A new species of the genus Leptolalax (Anura: Megophryidae) from southern Vietnam

Abstract

We describe a new species of megophryid frog from Phu Yen Province in southern Vietnam. Leptolalax macrops sp. nov. is distinguished from its congeners by a combination of the following morphological attributes: (1) body size medium (SVL 28.0-29.3 mm in three adult males, 30.3 mm in single adult female); (2) supra-axillary glands present, creamy white; ventrolateral glands indistinct; (3) tympanum externally distinct; (4) dorsal skin roughly granular with larger tubercles, dermal ridges on dorsum absent; (5) rudimentary webbing present between fingers I-II and II-III; rudimentary webbing between all toes; fingers and toes without dermal fringes; (6) in life ventral surface greyish-violet with white speckling; (7) supratympanic fold distinct, dark brown in life; (8) iris bicolored, typically golden in upper half, fading to golden green in lower half; (9) tibia short (TbL/SVL 0.44-0.45 in males); and (10) eyes large and protuberant (ED/SVL 0.15-0.16 in males). From all congeners for which comparable sequences are available, the new species differs markedly in the 16S rRNA mitochondrial gene sequence (P-distance>5.7%). The new species is currently known only from montane evergreen tropical forests of Song Hinh District, Phu Yen Province, and M'Drak District of Dak Lak Province at elevations of 470-630 m a.s.l. We suggest the new species should be considered as Data Deficient following the IUCN's Red List categories. We also report a previously unknown Leptolalax mtDNA lineage from an evergreen tropical forest in the Hoa Thinh District of Phu Yen Province, which may also represent an undescribed species.

Full text

ZOOLOGICAL RESEARCH
A new species of the genus Leptolalax (Anura:
Megophryidae) from southern Vietnam
Tang Van Duong1,2, Dang Trong Do3, Chung Dac Ngo4, Truong Quang Nguyen5,6, Nikolay A. Poyarkov, Jr.2,7,*
1Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, Hanoi, Vietnam
2Department of Vertebrate Zoology, Biological Faculty, Lomonosov Moscow State University, Leninskiye Gory, GSP-1, Moscow 119234,
Russia
3Faculty of Natural Sciences, Phu Yen University, Tuy Hoa, Phu Yen, Vietnam
4College of Education, Hue University, Hue, Vietnam
5Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, Hanoi, Vietnam
6Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Cau Giay, Hanoi, Vietnam
7Joint Russian-Vietnamese Tropical Research and Technological Center, Nghia Do, Cau Giay, Hanoi, Vietnam
ABSTRACT
We describe a new species of megophryid frog from
Phu Yen Province in southern Vietnam. Leptolalax
macrops
sp. nov.
is distinguished from its congeners
by a combination of the following morphological
attributes: (1) body size medium (SVL 28.0–29.3
mm in three adult males, 30.3 mm in single adult
female); (2) supra-axillary glands present, creamy
white; ventrolateral glands indistinct; (3) tympanum
externally distinct; (4) dorsal skin roughly granular
with larger tubercles, dermal ridges on dorsum
absent; (5) rudimentary webbing present between
fingers I–II and II–III; rudimentary webbing between
all toes; fingers and toes without dermal fringes;
(6) in life ventral surface greyish-violet with white
speckling; (7) supratympanic fold distinct, dark brown
in life; (8) iris bicolored, typically golden in upper
half, fading to golden green in lower half; (9) tibia
short (TbL/SVL 0.44–0.45 in males); and (10) eyes
large and protuberant (ED/SVL 0.15–0.16 in males).
From all congeners for which comparable sequences
are available, the new species differs markedly
in the 16S rRNA mitochondrial gene sequence
(P-distance>5.7%). The new species is currently
known only from montane evergreen tropical forests
of Song Hinh District, Phu Yen Province, and
M’Drak District of Dak Lak Province at elevations
of 470–630 m a.s.l. We suggest the new species
should be considered as Data Deficient following
the IUCN’s Red List categories. We also report a
previously unknown Leptolalax mtDNA lineage from
an evergreen tropical forest in the Hoa Thinh District
of Phu Yen Province, which may also represent an
undescribed species.
Keywords:
Leptolalax macrops
sp. nov.
; Phu Yen
Province; Dak Lak Province; Southern coastal region
of Vietnam
INTRODUCTION
Members of the genus Leptolalax Dubois, 1983
(Megophryidae Bonaparte, 1850) are widely distributed
from northeastern India and southern China southward to
the Southeast Asian mainland and Borneo. Knowledge
about Leptolalax species diversity has strikingly increased
in recent decades, from only four in 1983 (Dubois, 1983)
to 53 recognized species at present, 31 of which (~60%
of total species) have been described in the last 10 years
(Frost, 2017). In Vietnam, the number of Leptolalax species
has increased remarkably from six (Nguyen et al., 2009) to
23 species (Rowley et al., 2016, 2017a, 2017b) within the
last decade. However, considering the high morphological
similarity of many species within the genus (Rowley et al.,
2016) and the poor level of biological exploration of many
parts of Indochina, additional taxa likely remain undescribed.
The L. applebyi species group is a monophyletic lineage
of small to medium-sized frogs (adult SVL<40 mm) inhabiting
the southern and central parts of the Annamite (or Truong
Son) Mountains in southern Indochina (Poyarkov et al., 2015a;
Rowley et al., 2015a, 2016). The group is characterized
by morphological similarity of its members and widespread
microendemism, with distribution of several lineages restricted
Received: 03 September 2017; Accepted: 19 March 2018; Online: 10
April 2018
Foundation items: The work was partially supported by the
Russian Foundation of Basic Research (RFBR 15-29-02771) to
N.A.P.; Molecular and phylogenetic analyses were completed with
partial financial support from the Russian Science Foundation (RSF
14-50-00029) to N.A.P.
*Corresponding author, E-mail: n.poyarkov@gmail.com
DOI: 10.24272/j.issn.2095-8137.2018.009
Science Press Zoological Research 38(3): 1-16, 2018 1

to watershed basins (Rowley et al., 2015a). The L. applebyi
species group currently comprises nine species distributed in
the mountains of southern and central Vietnam and adjacent
northeastern Cambodia, and include L. applebyi Rowley &
Cao; L. ardens Rowley, Tran, Le, Dau, Peloso, Nguyen,
Hoang, Nguyen & Ziegler; L. bidoupensis Rowley, Le, Tran, &
Hoang; L. kalonensis Rowley, Tran, Le, Dau, Peloso, Nguyen,
Hoang, Nguyen & Ziegler; L. maculosus Rowley, Tran, Le,
Dau, Peloso, Nguyen, Hoang, Nguyen & Ziegler; L. melicus
Rowley, Stuart, Neang & Emmett; L. pallidus Rowley, Tran, Le,
Dau, Peloso, Nguyen, Hoang, Nguyen & Ziegler; L. pyrrhops
Poyarkov, Rowley, Gogoleva, Vassilieva, Galoyan, & Orlov;
and L. tadungensis Rowley, Tran, Le, Dau, Peloso, Nguyen,
Hoang, Nguyen & Ziegler (Rowley et al., 2016). Moreover,
recent molecular, morphological, and acoustic analyses of the
L. applebyi species group (Rowley et al., 2015a) revealed
another potential new species in the northeastern edges of the
Langbian Plateau, indicating that our current understanding of
Leptolalax diversity in Vietnam is far from complete.
In 2015, during field surveys in the Tay Hoa and Song
Hinh districts of the southern area of Phu Yen Province,
we encountered two previously unknown populations of
Leptolalax sp., which also represent the first records of the
genus from Phu Yen Province. Morphologically, the newly
discovered populations resembled species of the L. applebyi
group. Consequently, analyses of mtDNA sequences and
diagnostic morphological characters suggested that these
two populations corresponded to two previously undescribed
species of the L. applebyi group, one of which is described
herein as a new species.
MATERIALS AND METHODS
Sample collection
Field surveys were conducted in the forests near Hon Den
Mountain, Ea Ly commune, Song Hinh District, and Hoa Thinh
commune, Phu Thu Township, Tay Hoa District, in Phu Yen
Province, southern Vietnam by Dang Trong Do between July
and August 2015 (Figure 1). Specimens were collected by
hand from 1900 h to 2300 h. Specimens were photographed
in life, then euthanized in a closed vessel with a piece of
cotton wool containing ethyl acetate (Simmons, 2002), fixed
in 80% ethanol for 5 h, and later transferred to 70% ethanol
for permanent storage. Femoral muscle tissue samples were
taken prior to preservation for genetic analysis and stored in
96% ethanol. Preserved specimens were deposited in the
zoological collection of the Phu Yen University (PYU), Phu
Yen Province, Institute of Ecology and Biological Resources
(IEBR), Hanoi, Vietnam, and in the herpetological collection
of the Zoological Museum of Lomonosov Moscow State
University (ZMMU), Moscow, Russia.
