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A new species of the endemic Himalayan genus Liurana (Anura, Ceratobatrachidae) from southeastern Tibet, China, with comments on the distribution, reproductive biology, and conservation of the genus

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  • Chengdu Institute of Biology CAS

Abstract and Figures

A new species of the genus Liurana Dubois, 1986 is described from Medog County, Tibet, China, based on morphological and molecular data. The new species can be differentiated from all other congeners by the following combination of characters: (1) head wider than long; (2) tympanum distinct and large; (3) hindlimb long, tibiotarsal articulation beyond tip of snout when adpressed; (4) belly with flat tubercles, cloacal region with small tubercles; (5) transverse bands distinctly on dorsal limbs, four bands on thigh and three on tibia; and, (6) dark brown marbled patterns or speckles on white belly. Here, we also discuss the distribution pattern of Liurana in the East Himalaya region, the role of the Yarlung Tsangpo River in the speciation and genetic isolation of congeners, the direct developmental mode of reproduction, and the two different ecotypes of the genus. Lastly, we provide conservation recommendations for the genus in southeastern Tibet.
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ZOOLOGICAL RESEARCH
A new species of the endemic Himalayan genus
Liurana (Anura, Ceratobatrachidae) from southeastern
Tibet, China, with comments on the distribution,
reproductive biology, and conservation of the genus
Ke Jiang1,#, Kai Wang1,2,#, Yu-Fan Wang3, Cheng Li4, Jing Che1,5,*
1
State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan
650223, China
2Sam Noble Oklahoma Museum of Natural History and Department of Biology, University of Oklahoma, Norman OK 73072-7029, USA
3Zhejiang Forest Resource Monitoring Center, Hangzhou Zhejiang 310020, China
4Funsome Nature Center, Shenzhen Guangdong 518067, China
5Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences, Yezin Nay Pyi Taw 05282, Myanmar
ABSTRACT
A new species of the genus Liurana Dubois, 1986 is
described from Medog County, Tibet, China, based on
morphological and molecular data. The new species
can be differentiated from all other congeners by the
following combination of characters: (1) head wider
than long; (2) tympanum distinct and large; (3) hindlimb
long, tibiotarsal articulation beyond tip of snout when
adpressed; (4) belly with flat tubercles, cloacal region
with small tubercles; (5) transverse bands distinctly on
dorsal limbs, four bands on thigh and three on tibia;
and, (6) dark brown marbled patterns or speckles on
white belly. Here, we also discuss the distribution pattern
of Liurana in the East Himalaya region, the role of the
Yarlung Tsangpo River in the speciation and genetic
isolation of congeners, the direct developmental mode of
reproduction, and the two different ecotypes of the genus.
Lastly, we provide conservation recommendations for the
genus in southeastern Tibet.
Keywords:
Advertisement call; Biogeography; Ecology;
Natural history; Tibet; Taxonomy
INTRODUCTION
As a wide-spread amphibian family in Southeast Asia, species
of the family Ceratobatrachidae are distributed from the southern
foothills of the Himalaya to the tropics of Southeast Asia (Yan et
al., 2016). Members of the family are characterized as tropical
specialists, with many species undergoing direct development
without reliance on standing water bodies for breeding (Brown
& Alcala, 1982; Brown et al., 2015). Within this family, frogs
of the genus Liurana represent an understudied yet fascinating
endemic group from the East Himalaya region.
First considered as a subgenus of Ingerana by Dubois
(1986), Liurana was established based on the type species
Cornufer xizangensis Hu, 1987, with the subgenus later
elevated to full genus based on morphological evidence (Fei
et al., 1997). This taxonomic elevation is supported by
recent phylogenetic studies, where Liurana was recovered as
a distinct monophyletic clade from Ingerana,Platymantis, and
Cornufer (Yan et al., 2016). However, despite research efforts
on the higher-level systematic relationships of the genus Liurana,
Received: 12 October 2018; Accepted: 24 October 2018; Online: 20
March 2019
Foundation items: This project was supported by the Strategic
Priority Research Program (A) Grant XDB13020200 of the Chinese
Academy of Sciences (CAS), Ministry of Science and Technology
of China (2014FY210200), China’s Biodiversity Observation Network
(Sino-BON), Nanjing Institute of Environmental Sciences, Ministry
of Environmental Protection of China, and Animal Branch of
the Germplasm Bank of Wild Species of CAS (Large Research
Infrastructure Funding)
#Authors contributed equally to this work
*Corresponding author, E-mail: chej@mail.kiz.ac.cn
DOI: 10.24272/j.issn.2095-8137.2019.025
Science Press Zoological Research 40(3): 175–184, 2019 175
little attention has been given to the species level taxonomy of the
group in the Himalaya. To date, only a few studies have focused
on species level taxonomy of the genus, with just a single study
conducted in the last decade (Borah et al., 2013; Fei et al., 1997;
Huang & Ye, 1997; Sichuan Biological Research Institute, 1977).
As a result, all recognized species of Liurana are known from
only a few vouchered specimens, and little is understood about
their morphological variation and population structure. Based on
limited studies, three species have been recognized in the genus
to date, including L. alpina Huang, Ye, 1997, L. medogensis Fei,
Ye, Huang, 1997, and L. xizangensis (Hu, 1987).
During herpetological surveys in southeastern Tibet from
2012 to 2016, 20 specimens of the genus Liurana were
collected from Bomê and Medog Counties of the Nyingchi
Prefecture, Tibet, China. Combining phylogenetic and
morphological datasets, we describe here a new species of the
genus from the tropical rainforest of Medog County, Nyingchi
Prefecture, Tibet, China. Fur thermore, we comment on the
evolution, ecology, natural history, and conservation of the
genus Liurana in China.
MATERIALS AND METHODS
Taxon sampling
A total of 20 individuals of the recognized species of the genus
Liurana were collected from different localities in the Bomê
and Medog Counties, Nyingchi Prefecture, southeastern Tibet,
China, and were comprised of 18 individuals of L. alpina,L.
medogensis, and L. xizangensis and two individuals (one adult
male and one adult female) of the new species (Figure 1;
Appendix I). Following euthanasia, tissue samples were taken
and preserved in 95% ethanol, with the specimens then fixed in
10% buffered formalin solution and transferred to 75% ethanol
after fieldwork. All specimens were deposited in the Museum
of the Kunming Institute of Zoology, Chinese Academy of
Sciences (KIZ) (Appendix II).
