zoological Research 39(3): 130-155, 2018 145

Figure 8 Male holotype of Vietnamophryne orlovi sp. nov. (ZMMU A-5821) in life
A: Dorsal view; B: Ventral view; C: Lateral view of head; D: Palmar view of left hand; E: Thenar view of left foot. Photos by N. A. Poyarkov.
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. Tips of three outer fingers II–IV
rounded, not dilated, finger disks absent, terminal grooves
absent; longitudinal furrow on dorsal surface of fingers absent;
subarticular tubercles under fingers indistinct; nuptial pad
absent; two palmar tubercles: inner palmar tubercle small,
rounded; outer palmar tubercle rounded, slightly longer than
inner palmar tubercle (IPTL/OPTL 90.9%); palmar surface
smooth, supernumerary palmar tubercles absent.
Hindlimbs short and thick, tibia length less than half of
snout-vent length (TL/SVL 46.0%); tibiotarsal articulation of
adpressed limb reaching eye level; foot length equal to tibia
length (FL/TL 100.7%); relative toe lengths: I<II<V<III<IV;
tarsus smooth, tarsal fold absent; tips of toes rounded, tips of
toes III and IV slightly dilated (Figure 8E), terminal grooves or
dermal fringes on toes absent; toes rounded in cross-section;
toe webbing absent between all toes; subarticular tubercles
under toes indistinct; single metatarsal tubercle: inner
metatarsal tubercle rounded, flattened (IMTL/SVL ratio 4.2%).
Skin on anterior dorsal and dorsolateral surfaces smooth,
shagreened on posterior dorsum and dorsal surfaces of
hindlimbs; small flat tubercles loosely scattered on sacral
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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).
Figure 9 Macrohabitat (A) and microhabitat (B) at type locality of Vietnamophryne orlovi sp. nov. in Phia Oac-Phia Den N.P., Cao
Bang Province, Vietnam (Photos by Le Xuan Son)
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.
The polydominant subtropical forests in Phia Oac-Phia
Den National Park at elevations of 1 200–1 400 m a.s.l.
show thick bamboo undergrowth and are dominated by
trees from the families Fagaceae (Lithocarpus,Castanopsis),
Sapindaceae (Acer ), Platanaceae (Platanus), Elaeocarpaceae
(Elaeocarpus), Ericaceae (Rhododendron), Lauraceae
(Cinnamomum), and Theaceae (Schima), with thick layers of
moss and numerous epiphytic plants (Orchidaceae, Ericaceae,
Pteridophyta) (Figure 9).
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
Zoological Research 39(3): 130-155, 2018 147