1
Figure 1 Map of Tay Nguyen Plateau (=Central Highlands) showing distribution of the members of the Leptolalax applebyi species
group and sampling locations examined in this study
Color of locality icons correspond to color bars marking Leptolalax species in Figure 2; dot in a center of a circle indicate the type locality of a species.
2www.zoores.ac.cn

Morphological characters
Morphological data were recorded from preserved specimens.
Measurements were taken using a digital caliper to the
nearest 0.1 mm; the morphometrics of adults and character
terminology follow Mahony (2011), Mahony et al. (2013),
and Poyarkov et al. (2017). Morphometric abbreviations
are: snout to vent length (SVL); head width (HW); head
length (HL); eye diameter (ED); tympanum diameter (TYD);
eye to tympanum distance (E-T); snout length, measured
from snout tip to the anterior corner of the eye (E-S); eye to
nare distance (E-N); nare to snout distance (N-S); interorbital
distance, measured as the narrowest distance between upper
eyelids (IO); internarial distance (IN); upper eyelid width
(ELW); forearm length (FAL); hand length (HAL); first finger
length (FIL); second finger length (FIIL); third finger length
(FIIIL); fourth finger length (FIVL); tibia length (TbL); femur
length (FeL); foot length (FOL); tibiotarsal articulation to tip
of fourth toe distance (TFOL); and inner metatarsal tubercle
length (IMT). Additionally, for description of the type series
we measured the distance between anterior orbital borders
(IFE); distance between posterior orbital borders (IBE); and
length of toes I–V (TI–VL). All measurements were taken on
the right side of the examined specimen. Sex was determined
by gonadal inspection following dissection.
Statistical analyses were performed with Statistica 6.0
(StatSoft, Inc.). Sexes were separated for subsequent
comparisons among samples. One-way analysis of variance
(ANOVA) and Duncan’s post-hoc tests were used for
morphometric comparisons. A significance level of 95% was
used of all statistical tests.
Comparative morphological data were obtained from
museum specimens of Leptolalax and (when available)
photographs of these specimens in life (Appendix). Data
on the morphological characters of Leptolalax species are
also available from the following literature: L. aereus Rowley,
Stuart, Richards, Phimmachak & Sivongxay (Rowley et al.,
2010b); L. alpinus Fei, Ye & Li (Fei et al., 1990, 2009, 2010), L.
applebyi (Rowley & Cao, 2009; Rowley et al., 2016); L. arayai
Matsui (Matsui, 1997), L. ardens (Rowley et al., 2016); L.
bidoupensis (Rowley et al., 2011, 2016); L. botsfordi Rowley,
Dau & Nguyen (Rowley et al., 2013); L. bourreti Dubois
(Dubois, 1983; Ohler et al., 2011); L. croceus Rowley, Hoang,
Le, Dau & Cao (Rowley et al., 2010c); L. dringi Dubois
(Dubois, 1987; Inger et al., 1995); L. eos Ohler, Wollenberg,
Grosjean, Hendrix, Vences, Ziegler & Dubois (Ohler et al.,
2011); L. firthi Rowley, Hoang, Dau, Le & Cao (Rowley
et al., 2012); L. fritinniens Dehling & Matsui (Dehling &
Matsui, 2013) L. fuliginosus Matsui (Matsui, 2006); L. gracilis
(Günther) (Günther, 1872; Inger & Stuebing, 2005); L. hamidi
Matsui (Matsui, 1997); L. heteropus (Boulenger) (Boulenger,
1900); L. isos Rowley, Stuart, Neang, Hoang, Dau, Nguyen
& Emmett (Rowley et al., 2015b); L. kajangensis Grismer,
Grismer & Youmans (Grismer et al., 2004); L. kalonensis
(Rowley et al., 2016); L. kecil Matsui, Belabut, Ahmad &
Yong (Matsui et al., 2009); L. khasiorum Das, Tron, Rangad &
Hooroo (Das et al., 2010); L. lateralis (Anderson) (Anderson,
1871; Humtsoe et al., 2008), L. laui Sung, Yang & Wang
(Sung et al., 2014), L. liui Fei & Ye (Fei et al., 1990, 2009,
2010); L. maculosus (Rowley et al., 2016); L. maoershanensis
Yuan, Sun, Chen, Rowley & Che (Yuan et al., 2017); L.
marmoratus Matsui, Zainudin & Nishikawa (Matsui et al.,
2014b); L. maurus Inger, Lakim, Biun & Yambun (Inger et
al., 1997); L. melanoleucus Matsui (Matsui, 2006); L. melicus
(Rowley et al., 2010a, 2016); L. minimus (Taylor) (Taylor,
1962; Ohler et al., 2011); L. nahangensis Lathrop, Murphy,
Orlov & Ho (Lathrop et al., 1998); L. nokrekensis (Mathew
& Sen) (Mathew & Sen, 2009); L. nyx Ohler, Wollenberg,
Grosjean, Hendrix, Vences, Ziegler & Dubois (Ohler et al.,
2011); L. oshanensis (Liu) (Fei et al., 2009, 2010; Liu, 1950);
L. pallidus (Rowley et al., 2016); L. pelodytoides Boulenger
(Boulenger, 1893, 1908; Ohler et al., 2011); L. petrops Rowley,
Dau, Hoang, Le, Cutajar & Nguyen (Rowley et al., 2017a);
L. pictus Malkmus (Malkmus, 1992; Malkmus et al., 2002);
L. platycephalus Dehling (Dehling, 2012); L. pluvialis Ohler,
Marquis, Swan & Grosjean (Ohler et al., 2000, 2011), L.
puhoatensis Rowley, Dau & Cao (Rowley et al., 2017b); L.
pyrrhops (Poyarkov et al., 2015a); L. sabahmontanus Matsui,
Nishikawa & Yambun (Matsui et al., 2014a), L. solus Matsui
(Matsui, 2006); L. sungi Lathrop, Murphy, Orlov & Ho (Lathrop
et al., 1998); L. tadungensis (Rowley et al., 2016); L. tamdil
Sengupta, Sailo, Lalremsanga, Das & Das (Sengupta et al.,
2010); L. tengchongensis Yang, Wang, Chen & Rao (Yang
et al., 2016); L. tuberosus Inger, Orlov & Darevsky (Inger
et al., 1999; Rowley et al., 2010c); L. ventripunctatus Fei,
Ye & Li (Fei et al., 1990, 2009, 2010; Ohler et al., 2011);
and L. zhangyapingi Jiang, Yan, Suwannapoom, Chomdej &
Che (Jiang et al., 2013). However, due to the considerable
undiagnosed diversity within the family Megophryidae (Chen
et al., 2017; Mahony et al., 2017; Rowley et al., 2015a, 2016),
we relied on examination of topotypic material and/or original
species descriptions.
DNA isolation, PCR, and sequencing
Total DNA was extracted from muscle tissue using standard
phenol-chloroform extraction (Hillis et al., 1996), followed by
isopropanol precipitation. We amplified a 454–474-bp length
fragment of the 16S rRNA mitochondrial gene, which has
been successfully applied for DNA-identification of cryptic
diversity within the genus Leptolalax (Poyarkov et al., 2015a;
Rowley et al., 2015a, 2016, 2017a, 2017b). The 16S
rRNA-gene fragment was amplified using ScreenMix-HS
(Evrogen, Russia) following the manufacturer’s instructions.
The PCR contained 6 µL of ScreenMix-HS, 21 µL of water,
0.9 µL of each primer at a concentration of 10 pmol/µL, and
1.2 µL of template DNA at a concentration up to ca. 100 ng
DNA/µL in a 30 µL reaction volume.
The primers used for PCR and sequencing were: 16SL-1
(50-CTGACCGTGCAAAGGTAGCGTAATCACT-30; forward)
and 16SH-1 (50-CTCCGGTCTGAACTCAGATCACGTAGG-30;
reverse) (Hedges, 1994). The PCR conditions followed
Poyarkov et al. (2015b). The amplification protocols included:
94 ◦C for 5 min of initial denaturation; followed with 35 cycles
of denaturation at 94 ◦C for 1 min, annealing at 55 ◦C for 1
min, and extension at 72 ◦C for 1 min; and a final extension
at 72 ◦C for 10 min. The obtained PCR products were loaded
onto 1% agarose gels and visualized in the presence of
ethidium bromide in a Dark Reader Transilluminator (Clare
Chemical, USA). If distinct bands were produced, they were
sent to Evrogen (Moscow, Russia) for subsequent purification
and sequencing in both directions. The obtained sequences
were checked by eye using chromatogram editor software
DNA Baser v4.20.0; primer sequences were removed, and
the edited sequences were submitted to GenBank under the
Zoological Research 38(3): 1-16, 2018 3

accession numbers MG787987–MG787993 (Table 1).
Phylogenetic analyses
For phylogenetic analyses of the L. applebyi species group,
we used 32 published sequences of 16S rRNA (Poyarkov
et al., 2015a; Rowley et al., 2015a, 2016) and seven newly
obtained sequences of Leptolalax sp. from Phu Yen Province
(Table 1). In total, a dataset of 39 ingroup sequences was
used for the analyses. Sequences of L. ventripunctatus,L.
bourreti,L. pluvialis,L. firthi, and L. pictus, representing
different species groups within Leptolalax, were used as
outgroup taxa following Rowley et al. (2016).