Morphological data
We confirmed the sex of each specimen by anatomical
observation, with an incision made on the left side. All
measurements were carried out by Ke Jiang using a digital
caliper to the nearest 0.1 mm. Morphological characters and
their measurement followed Fei et al. (2009) and included:
snout–vent length (SVL); head length (HL), measured from
posterior corner of mandible to tip of snout; head width (HW),
measured at the angle of the jaw; snout length (SL), measured
from tip of snout to anterior corner of eye; internarial distance
(IND); interorbital distance (IOD), measured at the shortest
distance between upper eyelid; maximum width of upper
eyelid (UEW); eye diameter (ED), measured as the horizontal
diameter of eye); tympanum diameter (TD), measured as the
maximum horizontal diameter of tympanum); length of lower
arm and hand (LAHL), measured from the elbow joint to the tip
of the longest finger; largest diameter of lower arm (LAD); hand
length (HAL), measured from the base of the outer metacarpal
tubercle to the tip of finger III; femur length (FML), measured as
the linear distance between the insertion of the leg to the knee
joint; tibia length (TL), measured as the linear distance between
the knee joint and tibiotarsal articulation; length of tarsus and
foot (TFL), measured from the tibiotarsal articulation to the tip
of toe IV; and foot length (FL), measured from the base of the
inner metatarsal tubercle to the tip of toe IV.
Figure 1 Distribution of Liurana species in East Himalaya
Blue star represents type locality of the new species, Liurana vallecula sp. nov.
Rectangles represent type localities of recognized species and circles represent
additional known localities of recognized species. Different species are color-coded as
follows: L. alpina (red), L. medogensis (purple), and L. xizangensis (yellow). For L.
xizangensis, the two additional localities are (1) Tongmai, Bomê County, Tibet, China;
and (2) 62K, Medog County, Tibet, China. The two light blue localities represent
reported records of Liurana cf. medogensis from Southern Tibet (see discussion for
details).
Figure 1 Distribution of Liurana species in the East Himalaya
Blue star represents type locality of the new species, Liurana vallecula sp. nov. Rectangles represent type localities of recognized species and circles represent
additional known localities of recognized species. Different species are color-coded as follows: L. alpina (red), L. medogensis (purple), and L. xizangensis
(yellow). For L. xizangensis, the two additional localities are (1) Tongmai, Bomê County, Tibet, China; and (2) 62K, Medog County, Tibet, China. The two light
blue localities represent reported records of Liurana cf. medogensis from Southern Tibet (see discussion for details).
176 www.zoores.ac.cn
In addition to the newly obtained data, morphological data
of congeners were also obtained from published literature for
comparison (Borah et al., 2013; Fei et al., 1997, 2009; Huang
& Ye, 1997).
Molecular analysis
Genomic DNA was extracted from tissue samples using
standard phenol-chloroform protocols (Sambrook et al., 1989).
Fragments of a single mitochondrial DNA locus (cytochrome
c oxidase subunit I, COI) and three nuclear loci, including
recombination activating protein 1 (Rag1), tyrosinase (Tyr),
and rhodopsin (Rhod), were targeted and amplified following
published primers and protocols (Che et al., 2011; Yan et
al., 2016). The products were purified with a Gel Extraction
Mini Kit (Watson Biotechnologies, Shanghai, China) and
sequenced on an ABI 3730xI DNA automated sequencer
(Applied Biosystems, UK).
Additional sequences of congeners and closely related
outgroups were obtained through GenBank (Appendix II).
Sequences were aligned using MUSCLE implemented in
Geneious R10.0.6. Uncorrected genetic distances of the
mitochondrial locus COI were calculated using PAUP* v4.0b10.
To assess phylogenetic congruence between the mitochondrial
and nuclear data, the phylogeny for each gene was inferred
independently using Bayesian analyses. As no strongly
supported incongruences between mitochondrial and nuclear
data were observed visually, two datasets were concatenated
for the final analyses.
Phylogenetic relationships were inferred using both
partitioned Bayesian (MrBayes v3.2.1; Ronquist &
Huelsenbeck, 2003) and maximum likelihood analyses
(RAXML VI-HPC v8.2.10; Stamatakis, 2014). For Bayesian
analyses, all protein-coding genes were partitioned by codon
positions, and the best models of nucleotide substitution were
selected for each partition by the Akaike Information Criterion
(AIC), as implemented in jModelTest v2.1.10 (Darriba et al.,
2012; Guindon & Gascuel, 2003). A rate multiplier model
was used to allow substitution rates to vary among subsets.
Default settings were used for all other model parameters.
Two independent Markov chain Monte Carlo analyses were
run, each with four Metropolis-coupled chains, a melting
temperature of 0.02, and an exponential distribution with a rate
parameter of 25 as the prior branch lengths (Marshall, 2010).
All Bayesian analyses were run for 6000 000 generations, with
parameters and topologies sampled every 3 000 generations.
Stationarity and convergence were assessed with TRACER
v1.6.0 (Rambaut et al., 2013).
Partitioned maximum likelihood analyses were conducted on
the concatenated dataset using the same partitioning strategy
as for Bayesian analysis. The more complex model (GTR+Γ)
was applied for all subsets (Table 1), with 1 000 replicate ML
inferences. Each inference was initiated with a random starting
tree, and nodal support was assessed with 1 000 bootstrap
pseudoreplicates (Stamatakis et al., 2008).
The resulting phylogenetic trees were rooted using the clade
containing Ingerana and Limnonectes, following recent studies
on phylogenetic relationships of the focal group (Yan et al., 2016).
Table 1 Partition strategies and best evolutionary models
selected for each partition
Gene Codon Fragment length (bp) Model selected
COI 1st 190 GTR
2nd 190 HKY+Γ
3rd 190 HKY
Rag1 1st 184 F81
2nd 184 HKY
3rd 184 HKY
Rod 1st 105 JC
2nd 105 F81
3rd 105 GTR
Tyr 1st 177 GTR+Γ
2nd 177 HKY+Γ
3rd 177 HKY+Γ
RESULTS
Morphology
Morphometric variation of the examined Liurana species is
summarized in Table 2. The unidentified specimens of Liurana
were morphologically most similar to L. medogensis and could
be differentiated from all recognized species by having a
relatively wider head (HW/HL >100% vs. <100%) and longer
hindlimbs, with tibiotarsal articulation reaching beyond tip of
snout when adpressed (vs. reaching anterior corner of eye
only). Furthermore, the unidentified Liurana specimens from
Medog possessed distinct tubercles near the cloaca, which are
absent or indistinct in all recognized congeners except for L.
xizangensis.