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).
Other species of amphibians recorded syntopically with
the new species at the type locality include Tylototriton
ziegleri Nishikawa, Matsui & Nguyen, 2013, Raorchestes
parvulus (Boulenger, 1893), Kurixalus odontotarsus (Ye &
Fei, 1993), Gracixalus gracilipes (Bourret, 1937), Gracixalus
jinxiuensis (Hu, 1978), Polypedates mutus (Smith, 1940), and
Ophryophryne microstoma Boulenger, 1903.
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.
Vietnamophryne orlovi sp. nov. can be distinguished from
Vietnamophryne occidentalis sp. nov. (known from Chiang
Rai Province, northern Thailand, described below) based on
the following combination of morphological features: smaller
body size, SVL 15.4 mm in single male holotype (vs. larger
SVL 20.5 in single male holotype), lemon-yellow belly with
dark brown marbling (vs. bright orange-red belly with sparse
dark brown marbling), head longer than wide, HW/HL 86.5%
(vs. head length almost equal to head width, HW/HL 99.0%),
snout notably longer than eye length, SL/EL 141.3% (vs. snout
length notably shorter than eye length, SL/EL 85.5%), eye to
nostril distance almost equal to eye length, N-EL/EL 109.5%
(vs. eye to nostril distance twice as short as eye length,
N-EL/EL 55.2%), and slightly shorter forelimb, FLL/SVL 53.2%
(vs. comparatively longer forelimb, FLL/SVL 62.7%).
Distribution and biogeography: At present, Vietnamophryne
orlovi sp. nov. is known only from the type locality on Phia
Oac Mt., in the montane subtropical forest of Phia Oac-Phia
Den National Park, Cao Bang Province, northern Vietnam at an
elevation of ca. 1 200 m a.s.l. Phi Oac Mt. is the highest peak of
the Ngan Son-Yen Lac Mountain Ridge located in northeastern
Vietnam (Cao Bang, Bak Kan, and Thai Nguyen provinces);
the occurrence of this species in other montane forest areas
of the Ngan Son-Yen Lac Mountain Ridge at similar elevations
is considered likely.
Conservation status: At present, Vietnamophryne orlovi sp.
nov. is only known from a single specimen, possibly due
to the secretive semi-fossorial biology of the species. The
distribution and population status of Vietnamophryne orlovi
sp. nov. are unknown and additional surveys in other areas
of the Dong Bac (north-east) region of Vietnam are essential
for elucidating the biology of the new species and clarifying
its distribution. Given the available information, we suggest
Vietnamophryne orlovi 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 “orlovi” is a Latinized patronymic
in genitive singular; the name of the new species is given in
honor of Dr. Nikolai L. Orlov (ZISP, St. Petersburg, Russia) for
recognition of his outstanding contribution to the knowledge of
herpetofauna of Indochina.
Suggested common names: We recommend the following
common names for the new species: “Orlov’s Dwarf Frog”
(English) and “Nhái Lùn Ðông Bac” (Vietnamese).
Vietnamophryne occidentalis sp. nov.
Figure 5C, Figure 10, Figure 11; Table 3.
Holotype: ZMMU A-5822, adult male in poor state of
preservation, from a primary montane subtropical forest on
limestone outcrops of Doi Tung Mt., Pong Ngam District,
Chaing Rai Province, northern Thailand (N20.344°, E99.830°;
elevation 1 050 m a.s.l.); collected on 5 April 2017 by Parinya
Pawangkhanant at 1400 h from soil and leaf litter on the
watershed of a steep mountain slope (Figure 11A) near a
forest trail far from streams or rivers (Figure 11B).
Diagnosis: Assigned to the genus Vietnamophryne Gen. nov.
based on morphological character traits and phylogenetic
position in mtDNA genealogy (see Diagnosis of the new
genus and Results). Vietnamophryne occidentalis sp. nov.
can be distinguished from other congeners by the following
combination of morphological features: (1) body size small,
SVL of single male 20.5 mm; (2) body habitus stout, FLL/SVL
and HLL/SVL ratios 62.7% and 140.3%, respectively; (3)
head as long as wide, HW/HL ratio 99.0%; (4) snout short,
obtuse in dorsal view, rounded in lateral view, shorter than
eye length (85.5% of eye length); (5) eye medium-sized, eye
length/snout-vent length ratio 12%; eye to nostril distance 6.7%
of SVL; (6) tympanum comparatively small, rounded, 5.0% of
SVL; located very close to eye (TED/SVL ratio 1.8%); (7) tips of
digits rounded, not expanded in F1–F4, T1, T2, and T5, weakly
expanded in T3 and T4; (8) first finger (F1) well developed,
half of F2 length (1FL/2FL ratio 43.0%), relative finger lengths:
I<II<IV<III, relative toe lengths: I<II<V<III<IV; (9) subarticular
tubercles under fingers and toes weak, indistinct; (10) outer
metatarsal tubercle absent, inner metatarsal tubercle small,
rounded (4.2% of SVL); (11) skin of ventral surface completely
smooth, skin of dorsal and lateral surfaces smooth, posteriorly
with loosely scattered small flat tubercles present on dorsal
surfaces of posterior dorsum and hindlimbs; (12) dorsomedial
vertebral skin ridge distinct, well discernable on midline of
dorsum and head; (13) dorsally dark brick-brown, lateral sides
of head dark brown to black; ventrally orange-red with few dark
brown flecks.
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,
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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.
Figure 10 Male holotype of Vietnamophryne occidentalis sp. nov. (ZMMU A-5822) in life
A: Dorsal view; B: Ventral view; C: Palmar view of right hand; D: Thenar view of left foot. Photos by P. Pawangkhanant.
Forelimbs comparatively long, almost half hindlimb length
(FLL/HLL 44.7%); hand much shorter than lower arm,
less than half forelimb length (HAL/FLL 43.9%); fingers
comparatively long, slender, round in cross-section, first
finger well developed, length slightly less than half of second
finger (1FL/2FL 42.7%); relative finger lengths: I<II<IV<III
(Figure 10C). Finger webbing and dermal fringes on fingers
absent. First finger tip rounded, first finger well developed.
Tips of three outer fingers II–IV rounded, not dilated, finger
disks absent, terminal grooves absent; longitudinal furrow
on dorsal surface of fingers absent; subar ticular tubercles
under fingers indistinct; nuptial pad absent; two palmar
tubercles: inner palmar tubercle small, rounded; outer palmar
tubercle rounded, slightly shorter than inner palmar tubercle
(IPTL/OPTL 109.7%); palmar surface smooth, supernumerary
palmar tubercles absent.
Hindlimbs short and thick, tibia length almost half of
snout-vent length (TL/SVL 49.0%); tibiotarsal articulation
of adpressed limb reaching eye level; foot length notably
shorter than tibia length (FL/TL 82.0%); relative toe lengths:
Zoological Research 39(3): 130-155, 2018 149