Table 1 Specimens, localities, museum voucher IDs, and GenBank accession numbers (AN) of the Leptolalax applebyi group
members and Leptolalax species outgroup used for molecular analyses
Species Locality Voucher No. GenBank AN
Leptolalax applebyi Song Thanh, Quang Nam Prov., Vietnam AMS R171703 HM133597
L. applebyi Ngoc Linh Mt., Kon Tum Prov., Vietnam AMS R173778 KR018108
L. applebyi Ngoc Linh Mt., Kon Tum Prov., Vietnam AMS R173737 KU530188
L. applebyi Ngoc Linh Mt., Kon Tum Prov., Vietnam AMS R173735 KU530189
L. ardens Kon Ka Kinh, Gia Lai Prov., Vietnam AMS R176454 KR018109
L. ardens Kon Ka Kinh, Gia Lai Prov., Vietnam AMS R176463 KR018110
L. ardens Kon Ka Kinh, Gia Lai Prov., Vietnam AMS R176467 KR018111
L. bidoupensis Hon Giao Mt., Lam Dong Prov., Vietnam ASM R173133 HQ902880
L. bidoupensis Hon Giao Mt., Lam Dong Prov., Vietnam AMS R173134 HQ902881
L. bidoupensis Hon Giao Mt., Lam Dong Prov., Vietnam NCSM77320 HQ902882
L. bidoupensis Hon Giao Mt., Lam Dong Prov., Vietnam NCSM77321 HQ902883
L. bidoupensis Bidoup Mt., Lam Dong Prov., Vietnam ZMMU NAP- 01453 KP017573
L. bidoupensis Bidoup Mt., Lam Dong Prov., Vietnam ZMMU NAP-01458 KP017574
L. kalonensis Song Luy, Binh Thuan Prov., Vietnam IEBRA2014.15 KR018114
L. kalonensis Song Luy, Binh Thuan Prov., Vietnam AMNHA191762 KR018115
L. kalonensis Song Luy, Binh Thuan Prov., Vietnam IEBRA2014.16 KR018116
L. kalonensis Song Luy, Binh Thuan Prov., Vietnam AMNHA191765 KR018117
L. macrops sp. nov. Dak Lak Prov., Vietnam AMS R177663 KR018118
L. macrops sp. nov. Hon Den Mt., Phu Yen Prov., Vietnam IEBR A.2017.9 MG787990
L. macrops sp. nov. Hon Den Mt., Phu Yen Prov., Vietnam PYU DTD-508 MG787991
L. macrops sp. nov. Hon Den Mt., Phu Yen Prov., Vietnam PYU DTD-509 MG787992
L. macrops sp. nov. Hon Den Mt., Phu Yen Prov., Vietnam ZMMU A-5823 MG787993
L. maculosus Phuoc Binh, Ninh Thuan Prov., Vietnam AMS R177660 KR018119
L. maculosus Phuoc Binh, Ninh Thuan Prov., Vietnam ZFMK 96600 KR018120
L. melicus Virachey, Ratanakiri Prov., Cambodia MVZ 258197 HM133599
L. melicus Virachey, Ratanakiri Prov., Cambodia MVZ 258198 HM133600
L. melicus Virachey, Ratanakiri Prov., Cambodia MVZ 258199 HM133601
L. pallidus Gia Rich, Lam Dong Prov., Vietnam USN00510 KR018112
L. pallidus Gia Rich, Lam Dong Prov., Vietnam USN00512 KR018113
L. pallidus Gia Rich, Lam Dong Prov., Vietnam USN00511 KU530190
L. pyrrhops Loc Bac, Lam Dong Prov., Vietnam ZMMU A-5208 KP017575
L. pyrrhops Loc Bac, Lam Dong Prov., Vietnam ZMMU A-4873-1 KP017576
L. pyrrhops Loc Bac, Lam Dong Prov., Vietnam ZMMU A-4873-2 KP017577
L. pyrrhops Loc Bac, Lam Dong Prov., Vietnam ZMMU A-4873-3 KP017578
L. tadungensis Ta Dung, Dak Nong Prov., Vietnam USN00515 KR018121
L. tadungensis Ta Dung, Dak Nong Prov., Vietnam USN00517 KR018122
Leptolalax sp. Hoa Thinh, Phu Yen Prov., Vietnam PYU DTD-488 MG787987
Leptolalax sp. Hoa Thinh, Phu Yen Prov., Vietnam ZMMU A-5824 MG787988
Leptolalax sp. Hoa Thinh, Phu Yen Prov., Vietnam PYU DTD-490 MG787989
L. bourreti Lao Cai Prov., Vietnam AMS R177673 KR018124
L. firthi Kon Tum Prov., Vietnam AMS R 176524 JQ739206
L. pictus Malaysia, Borneo UNIMAS 8705 KJ831295
L. pluvialis Lao Cai Prov., Vietnam MNHN1999.5675 JN848391
L. ventripunctatus Phongsaly Prov., Laos MNHN 2005.0116 JN848410
4www.zoores.ac.cn

Sequences of 44 specimens of Leptolalax representatives, with
a total length of up to 1 046 bp, were included in the final
alignment and subjected to phylogenetic analyses. Sequences
were initially aligned using ClustalW (Thompson et al., 1997)
in Bioedit 7.0.5 (Hall, 1999) with default parameters. Mean
uncorrected genetic distances (P-distances) between sequences
and species were calculated using MEGA 7.0 (Kumar et al.,
2016). PartitionFinder v.1.1.0 (Lanfear et al., 2012) was applied
to estimate the optimal evolutionary models used for dataset
analysis. The best-fitting model was the GTR+I+G model
of DNA evolution, as suggested by the Akaike Information
Criterion (AIC), corrected Akaike Information Criterion (AICc),
and Bayesian Information Criterion (BIC).
The matrilineal genealogy was inferred using Bayesian
inference (BI) and maximum likelihood (ML) algorithms.
The BI analyses were conducted in MrBayes v.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 ten million generations and sampled
every 1 000 generations. Five independent MCMCMC runs
were performed and 1 000 trees were discarded as burn-in.
Confidence in topology was assessed by posterior probability
(BI PP, Huelsenbeck & Ronquist, 2001). The ML analyses
were conducted using Treefinder (Jobb et al., 2004) and
confidence in node topology was tested by non-parametric
bootstrapping with 1 000 replicates (ML BS, Felsenstein,
1985). We a priori regarded tree nodes with bootstrap (ML BS)
values of 70% or greater and Bayesian posterior probabilities
(BI PP) values over 0.95 as sufficiently resolved (Felsenstein,
2004; Hillis & Bull, 1993; Huelsenbeck & Hillis, 1993). The
ML BS values between 70% and 50% (BI PP between 0.95
and 0.90) were treated as tendencies and nodes with ML
BS values below 50% (BI PP below 0.90) were regarded as
unresolved.
RESULTS
Sequence variation
The 16S rRNA dataset contained 39 ingroup and five outgroup
Leptolalax sequences. The final alignment consisted of 1 075
sites, with 617 conserved sites and 413 variable sites, 139 of
which were parsimony-informative; the transition-transversion
bias (R) was estimated as 2.14 (all data given for ingroup
only). Substitution rates were estimated under the General
Time Reversible (GTR) model (+I+G). Nucleotide frequencies
were A=31.23%, T=24.47%, C=24.27%, and G=20.03%.
Phylogenetic relationships
Phylogenetic analysis results of the 16S rRNA gene fragment
are shown in Figure 2. The ML and BI phylogenetic analyses
showed essentially similar topologies, which only differed
slightly in associations at poorly supported basal nodes.
Figure 2 Bayesian inference (BI) phylogram for Leptolalax applebyi species group based on analysis of 16S rRNA sequences
Figure 2 Bayesian inference (BI) phylogram for Leptolalax applebyi species group based on analysis of 16S rRNA sequences
Values at nodes correspond to BI PP / ML BS support values, respectively. Sequences of L. pictus, L. pluvialis, L. ventripunctatus, L. firthi, and L. bourreti
were used as outgroups.
In general, the topology of the BI cladogram was consistent
with results reported in previous work (Poyarkov et al., 2015a;
Rowley et al., 2015a, 2016), suggesting monophyly of the L.
applebyi species group (node support values 1.0/99, hereafter
given for BI PP/ML BS, respectively) and the presence of two
major lineages within it. Clade I encompassed three species
inhabiting the Tay Nguyen (Kon Tum) Plateau in central Vietnam
and northeastern Cambodia: namely, L. applebyi,L. ardens,
and L. melicus (Figure 2). Clade II comprised the remaining L.
applebyi group species from the Langbian (Da Lat) Plateau of
Zoological Research 38(3): 1-16, 2018 5

the Southern Annamite Mountains. Phylogenetic relationships
within Clade II were not sufficiently resolved: there was a
tendency toward a more distant position for L. pyrrhops and L.
maculosus, with the remaining lineages forming a monophyletic
group (0.95/70). The two newly discovered populations of
Leptolalax sp. from Phu Yen Province formed two independent
mtDNA matrilines: that is, the Hon Den Mt. lineage and Hoa
Thinh lineage. The sequence of Leptolalax sp. from Dak Lak
Province (indicated as “molecular lineage 7” in Rowley et al.,
2015a) shared the same mtDNA haplotype as the Leptolalax
sp. population from Hon Den Mt., Phu Yen Province, suggesting
that these two populations are conspecific.