Coloration and ornamentation were highly variable among
the examined specimens of L. alpina and L. xizangensis,
ranging from uniform bright orange reddish to brownish gray
with blackish speckles (Figure 3). For L. medogensis and
the two unidentified individuals from Medog, the coloration
and ornamentation were less variable. A single individual
of L. medogensis (KIZ05587) possessed a broad, yellowish
dorsal vertebral stripe from snout to vent, whereas the other
individuals of the same species exhibited a much darker
vertebral stripe in light reddish brown.
Phylogeny
The phylogenetic placement of the genus Liurana is similar to
previous results (Yan et al., 2016), with the genus recovered as
monophyletic with strong support (Bayesian posterior probability
(PP)=1.00; ML bootstrap support (BS)=100) (Figure 2). Within
the genus, L. xizangensis and L. alpina form a monophyletic
group with strong supports (PP=0.99, BS=100), which is sister
to L. medogensis (PP=1.00, BS=100). The two unidentified
individuals (Liurana sp.) collected from Medog County form a
distinct, monophyletic clade (PP=1.00, BS=100), which is basal
with respect to all other Liurana congeners (Figure 2).
Zoological Research 40(3): 175–184, 2019 177
For the obtained 570 bp fragment of COI data, the
uncorrected genetic distance between the two unidentified
Liurana individuals is 0.88% only, while such distance
is 22.19%–22.22% to its morphologically most similar
congener, L. medogensis, 21.67%–22.63% to L. alpina, and
20.19%–21.30% to L. xizangensis (Table 3). The observed
intraspecific variation among recognized Liurana species is
0%–0.183% within conspecifics of L. alpina. For L. xizangensis,
the greatest genetic divergence (3.12%) is observed between
individuals from Tongmai, Bomê County, Tibet, China (Locality
#1) and 62K, Medog County, Tibet, China (Locality #2), which
are on different sides of the Yarlung Tsangpo River (Figure 1).
Because the two unidentified specimens of Liurana
from Medog possess a suite of distinct morphological
characteristics, high genetic divergence from congeners, and
monophyletic and distinct phylogenetic position, we here
describe them as a new species.
Taxonomy
Liurana vallecula sp. nov. Jiang, Wang, Wang, Li, and Che
Figures 1–5.
Holotype: KIZ014083, adult female, from the side of Yarlung
Zangbo River near Xirang (N29.1776°, E95.0057°, elevation
550 m a.s.l.; Figure 3), Beibeng, Medog County, southeastern
Tibet, China. Collected by Cheng Li on 17 April 2016.
Table 2 Morphological comparisons for four species of the genus Liurana
Liurana vallecula sp. nov. L. alpina L. medogensis L. xizangensis
SVL (mm) 14.6 (1
Table 2 Morphological comparisons for four species of the genus Liurana
Liurana vallecula sp. nov. L. alpina L. medogensis L. xizangensis
SVL (mm) 14.6 (1♂)
20.4 (1♀)
23.2–24.9 (3♂)
25.5 (1♀)
13.1–19.0 (3♂) 20.6–22.5 (9♂)
29.4–30.5 (2♀)
HL/HW <100% >100% >100% >100%
Tibiotarsal articulation Beyond tip of snout Reaching anterior corner of
eye
Reaching anterior corner of
eye
Reaching anterior corner of
eye
Flat tubercles on belly Absent Absent Absent Present
Tubercles around cloaca Present Absent Indistinct Present
Ventral pattern Thin, marbled-patterns Thin, marbled-patterns Thick , vermiculated stripes Thin, marbled-patterns
Morphological abbreviations are listed in the methods.
)
20.4 (1
Table 2 Morphological comparisons for four species of the genus Liurana
Liurana vallecula sp. nov. L. alpina L. medogensis L. xizangensis
SVL (mm) 14.6 (1♂)
20.4 (1♀)
23.2–24.9 (3♂)
25.5 (1♀)
13.1–19.0 (3♂) 20.6–22.5 (9♂)
29.4–30.5 (2♀)
HL/HW <100% >100% >100% >100%
Tibiotarsal articulation Beyond tip of snout Reaching anterior corner of
eye
Reaching anterior corner of
eye
Reaching anterior corner of
eye
Flat tubercles on belly Absent Absent Absent Present
Tubercles around cloaca Present Absent Indistinct Present
Ventral pattern Thin, marbled-patterns Thin, marbled-patterns Thick , vermiculated stripes Thin, marbled-patterns
Morphological abbreviations are listed in the methods.
)
23.2–24.9 (3
Table 2 Morphological comparisons for four species of the genus Liurana
Liurana vallecula sp. nov. L. alpina L. medogensis L. xizangensis
SVL (mm) 14.6 (1♂)
20.4 (1♀)
23.2–24.9 (3♂)
25.5 (1♀)
13.1–19.0 (3♂) 20.6–22.5 (9♂)
29.4–30.5 (2♀)
HL/HW <100% >100% >100% >100%
Tibiotarsal articulation Beyond tip of snout Reaching anterior corner of
eye
Reaching anterior corner of
eye
Reaching anterior corner of
eye
Flat tubercles on belly Absent Absent Absent Present
Tubercles around cloaca Present Absent Indistinct Present
Ventral pattern Thin, marbled-patterns Thin, marbled-patterns Thick , vermiculated stripes Thin, marbled-patterns
Morphological abbreviations are listed in the methods.
)
25.5 (1
Table 2 Morphological comparisons for four species of the genus Liurana
Liurana vallecula sp. nov. L. alpina L. medogensis L. xizangensis
SVL (mm) 14.6 (1♂)
20.4 (1♀)
23.2–24.9 (3♂)
25.5 (1♀)
13.1–19.0 (3♂) 20.6–22.5 (9♂)
29.4–30.5 (2♀)
HL/HW <100% >100% >100% >100%
Tibiotarsal articulation Beyond tip of snout Reaching anterior corner of
eye
Reaching anterior corner of
eye
Reaching anterior corner of
eye
Flat tubercles on belly Absent Absent Absent Present
Tubercles around cloaca Present Absent Indistinct Present
Ventral pattern Thin, marbled-patterns Thin, marbled-patterns Thick , vermiculated stripes Thin, marbled-patterns
Morphological abbreviations are listed in the methods.