I<II<V<III<IV; tarsus smooth, tarsal fold absent; tips of
toes rounded, tip of toe III slightly dilated, tip of toe IV
notably dilated (Figure 10D), terminal grooves or dermal
fringes on toes absent; toes rounded in cross-section; toe
webbing absent between all toes; subarticular tubercles under
toes indistinct; single metatarsal tubercle: inner metatarsal
tubercle rounded, flattened (IMTL/SVL ratio 4.2%).
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).
Figure 11 Macrohabitat (A) and microhabitat (B) at type locality of Vietnamophryne occidentalis sp. nov. in Doi Tung Mt., Chiang
Rai Province, Thailand (Photos by P. Pawangkhanant and M. Naidaungchan)
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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. Temperatures are lowest from November
to February, with an average minimum at 500 m of 13 °C
in January–February and 21 °C from June–August (Maxwell,
2007). At elevations above 1 000 m a.s.l., the typical montane
forest canopy trees include: Schima wallichii (Theaceae),
Sarcosperma arboretum (Sapotaceae), Cinnamomum iners
(Lauraceae), Balakata baccata (Euphorbiaceae), and several
Fagaceae (Castanopsis armata,C. tribuloides, and Lithocarpus
elegans) (Maxwell, 2007).
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.
The associated species of amphibians and reptiles recorded
in the area include: Microhyla berdmorei (Blyth, 1856),
Microhyla heymonsi Vogt, 1911, Sylvirana nigrovittata (Blyth,
1856), Rhacophorus rhodopus Liu & Hu, 1960, Theloderma
albopunctatum (Liu & Hu, 1962), Theloderma gordoni Taylor,
1962, Acanthosaura lepidogaster (Cuvier, 1829), Pseudocalotes
microlepis (Boulenger, 1888), Tropidophorus thai Smith, 1919,
Oreocryptophis porphyraceus cf. porphyraceus (Cantor, 1839),
and Ovophis monticola (Günther, 1864).
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
Zoological Research 39(3): 130-155, 2018 151

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.
COMPETING INTERESTS
The authors declare that they have no competing interests.
AUTHORS’ CONTRIBUTIONS
N.A.P. and J.C. designed the study. N.A.P., C.S., P.P., and A.A. collected
data. N.A.P. and T.V.D. performed molecular experiments. D.V.K performed
micro-CT scanning. N.A.P. and D.V.K. examined morphology. N.A.P.
supervised analyses. N.A.P. wrote the manuscript, N.A.P., C.S., A.A.,
and J.C. revised the manuscript. All authors read and approved the final
manuscript.
152 www.zoores.ac.cn

ACKNOWLEDGEMENTS
Fieldwork in Vietnam was funded by the Joint Russian-Vietnamese Tropical
and Technological Center and was conducted under permission of the
Department of Forestry, Ministry of Agriculture and Rural Development
of Vietnam (permit numbers 547/TCLN-BTTN, issued 21.04.2016,
432/TCLN-BTTN, issued 30.03.2017). The Forest Protection Department
of the Peoples’ Committee of Gia Lai Province provided permits for
fieldwork and sample collection in Kon Chu Rang Nature Reserve (permit
numbers 1951/UBND-NV, issued 04.05.2016, and 142/SNgV-VP, issued
11.04.2017). The Forest Protection Department of the Peoples’ Committee
of Cao Bang Province provided permits for fieldwork and sample collection
in Phia Oac-Phia Den National Park (permit numbers 1659/UBND-NC,
issued 06.06.2017). The authors are grateful to Andrey N. Kuznetsov,
Vyacheslav V. Rozhnov, and Leonid P. Korzoun for permanent support and
organization of fieldwork. Fieldwork in Thailand and specimen collection
and exportation were authorized by the Animal Research Centre, University
of Phayao, Thailand, and by the Institute of Animals for Scientific Purpose
Development (IAD), Bangkok, Thailand, permit no. U1-01205-2558
(Thailand). Animal use was approved by the University of Phayao,
Phayao, Thailand, No. UP-AE59-01-04-0022 issued to Chatmongkon
Suwannapoom. We would like to express our deep gratitude to all the
authorities in Vietnam and Thailand for fieldwork permission and support.
We are grateful to Kawin Jiaranaisakul (Bangkok, Thailand), Mali
Naidaungchan (Tavoy, Myanmar), and Le Xuan Son (Hanoi, Vietnam)
for help during field work. NAP thanks Nguyen Thanh Luan (Asian
Turtle Program-Indo-Myanmar Conservation, Hanoi, Vietnam) and Nguyen
Van Tan (Save Vietnam’s Wildlife, Cuc Phuong National Park, Ninh Binh
Province, Vietnam) for help and discussion, Vladislav Gorin (Biological
Faculty, Lomonosov Moscow State University, Moscow, Russia) for help
and assistance in the lab and with phylogenetic analyses, and Alexandra A.
Elbakyan for assistance with accessing required literature. We are indebted
to Evgeniya N. Solovyeva (Zoological Museum of Moscow University,
Moscow, Russia) for help with primer design. For permission to study
specimens under their care and permanent support we are most obliged
and thank Valentina F. Orlova (ZMMU). Alina V. Alexandrova and Le Xuan
Son provided habitat photos. We are deeply grateful to Egill Scallagrimsson
for proofreading the manuscript. The authors express gratitude to two
anonymous reviewers for useful comments on earlier versions of the paper,
and to Mark D. Scherz for his thorough work on revising the paper, which
helped us to significantly improve the manuscript.
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