Genetic distances
The uncorrected P-distances among and within the 16S rRNA
gene fragment sequences of the studied Leptolalax species
are shown in Table 2. The observed interspecific distances
within the L. applebyi group members ranged from 4.4%
(between L. kalonensis and L. pallidus) to 10.3% (between
L. applebyi and L. pyrrhops) of substitutions. The uncorrected
genetic P-distances in the ingroup and outgroup comparisons
partly overlapped: genetic distances between the L. applebyi
group members versus the Leptolalax taxa outgroup ranged
from 9.2% (between L. maculosus and L. pluvialis) to 15.4%
(between L. kalonensis and L. firthi ).
Table 2 Uncorrected P-distances (percentages) between the examined 16S rRNA sequences of the Leptolalax applebyi group
members (1–11) and Leptolalax species outgroup (12–13)
Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1Leptolalax macrops sp. nov. 0.1
2L. ardens 6.4 0.0
3L. bidoupensis 5.7 7.3 0.0
4L. kalonensis 6.7 8.7 4.9 0.4
5L. maculosus 6.3 5.9 6.3 6.6 0.2
6L. melicus 8.2 4.2 6.9 9.2 7.7 0.0
7L. pallidus 5.8 6.9 4.5 4.4 6.3 8.0 0.0
8L. pyrrhops 7.2 7.4 7.7 6.9 5.5 10.2 6.3 0.2
9L. tadungensis 6.5 7.0 5.3 5.3 6.0 7.9 5.5 6.0 0.2
10 L. applebyi 8.7 5.3 6.9 9.4 8.2 4.9 7.6 10.3 8.0 0.0
11 Leptolalax sp. 5.7 8.3 4.5 5.8 7.3 8.3 4.5 7.4 5.9 8.5 0.0
12 L. bourreti 12.5 10.4 12.2 13.3 11.1 10.4 12.4 13.3 12.9 10.9 13.6 —
13 L. pluvialis 10.8 10.4 10.7 11.3 9.2 10.0 10.8 11.1 9.8 10.0 11.0 7.6 —
14 L. pictus 12.1 11.3 13.0 12.9 12.6 12.0 12.0 13.7 12.8 11.8 14.3 14.0 12.3 —
15 L. ventripunctatus 12.5 10.5 12.8 13.2 10.3 11.2 12.4 11.2 11.7 11.4 13.2 8.5 5.4 11.5 —
16 L. firthi 13.5 12.5 13.4 15.4 12.6 12.7 13.7 13.7 12.7 13.0 14.2 10.4 10.3 12.5 8.8 —
Mean uncorrected intraspecific P-distances of the ingroup are shown on the diagonal.
The newly discovered population of Leptolalax sp. from
Hon Den Mt., Song Hinh District, was clearly distinct from all
other group members in the examined 16S rRNA fragment
sequences and appeared to be most closely related to L.
bidoupensis from the eastern edges of Langbian Plateau
(Lam Dong and Khanh Hoa provinces) and to a Leptolalax
sp. population from Hoa Thinh, Tay Hoa District (Phu Yen
Province) (P-distance=5.7% for both comparisons). The
Leptolalax sp. population from Hoa Thinh was genetically
closer to L. bidoupensis and L. pallidus, with a P-distance
value of 4.5% (both species from eastern Langbian Plateau).
The observed pairwise divergence in 16S rRNA was
greater than that usually seen among species of anurans
(Vences et al., 2005a, 2005b; Vieites et al., 2009) and
was higher than distances between some other recognized
species of the 1L. applebyi group (e.g., 4.4% between L.
pallidus and L. kalonensis and 4.2% between L. ardens and
L. melicus) (Table 2).
Intraspecific genetic P-distances were 0.0% in the
Leptolalax sp. population from Hoa Thinh, and 0.1% in
the Leptolalax sp. from Hon Den Mt.; the five examined
specimens of the latter species of Leptolalax from Dak Lak
and Phu Yen provinces had two haplotypes of the 16S rRNA
gene fragment.
Taxonomy
Our molecular data clearly indicated that the two recently
discovered populations of Leptolalax sp. from Song Hinh
(Hon Den Mt.) and Tay Hoa (Hoa Thinh) districts of Phu Yen
Province belong to two independent mtDNA lineages, clearly
distinct from each other and from the remaining members of
the L. applebyi species group. Despite geographical proximity
(~30 km between Hon Den Mt. and Hoa Thinh), these
two localities cradle distinct species of Leptolalax, and both
appear to be new to science. These two potentially new
species were assigned to the Langbian Plateau clade of the L.
applebyi species group and appear to be closely related to L.
pallidus,L. kalonensis, and L. bidoupensis. At the same time,
the population of Hon Den Mt. appears to be conspecific to a
Leptolalax sp. found in the eastern part of Dak Lak Province
(~30 km between localities).
Lacking enough material for morphological comparisons,
we tentatively indicate the Leptolalax sp. population of Hoa
Thinh (Tay Hoa District) as a candidate new species sensu
Vieites et al. (2009); further morphological and acoustic
studies are necessary to clarify its taxonomic status. Based on
genetic differentiation, phylogenetic analyses of a 16S rRNA
fragment of mtDNA, and analyses of diagnostic morphological
characters (see below in “Comparisons”), the population of
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Leptolalax from Hon Den Mt. in Phu Yen Province of southern
Vietnam clearly represents a new species, which we describe
as follows.
Leptolalax macrops sp. nov.
Figures 3–5, Table 3.
Chresonymy: Leptolalax sp. [molecular lineage 7] — Rowley
et al., 2015a: 10, 12.
Holotype: PYU DTD-508 (field no. DTD-0508), adult male
collected from Suoi Khi Stream, Hon Den Mt., Ea Ly and Ea
Trol Commune border, Song Hinh District, Phu Yen Province,
Vietnam (N12◦52047.000, E108◦49051.100 ; at an elevation of
500 m a.s.l.), collected by Dang Trong Do on 18 August 2015
at 2100 h.
Paratypes: IEBR A.2017.9 (field no. DTD-507) and ZMMU
A-5823 (field no. DTD-510), two adult males, and PYU
DTD-509 (field no. DTD-509), one adult female, collected from
the same locality as the holotype at elevations between 471
and 630 m a.s.l. by Dang Trong Do on 18 August 2015 from
1900 h to 2300 h.
Diagnosis: The species is assigned to the genus
Leptolalax based on the following characters: (1) finger tips
rounded; (2) elevated inner metacarpal tubercle present, not
continuous onto thumb; (3) body with macroglands (including
supra-axillary, pectoral, and femoral glands); (4) vomerine
teeth absent; (5) tubercles on eyelids present; and (6)
anterior tip of snout with whitish vertical bar (Delorme et
al., 2006; Dubois, 1980, 1983; Lathrop et al., 1998; Matsui,
1997, 2006; Rowley et al., 2013). Leptolalax macrops sp.
nov. is distinguished from its congeners by a combination
of the following morphological characters: (1) body size
medium (SVL 28.0–29.3 mm in three adult males, 30.3 mm
in single adult female); (2) supra-axillary glands present,
creamy white; ventrolateral glands indistinct; (3) tympanum
externally distinct; (4) dorsal skin roughly granular with larger
tubercles, dermal ridges on dorsum absent; (5) rudimentary
webbing present between fingers I–II and II–III; rudimentary
webbing between all toes; fingers and toes without dermal
fringes; (6) in life ventral surface greyish-violet with rare
white speckling; (7) supratympanic fold dark brown; (8) iris
bicolored, typically golden in upper half, fading to golden
green in lower half; (9) tibia short (TbL/SVL 0.44–0.45); and
(10) eyes large and protuberant (ED/SVL 0.15–0.16). The
new species is also markedly distinct from all congeners for
which comparable 16S rRNA mitochondrial gene sequences
are available (uncorrected genetic P-distance>5.7%).
Etymology: Specific epithet “macrops” is a noun in the
nominative case, derived from Greek “macros” for “large” and
“ops” for “eye”, in reference to its comparatively large eye size.
Recommended vernacular names: We recommend
“Big-eyed Litter Frog” as the common English name of the new
species and the common name in Vietnamese as “Cóc mày
m´
˘
at to”.