)13.1–19.0 (3
Table 2 Morphological comparisons for four species of the genus Liurana
Liurana vallecula sp. nov. L. alpina L. medogensis L. xizangensis
SVL (mm) 14.6 (1♂)
20.4 (1♀)
23.2–24.9 (3♂)
25.5 (1♀)
13.1–19.0 (3♂) 20.6–22.5 (9♂)
29.4–30.5 (2♀)
HL/HW <100% >100% >100% >100%
Tibiotarsal articulation Beyond tip of snout Reaching anterior corner of
eye
Reaching anterior corner of
eye
Reaching anterior corner of
eye
Flat tubercles on belly Absent Absent Absent Present
Tubercles around cloaca Present Absent Indistinct Present
Ventral pattern Thin, marbled-patterns Thin, marbled-patterns Thick , vermiculated stripes Thin, marbled-patterns
Morphological abbreviations are listed in the methods.
)20.6–22.5 (9
Table 2 Morphological comparisons for four species of the genus Liurana
Liurana vallecula sp. nov. L. alpina L. medogensis L. xizangensis
SVL (mm) 14.6 (1♂)
20.4 (1♀)
23.2–24.9 (3♂)
25.5 (1♀)
13.1–19.0 (3♂) 20.6–22.5 (9♂)
29.4–30.5 (2♀)
HL/HW <100% >100% >100% >100%
Tibiotarsal articulation Beyond tip of snout Reaching anterior corner of
eye
Reaching anterior corner of
eye
Reaching anterior corner of
eye
Flat tubercles on belly Absent Absent Absent Present
Tubercles around cloaca Present Absent Indistinct Present
Ventral pattern Thin, marbled-patterns Thin, marbled-patterns Thick , vermiculated stripes Thin, marbled-patterns
Morphological abbreviations are listed in the methods.
)
29.4–30.5 (2
Table 2 Morphological comparisons for four species of the genus Liurana
Liurana vallecula sp. nov. L. alpina L. medogensis L. xizangensis
SVL (mm) 14.6 (1♂)
20.4 (1♀)
23.2–24.9 (3♂)
25.5 (1♀)
13.1–19.0 (3♂) 20.6–22.5 (9♂)
29.4–30.5 (2♀)
HL/HW <100% >100% >100% >100%
Tibiotarsal articulation Beyond tip of snout Reaching anterior corner of
eye
Reaching anterior corner of
eye
Reaching anterior corner of
eye
Flat tubercles on belly Absent Absent Absent Present
Tubercles around cloaca Present Absent Indistinct Present
Ventral pattern Thin, marbled-patterns Thin, marbled-patterns Thick , vermiculated stripes Thin, marbled-patterns
Morphological abbreviations are listed in the methods.
)
HL/HW <100% >100% >100% >100%
Tibiotarsal articulation Beyond tip of snout Reaching anterior corner of eye Reaching anterior corner of eye Reaching anterior corner of eye
Flat tubercles on belly Absent Absent Absent Present
Tubercles around cloaca Present Absent Indistinct Present
Ventral pattern Thin, marbled-patterns Thin, marbled-patterns Thick, vermiculated stripes Thin, marbled-patterns
Morphological abbreviations are listed in the methods.
Table 3 Uncorrected genetic distances of the COI fragment obtained (570 bp) among Liurana species
1L. sp. nov. YPX47504 –
2L. sp. nov. YPX47527 0.00877 –
3L. medogensis KIZ 010955 0.22191 0.222
4L. alpina KIZ 07357 0.22029 0.21666 0.13394 –
5L. alpina KIZ 011140 0.22632 0.22281 0.13207 0.00178 –
6L. alpina KIZ 011141 0.22216 0.21853 0.13211 0.00183 0
7L. xizangensis KIZ 06707 (Locality #2) 0.2129 0.21297 0.14312 0.09541 0.09345 0.09358 –
8L. xizangensis KIZ 09956 (Locality #2) 0.2129 0.21297 0.14312 0.09541 0.09345 0.09358 0
9L. xizangensis KIZ 012705 (Locality #2) 0.2129 0.21297 0.14312 0.09541 0.09345 0.09358 0 0
10 L. xizangensis KIZ 012706 (Locality #2) 0.2129 0.21297 0.14312 0.09541 0.09345 0.09358 0 0 0
11 L. xizangensis KIZ 011104 (Locality #2) 0.2129 0.21297 0.14312 0.09541 0.09345 0.09358 0 0 0 0
12 L. xizangensis KIZ 012707 (Locality #2) 0.21107 0.21114 0.14495 0.09725 0.09535 0.09541 0.00183 0.00183 0.00183 0.00183 0.00183 –
13 L. xizangensis KIZ 012704 (Topotype) 0.20372 0.20379 0.12661 0.08624 0.08436 0.0844 0.02752 0.02752 0.02752 0.02752 0.02752 0.02936 –
14 L. xizangensis KIZ 014046 (Locality #1) 0.20187 0.20195 0.12844 0.08807 0.0862 0.08624 0.02936 0.02936 0.02936 0.02936 0.02936 0.03119 0.00183 –
Paratype: KIZ014106, adult male, from the side of Baimaxilu
River (a tributary of the Yarlung Zangbo River) near Maniweng
(N29.2667°, E95.1667°, elevation 850 m a.s.l.), Beibeng,
Medog County, southeastern Tibet, China. Collected by Ke
Jiang and Yu-Fan Wang on 29 April 2016.
Diagnosis: Liurana vallecula sp. nov. is assigned to the
genus Liurana by its phylogenetic position and the following
morphological characters: (1) body size small (SVL 14.6–20.4 mm,
n=2); (2) tips of fingers and toes not expanded, (3) grooves absent
on tips of fingers and toes; (4) webbing absent on all digits; (5)
metacarpal tubercles and metatarsal tubercles absent; (6) tarsal
fold absent; and (7) vocal sac and vocal sac openings absent.
Liurana vallecula sp. nov. can be distinguished from
all currently recognized congeners by a combination of the
following morphological characters: (1) head wider than long
HW/HL 108%–113%; (2) tympanum distinct, large; (3) hind limbs
long, with tibiotarsal articulation reaching beyond tip of snout
when adpressed; (4) flat tubercles present on ventral surface of
body; (5) small tubercles present on cloacal region; and (6) dark
brown marbled pattern present on ventral surface of body.
Description of holotype: Body size small, SVL 20.4 mm.
Head wider than long (HW/HL=108%); snout rounded, slightly
projecting over lower jaw; canthus rostralis distinct, relatively
sharp; nostril closer to tip of snout than to eye; loreal region
slightly concave and oblique; eye relatively large (ED/HL=40%);
internarial distance larger than interorbital distance and width
of upper eyelid; tympanum oval, concealed under skin,
with distinct, elevated rim, slightly less than half of eye
diameter (TD/ED=45%); supratympanic fold distinct, extending
posterior-inferiorly to area above forelimb insertion; pair of
distinct skin folds present on head from posterior orbit dorsally
to pectoral region, symmetrical along vertebral line, extending
toward medial line posteriorly.
178 www.zoores.ac.cn
1
Figure 2 Phylogenetic relationships among Liurana species based on maximum likelihood and Bayesian analyses of one
mitochondrial gene (COI) and three nuclear genes (Rag1,Rod, and Tyr)
Maximum likelihood bootstrap and Bayesian posterior probability values are given at all nodes (in such order respectively), except the internal nodes within L.
xizangensis from Locality #2, which have short branch lengths and resemble polytomy.
Figure 3 Holotype of Liurana vallecula sp. nov. in life (adult female, KIZ014083)
A: Dorsolateral view; B: Ventral view; C: Ventral close-up of hand; D: Ventral close-up of feet. Photos by Yu-Fan Wang.
Zoological Research 40(3): 175–184, 2019 179
Forelimb slender; forearm and hand length less than half of
body length (LAHD/SVL=44%); fingers compressed vertically,
tips rounded but not expanded, transverse grooves absent,
relative finger lengths I<II<IV<III; subarticular tubercles absent;
three metacarpal tubercles present, flat and indistinct. Hind
limb slender, tibiotarsal articulation reaching beyond tip of
snout when adpressed; heels overlapped when flexed and
held perpendicular to body; tibia length larger than half
of body length (TL/SVL=58%); toes compressed vertically,
tips slightly expanded, transverse grooves absent; relative
toe lengths I<II<V<III<IV; toe webbing absent; subar ticular
tubercles indistinct; inner metatarsal tubercle oval, indistinct;
outer metatarsal tubercle indistinct; tarsal fold absent.
Relatively weak, discontinuous folds present on dorsolateral
body from shoulder to about two thirds of trunk on each
side of body; single skin fold present along vertebrate from
snout to vent, much weaker than discontinuous folds laterally;
dorsal surface relatively rough, tubercles randomly scattered
on dorsal and lateral head, body, and limbs as well as around
cloaca; tubercles much finer on ventrolateral region of body.
Ventral head, body, and limbs mostly smooth, except several
flat tubercles on base of ventral thigh and small tubercles
on ventral surface of tarsus and metatarsus. Vomerine
teeth absent; tongue large, elongated oval, deeply notched
posteriorly, small papillae scattered on tongue.
Coloration of holotype in life: The dorsal surfaces of the
head and body are reddish brown. Dark brown streaks and
marble patterns are present on dorsal head, body, and limbs,
including a transverse streak between orbit on dorsal surface of
head, a X-shaped pattern on pectoral region of dorsum, irregular
marble patterns on lateral body, and transverse streaks across
dorsal limbs (more distinct on the dorsal hind limbs). The lower
parts of canthus rostralis and supratympanic fold are blackish
brown. The ventrolateral surface of the body is dark brown, with
small white spots scattered across. Ventral surfaces of limbs,
head, and body are light grayish brown; white marble patterns are
present on ventral surfaces of head, body, and ventral forelimbs,
and ventrolateral surfaces of thigh. The white marble patterns are
finer and much smaller on ventral head comparing to ventral body,
giving a speckle impression; white marble patterns on ventral
body are mostly interconnected. A few smaller white spots are
also present on ventral thigh, tibia, and femur.
Coloration of holotype in preservative: Ornamentation
patterns remain after preservation. However, coloration
changes after preservation, include: (1) dorsal surfaces of
head, limbs, and body become grayish brown, with dark gray
patches; (2) lower parts of canthus rostralis and supratympanic
fold, transverse bands on dorsal limbs, as well as lower part
of lateral body become dark gray; (3) ventral surfaces of throat
and limbs become brown, with grayish white spots.
Variation: Morphometric variations of the type series are
shown in Table 4. Most external morphological characters
are identical between the two individuals, but the paratype is
smaller than the holotype (SVL 14.6 mm in paratype male vs.
20.4 mm in holotye female), as well as in having rather paler dorsal
coloration and more dense spots on ventral body. No secondary
sexual characters, such as vocal sac or nuptial pad, are observed
in the paratype male, but a single black testicle was observed
on the left side of the male paratype, which is oval shaped and
relatively large, with a longitudinal length of about 1.5 mm.
Ecological and natural history notes: Liurana vallecula
sp. nov. is a terrestrial, leaf-litter specialist, inhabiting the
forest floor of tropical broad-leaf forest at low elevations (below
1 000 m a.s.l.) near Yarlung Zangbo River and its immediate
tributaries. The female holotype had about five immature eggs
in the left ovary, which were well developed and relatively large.
Distribution: Currently the new species is known only from the
type localities of Xirang and Maniweng of Beibeng, Medog County,
Nyingchi Prefecture, Tibet, China. The new species likely inhabits
other nearby regions in southern Tibet (see Discussion below).
Etymology: The specific epithet of the new species, “vallecula
means “valley inhabitor”, in reference to the habitat of this
species in the lower river valley of Yarlung Zangbo Grand
Canyon. We suggest Valley Papilla-tongued Frog as its English
common name and He Gu She Tu Wa () as its
Chinese common name.