Description of holotype: Medium-sized Leptolalax
specimen (SVL 28.0 mm); body and head in good state of
preservation, fingers and toes partially dehydrated due to
ethanol preservation (Figure 3A,B). Left thigh of holotype
damaged, skin on ventral surface of thigh dissected, with
a significant portion of femoral muscle removed, dissection
length ca. 10 mm. Belly also dissected medially, dissection
length ca. 9 mm, testes can be seen through dissection.
Head: Head flattened, slightly longer than wide (HW/HL
92.7%), top of head weakly concave; snout short (E-S/HL
34.2%), slightly projecting beyond margin of lower jaw; slightly
truncated in dorsal view (Figure 3A), obtusely rounded in
ventral view (Figure 3B), gently sloping and rounded in profile
(Figure 3C); nostril ovoid, oblique, slightly closer to tip of snout
than to eye (Figure 3C; N-S/E-N 72.1%); canthus rostralis
distinct, bluntly rounded; loreal region slightly concave; eyes
very large (ED/HL 40.3%), eye diameter greater than snout
length (ED/E-S 117.8%), notably protuberant in dorsal view
in life (eyeballs depressed down in preserved holotype, Figure
3C); pupil vertical, diamond-shaped; tympanum distinct, round
with vertical diameter equal to horizontal diameter; tympanum
small, less than half eye diameter (TyD/ED 44.0%); tympanic
rim indistinct, not elevated relative to skin of temporal region;
pineal ocellus absent; vomerine teeth absent; vocal sac gular,
vocal sac openings small, oval, and slit-like, located laterally in
corners of mouth floor; tongue long, wide, with free posterior
end, heart-shaped with shallow medial notch at posterior tip;
supratympanic fold well-developed forming distinct glandular
ridge, running from posterior corner of eye posteriorly toward
dorsal edge of tympanum, gently curving ventrally toward
axilla, bearing several flat tubercles (Figure 3C).
Forelimbs: Forelimbs thin, slender; finger tips in life rounded,
but appear slightly enlarged and truncate in preservative due
to partial dehydration, finger tips approximately same width
as distal finger articulation; relative finger lengths: IV=I<II<III;
nuptial pad indistinct; subarticular tubercles absent, replaced
with low dermal ridges prominent on fingers II–IV; inner
metacarpal tubercle large, fused with outer one, forming
single bulging callous structure, prominent on palmar surface
(maximal length 1.3 mm); border between inner and outer
metacarpal tubercles indistinct; fingers in life lack dermal
fringing, basal webbing present between fingers I and II
and fingers II and III, absent between fingers III and IV
(Figures 3D, 4A).
Hindlimbs: Hindlimbs slender, short, tibia less than half
snout-vent length (TbL/SVL 44.5%); tibiotarsal articulation of
adpressed limb reaching eye-level; toe tips round in life, slightly
truncate in ethanol preservative due to partial dehydration;
relative toe lengths: I<V<II<III<IV; subarticular tubercles absent,
replaced by dermal ridges, distinct on all toes and continuing
to metatarsus of toes III–V; inner metatarsal tubercle large,
oval-shaped, nearly two times longer than wide (IMT to width
of inner metatarsal tubercle ratio 186.4%), outer metatarsal
tubercle absent; toes without lateral dermal fringes; basal
webbing present between all five toes, webbing well-developed
between toes I and II, II and III, and III and IV (reaching level
of proximal finger articulation), and somewhat reduced between
toes IV and V (Figures 3E, 4B).
Skin texture and skin glands: Skin on entire dorsum
roughly granular, covered in tubercles of varying sizes, smaller
dorsolaterally; upper eyelids with numerous small rounded
tubercles (flattened in preservative, Figure 3C), snout smooth;
ventral skin smooth; pectoral gland distinct in preservative and
Zoological Research 38(3): 1-16, 2018 7

in life, round, located near axilla, 0.9 mm in diameter (Figure
3B); femoral gland oval, small, 0.7 mm in diameter, located on
posteroventral surface of thigh, approximately five times closer
to knee than to vent; supra-axillary gland present, protuberant,
creamy white, located in axillary region dorsally from insertion
of forelimb, 0.9 mm in diameter; ventrolateral glands indistinct.
Figure 3 Male holotype of Leptolalax macrops sp. nov. (PYU DTD-508) in preservative
(Photos by Nikolay A. Poyarkov)
A: Dorsal view of body; B: Ventral view of body; C: Lateral view of head; D: Volar view
of left hand; E: Plantar view of right foot.
Figure 3 Male holotype of Leptolalax macrops sp. nov. (PYU DTD-508) in preservative (Photos by Nikolay A. Poyarkov)
A: Dorsal view of body; B: Ventral view of body; C: Lateral view of head; D: Volar view of left hand; E: Plantar view of right foot.
Coloration in life: Dorsal surfaces of head and trunk dark
brownish-grey with indistinct dark brown blotches scattered
on posterior part of dorsum and between eyes; interorbital
region with dark bar with indistinct edges; several light
brown blotches of irregular shape and indistinct edges on
anterior part of upper eyelids, scapular region, and sacrum.
Dorsal surfaces of forelimbs and hindlimbs brownish-grey,
elbows and upper arms dorsally much lighter with coppery
orange background. Dark brown line running along canthus
rostralis through eye, and continuing below supratympanic
fold, terminating above axilla, encompassing nare, loreal
region but not tympanum; tympanum lighter than surrounding
skin of temporal region. Faint transverse dark brown bars
on dorsal surface of thighs, tibia, tarsus, lower arms, fingers,
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and toes. Small indistinct dark brown blotches on flanks.
Tiny whitish flecks scattered on dorsolateral sides of body
from groin to axilla. Belly and chest greyish-violet with rare
white speckling on entire ventral surface, including throat,
arms, and legs. Supra-axillary gland creamy white; femoral
glands whitish; pectoral glands white. Iris bright orange-gold
with greenish tint in lower half and fine black reticulations
throughout. Iris periphery lined with black. Sclera light
yellowish-green.
Coloration in preservative: In preservative, coloration of
holotype significantly faded to light brown on dorsum and
flanks, with slightly paler limbs and beige on ventral sides
(Figure 3B); dark markings on dorsal surfaces brownish, dark
banding on dorsal surface of tibiotarsus, antebrachium, hands,
and feet well-discernable (Figure 3A). Elbows and upper arms
pale brown. White speckles on ventral surface not discernable.
Macroglands creamy white.
Measurements of holotype (in mm): SVL 28.0; HW 9.9; HL
10.7; ED 4.3; TyD 1.9; E-T 0.8; E-S 3.7; E-N 2.0; N-S 2.1; IO
2.9; IN 2.3; ELW 2.7; FAL 7.1; HAL 6.2; FIL 1.8; FIIL 2.6; FIIIL
4.5; FIVL 1.8; TbL 12.5; FeL 11.9; FOL 11.2; TFOL 17.0; IMT
1.8; IFE 5.0; IBE 8.5; TIL 1.1; TIIL 3.3; TIIIL 4.2; TIVL 6.3; TVL
3.0.
Variation: All individuals in the type series were generally
similar in morphology and body proportions; measurements
of the type series are shown in Table 3 and representative
photograph of male paratype in life is shown in Figure 5. Eyes
were notably protuberant in living specimens (Figure 5). All
specimens showed certain variation in darker brown patterns
on dorsum and dark bands on shanks, forearms, hands, and
feet. The single known female (PYU DTD-509) was slightly
larger (SVL 30.3 mm) than the holotype and two paratype
males. Skin texture appeared to be much less tuberculate
in preservative (Figure 3) than in life (Figure 5).
Figure 4 Volar surface of left hand and plantar surface of right foot of preserved holotype
of Leptolalax macrops sp. nov. (PYU DTD-508) (Drawings by Valentina D. Kretova)
Scale bar: 2 mm.
Figure 4 Volar surface of left hand and plantar surface of right
foot of preserved holotype of Leptolalax macrops sp. nov.
(PYU DTD-508) (Drawings by Valentina D. Kretova)
Scale bar: 2 mm.
Figure 5 Male paratype of Leptolalax macrops sp. nov. (IEBR A.2017.9) in life (Photo taken in situ. Photo by Dang Trong Do)
Figure 5 Male paratype of Leptolalax macrops sp. nov. (IEBR A.2017.9) in life (Photo taken in situ. Photo by Dang Trong Do)
Zoological Research 38(3): 1-16, 2018 9

Table 3 Measurements of the type series of Leptolalax macrops sp. nov.