Comparisons: Liurana vallecula sp. nov. differs from
the three congeners by having a much wider head (HW/HL
>100% vs. <100%) and longer hindlimbs (tibiotarsal ar ticulation
reaching beyond tip of snout when adpressed vs.reaching only
anterior corner of eye when adpressed). In addition, Liurana
vallecula sp. nov. can be further distinguished from L. alpina
and L. xizangensis by having a smaller body size (SVL 14.6
mm in male, 20.4 mm in female vs. SVL 23.2–24.9 mm in
males, 25.5 mm in female for L. alpina; 20.6–24.5 mm in
males, 29.4–30.5 mm in females for L. xizangensis); and from
L. medogensis by its different ventral pattern (small, marbled
patterns or speckles vs. broad, vermiculated stripes).
DISCUSSION
Liurana medogensis from Southern Tibet
Recently, Borah et al. (2013) reported L. medogensis from
the eastern part of Southern Tibet, which they recognized as
Limnonectes (Taylorana)medogensis in the study (localities
1 and 2 in light blue; Figure 1) (all abbreviations of “L.” in
this paragraph refer to Liurana, not Limnonectes). Although
one of the reported specimens (ZSI a11549) resembles the
external appearance of L. medogensis based on the figure
in the manuscript (Borah et al., 2013; Figure 1), according to
the description, it possesses morphological characteristics that
differ from the diagnosis of the genus Liurana, including having
a distinct vomerine ridge, prominent vomerine teeth, and
rudimentary webbing on toes (vs. vomerine ridge, vomerine
teeth, and toe webs absent in Liurana) (Fei et al., 2009, 2010;
Yan et al., 2016). Fur thermore, this specimen was collected at
a much higher elevation (2000–2 500 m a.s.l.) than the known
180 www.zoores.ac.cn
range for L. medogensis (about 1 400 m a.s.l.). For the second
reported specimen of L. cf. medogensis (BMHE a0081), based
on the description provided by the authors, it matches the
morphological diagnosis of our new species, Liurana vallecula,
including having a wide head (HW/HL>100%), but lacks the
broad, vermiculate patterns on the ventral surface of the
body (Borah et al., 2013). As we cannot examine these
specimens or obtain their genetic data, we cannot confirm the
taxonomic status of these two specimens reported by Borah
et al. (2013). Future taxonomic studies are needed to gain a
better understanding of Liurana diversity in this region.
Table 4 Measurements (in mm) of type series of Liurana
vallecula sp. nov.
Number Status Sex SVL HL HW SL IND IOD UEW
KIZ014083 Holotype Female 20.4 7.3 7.9 3.2 3.0 2.2 2.0
Ratio to 37.2 38.7 15.6 14.5 10.7 9.8
SVL (%)
KIZ014106 Paratype Male 14.6 5.6 6.3 2.5 2.3 1.5 1.5
Ratio to SVL (%) 38.1 43.2 17.5 15.7 10.4 10.0
Number ED TD LAHL LAD HAL FML TL TFL FL
KIZ014083 2.9 1.3 9.0 1.5 5.1 10.9 11.8 17.4 11.6
Ratio to SVL (%) 14.1 6.4 44.1 7.3 24.7 53.3 57.6 85.3 57.0
KIZ014106 2.0 1.0 6.3 1.1 3.3 7.7 8.5 11.2 7.1
Ratio to SVL (%) 13.7 43.6 7.4 22.4 52.7 58.4 76.8 49.0 43.6
See methods section for abbreviations. –: Not applicable.
Ecotypes
Liurana diversity can be divided into two major ecotypes:
the alpine ecotype that inhabits cool, moist mixed forests at
2 000–3 000 m a.s.l. (including L. alpina and L. xizangensis),
and the tropical ecotype that inhabits low-elevation tropical
rainforest below 2000 m a.s.l. (including L. medogensis
and Liurana vallecula). For the first ecotype, coloration of
individuals is highly variable, ranging from uniform bright
reddish orange to marbled purplish gray. Frogs of this ecotype
live under thick layers of moss on fallen tree trunks or rocks
along the forest edge. The second ecotype consists of leaf litter
specialists that inhabit the forest floor under pristine tropical
rainforest. Coloration of this ecotype is much more cryptic,
ranging from a single, wide, brownish orange dorsal stripe to
dark brown with darker marbling (Figures 4, 5).
Even though L. medogensis and Liurana vallecula both
belong to the second ecotype, they do not form a monophyletic
group according to our genetic data (Figure 2). Future
ecological studies are needed to further differentiate the
ecological niche of each species and investigate the evolution
of ecotypes in the genus Liurana.
Reproductive biology
Although the reproductive biology, particularly the direct
developing reproductive mode, has long been documented
within the family Ceratobatrachidae (Brown & Alcala, 1982;
Siler et al., 2010), little is known about the reproductive biology
of Liurana specifically. Considering the Tibetan Liurana species
as Platymantis at that time, Hu et al. (1987) first commented on
the reproductive mode of the Tibetan species and suggested
that they may go through direct development, similar to other
Platymantis species. It was only until 2010 when the eggs
of Liurana species in the alpine ecotype from Tibet were first
collected (Li et al., 2010). According to Li et al. (2010), eggs of
the unidentified Liurana species from 62K in the Medog County
(confirmed as L. xizangensis according to Yan et al. (2016) and
the present study) were large (with a diameter of about 3.5 mm)
and the clutch size was small. These egg characteristics are
similar to those of direct development in the same family (Siler
et al., 2010). Similar to Li et al. (2010), we also observed eggs
in a female L. xizangensis (KIZ014153) from the same locality,
where the female displayed 14 and 16 yellowish white eggs in
the left and right oviduct, respectively, though not all were fully
developed (Figure 6).
More recently, Borah et al. (2013) provided information
regarding the reproductive biology of the tropical ecotype
of Liurana from Southern Tibet. Embryos displayed
characteristics of the direct developing species in the family
Ceratobatrachidae, including having a few embryos, large
individual embryo size, and whitish coloration (Borah et al.,
2013). Based on these similarities, the authors claim that
this Liurana species reproduces through direct development
without the larval stage (Borah et al., 2013). However, such
conclusions on direct development in Liurana are still based
on indirect inferences of egg characteristics. Therefore, we
recommend future studies focus on the reproductive biology of
the genus to clarify the reproductive mode of Liurana.
Acoustic signals
In addition to the differences in general habitat preferences and
external morphology, the two ecotypes of Liurana also differ in
their acoustic signals. In fact, extensive acoustic signals have
been observed in the alpine ecotype only (Fei et al., 2009; Hu et
al., 1987; present study). Such differences in acoustic signals
may be explained by the relative cost-benefit ratios of calling
in each specific environment (Ryan, 1988). Future ecological
and behavioral studies are needed to confirm this hypothesis
in the field and investigate the communication strategies of the
tropical ecotype.
Evolution
Frogs of the genus Liurana are endemic to eastern Himalaya,
and all are found in the Yarlung Zangbo Drainage in Southern
Tibet (Figure 1). Based on the available distribution data, L.
xizangensis is distributed on the eastern side of the Yarlung
Zangbo River, whereas L. alpina is found on the western
side of the river. As the two species are found in cool
environments at high elevation only (>2000 m a.s.l.), the hot
tropical valley of the Yarlung Zangbo River may serve as a
dispersal barrier, thereby shaping the genetic diversity and
facilitating the speciation processes of the two congeners. This
hypothesis is partly supported by our genetic data, where
closely distributed populations from two sides of the river
possessed higher genetic divergence than further distributed
populations from the same side (Table 3). Future studies
should expand population sampling of the two species along
both sides of Yarlung Zangbo River to examine its role in the
evolution of Liurana.
Zoological Research 40(3): 175–184, 2019 181
2
Figure 4 Comparisons of dorsal (1) and ventral (2) views of live individuals of Liurana xizangensis (A and B), L. alpina (C and D), L.
medogensis (E), and Liurana vallecula sp. nov. (F). Photos by Ke Jiang, Kai Wang, and Yu-Fan Wang
A: Dorsolateral view; B: Ventral view; C: Ventral close-up of hand; and D: Ventral
close-up of feet. Photos by Yu-Fan Wang.
Figure 4 Comparisons of dorsal (column 1 and 3) and ventral (column 2 and 4) views
of live individuals of Liurana xizangensis (A), L. alpina (B), L. medogensis (C), and
Liurana vallecula sp. nov. (D). Photos by Ke Jiang, Kai Wang, and Yu-Fan Wang
Figure 5 Habitat of Liurana vallecula sp. nov. (A) (Xirang, elevation 550 m a.s.l.), L.
medogensis (B) (Xigong Lake, elevation 1 300 m a.s.l.), L. alpina (C) (Dayandong,
elevation 3 000 m a.s.l.), and L. xizangensis (D) (62 K, elevation 2 800 m a.s.l.) in
Medog County, southeastern Tibet, China, respectively. Photos by Ke Jiang, Kai
Wang, and Shuai Wang
Figure 6 Gravid female (A) and developing eggs through transparent abdomen (B) of
Figure 5 Habitat of Liurana vallecula sp. nov. (A) (Xirang, elevation 550 m a.s.l.), L. medogensis (B) (Xigong Lake, elevation 1 300 m a.s.l.),
L. alpina (C) (Dayandong, elevation 3000 m a.s.l.), and L. xizangensis (D) (62K, elevation 2800 m a.s.l.) in Medog County, southeastern
Tibet, China, respectively. Photos by Ke Jiang, Kai Wang, and Shuai Wang
182 www.zoores.ac.cn
A: Dorsolateral view; B: Ventral view; C: Ventral close-up of hand; and D: Ventral
close-up of feet. Photos by Yu-Fan Wang.
Figure 4 Comparisons of dorsal (column 1 and 3) and ventral (column 2 and 4) views
of live individuals of Liurana xizangensis (A), L. alpina (B), L. medogensis (C), and
Liurana vallecula sp. nov. (D). Photos by Ke Jiang, Kai Wang, and Yu-Fan Wang
Figure 5 Habitat of Liurana vallecula sp. nov. (A) (Xirang, elevation 550 m a.s.l.), L.
medogensis (B) (Xigong Lake, elevation 1 300 m a.s.l.), L. alpina (C) (Dayandong,
elevation 3 000 m a.s.l.), and L. xizangensis (D) (62 K, elevation 2 800 m a.s.l.) in
Medog County, southeastern Tibet, China, respectively. Photos by Ke Jiang, Kai
Wang, and Shuai Wang
Figure 6 Gravid female (A) and developing eggs through transparent abdomen (B) of
Figure 6 Gravid female (A) and developing eggs through transparent abdomen (B) of Liurana xizangensis from 62K, Medog County,
Tibet, China. Photos by Ke Jiang
Conservation
As micro-endemic habitat specialists, Liurana species are
threatened by habitat destruction in southern Tibet. Based
on our continuous field surveys since 2012, considerable
habitat destruction has been observed at 62K in Medog
County, which is one of only three known localities for L.
xizangensis. Unregulated infrastructure developments have
destroyed the mossy fields along the forest edges, streams,
and wetlands, which constitute the core habitats not only for
L. xizangensis, but also other micro-endemic anuran species
such as Scutiger wuguanfui and S. spinosus (Jiang et al.,
2016). Similarly, continuous tourist development and road
construction along the hiking trail of Medog pose serious
threats to habitat at the only known locality of L. alpina.
Therefore, we recommend that local authorities and regional
governments take habitat conservation into account when
making developmental decisions, and we urge law enforcement
agencies to enforce the existing environmental regulations
of construction projects in the region, particularly in Medog
County.
COMPETING INTERESTS
The authors declare that they have no competing interests.
AUTHORS’ CONTRIBUTIONS
K.W., K.J., Y.W., and C.L. conducted fieldworks in Tibet. K.J. and K.W.
collected data and conducted the analyses. J.C. supervised the analyses.
K.J. and K.W. wrote the manuscript with the other authors’ inputs. K.W.
submitted molecular data to GenBank. K.J., K.W., and J.C. revised
the manuscript. All authors read and approved the final version of the
manuscript.
NOMENCLATURAL ACTS REGISTRATION
The electronic version of this article in portable document format will
represent a published work according to the International Commission on
Zoological Nomenclature (ICZN), and hence the new names contained
in the electronic version are effectively published under that Code from
the electronic edition alone (see Articles 8.5–8.6 of the Code). This
published work and the nomenclatural acts it contains have been registered
in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs
(Life Science Identifiers) can be resolved and the associated information can
be viewed through any standard web browser by appending the LSID to the
prefix http://zoobank.org/.
Publication LSID:
urn:lsid:zoobank.org:pub: 8D6905B9-0AEA-4091-BCA6-A7C849BD1848.
Liurana vallecula LSID:
urn:lsid:zoobank.org:act: 2AD61C3A-E263-46CC-A919-399992A558C8.