Specimen Holotype Paratype Paratype Mean(M) SD (M) Paratype
character PYU DTD-508 IEBR A.2017.9 ZMMU A-5823 PYU DTD-509
Sex M M M F
SVL 28.0 29.3 28.3 28.6 0.7 30.3
HW 9.9 10.6 10.1 10.2 0.4 11.2
HL 10.7 10.9 10.7 10.8 0.1 11.9
ED 4.3 4.5 4.5 4.4 0.1 4.8
TyD 1.9 1.9 1.9 1.9 0.0 2.1
E-T 0.8 0.8 0.8 0.8 0.0 1.0
E-S 3.7 4.0 4.1 3.9 0.2 4.3
E-N 2.0 2.0 2.1 2.0 0.1 2.4
N-S 1.4 1.4 1.5 1.5 0.1 1.9
IO 2.9 3.0 2.9 2.9 0.1 3.2
IN 2.3 2.3 2.4 2.3 0.0 2.5
ELW 2.7 2.9 2.8 2.8 0.1 3.1
FAL 7.1 7.7 7.3 7.3 0.3 7.9
HAL 6.2 6.9 6.4 6.5 0.4 7.1
FIL 1.8 1.9 1.9 1.9 0.1 2.0
FIIL 2.6 2.8 2.5 2.6 0.2 2.9
FIIIL 4.5 4.8 4.6 4.6 0.2 5.2
FIVL 1.8 2.3 2.3 2.2 0.3 3.0
TbL 12.5 13.3 12.8 12.8 0.4 14.3
FeL 11.9 13.1 12.6 12.5 0.6 13.8
FOL 11.2 12.5 11.5 11.7 0.7 13.9
TFOL 17.0 19.4 17.5 18.0 1.3 20.3
IMT 1.8 2.2 2.1 2.0 0.2 2.4
SVL/HL 2.6 2.7 2.6 2.6 0.0 2.5
HL/HW 1.1 1.0 1.1 1.1 0.0 1.1
SVL/TbL 2.2 2.2 2.2 2.2 0.0 2.1
ED/SVL 0.2 0.2 0.2 0.2 0.0 0.2
SD: standard deviation; M: male; F: female; for other abbreviations see the Materials and Methods. All measurements are in mm.
Comparisons: Leptolalax macrops sp. nov. differs from all
other Leptolalax species in mainland Southeast Asia based
on morphology.
Leptolalax macrops sp. nov. can be distinguished from all
congeners that are not members of the L. applebyi species
group in its overall morphology. Leptolalax macrops sp.
nov. can be differentiated from all Leptolalax species south
of the Isthmus of Kra currently assigned in the subgenus
Leptolalax (L. arayai,L. dringi,L. fritinniens,L. gracilis,L.
hamidi,L. heteropus,L. kajangensis,L. kecil,L. marmoratus,
L. maurus,L. pictus,L. platycephalus,L. sabahmontanus, and
L. solus) in having pectoral and ventrolateral macroglands
(vs. absent) and supra-axillary glands (vs. absent in most
species, except L. marmoratus). With its medium body
size (SVL 28.0–29.3 mm in adult males, 30.3 mm in single
adult female), the new species can be distinguished from
larger congeners, including L. bourreti (males 28.0–36.2 mm,
females 42.0–45.0 mm), L. eos (males 33.1–34.7 mm, female
40.7 mm), L. nahangensis (male 40.8 mm), L. platycephalus
(male 35.1 mm, female 46.0 mm), L. sungi (males 48.3–52.7
mm, females 56.7–58.9 mm), and L. zhangyapingi (males
47.6–50.7 mm); and from smaller-sized species, including L.
alpinus (males 24.0–26.4 mm), L. croceus (males 22.2–27.3
mm), L. isos (males 23.7–27.9 mm), L. kecil (males 19.3–20.5
mm, female 25 mm), L. khasiorum (males 24.5–27.3 mm),
L. laui (males 24.8–26.7 mm), L. pluvialis (males 21.3–22.3
mm), and L. tengchongensis (males 23.9–26.0 mm). With its
distinct tympanum, Leptolalax macrops sp. nov. differs from
L. tuberosus,L. croceus, and L. sungi (vs. tympanum hidden
in the latter species). With its roughly granular dorsum with
larger tubercles, Leptolalax macrops sp. nov. differs from
L. alpinus,L. bourreti,L. fuliginosus,L. gracilis,L. hamidi,L.
heteropus,L. isos,L. kajangensis,L. kalonensis,L. liui,L.
melanoleucus,L. minimus,L. nahangensis,L. oshanensis,L.
pelodytoides,L. pictus, and L. pluvialis (vs. mostly smooth
skin with or without skin ridges) and from L. croceus and
L. tuberosus (vs. highly tuberculate dorsum). With its
greyish-violet ventral surface with rare white speckling, the
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new species also differs from L. croceus (vs. orange belly);
from L. aereus,L. bourreti,L. eos,L. firthi,L. fuliginosus,
L. isos,L. khasiorum,L. lateralis,L. laui,L. liui,L. minimus,
L. nahangensis,L. nokrekensis,L. nyx,L. oshanensis,L.
pelodytoides,L. solus,L. sungi,L. tamdil,L. tuberosus, and
L. zhangyapingi (vs. mostly white, creamy white, or pale
grey ventral surfaces with or without dark spots or mottling);
from L. alpinus,L. maoershanensis,L. melanoleucus,L.
pluvialis,L. tengchongensis, and L. ventripunctatus (vs. large
patches of distinct brown/grey and white marbling or blotches);
from L. petrops (vs. pale pink and slightly translucent belly,
ventral surface of chest and abdomen immaculate white); and
from L. kecil (vs. uniformly dark venter with large, dark
orange pectoral glands). The new species can be further
distinguished from L. aereus,L. croceus,L. eos,L. firthi,L.
isos,L. laui, and L. tuberosus by having a supratympanic fold
with a distinct dark brown to black line (vs. dark supratympanic
line absent in the latter species). With its toes showing
basal webbing and no lateral fringing, Leptolalax macrops sp.
nov. can be diagnosed from L. aereus,L. eos,L. firthi,L.
isos,L. khasiorum and L. tamdil (vs. extensive toe webbing
and distinct lateral fringes on toes). The new species can
be further differentiated from L. botsfordi (Lao Cai Province,
northern Vietnam) by having a bicolored golden green iris
(vs. uniformly brownish-golden iris), greyish-violet ventral
coloration (vs. reddish-brown belly with white speckling), and
roughly granulate dorsum (vs. weakly shagreened dorsum in
L. botsfordi). Leptolalax macrops sp. nov. can be further
diagnosed from L. puhoatensis (Nghe An Province, northern
Vietnam) by its larger size in males (SVL 28.0–29.3 vs.
SVL 24.2–28.1 mm), roughly granulate dorsum, not forming
dermal ridges in life (vs. distinct dermal ridges present), and
tympanum lighter than supratympanic fold (vs. tympanum
completely dark).
Leptolalax macrops sp. nov. is most similar to members of
the L. applebyi species group inhabiting the Central Highlands
of central and southern Vietnam and the northeastern par t of
Cambodia, including L. applebyi,L. ardens,L. bidoupensis,L.
kalonensis,L. maculosus,L. melicus,L. pallidus,L. pyrrhops,
and L. tadungensis. Superficially, the new species resembles
L. pyrrhops, another medium-sized member of the L. applebyi
species group with large eyes, distributed in the western part
of Langbian Plateau (Lam Dong Province) (Poyarkov et al.,
2015a). Comparisons of the new species with members of
the L. applebyi species group are thus appropriate.
From other members of the L. applebyi species group,
Leptolalax macrops sp. nov. can be distinguished by a
combination of morphological characters (Rowley et al., 2016;
the following morphometric differences refer to males only). In
body size, Leptolalax macrops sp. nov. (SVL 28.0–29.3 mm,
mean 28.6 mm, n=3) differs from all other members of the
L. applebyi species group, except for L. kalonensis, including
smaller species L. applebyi,L. ardens,L. bidoupensis,L.
maculosus,L. melicus,L. pallidus, and L. tadungensis (vs.
SVL 19.6–22.3 mm, mean 20.8 mm, n=9, in L. applebyi ;
SVL 21.3–24.7 mm, mean 22.8 mm, n=16, in L. ardens;
SVL 18.5–25.4 mm, mean 23.6 mm, n=12, in L. bidoupensis;
SVL 24.2–26.6 mm, mean 25.5 mm, n=3, in L. maculosus;
SVL 19.5–22.7 mm, mean 20.7 mm, n=8, in L. melicus; SVL
24.5–27.7 mm, mean 25.6 mm, n=8, in L. pallidus; SVL
23.3–28.2 mm, mean 25.0 mm, n=10, in L. tadungensis), and
larger species L. pyrrhops (vs. SVL 30.8–34.3 mm, mean
33.2 mm, n=7, in L. pyrrhops). The new species differs
from other members of the L. applebyi species group, with
the exception of L. pyrrhops, in having a much larger eye
diameter (ED/SVL 0.15–0.16, mean 0.16, n=3, in the new
species vs. 0.10–0.13, mean 0.12, n=9, in L. applebyi ; vs.