ACKNOWLEDGEMENTS
We thank Mr. Tao Liang, Mr. Duan You, Mr. Ya-Di Huang, and Mr. Qi Liu for
their help in the field; Ms. Jie-Qiong Jin for her help in molecular lab works;
and Prof. Liang Fei and Prof. Chang-Yuan Ye (Chengdu Institute of Biology,
Chinese Academy of Sciences) for providing valuable information.
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APPENDIX I
Specimens examined for comparison. Museum abbreviations follow those
from the methods section.
Liurana alpina (n=4): KIZ011140–42 (males), KIZ07358 (Dayandong (type
locality)) Medog County, Tibet, China.
Liurana medogensis (n=3): KIZ010955, 05886, 05587 (males), (Xigong Lake
(type locality)) Medog County, Tibet, China.
Liurana xizangensis (n=11): KIZ012704, 014046 (males), (Tongmai (close to
type locality “Yi’ong”, =Yigong)) Bomê, Tibet, China; KIZ06707, 09954–56,
011105–06, 012706 (males); KIZ09953, 014153 (females), 62K, Medog
County, Tibet, China.
APPENDIX II GenBank sequences used and accession Nos. of novel sequences
Gene and GenBank accession No.
Genus Species Voucher or tissue number Locality COI Rag1 Rod Tyr
Alcalus tasanae CAS232349 Myanmar KU243096 KU243106 KU243116
Alcalus tasanae CAS247243 Myanmar KU243097 KU243107 KU243117
Alcalus tasanae THNHM20534 Myanmar KU243098 KU243108 KU243118
Ceratobatrachus guentheri VUB1017(SR5543) Solomon Islands AY883979 DQ347272 DQ347391 DQ347179
Cornufer boulengeri BPBM22329 Papua New Guinea HQ844999 KP298309 KP298385
Ingerana tenasserimensis CAS205064/ TADP918 Myanmar/Thailand KR087736 DQ347258 AY322236 KP298327
Limnonectes limgorgi VUB1218 Laos DQ347286 DQ347407 DQ347194
Liurana alpina KIZ011140 Medog, Tibet, China MK462138 KU243094 KU243104 KU243114
Liurana alpina KIZ011141 Medog, Tibet, China MK462139 KU243095 KU243105 KU243115
Liurana alpina KIZ07357 Medog, Tibet, China MK462137
Liurana medogensis KIZ010955 Medog, Tibet, China MK462136 KU243103 KU243113
Liurana vallecula KIZ014083 Medog, Tibet, China MK462134 MK315115 MK462150 MK462155
Liurana vallecula KIZ014106 Medog, Tibet, China MK462135 MK462151 MK462156
Liurana xizangensis KIZ06707 Medog, Tibet, China MK462140 KU243092 KU243101 KU243111
Liurana xizangensis KIZ09956 Medog, Tibet, China MK462141
Liurana xizangensis KIZ011104 Medog, Tibet, China MK462144
Liurana xizangensis KIZ012705 Medog, Tibet, China MK462142
Liurana xizangensis KIZ012706 Medog, Tibet, China MK462143
Liurana xizangensis KIZ012707 Medog, Tibet, China MK462145
Liurana xizangensis KIZ012704 Medog, Tibet, China MK462146
Liurana xizangensis KIZ014046 Medog, Tibet, China MK462147
Platymantis hazelae TNHC62160 & CMNH-RSK3918 Phillipines DQ347248 DQ347369 DQ347153
–: Not available.
184 www.zoores.ac.cn
... Before 2019, only three species were recognized in Liurana, including L. alpina Huang and Ye, 1997, L. medogensis Fei, Ye, and Huang, 1997, and L. xizangensis (Hu, 1977, all of which were found in Medog, Xizang, China (Fei et al., 1997;Huang and Ye, 1997;Sichuan Biological Research Institute, 1977). In 2019, a new species of the genus Liurana, i.e., L. vallecula Jiang, Wang, Wang, Li, and Che, 2019 was described from Medog, Xizang, China, based on morphological and molecular data (Jiang et al., 2019). Also in 2019, three other species of this genus, i.e., L. himalayana Saikia andSinha, 2019, L. indica Saikia andSinha, 2019 and L. minuta Saikia and Sinha, 2019 were described based on only morphological data (Saikia and Sinha, 2019). ...
... However, the amphibian diversity in this area has not yet been fully explored. In recent years, some new amphibian species in this region have been reported, such as L. vallecula (Jiang et al., 2019); Jingophrys zhoui (Shi, Zhang, Xie, Jiang, Liu, Ding, Luan, and Wang, 2020) and J. yeae (Shi, Zhang, Xie, Jiang, Liu, Ding, Luan, and Wang, ...
... The genus Liurana is an understudied yet fascinating endemic group from the East Himalayan region (Jiang et al., 2019). , 1977), and knowledge on the ecology, behaviour, evolution, and other aspects of these species is still lacking. ...
... The following literatures were consulted for comparative data of all the known congeners of the genus: Hu (1977); Huang and Ye (1997); Fei et al., (1997); Borah et al., (2013), Fei et al., (2012), Yan et al., (2016), Roy et al., (2018) and Jiang et al., 2019. ...
... large and distinct; near to eye), snout shape appears like an arrow head (vs. none), On the Liurana (Anura: Ceratobatrachidae) of India with the description of three new species from Talley ... Table 2. Comparative data of morphological characters for L. alpina, L. medogensis, L. vallecula and L. xizangensis based on Hu (1977), Hunag & Ye (1997), Fei et al., (1997), Borah et al., (2013), Fei et al., (2012) and Fei and Ye (2016), Jiang et al., (2019 lighter blotch), belly with marbled markings (vs. none), fourth toe less than one-third of SVL (vs. ...
... While reporting L. medogensis from India, Borah et al., (2013) mentioned the diagnostic characters of Liurana genus (then sub-genus of Ingerana) from Ingerana by the presences of grooves, enlarged toe pads, extension of webbings and presence of lingual papilla. However, contradicting Borah et al., (2013), Yan et al., (2016) and Jiang et al., (2019) reported about the absence of grooves on the finger and toe tips and absence of interdigital webbings. The genus Liurana was erected by Dubois (1987) where he had highlighted the following characteristics: (1) fingertips and toe-tips not dilated, without a clearly differentiated circummarginal groove, (2) webbing absent or reduced and (3) lingual papilla present. ...
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