0.12–0.14, mean 0.13, n=16, in L. ardens; vs. 0.11–0.14,
mean 0.12, n=12, in L. bidoupensis; vs. 0.12–0.15, mean
0.13, n=16, in L. kalonensis; vs. 0.12–0.14, mean 0.13, n=3,
in L. maculosus; vs. 0.13–0.14, mean 0.13, n=8, in L. melicus;
vs. 0.12–0.14, mean 0.13, n=8, in L. pallidus; vs. 0.11–0.14,
mean 0.13, n=10, in L. tadungensis). Leptolalax macrops
sp. nov. has a narrower head than that of L. maculosus
(HW/SVL 0.35–0.36, mean 0.36, n=3 vs. 0.37–0.38, mean
0.37, n=3) and L. pyrrhops (HL/HW 1.03–1.08, mean 1.06,
n=3 vs. 1.12–1.31, mean 1.14, n=7). The new species has
a shorter eye-tympanum distance (E-T/SVL 0.03–0.03, mean
0.03, n=3) than L. maculosus (vs. E-T/SVL 0.04–0.05, mean
0.04, n=3), L. pallidus (vs. E-T/SVL 0.04–0.06, mean 0.05,
n=8), and L. pyrrhops (vs. E-T/SVL 0.04–0.06, mean 0.05,
n=7). Leptolalax macrops sp. nov. has a shorter tibia
(TbL/SVL 0.44–0.45, mean 0.45, n=3, in the new species)
than that of L. kalonensis (vs. TbL/SVL 0.45–0.52, mean 0.48,
n=16), L. maculosus (vs. TbL/SVL 0.48–0.50, mean 0.50,
n=3), L. pallidus (vs. TbL/SVL 0.45–0.51, mean 0.49, n=8),
and L. pyrrhops (vs. TbL/SVL 0.48–0.50, mean 0.50, n=7).
Leptolalax macrops sp. nov. is unique among members of
the L. applebyi species group in having rudimentary webbing
between fingers I–II and II–III (vs. finger webbing absent in
other species) and in having rudimentary webbing between
its toes (vs. toe webbing absent in L. ardens,L. kalonensis,
L. maculosus,L. pallidus,and L. tadungensis). Leptolalax
macrops sp. nov. can be further distinguished from most
L. applebyi species group members, except for L. pallidus
and L. pyrrhops, in having roughly granulate skin on dorsum
with larger tubercles (vs. smooth to weakly shagreened skin
in L. applebyi,L. ardens,L. bidoupensis,L. kalonensis,L.
maculosus,L. melicus, and L. tadungensis); L. pallidus has
tuberculate skin and L. pyrrhops has dorsum varying from
finely shagreened to tuberculate. By lacking dermal fringes
on its toes, Leptolalax macrops sp. nov. can be distinguished
from L. bidoupensis,L. maculosus, and L. tadungensis (vs.
weak or distinct lateral fringes on toes). In having creamy
white supra-axillary glands, the new species differs from
other species of the L. applebyi species group, except for
L. applebyi and L. bidoupensis, all of which have copper
to orange supra-axillary glands. The new species can be
further distinguished from L. pallidus by having a distinct black
supratympanic line (vs. black supratympanic line absent).
With its golden/greenish bicolored iris, Leptolalax macrops sp.
nov. can be further distinguished from L. applebyi,L. ardens,
L. melicus, and L. tadungensis (vs. uniform coloration of iris).
Distribution: The new species is currently known from only
two sites (~30 km from each other) in the tropical evergreen
forests of Phu Yen Province (Hon Den Mt., Ea Ly and Ea
Trol Commune border, Song Hinh District) and Dak Lak
Province (Chu Mu Mt., M’Drak District, based on molecular
data from Rowley et al., 2015a). The new species inhabits
the northeastern outcrops of the Langbian Plateau. In Phu
Yen Province, Leptolalax macrops sp. nov. was recorded at
elevations between 471 and 630 m a.s.l. The distribution of
Zoological Research 38(3): 1-16, 2018 11

the new species may be quite narrow, possibly restricted to a
small mountain ridge located on the border of Dak Lak, Khanh
Hoa, and Phu Yen provinces.
Ecological notes: Leptolalax macrops sp. nov. is currently
known only from evergreen tropical forests on the border of
Phu Yen, Dak Lak, and Khanh Hoa provinces. All specimens
of the new species were found along cascade rocky streams at
elevations between 471–630 m a.s.l. (Figure 6). Surrounding
habitat was evergreen tropical forest of large and medium
hardwoods and shrubs, with varying degrees of disturbance.
Animals were collected at night between 1900–2300 h. Both
males and females were found on rocks mid-stream and up to
1 m from the streams.
Leptolalax macrops sp. nov. is known to occur in
syntopy with Microhyla berdmorei (Blyth) (Do et al., 2017b).
Other anuran species recorded in Song Hinh District include
Ophryophryne cf. synoria Stuart, Sok & Neang (indicated
as O. hansi Ohler by Do et al., 2017b, identification
following Poyarkov et al., 2017); Calluella guttulata (Blyth);
Kalophrynus cf. honbaensis Vassilieva, Galoyan, Gogoleva
& Poyarkov (Vassilieva et al., 2014); Kaloula indochinensis
Chan, Blackburn, Murphy, Stuart, Emmett, Ho & Brown;
Microhyla mukhlesuri Hasan, Islam, Kuramoto, Kurabayashi
& Sumida (identification following Poyarkov et al., 2014,
Yuan et al., 2016); Microhyla pulchra (Hallowell), Fejervaria
limnocharis (Gravenhorst); Limnonectes cf. bannaensis Ye,
Fei & Jiang; Limnonectes poilani (Bourret); Occidozyga lima
(Gravenhorst); Sylvirana nigrovittata (Blyth); Odorrana cf.
morafkai (Bain, Lathrop, Murphy, Orlov & Ho); Polypedates
mutus (Smith), and Rhacophorus annamensis Smith (data
from Do et al., 2015, 2017b).
Conservation status: To date, the new species is only
known from a small montane area on the border of Dak Lak,
Khanh Hoa, and Phu Yen provinces. It is likely that the
range of Leptolalax macrops sp. nov. is quite narrow. The
species probably inhabits Ea So Nature Reserve (Dak Lak
Province); however, additional research in this area is needed.
The new species appears to require closed evergreen forest
along the streams where it occurs. Areas of low to middle
elevation montane tropical forest are greatly endangered in
the southern coastal areas of Vietnam, including Phu Yen
Province. Given the available information, we suggest the
species should be considered as Data Deficient following the
IUCN’s Red List categories (IUCN, 2001) until the distribution
and habitat requirements of the new species are more fully
documented.
Figure 6 Typical habitat (A) and microhabitat (B) of Leptolalax macrops sp. nov. in type locality: Suoi Khi Stream, Hon Den Mt., Ea Ly and
Ea Trol commune border, Song Hinh District, Phu Yen Province, Vietnam (Photos by Dang Trong Do)
Frogs were recorded along a cascading stream.
Figure 6 Typical habitat (A) and microhabitat (B) of Leptolalax macrops sp. nov. in type locality: Suoi Khi Stream, Hon Den Mt.,
Ea Ly and Ea Trol commune border, Song Hinh District, Phu Yen Province, Vietnam (Photos by Dang Trong Do)
Frogs were recorded along a cascading stream.
DISCUSSION
Our molecular data revealed hidden diversity of the L. applebyi
species group, with additional herpetological surveys in
mountain areas of Indochina possibly leading to the discovery
of further new lineages and species of Leptolalax. Our
finding brings the number of recognized species of the genus
Leptolalax to 54, and the number of Leptolalax species known
from Vietnam to 24.
The herpetofauna of the Phu Yen Province is poorly
studied compared to the adjacent provinces of Dak Lak and
Khanh Hoa. Nguyen et al. (2009) recorded 12 species
of reptiles and only five species of amphibians from this
province. Ziegler et al. (2013) described a new species
of bent-toed gecko, Cyrtodactylus kingsadai Ziegler, Phung,
Le & Nguyen, from Tuy Hoa District in the eastern part of
Phu Yen Province. Recently, Do et al. (2017a) repor ted
Lycodon cardamomensis Daltry & Wüster for the first time
from Vietnam based on a single specimen collected from Phu
Yen Province. More recently, Do et al. (2017b) reviewed
the available data on amphibian species found in Phu Yen
Province, and added eight new provincial records and listed
33 species of amphibians for the province. The present paper
describes a new species of Leptolalax from the Song Hinh
District. We also recorded a previously undescribed lineage
12 www.zoores.ac.cn

of Leptolalax sp. from Tay Hoa District of Phu Yen Province;
however, further morphological and molecular research is
required to clarify the taxonomic status of this population.
Tropical forests are greatly endangered throughout
Southeast Asia, including Vietnam. Compared with the
hard-to-access montane tropical forests in the Annamite
Mountains, evergreen tropical forests in lowland and foothill
areas of the southern coastal region of Vietnam are more
endangered; most areas of lowland tropical forest are already
destroyed due to logging and other human activities (De
Koninck, 1999; Laurance, 2007; Meijer, 1973; Meyfroidt &
Lambin, 2008). However, despite their accessibility, the many
remaining patches of tropical forest could cradle still unknown
biodiversity, which makes the need for biological exploration in
this region even more urgent.
ADDENDUM
During the revision process of the present manuscript, a
new paper by Nguyen et al. (2018) was published providing
description of Leptolalax rowleyae: Nguyen, Poyarkov, Le,
Vo, Phan, Duong, Murphy & Nguyen, 2018, a new species
of the L. applebyi group from the Son Tra Peninsula in Da
Nang City, central Vietnam (published on 1 March 2018).
We were unable to include L. rowleyae in the comparisons
section or phylogenetic analysis in the present manuscript;
however, it is markedly distinct from the new species in a
number of morphological attributes: by smaller body size:
SVL 23.4–25.4 mm in males and 27–27.8 mm in females (vs.
SVL 28.0–29.3 mm in adult males and 30.3 mm in single
adult female of the new species); by pinkish milk-white to
light brown ventral surface with numerous white speckles (vs.
greyish-violet ventral surface with rare white speckling in the
new species); and by much smaller eyes, ED/SVL 0.08–0.11
in males (vs. ED/SVL 0.15–0.16 in males of the new species)
(data from Nguyen et al., 2018). The new species can also be
distinguished from L. rowleyae by deep divergence in the 16S
rRNA mtDNA gene (P-distance 12.60%) and phylogenetic
position (the new species is mentioned as “Leptolalax sp.” in
the work of Nguyen et al., 2018: Figure 1).
In addition, a recently accepted manuscript by Chen et
al. (2018) (published online on 10 March 2018) provides
a novel multi-locus phylogenetic hypothesis for the genus
Leptolalax, describing the latter as a synonym of the genus
Leptobrachella Smith, 1925. Due to the simultaneous review
period of the present paper and the work of Chen et al. (2018),
we were unable to implement the new taxonomy at the stage
of submission and reviewing process. We suggest that the
new species Leptolalax macrops sp. nov. should hereafter
be referred to as Leptobrachella macrops Duong, Do, Ngo,
Nguyen & Poyarkov to reflect the revised taxonomy.
COMPETING INTERESTS
The authors declare that they have no competing interests.
AUTHORS’ CONTRIBUTIONS
N.A.P. and T.Q.N. designed the study. T.V.D., D.T.D. and C.D.N. collected
data. T.V.D. preformed molecular experiments. D.T.D. and N.A.P. examined
morphology. N.A.P. supervised the analyses. N.A.P. and T.V.D. wrote the
manuscript, D.T.D., N.A.P. and T.Q.N. revised the manuscript. All authors
read and approved the final manuscript.
ACKNOWLEDGEMENTS
We would like to thank the directorates of the Forest Protection Department
of the Phu Yen Province for supporting our field work and issuing relevant
permits. NAP thanks Dr. Andrei N. Kuznetsov and Prof. Leonid P. Korzoun
for organizing his work in Vietnam and Luan T. Nguyen for help and useful
discussions. We would like to express our gratitude to Dr. Jian-Huan Yang,
Prof. Ying-Yong Wang, and two anonymous reviewers for useful comments
on the earlier version of this manuscript.
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Zoological Research 38(3): 1-16, 2018 15

APPENDIX
Examined material, museum IDs given in bold.
Abbreviations: VNMN: Vietnam National Museum of Nature, Hanoi
(Vietnam); ZMMU: Zoological Museum of Lomonosov Moscow State
University, Moscow (Russia); ZISP: Zoological Institute R.A.S., St.
Petersburg (Russia).
Leptolalax aereus:ZISP 12042–12045 (Vietnam, Quang Binh Pr., Phong
Nha – Ke Bang N.P.; 4 sp.); ZMMU A-5214 (Vietnam, Quang Binh Pr.,
Phong Nha – Ke Bang N.P.; 2 sp.).
Leptolalax applebyi:ZMMU A-5529 (Vietnam, Thua Thien - Hue Pr., A
Roang area; 15 sp.); ZMMU A-5556 (Vietnam, Thua Thien - Hue Pr., A
Roang area; 1 sp.).
Leptolalax ardens:ZMMU NAP-06099–06100 (Vietnam, Gia Lai Pr., Kon
Ka Kinh N.P., 2 sp.).
Leptolalax bidoupensis:ZMMU A-4717 (Vietnam, Lam Dong Pr., Bidoup –
Nui Ba N.P., Bidoup Mt.; 1 sp.); ZMMU A-4797 (Vietnam, Lam Dong Pr.,
Bidoup – Nui Ba N.P., Bidoup Mt., Hon Giao Mt.; 4 sp.); ZMMU A-5211
(Vietnam, Lam Dong Pr., Bidoup – Nui Ba N.P., Bidoup Mt.; 1 sp.).
Leptolalax bourreti:ZISP 12046,ZISP 12048 (Vietnam, Lao Cai Pr., Sa
Pa, Hoang Lien N.P.; 2 sp.); ZMMU A-5220 (Vietnam, Lao Cai Pr., Sa Pa,
Hoang Lien N.P.; 1 sp.); ZISP 12048–12050 (Vietnam, Lao Cai Pr., Van
Ban N.R.; 3 sp.); ZMMU A-5219 (Vietnam, Lao Cai Pr., Van Ban N.R.; 1
sp.); ZMMU A-5031 (Vietnam, Lao Cai Pr., Sa Pa, Tram Don, Fansipan Mt.,
Hoang Lien N.P.; 6 sp.).
Leptolalax firthi :ZISP 12091,ZISP 12058,ZISP 12051–12057,ZISP
12092–12093 (Vietnam, Kon Tum, Ngoc Linh N.P., Dac Glei; 11 sp.); ZMMU
A-5210 (Vietnam, Kon Tum, Ngoc Linh N.P., Dac Glei; 5 sp.).
Leptolalax heteropus:ZMMU NAP-06788 (Malaysia, Perak, Larut hills; 1
sp.).
Leptolalax nahangensis:ZMMU VNH10, ZMMU VNH16 (Vietnam, Bak Kan
Pr., Na Hang; 2 sp.).
Leptolalax nyx:ZISP12059–12061 (Vietnam, Ha Giang Pr., Ha Giang;
3 sp.).
Leptolalax pallidus:ZMMU NAP-01740 (Vietnam, Lam Dong Pr., Bidoup –
Nui Ba N.P., Giang Ly St.; 1 sp.); ZMMU ABV-00453 (Vietnam, Lam Dong
Pr., Bidoup – Nui Ba N.P., Bidoup Mt.; 1 sp.).
Leptolalax petrops:ZMMU NAP-06537;ZMMU NAP-06565;ZMMU
NAP-06567 (Vietnam, Phu Tho Pr., Xuan Son N.P.).
Leptolalax pluvialis:ZISP 12075–12081 (Vietnam, Lao Cai Pr., Sa Pa,
Hoang Lien N.P., Tram Don; 7 sp.); ZMMU A-5209 (Vietnam, Lao Cai Pr.,
Sa Pa, Hoang Lien N.P., Tram Don; 4 sp.); ZMMU A-5222 (Vietnam, Lao
Cai Pr., Sa Pa, Tram Don, Fansipan Mt.; 8 sp.).
Leptolalax pyrrhops:ZMMU A-5208 (Vietnam, Loc Bac Forest Enterprise,
Loc Bao Comm., Bao Lam Distr., Lam Dong Pr., Vietnam; holotype); ZMMU
A-4873 (Vietnam, Loc Bac Forest Enterprise, Loc Bao Comm., Bao Lam
Distr., Lam Dong Pr., Vietnam; 6 sp., paratypes); ZISP 12041 (Vietnam,
Loc Bac Forest Enterprise, Loc Bao Comm., Bao Lam Distr., Lam Dong Pr.,
Vietnam; paratype); VNMN A2015.02 (Vietnam, Loc Bac Forest Enterprise,
Loc Bao Comm., Bao Lam Distr., Lam Dong Pr., Vietnam; paratype).
Leptolalax sungi:ZMMU A-4349 (Vietnam, Ha Giang Pr., Ha Giang; 3 sp.).
Leptolalax tuberosus:ZMMU A-4110 (Vietnam, Kon Tum Pr., Kon Plong; 1
sp.); ZISP 12094–12095 (Vietnam, Kon Tum Pr., Kon Plong; 2 sp.); ZMMU
A-5213 (Vietnam, Kon Tum Pr., Kon Plong; 1 sp.).
Leptolalax ventripunctatus:ZMMU A-5223 (Vietnam, Dien Bien Pr.,
Muong Nhe N.R., Sin Hau St.; 12 sp.); ZMMU A-5156 (Vietnam, Dien Bien
Pr., Muong Nhe N.R., Sin Hau St.; 5 sp.); ZMMU A-5225 (Vietnam, Phu
Tho, Xuan Son N.P.; 3 sp.); ZMMU A-5224 (Vietnam, Phu Tho, Xuan Son
N.P.; 1 sp.); ZISP 12062–12074 (Vietnam, Vinh Phuc Pr., Tam Dao N.P.; 13
sp.); ZMMU A-5212 (Vietnam, Vinh Phuc Pr., Tam Dao N.P.; 5 sp.).
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